Microorganisms and methods for the biosynthesis of adipate, hexamethylenediamine and 6-aminocaproic acid

This application is filed in U.S. Provisional Patent Application No. 61/176,196 filed May 7, 2009, U.S. Provisional Patent Application No. 61/219,365 filed June 22, 2009, U.S. Provisional Patent Application No. 61/244,844 filed on September 29, 2009, U.S. Provisional Patent Application No. 61/246,973 filed on September 30, 2009 61/247,533, each of which is incorporated herein by reference in its entirety.

The present invention relates generally to biosynthetic processes, and in particular to organisms capable of biosynthesizing adipate, hexamethylene diamine, 6-aminocaproic acid, and caprolactam.

Adipic acid, a dicarboxylic acid, has a molecular weight of 146.14. It can be used to produce nylon 6,6, a linear polyamide made by condensing hexamethylene diamine and adipic acid. It is used to produce various types of fibers. Other uses of adipic acid include its use in plasticizers, unsaturated polyesters, and polyester polyols. Further uses include polyurethanes, for the production of lubricating ingredients, and food ingredients as flavorants and gelling aids.

Historically, adipic acid was prepared from a wide variety of fats using oxidation. Some current processes for adipic acid synthesis use an excess of strong nitric acid, a mixture of cyclohexanone which is the ketone or K component and cyclohexanol which is the alcohol or A component of KA oil or pure cyclohexanol. depends on the oxidation of There are several variations of this main component, with different routes of production of KA or cyclohexanol.

For example, phenol represents an alternative raw material in KA oil production, and a process for the synthesis of adipic acid from phenol has been described. Other versions of this process tend to use oxidizing agents other than nitric acid, such as hydrogen peroxide, air, or oxygen.

In addition to hexamethylenediamine (HMDA) being used in the production of nylon-6,6, as described above, it is also a monomer feedstock used in the production of polyurethane. It is also used to make methylene diisocyanate. This diamine also serves as a crosslinking agent in epoxy resins.

HMDA is currently produced by hydrogenation of adiponitrile.

Caprolactam is an organic compound that is a lactam of 6-aminohexanoic acid (ε-aminohexanoic acid, 6-aminocaproic acid). It can alternatively be considered a cyclic amide of caproic acid. One use of caprolactam is as a monomer in the production of nylon-6. Caprolactam can be synthesized from cyclohexanone via an oximation process using hydroxylammonium sulfate followed by catalytic rearrangement using a Beckmann rearrangement process step.

Methods for efficiently producing commercial quantities of compounds such as hexamethylene diamine, 6-aminocaproic acid, levulinic acid, caprolactam, and the like are described herein and include associated advantages.

The present invention provides non-naturally occurring microbial organisms having a 6-aminocaproic acid, caprolactam, or hexamethylene diamine pathway. The microbial organism contains at least one exogenous nucleic acid encoding an enzyme in each of the 6-aminocaproic acid, caprolactam, hexamethylene diamine, or levulinic acid pathways. The invention further provides a method for producing 6-aminocaproic acid, caprolactam, or hexamethylene diamine. The method can include culturing a microbial organism that produces 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid, wherein the microbial organism produces 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. or at least one exogenous nucleic acid encoding a levulinic acid pathway enzyme in an amount sufficient to produce the respective product, for producing 6-aminocaproic acid, caprolactam, hexamethylene diamine, or levulinic acid. Expressed under the conditions and for a sufficient period of time.

<figref num="1">FIG. 2 shows an exemplary pathway for adipate degradation in peroxisomes of Penicillium chrysogenum.</figref><figref num="2">FIG. 2 illustrates an exemplary route for adipate formation via the reverse decomposition pathway. Several options are offered for the final conversion of adipyl-CoA to adipate.</figref><figref num="3">FIG. 3 shows an exemplary route for adipate formation via the 3-oxoadipate pathway.</figref><figref num="4">FIG. 3 shows the 3-oxoadipate pathway for adipate synthesis and the analogous enzymatic chemistry of the last three steps of the reductive TCA cycle.</figref><figref num="5">FIG. 2 shows an exemplary route for the synthesis of adipic acid from glucose via cis,cis-muconic acid. Biosynthetic intermediates (abbreviations): D-erythrose 4-phosphate (E4P), phosphoenolpyruvate (PEP), 3-deoxy-D-arabinoheptulosonic acid 7-phosphate (DAHP), 3-dehydroquina acid (DHQ), 3-dehydroshikimic acid (DHS), protocatechuic acid (PCA). Enzymes (encoding genes) or reaction conditions: (a) DAHP synthase (aroFFBR), (b) 3-dehydroquinate synthase (aroB), (c) 3-dehydroquinate dehydratase (aroD), (d) DHS dehydratase (aroZ) , (e) protocatechuate decarboxylase (aroY), (f) catechol 1,2-dioxygenase (catA), (g) 10%Pt/C, H₂, 3400kPa, 25°C. Figure taken from Niu et al., Biotechnol. Prog. 18:201-211 (2002)).</figref><figref num="6">FIG. 2 shows an exemplary route for adipate synthesis via alpha-ketoadipate using alpha-ketoglutarate as a starting point.</figref><figref num="7">FIG. 2 shows an exemplary route for the synthesis of adipate using lysine as a starting point.</figref><figref num="8">FIG. 2 shows an exemplary caprolactam synthesis route using adipyl-CoA as a starting point.</figref><figref num="9">FIG. 2 shows an exemplary adipate synthesis route using alpha-ketoadipate as a starting point.</figref><figref num="10">FIG. 3 shows exemplary routes from succinyl-CoA and acetyl-CoA to hexamethylene diamine (HMDA) and caprolactam. Pathways for the production of adipate, 6-aminocaproate, caprolactam, and hexamethylene diamine from succinyl-CoA and acetyl-CoA are represented. Abbreviations: A) 3-oxoadipyl-CoA thiolase, B) 3-oxoadipyl-CoA reductase, C) 3-hydroxyadipyl-CoA dehydratase, D) 5-carboxy-2-pentenoyl-CoA reductase, E) 3-oxoadipyl- CoA/acyl-CoA transferase, F) 3-oxoadipyl-CoA synthase, G) 3-oxoadipyl-CoA hydrolase, H) 3-oxoadipate reductase, I) 3-hydroxyadipate dehydratase, J) 5-carboxy-2-penta noate reductase, K) adipyl-CoA/acyl-CoA transferase, L) adipyl-CoA synthase, M) adipyl-CoA hydrolase, N) adipyl-CoA reductase (aldehyde formation), O) 6-aminocaproate transaminase, P) 6-aminocaproyl-CoA/acyl-CoA transferase, Q) 6-aminocaproyl-CoA/acyl-CoA transferase, R) 6-aminocaproyl-CoA synthase, S) amidohydrolase, T) spontaneous cyclization, U )6-aminocaproyl-CoA reductase (aldehyde formation), V) HMDA transaminase, W) HMDA dehydrogenase.</figref><figref num="11">FIG. 4 shows exemplary routes from 4-aminobutyryl-CoA and acetyl-CoA to hexamethylene diamine and caprolactam. A pathway for the production of 6-aminocaproate, caprolactam, and hexamethylene diamine from 4-aminobutyryl-CoA and acetyl-CoA is represented. Abbreviations: A) 3-oxo-6-aminohexanoyl-CoA thiolase, B) 3-oxo-6-aminohexanoyl-CoA reductase, C) 3-hydroxy-6-aminohexanoyl-CoA dehydratase, D) 6 -aminohex-2-enoyl-CoA reductase, E) 3-oxo-6-aminohexanoyl-CoA/acyl-CoA transferase, F) 3-oxo-6-aminohexanoyl-CoA synthase, G) 3-oxo -6-aminohexanoyl-CoA hydrolase, H) 3-oxo-6-aminohexanoate reductase, I) 3-hydroxy-6-aminohexanoate dehydratase, J) 6-aminohexanoate reductase , K) 6-aminocaproyl-CoA/acyl-CoA transferase, L) 6-aminocaproyl-CoA synthase, M) 6-aminocaproyl-CoA hydrolase, N) 6-aminocaproyl-CoA reductase (aldehyde formation), O) HMDA transaminase, P) HMDA dehydrogenase, Q) spontaneous cyclization, R) amidohydrolase.</figref><figref num="12">FIG. 3 shows the route to 6-aminocaproate from pyruvate and succinic semialdehyde. The enzymes are: A) HODH aldolase, B) OHED hydratase, C) OHED reductase, D) 2-OHD decarboxylase, E) adipate semialdehyde aminotransferase and/or adipate semialdehyde oxidoreductase (amination), F) OHED decarboxylase. Carboxylase, G) 6-OHE reductase, H) 2-OHD aminotransferase and/or 2-OHD oxidoreductase (amination), I) 2-AHD decarboxylase, J) OHED aminotransferase and/or OHED oxidoreductase (amino K) 2-AHE reductase, L) HODH formate lyase and/or HODH dehydrogenase, M) 3-hydroxyadipyl-CoA dehydratase, N) 2,3-dehydroadipyl-CoA reductase, O) adipyl-CoA Dehydrogenase, P) OHED formate lyase and/or OHED dehydrogenase, Q) 2-OHD formate lyase and/or 2-OHD dehydrogenase. Abbreviations are: HODH=4-hydroxy-2-oxoheptane-1,7-dioate, OHED=2-oxohept-4-ene-1,7-dioate, 2-OHD=2-oxoheptane-1,7-dioate , 2-AHE=2-aminohept-4-ene-1,7-dioate, 2-AHD=2-aminoheptane-1,7-dioate, and 6-OHE=6-oxohex-4-enoate.</figref><figref num="13">FIG. 2 is a diagram showing the route from 6-aminocaproate to hexamethylene diamine. The enzymes are: A) 6-aminocaproate kinase, B) 6-AHOP oxidoreductase, C) 6-aminocaproate semialdehyde aminotransferase and/or 6-aminocaproate semialdehyde oxidoreductase (amination), D) 6 - aminocaproate N-acetyltransferase, E) 6-acetamidohexanoate kinase, F) 6-AAHOP oxidoreductase, G) 6-acetamidohexanal aminotransferase and/or 6-acetamidohexanal oxidoreductase (amination), H) 6-acetamidohexanamine N-acetyltransferase and/or 6-acetamidohexanamine hydrolase (amide), I) 6-acetamidohexanoate CoA transferase and/or 6-acetamidohexanoate CoA ligase, J) 6- acetamidohexanoyl-CoA oxidoreductase, K) 6-AAHOP acyltransferase, L) 6-AHOP acyltransferase, M) 6-aminocaproate CoA transferase and/or 6-aminocaproate CoA ligase, N) 6- Aminocaproyl-CoA oxidoreductase. The abbreviations are: 6-AAHOP=[(6-acetamidohexanoyl)oxy]phosphonate and 6-AHOP=[(6-aminohexanoyl)oxy]phosphonate.</figref><figref num="14">A) Acetyl-CoA cycle of arginine biosynthesis. Reactions (1) and (2) are catalyzed by ornithine acetyltransferase, which has the functionality of acetylglutamate synthase and ornithine acyltransferase. Reaction 3 is a packaged reaction catalyzed by acetylglutamate kinase, N-acetylglutamyl phosphate reductase, and acetylornithine aminotransferase; B) Acetyl-CoA cycle of HMDA biosynthesis. Reactions (1) and (2) are catalyzed by HMDA acetyltransferase. Reaction (3) is an all-in-one reaction including the entire route from 6-acetamidohexanoate to 6-acetamidohexanamine shown in FIG.</figref><figref num="15">FIG. 3 shows the growth of E. coli in media containing various concentrations of 6-ACA. E. coli was inoculated into culture medium and grown in aerobic (left and right bars) or anaerobic (middle bar) conditions. Cultures were grown under aerobic conditions (right bar) for 48 h during the first test and 30 h for the second test.</figref><figref num="16">FIG. 3 is a diagram showing the resistance of E. coli when exposed to 6-ACA. Mid-log phase (OD600=0.3, below the dotted line) or early quiescent (OD600=0.6, above the dotted line) cells were spun down and re-incubated in fresh M9-glucose medium with varying concentrations of 6-ACA. Suspended. After overnight growth, cultures were measured for growth by measuring OD600.</figref><figref num="17">FIG. 2 shows ethanol production from cultures exposed to various concentrations of 6-ACA. Mid-log phase or early quiescent cells were spun down and resuspended in fresh M9-glucose medium with varying concentrations of 6-ACA. After overnight growth, cultures were measured for growth by measuring OD600 and metabolic activity was assayed by ethanol production.</figref><figref num="18">Panels A and B show growth on various concentrations of 6-ACA with and without glycine betaine. Panel A. OD600 measurements of media inoculated with mid-log phase cultures of E. coli with various concentrations of 6-ACA with (right bar) and without (left bar) 2mM glycine betaine. Panel B. Photograph showing growth of the same culture in anaerobic bottles.</figref><figref num="19">FIG. 3 shows LC/MS analysis of an in vitro thiolase reaction. Succinyl-CoA and acetyl-CoA were added to the His-tagged purified thiolase at a ratio of 2:1 (succinyl-CoA:acetyl-CoA). Reactions were analyzed by LC/MS and quantified by comparison to standards for acetoacetyl-CoA or by determined peak areas for 3-oxoadipyl-CoA (β-ketoadipyl-CoA).</figref><figref num="20">FIG. 2 shows an exemplary route from glutamic acid to hexamethylene diamine (HMDA) and 6-aminocaproate. Enzymes are designated as: A) glutamyl-CoA transferase and/or ligase, B) beta-ketothiolase, C) 3-oxo-6-aminopimeloyl-CoA oxidoreductase, D) 3-hydroxy-6-aminopimeloyl. -CoA dehydratase, E) 6-amino-7-carboxyhept-2-enoyl-CoA reductase, F) 6-aminopimeloyl-CoA reductase (aldehyde formation), G) 2-amino-7-oxoheptanoate aminotransferase and / or aminating oxidoreductase, H) homolysine decarboxylase, I) 6-aminopimeloyl-CoA hydrolase, transferase, and/or ligase, J) 2-aminopimelate decarboxylase. The enzyme commission number indicated for each reaction is described below in Example XXVI.</figref><figref num="21">FIG. 3 shows an exemplary route from glutaryl-CoA to hexamethylene diamine (HMDA) and 6-aminocaproate. Enzymes are designated as: A) glutaryl-CoA beta-ketothiolase, B) 3-oxopimeloyl-CoA hydrolase, transferase, and/or ligase, C) 3-oxopimelate reductase, D) 3-oxopimelate reductase. -1-carboxyheptanal 7-aminotransferase and/or 7-aminated oxidoreductase, E) 3-oxo-7-aminoheptanoate 3-aminotransferase and/or 3-aminated oxidoreductase, F) 3- Oxopimelate kinase, G) 5-oxopimeloylphosphonate reductase, H) 3-oxopimelate-CoA transferase and/or ligase, I) 5-oxopimelate-CoA reductase (aldehyde formation), J) 3-oxopimelate 3-amino transferase and/or 3-aminating oxidoreductase, K) 3-aminopimelate-CoA transferase and/or ligase, L) 5-aminopimeloyl-CoA reductase (aldehyde formation), M) 3-aminopimelate kinase, N) 5- Aminopimeloylphosphonate reductase, O)3-aminopimelate reductase, P)3-amino-7-oxoheptanoate 2,3-aminomutase, Q)2-amino-7-oxoheptanoate 7- Aminotransferase and/or aminating oxidoreductase, R) 3,7-diaminoheptanoate 2,3-aminomutase, S) Homolysine decarboxylase, T) 3-aminopimelate 2,3-aminomutase, U) 2- Aminopimelate kinase, V) 2-aminopimelate CoA transferase and/or ligase, W) 2-aminopimelate reductase, X) 6-aminopimeloylphosphonate reductase, Y) 6-aminopimelate-CoA reductase (aldehyde formation) ), Z) 3-amino-7-oxoheptanoate 7-aminotransferase and/or 7-aminated oxidoreductase, AA) 2-aminopimelate decarboxylase, and AB) 3-oxo-1-carboxyheptanoate Nal 3-aminotransferase and/or 3-aminating oxidoreductase. The enzyme commission number indicated for each reaction is listed below in XXVI.</figref><figref num="22">FIG. 2 shows an exemplary route from pyruvate and 4-aminobutanal to hexamethylene diamine (HMDA). The enzymes are designated as: A) 2-oxo-4-hydroxy-7-aminoheptanoate aldolase, B) 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase, C) 2- Oxo-7-aminoheptanoate reductase, D) 2-oxo-7-aminoheptanoate aminotransferase and/or aminating oxidoreductase, E) Homolysine decarboxylase, F) 2-oxo-7-amino Heptanoate decarboxylase, G) 6-aminohexanal aminotransferase and/or 6-aminohexanal aminating oxidoreductase. The enzyme commission number indicated for each reaction is described below in Example XXVI.</figref><figref num="23">FIG. 2 shows an exemplary route from homolysine to 6-aminocaproate. Step A is catalyzed by homolysine 2-monooxygenase. Step B is a dilute acid or base catalyzed hydrolysis.</figref><figref num="24">FIG. 2 shows an exemplary route from 6-aminocaproate to hexamethylene diamine. This figure also represents a further route to that shown in FIG. 13. The enzymes are designated as: A) 6-aminocaproate kinase, B) 6-AHOP oxidoreductase, C) 6-aminocaproic acid semialdehyde aminotransferase and/or 6-aminocaproic acid semialdehyde oxidoreductase ( amination), D) 6-aminocaproate N-acetyltransferase, E) 6-acetamidohexanoate kinase, F) 6-AAHOP oxidoreductase, G) 6-acetamidohexanal aminotransferase and/or 6-acetamidohexanal Oxidoreductase (amination), H) 6-acetamidohexanamine N-acetyltransferase and/or 6-acetamidohexanamine hydrolase (amide), I) 6-acetamidohexanoate CoA transferase and/or 6-acetamidohexanoate CoA ligase, J) 6-acetamidohexanoyl-CoA oxidoreductase, K) 6-AAHOP acyltransferase, L) 6-AHOP acyltransferase, M) 6-aminocaproate CoA transferase and/or 6-aminocaproate CoA ligase, N) 6-aminocaproyl-CoA oxidoreductase, O) 6-aminocaproate reductase, and P) 6-acetamidohexanoate reductase. The abbreviations are: 6-AAHOP=[(6-acetamidohexanoyl)oxy]phosphonate and 6-AHOP=[(6-aminohexanoyl)oxy]phosphonate. The enzyme commission number indicated for each reaction is described below in Example XXVI.</figref><figref num="25">FIG. 3 shows exemplary routes from succinyl-CoA and acetyl-CoA to hexamethylene diamine (HMDA), caprolactam, or levulinic acid. Pathways for the production of adipate, 6-aminocaproate, caprolactam, hexamethylenediamine, and levulinic acid from succinyl-CoA and acetyl-CoA are represented. This figure also represents a further route to that shown in FIG. The enzymes are designated as: A) 3-oxoadipyl-CoA thiolase, B) 3-oxoadipyl-CoA reductase, C) 3-hydroxyadipyl-CoA dehydratase, D) 5-carboxy-2-pentenoyl-CoA. reductase, E) 3-oxoadipyl-CoA/acyl-CoA transferase, F) 3-oxoadipyl-CoA synthase, G) 3-oxoadipyl-CoA hydrolase, H) 3-oxoadipate reductase, I) 3-hydroxyadipate dehydratase, J )5-carboxy-2-pentanoate reductase, K) adipyl-CoA/acyl-CoA transferase, L) adipyl-CoA synthase, M) adipyl-CoA hydrolase, N) adipyl-CoA reductase (aldehyde formation), O) 6-aminocaproate transaminase, P) 6-aminocaproate dehydrogenase, Q) 6-aminocaproyl-CoA/acyl-CoA transferase, R) 6-aminocaproyl-CoA synthase, S) amidohydrolase, T ) spontaneous cyclization, U) 6-aminocaproyl-CoA reductase (aldehyde formation), V) HMDA transaminase, W) HMDA dehydrogenase, X) adipate reductase, Y) adipate kinase, Z) adipyl phosphate reductase, and AA) 3-oxoadipate decarboxylase.</figref><figref num="26">FIG. 2 shows an exemplary route from 2-amino-7-oxosubarate to hexamethylene diamine (HMDA) and 6-aminocaproate. The enzymes are designated as: A) 2-amino-7-oxosabarate ketoacid decarboxylase, B) 2-amino-7-oxoheptanoate decarboxylase, C) 6-aminohexanalaminated oxide. reductase and/or 6-aminohexanal aminotransferase, D) 2-amino-7-oxoheptanoate oxidoreductase, E) 2-aminopimelate decarboxylase, F) 6-aminohexanal oxidoreductase, G) 2- Amino-7-oxoheptanoate decarboxylase, H) homolysine decarboxylase, I) 2-amino-7-oxosabarate amino acid decarboxylase, J) 2-oxo-7-aminoheptanoate aminating oxidoreductase and / or 2-oxo-7-aminoheptanoate aminotransferase, K) 2-amino-7-oxosubarate aminating oxidoreductase and/or 2-amino-7-oxosubarate aminotransferase, L) 2,7 -diaminosabarate decarboxylase, and M) 2-amino-7-oxoheptanoate aminating oxidoreductase and/or 2-amino-7-oxoheptanoate aminotransferase. The enzyme commission number indicated for each reaction is described below in Example XXVI.</figref><figref num="27">FIG. 2 shows an exemplary route from glutamic acid-5-semialdehyde to 2-amino-7-oxosabarate. The enzyme is designated as: A) 2-amino-5-hydroxy-7-oxosabarate aldolase, B) 2-amino-5-hydroxy-7-oxosabarate dehydratase, C) 2-amino- 5-ene-7-oxosabare reductase.</figref><figref num="28">FIG. 6 shows the growth yield for 6-ACA versus ADHHer, LDH_D mutant.</figref><figref num="29">FIG. 3 shows growth yields for 6-ACA versus ADHHer, MDH, ASPT, LDH_D mutants.</figref><figref num="30">FIG. 3 shows growth yields for 6-ACA versus ADHHer, MDH, ASPT, LDH_D, THD2 mutants.</figref><figref num="31">FIG. 6 shows growth yields for 6-ACA versus ADHHer, MDH, ASPT, LDH_D, GLUDy mutants.</figref><figref num="32">FIG. 6 shows growth yields for 6-ACA versus PGI, EDA mutants or PGI, PGDHy mutants.</figref><figref num="33">FIG. 6 shows growth yields for 6-ACA versus PGI, EDA, ADHEr mutants or PGI, PGDHy, ADHEr mutants.</figref><figref num="34">FIG. 3 shows growth yields for 6-ACA versus ADHHer, PGI, HEX1 mutants.</figref><figref num="35">FIG. 3 shows the growth-related adipate production characteristics of the top strain design (gray) compared to wild-type E. coli (black). A glucose uptake rate of 10 mmol/gDW/Hr is assumed.</figref><figref num="36">FIG. 3 shows the activity of CAR889 and 891 using 20mM adipate. Activity is expressed as units per mg of total protein in the crude lysate.</figref><figref num="37">FIG. 3 shows the activity of CAR720, 889, 890, 891 using 50 mM 6-aminocaproate. Activity is expressed as units per mg of total protein in the crude lysate.</figref>

The present invention relates to the design and production of cells and organisms capable of biosynthetic production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. The results described herein demonstrate that metabolic pathways are designed and assembled to achieve the biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid in E. coli, other cells or organisms. This shows that it is possible to genetically manipulate genes by changing them. Biosynthetic production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid can be confirmed by construction of strains with designed metabolic genotypes. These metabolically engineered cells or organisms may also be used to further increase 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthesis, including under conditions approaching theoretical maximal growth. can be subjected to adaptive evolution.

As disclosed herein, a number of metabolic pathways are described for the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. Two routes, the reverse adipate decomposition pathway and the 3-oxoadipate pathway, reduce (i) adipate yield (92% molar yield based on glucose), (ii) insufficient oxygen demand for adipate synthesis, and (iii) ) associated energetic properties, and (iv) the theoretical ability to produce adipate as the sole fermentation product. Metabolic routes for adipate production via α-ketoadipate or lysine have also been described, but have low yields and require aeration to maximize production. Also disclosed herein are routes for producing either or both of 6-aminocaproate and caprolactam from adipyl-CoA, a precursor in the reverse degradation pathway.

As disclosed herein, a number of exemplary routes for adipate biosynthesis are described. An exemplary first pathway involves adipate synthesis via a route that relies on the reversibility of adipate degradation described in organisms such as P. chrysogenum (see Examples I and II). A second exemplary route involves the formation of 3-oxoadipate, followed by its reduction, dehydration, and reduction again to form adipate (see Examples III and IV). The adipate yield using either of these two routes is 0.92 moles per mole of glucose consumed. Oxygen uptake is not necessary to achieve these theoretical maximum yields, and the energetic profile under anaerobic conditions favors growth and product secretion. A method for producing adipate from glucose-derived cis,cis-muconic acid was previously described (Frost et al., U.S. Pat. No. 5,487,987, issued January 30, 1996) (see Example V). I want to be). The advantages of the embodiments disclosed herein over this previously described method are discussed. Metabolic routes for adipate production that pass through α-ketoadipate (Example VI) or lysine (Example VII) precursors are low yielding and require aeration to maximize production. A route for producing either or both 6-aminocaproate and caprolactam from adipyl-CoA, a precursor in the reverse degradation pathway, is described (see Examples VIII and IX). Additional routes for producing adipate are described in Examples X and XI. Routes for producing any one, two, three, or all four of 6-aminocaproate, caprolactam, hexamethylenediamine, and levulinic acid from succinyl-CoA and acetyl-CoA are described in Examples XII, XXVIII It is described in . Several routes for the production of 6-aminocaproate from succinic semialdehyde and pyruvate are described in Example XIX. Several routes for the production of hexamethylene diamine from 6-aminocaproate are described in Examples XX and XXVII. A route for producing either or both 6-aminocaproate and hexamethylene diamine from glutamate esters is described in Examples XXIV and XXV. Several routes for the production of hexamethylene diamine from glutaryl-CoA and at least one route for the production of 6-aminocaproate from glutaryl-CoA are described in Examples XXIV and XXV. A route for producing 6-aminocaproate from homolysine is described in Example XXV. A route for producing hexamethylene diamine from 2-amino-7-oxosubarate is described in Example XXIV. Several routes for producing 6-aminocaproate are described in Example XXV. Exemplary genes and enzymes required to construct microorganisms with these capabilities and methods for cloning and transformation, methods for monitoring product formation, and using genetically engineered microorganisms in production. A method for doing so is described.

As disclosed herein, six different routes for adipic acid synthesis using glucose/sucrose as the carbon substrate are described. For all maximal yield calculations, missing reactions in a given pathway are performed using E. coli chemistry in SimPheny similar to that previously described (Reed et al., Genome Biol. 4:R54 (2003)). Added to stoichiometric networks.

It was hypothesized that adipate becomes a charged molecule under physiological conditions and requires energy in the form of a proton-based symport system to be secreted from the network. Such a transport system is thermodynamically viable if the fermentation is carried out at neutral or near-neutral pH. Low pH adipic acid formation would require an ATP-dependent transport mechanism, such as the ABC system as opposed to proton symport. Reactions in the routes and methods of embodiments of these routes are described in Examples I-XI.

As used herein, the term "non-naturally occurring" when used with respect to a microbial organism or microorganisms of the invention means that the microbial organism is one of the above-mentioned species, including wild-type strains of the above-mentioned species. is intended to mean having at least one genetic variation not normally found in naturally occurring strains of the species. Genetic variations include, for example, modifications that introduce expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions, and/or other functional disruptions of the genetic material of the microorganism. Such modifications include, for example, coding regions and functional fragments thereof for polypeptides that are heterologous, homologous, or heterologous and homologous for the species mentioned above. Additional modifications include, for example, non-coding regulatory regions where the modification alters expression of the gene or operon. Exemplary metabolic polypeptides include enzymes within the 6-aminocaproic acid, caprolactam, hexamethylene diamine, or levulinic acid biosynthetic pathways.

Metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Thus, non-naturally occurring microorganisms may have genetic modifications to nucleic acids encoding metabolic polypeptides or functional fragments thereof. Exemplary metabolic modifications are disclosed herein.

As used herein, the term "isolated" when used with reference to a microbial organism means that the microbial organism described above is found in nature but is substantially free of at least one component. intended to mean living things. The term includes a microbial organism as it is found in its natural environment, but from which some or all components have been removed. The term also includes microbial organisms found in environments in which they do not naturally occur, but from which some or all components have been removed. As such, an isolated microbial organism is one that is found in nature or that grows, is preserved, or lives in an environment in which it does not naturally occur, but is partially or completely separated from other materials. has been done. Particular examples of isolated microbial organisms include partially pure microorganisms, substantially pure microorganisms, and microorganisms cultured in non-naturally occurring media.

As used herein, the term "microbial," "microbial organism," or "microorganism" refers to microscopic cells included within the scope of archaea, bacteria, or eukaryotes. Intended to mean any living thing in existence. As such, the term is intended to encompass prokaryotic or eukaryotic cells or organisms of microscopic size, such as all species of bacteria, archaea, and eubacteria, as well as yeasts and fungi. Includes eukaryotic microorganisms. The term also includes cell cultures of any species that can be cultured for the production of biochemicals.

As used herein, the term "CoA" or "Coenzyme A" refers to an organic cofactor whose presence is required for the activity of many enzymes (apoenzymes) to form active enzyme systems. Intended to mean a factor or a prosthetic group (the non-protein part of an enzyme). Coenzyme A functions in certain condensing enzymes, acting in acetyl or other acyl group transfers and in fatty acid synthesis and oxidation, pyruvate oxidation and other acetylations.

As used herein, the chemical formula -OOC-(CH₂)₄"Adipate" with -COO- (see Figure 2) (IUPAC name hexanedioate) is the ionized form of adipic acid (IUPAC name hexanedioate), and adipate and adipic acid in all its salt forms It is understood that it can be used interchangeably throughout to refer to a compound, including, in either its neutral or ionic form. It is understood by those skilled in the art that it will be understood that the particular form will depend on pH.

As used herein, the chemical formula -OOC-(CH₂)₅-NH₂"6-aminocaproate" with (see Figures 8 and 12) is the ionized form of 6-aminocaproic acid (IUPAC name 6-aminohexanoic acid), which includes 6-aminocaproate and 6-aminocaproate. It is understood that aminocaproic acid may be used interchangeably throughout to refer to the compound in either its neutral or ionized form, including any salt forms thereof. It is understood by those skilled in the art that it will be understood that the particular form will depend on pH.

As used herein, "caprolactam" (IUPAC name azepan-2-one) is a lactam of 6-aminohexanoic acid (see Figure 8).

As used herein, "hexamethylenediamine," also referred to as 1,6-diaminohexane or 1,6-hexanediamine, has the chemical formula H₂N(CH₂)₆N.H.₂(see Figures 10, 11 and 13).

As used herein, the term "substantially anaerobic" when used in reference to culture or growth conditions means that the amount of oxygen is less than about 10% of saturation with respect to dissolved oxygen in the liquid medium. is intended to mean. The term is also intended to include a closed chamber of liquid or solid media maintained with an atmosphere of less than about 1% oxygen.

As used herein, the term "osmoprotectant," when used in reference to culture or growth conditions, acts as an osmolyte that allows the microbial organisms described herein to Intended to mean compounds that help withstand osmotic stress. Osmoprotectants include, for example, betaine, amino acids, and the sugar trehalose. Non-limiting examples of such include glycine betaine, praline betaine, dimethyltetine, dimethylslfonioproprionate, methyl 3-dimethylsulfonio-2-propionate, pipecolic acid, dimethyl These include sulfonioacetic acid, choline, L-carnitine, and ectoine.

As used herein, the term "growth-associated" when used with respect to the production of biochemicals means that the biosynthesis of said biochemicals is produced during the growth phase of the microorganism. intended to mean. In certain embodiments, production in conjunction with growth may be unavoidable, meaning that the biosynthesis of the biochemicals described above is an unavoidable product produced during the growth phase of the microorganism.

As used herein, "metabolic modification" is intended to refer to a biochemical reaction that is altered from its naturally occurring state. Metabolic modification can include, for example, loss of biochemical reaction activity due to functional disruption of one or more genes encoding enzymes involved in the reaction. A set of exemplary metabolic modifications is described herein (see Example XXX).

As used herein, the term "gene disruption" or its grammatical equivalent is intended to mean a genetic mutation that renders the encoded gene product inactive. Genetic mutations can include, for example, deletion of an entire gene, deletion of regulatory sequences required for transcription or translation, deletion of a portion of a gene resulting in a truncated gene product, or a variety of mutations that inactivate the encoded gene product. It can be by any of a variety of mutation strategies. One particularly useful method of gene disruption is complete gene deletion, since it reduces or eliminates the occurrence of genetic reversion in the non-naturally occurring microorganisms of the invention.

"Exogenous" as used herein is intended to mean that the above-mentioned molecule or the above-mentioned activity is introduced into the host microbial organism. For example, the molecule can be introduced by introduction of an encoding nucleic acid into the host genetic material, such as by integration into the host chromosome, or as non-chromosomal genetic material, such as a plasmid. As such, the term, as used with respect to expression of a coding nucleic acid, refers to the introduction of a coding nucleic acid into an expressible form into a microbial organism. When used with respect to biosynthetic activity, the term refers to the activity that is introduced into the host reference organism. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the above-mentioned activity after introduction into the host microbial organism. The term "endogenous" therefore refers to the aforementioned molecules or activities that are present in the host. Similarly, the term when used in reference to expression of a coding nucleic acid refers to the expression of a coding nucleic acid contained within a microbial organism. The term "heterologous" refers to a molecule or activity that is derived from a source other than the species mentioned above. "Homologous" refers to molecules or activities that are derived from the host microbial organism. Thus, exogenous expression of the encoding nucleic acids of the invention can utilize either or both heterologous or homologous encoding nucleic acids.

If more than one exogenous nucleic acid is included in the microbial organism, the more than one exogenous nucleic acid may be associated with the aforementioned encoding nucleic acid or biosynthetic activity, as discussed above. be understood. As disclosed herein, more than one exogenous nucleic acid can be introduced into a host microbial organism on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof. It is further understood that a nucleic acid can be used and still be considered more than one exogenous nucleic acid. For example, as disclosed herein, a microbial organism can be genetically engineered to express two or more exogenous nucleic acids encoding desired pathway enzymes or proteins. In cases where two exogenous nucleic acids encoding desired activities are introduced into a host microbial organism, the two exogenous nucleic acids can be integrated into a single plasmid, e.g. on a single plasmid, on separate plasmids, or on separate plasmids. It is understood that the nucleic acid can be introduced as a single nucleic acid and can be integrated into the host chromosome at a single site or multiple sites and still be considered two exogenous nucleic acids. Similarly, more than two exogenous nucleic acids can be introduced into a host organism in any desired combination, e.g. on a single plasmid, on separate plasmids, at a single site or multiple It is understood that the exogenous nucleic acids can be integrated into the host chromosome at a site and still be considered more than one exogenous nucleic acid, such as three exogenous nucleic acids. Thus, the number of exogenous nucleic acids or biosynthetic activities mentioned above refers to the number of encoding nucleic acids or biosynthetic activities rather than the number of separate nucleic acids introduced into the host organism.

The non-naturally occurring microbial organisms of the invention can contain stable genetic variations, which refers to microorganisms that can be cultured for more than five generations without loss of variation. In general, stability genetic variations include modifications that persist for more than 10 generations; in particular, stability modifications will persist for more than about 25 generations; , there will be over 50 generations, including infinity.

In the case of gene disruption, a particularly useful stable genetic variation is gene deletion. The use of gene deletions to introduce stable genetic mutations is particularly useful in reducing the likelihood of reversion to the pre-genetic mutation phenotype. For example, growth-coupled production of biochemical stability can be achieved, for example, by deletion of a gene encoding an enzyme that catalyzes one or more reactions within a set of metabolic modifications. The stability of growth-associated production of biochemicals can further be enhanced through multiple deletions, significantly reducing the likelihood of multiple compensatory reversions occurring for each disrupted activity. let

Those skilled in the art will appreciate that the genetic variations, including metabolic modifications exemplified herein, can be implemented in suitable host organisms such as E. coli and their corresponding metabolic reactions or genes for desired metabolic pathways, etc. It will be understood that the description is made in terms of suitable source organisms for the desired genetic material. However, given the complete genome sequencing of a wide variety of organisms and the high level of technology in the field of genomics, one skilled in the art will be able to apply the teachings and guidance essentially provided herein to all other organisms. could be easily applied to For example, the E. coli metabolic variations exemplified herein can be easily applied to other species by incorporating the same or similar encoding nucleic acids from species other than those mentioned above. Such genetic variation includes, for example, genetic variation of species, homologs in general, orthologs, paralogs, or non-orthologous gene substitutions in particular.

Orthologs are gene(s) that are closely related by vertical lineage and perform substantially the same or identical functions in different organisms. For example, mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of epoxide hydrolysis. Genes are closely related by vertical descent, for example, if they share a sufficient amount of sequence similarity to show that they are the same species or are closely related by evolution from a common ancestor. . Genes are also classified if they share a sufficient amount of three-dimensional structure, not necessarily sequence similarity, to show that they evolved from a common ancestor to the extent that primary sequence similarity is not identifiable. It can also be considered an ortholog. Orthologous genes can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity. Genes encoding proteins that share less than 25% amino acid similarity can also be considered to have arisen by vertical descent if their three-dimensional structures also show similarity. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor.

Orthologs include genes or their encoded gene products that have diverged in structure or overall activity, eg, through evolution. For example, if a first species encodes a gene product that exhibits two functions, and such functions are separated into separate genes in the second species, then the three genes and their corresponding products are , are considered orthologs. For the production of biochemical products, those skilled in the art will understand that orthologous genes with metabolic activity to be introduced or disrupted will be selected for the construction of non-naturally occurring microorganisms. Will. An example of orthologs exhibiting separable activities is where the distinct activities are separated into distinct gene products between two or more species or within a single species. A particular example is the separation of two types of serine protease activities, elastase proteolysis and plasminogen proteolysis, into separate molecules as plasminogen activator and elastase.

A second example is the separation of Mycoplasma 5'-3' exonuclease and Drosophila DNA polymerase III activities. A DNA polymerase derived from a first species can be considered orthologous to an exonuclease and/or polymerase derived from a second species, and vice versa.

In contrast, paralogs are homologues that are closely related, eg, by duplication followed by evolutionary divergence, and have similar or common, although not identical, functions. Paralogs may occur or be derived from, for example, the same or different species. For example, microsomal epoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II) are two distinct entities that coevolved from a common ancestor, catalyzing distinct reactions and having distinct functions in the same species. Since it corresponds to an enzyme, it can be considered a paralog. Paralogs are proteins from the same species that have significant sequence similarity to each other, suggesting that they are the same species or are closely related through coevolution from a common ancestor. The group of paralogous protein families includes HipA homologs, luciferase genes, peptidases, and others.

A non-orthologous gene replacement is a non-orthologous gene from one species that can substitute for the above-mentioned gene function in a different species. Substitutions include, for example, those that can perform substantially the same or similar function in the species of origin as compared to the function described above in a different species. In general, non-orthologous gene substitutions can be identified as being structurally related to known genes encoding the functions mentioned above, as well as less structurally related but functionally similar genes and their The corresponding gene product would nevertheless still fall within the meaning of this term as it is used herein. Functional similarity requires, for example, at least some structural similarity in the active site or binding region of the non-orthologous gene product compared to the gene encoding the function to be replaced. As such, non-orthologous genes include, for example, paralogs or unrelated genes.

Therefore, in constructing and identifying non-naturally occurring microbial organisms of the present invention capable of biosynthesizing 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid, one skilled in the art will By applying the teachings and guidance provided herein, one will understand that the identification of metabolic alterations can include the identification and inclusion or inactivation of orthologs. To the extent that paralogous and/or non-orthologous gene substitutions encoding enzymes that catalyze similar or substantially similar metabolic reactions exist in the microorganisms mentioned above, one skilled in the art will also recognize that these are evolutionarily closely related. Certain genes can also be used. Gene disruption strategies include reducing or eliminating activity to ensure that any functional redundancy in the enzyme activity targeted for disruption does not interfere with the designed metabolic modification. Genes that are evolutionarily related can also be disrupted or deleted in the host microbial organism, paralog, or ortholog.

Orthologs, paralogs, and non-orthologous gene substitutions can be determined by methods familiar to those skilled in the art. For example, examination of the nucleic acid or amino acid sequences of two polypeptides will reveal sequence identity and similarity between the compared sequences. Based on such similarities, one skilled in the art can determine whether the similarities are sufficiently high to indicate that the proteins are closely related through evolution from a common ancestor. Align, BLAST, Clustal Algorithms well known to those skilled in the art, such as those of W. et al., compare and determine pure sequence similarity or identity and can also assign weights or scores to gaps in a sequence. Presence or significance is also determined. Such algorithms are also known in the art and are equally applicable for determining nucleotide sequence similarity or identity. The parameters for sufficient similarity to determine relatedness are well-known parameters for calculating statistical similarity in random polypeptides or the likelihood of finding similar matches and the significance of determined matches. Calculated based on the method Computer comparisons of two or more sequences can also be visually optimized by one of skill in the art, if desired. Related gene products or proteins can be expected to have a high degree of similarity, eg, 25% to 100% sequence identity. Unrelated proteins may have essentially the same identity as would be expected to occur by chance (about 5%) if a database of sufficient size is scanned. Sequences between 5% and 24% may or may not represent sufficient homology to conclude that the compared sequences are closely related. Further statistical analysis to determine the significance of such matches considering the size of the data set can be performed to determine the association of these sequences.

Exemplary parameters for determining the relatedness of two or more sequences using the BLAST algorithm can be, for example, as described below. Briefly, amino acid sequence alignments were performed using BLASTP version 2.0.8 (January 5, 1999) and the following parameters: matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50; expected: 10.0; Word size: 3; Filter: On. Nucleic acid sequence alignments were performed using BLASTN version 2.0.6 (September 16, 1998) and the following parameters: match: 1; mismatch: -2; gap open: 5; gap extension: 2; x_dropoff: 50; expected: 10.0; Can be run using Word Size: 11; Filter: Off.

Those skilled in the art will appreciate that any modifications can be made to the above parameters to increase or decrease the stringency of the comparison, e.g. to determine the relatedness of two or more sequences. .

Disclosed herein are non-naturally occurring microbial organisms capable of producing adipate, 6-aminocaproic acid, caprolactam, hexamethylene diamine, or levulinic acid. For example, the adipate pathway can be a reverse adipate decomposition pathway (see Examples I and II). For example, a non-naturally occurring microbial organism can have an adipate pathway that includes at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in amounts sufficient to produce adipate, the adipate pathway being , succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA synthetase or phosphotransadipyrase. (phosphotransadipylase)/adipate kinase or adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase. Additionally, the adipate route can be through the 3-oxoadipate route (see Examples III and IV). The non-naturally occurring microbial organism can have an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in sufficient quantities to produce adipate, the adipate pathway comprising succinyl -CoA: includes acetyl-CoA acyltransferase, 3-oxoadipyl-CoA transferase, 3-oxoadipate reductase, 3-hydroxyadipate dehydratase, and 2-enoate reductase.

Additionally, the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding a 6-aminocaproic acid pathway enzyme expressed in sufficient amounts to produce 6-aminocaproic acid pathway enzymes. The 6-aminocaproic acid pathway includes CoA-dependent aldehyde dehydrogenases and transaminases (see Examples VIII and IX). Alternatively, 6-aminocaproate dehydrogenase can be used to convert adipate semialdehyde to form 6-aminocaproate (see Figure 8). The non-naturally occurring microbial organism can also have a caprolactam pathway comprising at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in an amount sufficient to produce caprolactam, the caprolactam pathway comprising: Includes CoA-dependent aldehyde dehydrogenases, transaminases or 6-aminocaproate dehydrogenases, and amidohydrolases (see Examples VIII and IX).

As disclosed herein, microbial organisms that produce 6-aminocaproic acid or caprolactam can produce 6-aminocaproic acid and/or caprolactam from adipyl-CoA precursors (see Figure 8 and (See Examples VIII and IX). It is therefore understood that microbial organisms that produce 6-aminocaproic acid or caprolactam can further include a pathway for producing adipyl-CoA. For example, the adipyl-CoA pathway can include the enzyme of Figure 2 that utilizes succinyl-CoA and acetyl-CoA as precursors through the production of adipyl-CoA, i.e., to convert adipyl-CoA to adipate. Lacks enzyme for the final step. Thus, one exemplary adipyl-CoA pathway includes succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, and 5-carboxy-2-pentenoyl-CoA A reductase may be included.

Additionally, as shown in Figure 1, the adipate degradation pathway includes a step for converting adipate to adipyl-CoA by adipate-CoA ligase. Therefore, the adipyl-CoA pathway can be activated by, for example, either the adipate-CoA ligase activity in the first step of Figure 1 or the enzyme in the final step of Figure 2 carried out in the opposite direction, such as adipyl-CoA synthetase (adipate-CoA A ligase), phosphotransadipyrase/adipate kinase, adipyl-CoA:acetyl-CoA transferase, or adipyl-CoA hydrolase. It can be a route. An enzyme having adipate to adipyl-CoA activity can be endogenously active or can be provided as an exogenous nucleic acid encoding the enzyme, as disclosed herein. Therefore, it is understood that any adipate pathway can be utilized with adipate to adipyl-CoA enzymatic activity to obtain the adipyl-CoA pathway. Such a pathway can be included in a microbial organism that produces 6-aminocaproic acid or caprolactam to provide an adipyl-CoA precursor for 6-aminocaproic acid and/or caprolactam production.

A further exemplary adipate route utilizes alpha-ketoadipate as a precursor (see Figure 6 and Example VI). For example, a non-naturally occurring microbial organism can have an adipate pathway that includes at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in amounts sufficient to produce adipate, the adipate pathway being , homocitrate synthase, homoaconitase, homoisocitrate dehydrogenase, 2-ketoadipate reductase, alpha-hydroxyadipate dehydratase, and oxidoreductase. A further exemplary adipate pathway utilizes the lysine degradation pathway (see Figure 7 and Example VII). Other non-naturally occurring microbial organisms can have an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in amounts sufficient to produce adipate, the adipate pathway being , carbon-nitrogen lyases, oxidoreductases, transaminases, and oxidoreductases.

Other exemplary adipate routes utilize alpha-ketoadipate as a precursor (see Figure 9 and Examples X and XI). Thus, a non-naturally occurring microbial organism can have an adipate pathway that includes at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in amounts sufficient to produce adipate, and the adipate pathway is , alpha-ketoadipyl-CoA synthetase, phosphotransketoadipylase/alpha-ketoadipate kinase, or alpha-ketoadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydrogenase; 2-hydroxyadipyl -CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase, phosphotransadipyrase/adipate kinase, adipyl-CoA:acetyl-CoA transferase, or adipyl-CoA hydrolase. Additionally, the non-naturally occurring microbial organism can have an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in amounts sufficient to produce adipate, the adipate pathway being , 2-hydroxyadipate dehydrogenase; 2-hydroxyadipyl-CoA synthetase, phosphotranshydroxyadipylase/2-hydroxyadipylase, or 2-hydroxyadipyl-CoA:acetyl-CoA transferase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase, phosphotransadipyrase/adipate kinase, adipyl-CoA:acetyl-CoA transferase, or adipyl-CoA hydrolase.

As disclosed herein, the present invention comprises at least one exogenous nucleic acid encoding a 6-aminocaproic acid pathway enzyme expressed in an amount sufficient to produce 6-aminocaproic acid. - A non-naturally occurring microbial organism comprising a microbial organism having an aminocaproic acid pathway, wherein the 6-aminocaproic acid pathway is a 3-oxo-6-aminohexanoyl-CoA thiolase; Hexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoA dehydratase; 6-aminohex-2-enoyl-CoA reductase; and 6-aminocaproyl-CoA/acyl-CoA transferase, 6-aminocaproyl -CoA synthase, or 6-aminocaproyl-CoA hydrolase (see Examples XII and XIII; steps A/B/C/D/K of Figure 11). /L/M). The present invention further provides for a microbial organism having a 6-aminocaproic acid pathway comprising at least one exogenous nucleic acid encoding a 6-aminocaproic acid pathway enzyme expressed in an amount sufficient to produce 6-aminocaproic acid. 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA/acyl-CoA transferase; , 3-oxo-6-aminohexanoyl-CoA synthase, or 3-oxo-6-aminohexanoyl-CoA hydrolase; 3-oxo-6-aminohexanoate reductase; 3-hydroxy-6-aminohexanoate and 6-aminohex-2-enoate reductase (see Examples XII and XIV; steps A/E/F/G/H of Figure 11). /I/J).

In other embodiments, the invention provides a microbial organism with a caprolactam pathway comprising at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in an amount sufficient to produce caprolactam. The caprolactam pathway provides a non-naturally occurring microbial organism comprising 6-aminocaproyl-CoA/acyl-CoA transferase or 6-aminocaproyl-CoA synthase (Example See XII and XV; step K/L in Figure 11). Such non-naturally occurring microbial organisms that contain a caprolactam pathway can further include a 6-aminocaproic acid pathway (see Figure 11). Exemplary 6-aminocaproic acid pathways include CoA-dependent aldehyde dehydrogenases; and transaminases or 6-aminocaproate dehydrogenases or 3-oxo-6-aminohexanoyl-CoA thiolases; Oxo-6-aminohexanoyl-CoA/acyl-CoA transferase, 3-oxo-6-aminohexanoyl-CoA synthase, or 3-oxo-6-aminohexanoyl-CoA hydrolase; 3-oxo-6-aminohexanoyl-CoA Noate reductase; 3-hydroxy-6-aminohexanoate dehydratase; /I/J). It is understood that these or other exemplary 6-aminocaproic acid pathways disclosed herein can further be included in microbial organisms that have a caprolactam pathway, if desired. The present invention also provides a microbial organism having a hexamethylenediamine pathway comprising at least one exogenous nucleic acid encoding a hexamethylenediamine pathway enzyme expressed in an amount sufficient to produce hexamethylenediamine. In microbial organisms that are not present in ; and a non-naturally occurring microbial organism comprising hexamethylene diamine transaminase or hexamethylene diamine dehydrogenase (see Examples XII and XVI; steps K/L/N/O/P of Figure 11). Such non-naturally occurring microbial organisms that contain the hexamethylene diamine pathway can further include the 6-aminocaproic acid pathway (see Figure 11). Exemplary 6-aminocaproic acid pathways include CoA-dependent aldehyde dehydrogenases; and transaminases or 6-aminocaproate dehydrogenases or 3-oxo-6-aminohexanoyl-CoA thiolases; Oxo-6-aminohexanoyl-CoA/acyl-CoA transferase, 3-oxo-6-aminohexanoyl-CoA synthase, or 3-oxo-6-aminohexanoyl-CoA hydrolase; 3-oxo-6-aminohexanoyl-CoA Noate reductase; 3-hydroxy-6-aminohexanoate dehydratase; /I/J). It is understood that these or other exemplary 6-aminocaproic acid pathways disclosed herein can further be included in microbial organisms having a hexamethylenediamine pathway, if desired.

In still other embodiments, the present invention provides a non-naturally occurring microbial organism having a caprolactam pathway comprising at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in sufficient amount to produce caprolactam. 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoA dehydratase; -aminohex-2-enoyl-CoA reductase (see Examples XII and XVII; Steps A/B/C/D of Figure 11). A non-naturally occurring microbial organism having a hexamethylene diamine pathway comprising at least one exogenous nucleic acid encoding a hexamethylene diamine pathway enzyme expressed in an amount sufficient to produce hexamethylene diamine, comprising: The hexamethylenediamine pathway is 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoA dehydratase; 6-aminohexanoyl- Also provided are non-naturally occurring microbial organisms comprising 2-enoyl-CoA reductase; 6-aminocaproyl-CoA reductase (aldehyde formation); See XVIII; steps A/B/C/D/N/O/P in Figure 11).

In yet other embodiments, the invention provides at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in an amount sufficient to produce 6-aminocaproic acid (6-ACA). -A non-naturally occurring microbial organism having an ACA pathway, the 6-ACA pathway comprising 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) aldolase, 2-oxoheptane-4-ene -1,7-dioate (OHED) hydratase, 2-oxohept-4-ene-1,7-dioate (OHED) reductase, 2-oxoheptane-1,7-dioate (2-OHD) decarboxylase, adipate semialdehyde Aminotransferase, adipate semialdehyde oxidoreductase (amination), 2-oxohept-4-ene-1,7-dioate (OHED) decarboxylase, 6-oxohex-4-enoate (6-OHE) reductase, 2-oxoheptane -1,7-dioate (2-OHD) aminotransferase, 2-oxoheptane-1,7-dioate (2-OHD) oxidoreductase (amination), 2-aminoheptane-1,7-dioate (2-AHD) ) decarboxylase, 2-oxohept-4-ene-1,7-dioate (OHED) aminotransferase, 2-oxohept-4-ene-1,7-dioate (OHED) oxidoreductase (amination), 2-aminohept- 4-ene-1,7-dioate (2-AHE) reductase, 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) formate lyase, 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 2,3-dehydroadipyl-CoA reductase, adipyl-CoA dehydrogenase, 2-oxohept-4-ene-1,7-dioate (OHED) formate lyase, 2 -Oxoheptane-1,7-dioate (OHED) dehydrogenase, 2-oxoheptane-1,7-dioate (2-OHD) formate lyase, 2-oxoheptane-1,7-dioate (2-OHD) The non-naturally occurring microbial organism is provided comprising a dehydrogenase or pyruvate formate lyase activating enzyme (see Examples XIX and XXI; Steps A-Q of Figure 12). In further embodiments, the 6-ACA pathway comprises succinate semialdehyde dehydrogenase, alpha-ketoglutarate decarboxylase, or phosphoenolpyruvate (PEP) carboxykinase.

The invention further comprises a 6-ACA pathway comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in sufficient amounts to produce 6-aminocaproic acid (6-ACA). , a non-naturally occurring microbial organism in which the 6-ACA pathway produces HODH aldolase; OHED hydratase; OHED reductase; 2-OHD decarboxylase; (See Examples XIX and XXI; Steps A/B/C/D/E of Figure 12). In further embodiments, the 6-ACA pathway comprises succinate semialdehyde dehydrogenase, alpha-ketoglutarate decarboxylase, or phosphoenolpyruvate (PEP) carboxykinase. In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, the set comprising: HODH aldolase; OHED hydratase; OHED reductase; 2-OHD decarboxylase; and adipate semialdehyde aminotransferase or adipate semialdehyde oxidoreductase (amination).

The invention further comprises a 6-ACA pathway comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in sufficient amounts to produce 6-aminocaproic acid (6-ACA). , a non-naturally occurring microbial organism in which the 6-ACA pathway produces HODH aldolase; OHED hydratase; OHED decarboxylase; 6-OHE reductase; (See Examples XIX and XXI; Steps A/B/F/G/E of Figure 12). In further embodiments, the 6-ACA pathway comprises succinate semialdehyde dehydrogenase, alpha-ketoglutarate decarboxylase, or phosphoenolpyruvate (PEP) carboxykinase. In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, the set comprising: HODH aldolase; OHED hydratase; It encodes OHED decarboxylase; 6-OHE reductase; and adipate semialdehyde aminotransferase or adipate semialdehyde oxidoreductase (amination).

The invention further comprises a 6-ACA pathway comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in sufficient amounts to produce 6-aminocaproic acid (6-ACA). , a non-naturally occurring microbial organism, wherein the 6-ACA pathway comprises HODH aldolase; OHED hydratase; OHED aminotransferase or OHED oxidoreductase (amination); 2-AHE reductase; or 2-AHD decarboxylase. Provide a non-naturally occurring microbial organism (see Examples XIX and XXI; steps A/B/J/D/I of Figure 12). In further embodiments, the 6-ACA pathway comprises succinate semialdehyde dehydrogenase, alpha-ketoglutarate decarboxylase, or phosphoenolpyruvate (PEP) carboxykinase. In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, the set comprising: HODH aldolase; OHED hydratase; Encodes OHED aminotransferase or OHED oxidoreductase (amination); 2-AHE reductase; and 2-AHD decarboxylase.

The invention further comprises a 6-ACA pathway comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in sufficient amounts to produce 6-aminocaproic acid (6-ACA). , non-naturally occurring microbial organisms, in which the 6-ACA pathway produces HODH aldolase; OHED hydratase; OHED reductase; 2-OHD aminotransferase or 2-OHD oxidoreductase (amination); or 2-AHD decarboxylase. (See Examples XIX and XXI; Steps A/B/C/H/I of Figure 12). In further embodiments, the 6-ACA pathway comprises succinate semialdehyde dehydrogenase, alpha-ketoglutarate decarboxylase, or phosphoenolpyruvate (PEP) carboxykinase. In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, the set comprising: HODH aldolase; OHED hydratase; OHED reductase; 2-OHD Encodes aminotransferase or 2-OHD oxidoreductase (amination); and 2-AHD decarboxylase.

The invention further comprises a 6-ACA pathway comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in sufficient amounts to produce 6-aminocaproic acid (6-ACA). , a non-naturally occurring microbial organism in which the 6-ACA pathway is activated by HODH aldolase; HODH formate lyase and pyruvate formate lyase activating enzymes or HODH dehydrogenase; (See Examples XIX and XXI). ; Steps A/L/M/N/O/E in Figure 12). In further embodiments, the 6-ACA pathway comprises succinate semialdehyde dehydrogenase, alpha-ketoglutarate decarboxylase, or phosphoenolpyruvate (PEP) carboxykinase. In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, the set comprising HODH aldolase; HODH formate lyase and pyruvate formate lyase activities. 3-hydroxyadipyl-CoA dehydratase; 2,3-dehydroadipyl-CoA reductase; adipyl-CoA dehydrogenase; and adipate semialdehyde aminotransferase or adipate semialdehyde oxidoreductase (amination). .

The invention further comprises a 6-ACA pathway comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in sufficient amounts to produce 6-aminocaproic acid (6-ACA). , a non-naturally occurring microbial organism, in which the 6-ACA pathway includes HODH aldolase; OHED hydratase; OHED formate lyase and pyruvate formate lyase activating enzymes or OHED dehydrogenase; 2,3-dehydroadipyl-CoA reductase; or adipate semialdehyde aminotransferase or adipate semialdehyde oxidoreductase (amination) (see Examples XIX and XXI; Step A of Figure 12). /B/P/N/O/E). In further embodiments, the 6-ACA pathway comprises succinate semialdehyde dehydrogenase, alpha-ketoglutarate decarboxylase, or phosphoenolpyruvate (PEP) carboxykinase. In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, the set comprising: HODH aldolase; OHED hydratase; OHED formate lyase and pyruvate. It encodes formate lyase activating enzyme or OHED dehydrogenase; 2,3-dehydroadipyl-CoA reductase; adipyl-CoA dehydrogenase; and adipate semialdehyde aminotransferase or adipate semialdehyde oxidoreductase (amination).

The invention further comprises a 6-ACA pathway comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in sufficient amounts to produce 6-aminocaproic acid (6-ACA). , a non-naturally occurring microbial organism, in which the 6-ACA pathway is activated by HODH aldolase; OHED hydratase; OHED reductase; 2-OHD formate lyase and pyruvate formate lyase activating enzyme or 2-OHD dehydrogenase; ; or providing a non-naturally occurring microbial organism comprising adipate semialdehyde aminotransferase or adipate semialdehyde oxidoreductase (amination) (see Examples XIX and XXI; steps A/B/C of Figure 12). /Q/O/E). In further embodiments, the 6-ACA pathway comprises succinate semialdehyde dehydrogenase, alpha-ketoglutarate decarboxylase, or phosphoenolpyruvate (PEP) carboxykinase. In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, the set comprising: HODH aldolase; OHED hydratase; OHED reductase; 2-OHD It encodes formate lyase and pyruvate formate lyase activating enzyme or 2-OHD dehydrogenase; adipyl-CoA dehydrogenase; and adipate semialdehyde aminotransferase or adipate semialdehyde oxidoreductase (amination). The invention further comprises a 6-ACA pathway comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in sufficient amounts to produce 6-aminocaproic acid (6-ACA). , a non-naturally occurring microbial organism, in which the 6-ACA pathway is involved in glutamyl-CoA transferase, glutamyl-CoA ligase, beta-ketothiolase, 3-oxo-6-aminopimeloyl-CoA oxidoreductase, 3-hydroxy-6- Non-naturally occurring microbial organisms comprising aminopimeloyl-CoA dehydratase, 6-amino-7-carboxyhept-2-enoyl-CoA reductase, 6-aminopimeloyl-CoA reductase (aldehyde formation), or 2-aminopimelate decarboxylase (See Examples XXV and XXVI; steps A/B/C/D/E/I/J of Figure 20). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, the set comprising glutamyl-CoA transferase or glutamyl-CoA ligase; beta- Ketothiolase; 3-oxo-6-aminopimeloyl-CoA oxidoreductase; 3-hydroxy-6-aminopimeloyl-CoA dehydratase; 6-amino-7-carboxyhept-2-enoyl-CoA reductase; 6-aminopimeloyl-CoA reductase (aldehyde formation ); and encodes 2-aminopimelate decarboxylase.

The invention further comprises a 6-ACA pathway comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in sufficient amounts to produce 6-aminocaproic acid (6-ACA). , a non-naturally occurring microbial organism, the 6-ACA pathway has been shown to contain glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, 3-oxopimeloyl-CoA ligase, and 3-oxopimeloyl-CoA ligase. Provided is a non-naturally occurring microbial organism comprising a melate aminotransferase, a 3-oxopimelate aminating oxidoreductase, a 3-aminopimelate 2,3-aminomutase, or a 2-aminopimelate decarboxylase. See Examples XXV and XXVI; steps A/B/J/T/AA of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate 2,3-aminomutase; and 2- Encodes aminopimelate decarboxylase. The invention further comprises a 6-ACA pathway comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in sufficient amounts to produce 6-aminocaproic acid (6-ACA). , a non-naturally occurring microbial organism, in which the 6-ACA pathway provides a non-naturally occurring microbial organism containing homolysine 2-monooxygenase (see Examples XXV and XXVI; Step A). In a further embodiment, the 6-ACA pathway involves hydrolysis of the 6-aminohexanamide product with a dilute acid or base to convert the 6-aminohexanamide to 6-aminocaproate (see Examples XXV and XXVI). See step B in Figure 23).

The invention further comprises a 6-ACA pathway comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in sufficient amounts to produce 6-aminocaproic acid (6-ACA). , a non-naturally occurring microbial organism, in which the 6-ACA pathway includes adipate reductase, adipate kinase, or adipyl phosphate reductase (see Example XXVIII; steps X/Y/Z and Example XXXI). In further embodiments, the 6-ACA pathway includes adipate reductase. In other further embodiments, the 6-aminocaproic acid (6-ACA) pathway includes adipate kinase and adipyl phosphate reductase. In other embodiments, the microbial organism having a 6-ACA pathway described above further comprises an adipate pathway, a caprolactam pathway, and/or a hexamethylenediamine pathway as described herein (see Example XXVIII; Steps A to W in Figure 25).

In one embodiment, the invention provides a 6-ACA pathway enzyme comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in sufficient amounts to produce 6-aminocaproic acid (6-ACA). The 6-ACA pathway is a non-naturally occurring microbial organism that has a 2-amino-7-oxosabarate ketoacid decarboxylase, 2-amino-7-oxoheptanoate decarboxylase, 2-amino-7-oxoheptanoate decarboxylase, Amino-7-oxoheptanoate oxidoreductase, 2-aminopimelate decarboxylase, 6-aminohexanal oxidoreductase, 2-amino-7-oxoheptanoate decarboxylase, or 2-amino-7-oxosabalate A non-naturally occurring microbial organism is provided that includes an amino acid decarboxylase (see Examples XXV and XXVI; Steps A/B/D/E/F/G/I of Figure 26). In further embodiments, the microbial organism contains at least one exogenous enzyme encoding a 2-amino-7-oxosubarate pathway enzyme expressed in sufficient amount to produce 2-amino-7-oxosabarate. It has a 2-amino-7-oxosabarate pathway with nucleic acids, and the 2-amino-7-oxosabarate pathway is 2-amino-5-hydroxy-7-oxosabarate aldolase, 2-amino-5- hydroxy-7-oxosubarate dehydratase, or 2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; Steps A/B/C of Figure 27).

In another embodiment of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, the set comprising 2-amino-7-oxosabarate ketoacid. encodes decarboxylase; 2-amino-7-oxoheptanoate oxidoreductase; and 2-aminopimelate decarboxylase (see Example XXV; steps A/D/E of Figure 26). In another embodiment of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, the set comprising 2-amino-7-oxosabarate ketoacid. encodes decarboxylase; 2-amino-7-oxoheptanoate decarboxylase; and 6-aminohexanal oxidoreductase (see Example XXV; steps A/B/F of Figure 26). In another embodiment of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, the set comprising a 2-amino-7-oxosabalate amino acid encodes carboxylase; 2-amino-7-oxoheptanoate decarboxylase; and 6-aminohexanal oxidoreductase (see Example XXV; steps I/G/F of Figure 26). In a further aspect of each of the above embodiments, the microbial organism encodes a 2-amino-7-oxosabalate pathway enzyme expressed in an amount sufficient to produce 2-amino-7-oxosabalate. the 2-amino-7-oxosabarate pathway has a second set of exogenous nucleic acids that 2-amino-5-hydroxy-7-oxosabarate dehydratase; and 2-amino-5-en-7-oxosabarate reductase (see Examples XXV and XXVI; steps of FIG. 27). A/B/C).

In other embodiments, the present invention provides methods for producing naturally occurring hexamethylene diamine (HMDA) containing at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in sufficient amounts to produce HMDA. In microbial organisms that do not, the HMDA pathway includes 6-aminocaproate kinase, [(6-aminohexanoyl)oxy]phosphonate (6-AHOP) oxidoreductase, 6-aminocaproic acid semialdehyde aminotransferase, 6-aminocaproate semialdehyde aminotransferase, Aminocaproate semialdehyde oxidoreductase (amination), 6-aminocaproate N-acetyltransferase, 6-acetamidohexanoate kinase, [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) oxidoreductase, 6 -acetamidohexanal aminotransferase, 6-acetamidohexanal oxidoreductase (amination), 6-acetamidohexanamine N-acetyltransferase, 6-acetamidohexanamine hydrolase (amide), 6-acetamidohexanoate CoA transferase, 6-acetamidohexanamine Noate-CoA ligase, 6-acetamidohexanoyl-CoA oxidoreductase, [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) acyltransferase, [(6-aminohexanoyl)oxy]phosphonate (6-AHOP) Provide a non-naturally occurring microbial organism comprising an acyltransferase, a 6-aminocaproate CoA transferase, and a 6-aminocaproate CoA ligase (see Examples XX and XXI; Step A of Figure 13). ~N).

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in an amount sufficient to produce hexamethylene diamine (HMDA). In the body, the HMDA pathway is a naturally occurring enzyme that includes 6-aminocaproate kinase; 6-AHOP oxidoreductase; Provide free microbial organisms (see Examples XX and XXI; Steps A/B/C of Figure 13). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: 6-aminocaproate kinase; 6-AHOP oxidoreductase; and encodes 6-aminocaproic acid semialdehyde oxidoreductase (amination) or 6-aminocaproic acid semialdehyde aminotransferase.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in an amount sufficient to produce hexamethylene diamine (HMDA). 6-aminocaproate kinase; 6-AHOP acyltransferase; 6-aminocaproyl-CoA oxidoreductase; or 6-aminocaproic acid semialdehyde oxidoreductase (amination) A non-naturally occurring microbial organism is provided that includes an acid semialdehyde aminotransferase (see Examples XX and XXI; Steps A/L/N/C of Figure 13). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: 6-aminocaproate kinase; 6-AHOP acyltransferase; It encodes 6-aminocaproyl-CoA oxidoreductase; and 6-aminocaproic acid semialdehyde oxidoreductase (amination) or 6-aminocaproic acid semialdehyde aminotransferase.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in an amount sufficient to produce hexamethylene diamine (HMDA). 6-aminocaproate CoA transferase or 6-aminocaproate CoA ligase; 6-aminocaproyl-CoA oxidoreductase; or 6-aminocaproate semialdehyde oxidoreductase (amination). or 6-aminocaproic semialdehyde aminotransferase (see Examples XX and XXI; Steps M/N/C of Figure 13). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising 6-aminocaproate CoA transferase or 6-aminocaproate CoA transferase. It encodes ate-CoA ligase; 6-aminocaproyl-CoA oxidoreductase; and 6-aminocaproic acid semialdehyde oxidoreductase (amination) or 6-aminocaproic acid semialdehyde aminotransferase.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in an amount sufficient to produce hexamethylene diamine (HMDA). 6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate kinase; 6-AAHOP oxidoreductase; 6-acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase ); or providing a non-naturally occurring microbial organism comprising 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide) (see Examples XX and XXI; steps of FIG. 13 D/E/F/G/H). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: 6-aminocaproate N-acetyltransferase; 6-acetamide hexanoate kinase; 6-AAHOP oxidoreductase; 6-acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase (amination); and 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide). Code.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in sufficient amounts to produce hexamethylene diamine (HMDA). 6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate-CoA transferase or 6-acetamidohexanoate-CoA ligase; 6-acetamidohexanoyl-CoA oxidoreductase; Provided is a non-naturally occurring microbial organism comprising acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase (amination); or 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide) ( See Examples XX and XXI; steps D/I/J/G/H in Figure 13). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: 6-aminocaproate N-acetyltransferase; 6-acetamide Hexanoate CoA transferase or 6-acetamidohexanoate CoA ligase; 6-acetamidohexanoyl-CoA oxidoreductase; 6-acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase (amination); and 6-acetamidohexanamine N -Encodes acetyltransferase or 6-acetamidohexanamine hydrolase (amide). The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in sufficient amounts to produce hexamethylene diamine (HMDA). 6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate kinase; 6-AAHOP oxidoreductase; 6-acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase ); or providing a non-naturally occurring microbial organism comprising 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide) (see Examples XX and XXI; steps of FIG. 13 D/E/K/J/G). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: 6-aminocaproate N-acetyltransferase; 6-acetamide hexanoate kinase; 6-AAHOP oxidoreductase; 6-acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase (amination); and 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide). Code. The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in sufficient amounts to produce hexamethylene diamine (HMDA). The HMDA pathway includes glutamyl-CoA transferase, glutamyl-CoA ligase, beta-ketothiolase, 3-oxo-6-aminopimeloyl-CoA oxidoreductase, 3-hydroxy-6-aminopimeloyl-CoA dehydratase, 6-amino- 7-carboxyhept-2-enoyl-CoA reductase, 6-aminopimeloyl-CoA reductase (aldehyde formation), 2-amino-7-oxoheptanoate aminotransferase, 2-amino-7-oxoheptanoate aminating oxidoreductase , or a non-naturally occurring microbial organism comprising a homolysine decarboxylase (see Examples XXIV and XXVI; Steps A-H of Figure 20). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutamyl-CoA transferase or ligase; beta-ketothiolase; -6-aminopimeloyl-CoA oxidoreductase; 3-hydroxy-6-aminopimeloyl-CoA dehydratase; 6-amino-7-carboxyhept-2-enoyl-CoA reductase; 6-aminopimeloyl-CoA reductase (aldehyde formation); 2-amino -7-oxoheptanoate aminotransferase or aminating oxidoreductase; and homolysine decarboxylase.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in sufficient amounts to produce hexamethylene diamine (HMDA). The HMDA pathway includes glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate reductase, 3-oxo- 1-Carboxyheptanal aminotransferase, 3-oxo-1-carboxyheptanal aminating oxidoreductase, 3-oxo-7-aminoheptanoate 3-aminotransferase, 3-oxo-7-aminoheptanoate 3-amino oxidoreductase, 3-oxopimelate kinase, 5-oxopimeloylphosphonate reductase, 3-oxopimelate-CoA transferase, 3-oxopimelate ligase, 5-oxopimelate-CoA reductase (aldehyde formation), 3-oxopimelate-CoA transferase melate aminotransferase, 3-oxopimelate aminating oxidoreductase, 3-aminopimelate CoA transferase, 3-aminopimelate ligase, 5-aminopimeloyl-CoA reductase (aldehyde formation), 3-aminopimelate kinase, 5 -Aminopimeloylphosphonate reductase, 3-aminopimelate reductase, 3-amino-7-oxoheptanoate 2,3-aminomutase, 2-amino-7-oxoheptanoate 7-aminotransferase, 2- Amino-7-oxoheptanoate aminating oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomutase, homolysine decarboxylase, 3-aminopimelate 2,3-aminomutase, 2-aminopimelate kinase , 2-aminopimelate-CoA transferase, 2-aminopimelate-CoA ligase, 2-aminopimelate reductase, 6-aminopimeloylphosphonate reductase, 6-aminopimeloyl-CoA reductase (aldehyde formation), 3-amino-7-oxoheptano ate 7-aminotransferase, or 3-amino-7-oxoheptanoate aminating oxidoreductase (see Examples XXIV and XXVI; Figure 21).

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in sufficient amounts to produce hexamethylene diamine (HMDA). The HMDA pathway includes glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate reductase, 3-oxo-1 -Carboxyheptanal 7-aminotransferase, 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase, 3-oxo-7-aminoheptanoate 3-aminotransferase, 3-oxo-7-aminoheptanoate Provided are non-naturally occurring microbial organisms comprising a 3-aminated oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI). ;Steps A/B/C/D/E/R/S in Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate reductase; 3-oxo-1-carboxyheptanal 7-aminotransferase or 3-oxo-1-carboxyheptanal 7-amination Oxidoreductase; 3-oxo-7-aminoheptanoate 3-aminotransferase or 3-oxo-7-aminoheptanoate 3-amination oxidoreductase; 3,7-diaminoheptanoate 2,3-aminomutase; and encodes homolysine decarboxylase.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in an amount sufficient to produce hexamethylene diamine (HMDA). The HMDA pathway includes glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate kinase; 5-oxopimeloyl-CoA ylphosphonate reductase, 3-oxo-1-carboxyheptanal 7-aminotransferase, 3-oxo-1-carboxyheptanal 7-amination oxidoreductase, 3-oxo-7-aminoheptanoate 3-aminotransferase, 3 - Provides a non-naturally occurring microbial organism comprising oxo-7-aminoheptanoate 3-aminated oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomutase, or homolysine decarboxylase ( See Examples XXIV and XXVI; steps A/B/F/G/D/E/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate kinase; 5-oxopimeloylphosphonate reductase; 3-oxo-1-carboxyheptanal 7-aminotransferase or 3-oxo- 1-Carboxyheptanal 7-aminated oxidoreductase; 3-oxo-7-aminoheptanoate 3-aminotransferase or 3-oxo-7-aminoheptanoate 3-aminated oxidoreductase; 3,7-diaminohepta It encodes noate 2,3-aminomutase; and homolysine decarboxylase.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in sufficient amounts to produce hexamethylene diamine (HMDA). The HMDA pathway includes glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate-CoA transferase, 3-oxopimelate-CoA ligase, 5 -Oxopimeloyl-CoA reductase (aldehyde formation), 3-oxo-1-carboxyheptanal 7-aminotransferase, 3-oxo-1-carboxyheptanal 7-amination oxidoreductase, 3-oxo-7-aminoheptanoate Non-naturally occurring microorganisms containing 3-aminotransferase, 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomutase, or homolysine decarboxylase Provide the organism (see Examples XXIV and XXVI; steps A/B/H/I/D/E/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate-CoA transferase or 3-oxopimelate-CoA ligase; 5-oxopimeloyl-CoA reductase (aldehyde formation); 3-oxo-1-carboxyheptanal 7-amino transferase or 3-oxo-1-carboxyheptanal 7-amination oxidoreductase; 3-oxo-7-aminoheptanoate 3-aminotransferase or 3-oxo-7-aminoheptanoate 3-amination oxidoreductase; Encodes 3,7-diaminoheptanoate 2,3-aminomutase; and homolysine decarboxylase.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in an amount sufficient to produce hexamethylene diamine (HMDA). The HMDA pathway includes glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate reductase, 3-oxo-1 -Carboxyheptanal 3-aminotransferase, 3-oxo-1-carboxyheptanal 3-aminated oxidoreductase, 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate Provided are non-naturally occurring microbial organisms comprising a 7-aminated oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI). ; Steps A/B/C/AB/Z/R/S in Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate reductase; 3-oxo-1-carboxyheptanal 3-aminotransferase or 3-oxo-1-carboxyheptanal 3-amination oxidoreductase; 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; 3,7-diaminoheptanoate 2,3-aminomutase; and Encodes homolysine decarboxylase.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in an amount sufficient to produce hexamethylene diamine (HMDA). The HMDA pathway includes glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate kinase, and 5-oxopimeloyl-CoA ligase. phosphonate reductase, 3-oxo-1-carboxyheptanal 3-aminotransferase, 3-oxo-1-carboxyheptanal 3-amination oxidoreductase, 3-amino-7-oxoheptanoate 7-aminotransferase, 3 Provided is a non-naturally occurring microbial organism comprising -amino-7-oxoheptanoate 7-aminated oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomutase, or homolysine decarboxylase ( See Examples XXIV and XXVI; steps A/B/H/I/AB/Z/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate kinase; 5-oxopimeloylphosphonate reductase; 3-oxo-1-carboxyheptanal 3-aminotransferase or 3-oxo- 1-Carboxyheptanal 3-aminated oxidoreductase; 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminated oxidoreductase; 3,7-diaminohepta It encodes noate 2,3-aminomutase; and homolysine decarboxylase.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in an amount sufficient to produce hexamethylene diamine (HMDA). The HMDA pathway includes glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate-CoA transferase or 3-oxopimelate-CoA ligase, 5 -Oxopimeloyl-CoA reductase (aldehyde formation), 3-oxo-1-carboxyheptanal 3-aminotransferase, 3-oxo-1-carboxyheptanal 3-amination oxidoreductase, 3-amino-7-oxoheptanoate Non-naturally occurring microorganisms containing 7-aminotransferase, 3-amino-7-oxoheptanoate 7-aminating oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomutase, or homolysine decarboxylase Provide the organism (see Examples XXIV and XXVI; steps A/B/F/G/AB/Z/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate-CoA transferase or 3-oxopimelate-CoA ligase; 5-oxopimeloyl-CoA reductase (aldehyde formation); 3-oxo-1-carboxyheptanal 3-amino transferase or 3-oxo-1-carboxyheptanal 3-amination oxidoreductase; 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-amination oxidoreductase; Encodes 3,7-diaminoheptanoate 2,3-aminomutase; and homolysine decarboxylase.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in sufficient amounts to produce hexamethylene diamine (HMDA). The HMDA pathway includes glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate aminotransferase or 3-oxopimeloyl-CoA ligase. Melate aminating oxidoreductase, 3-aminopimelate reductase, 3-amino-7-oxoheptanoate 2,3-aminomutase, 2-amino-7-oxoheptanoate 7-aminotransferase, 2-amino -7-oxoheptanoate aminating oxidoreductase, or homolysine decarboxylase (see Examples XXIV and XXVI; Steps A/B// of Figure 21). J/O/P/Q/S). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate reductase; 3-amino-7-oxohepta It encodes noate 2,3-aminomutase; 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate 7-aminating oxidoreductase; and homolysine decarboxylase.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in sufficient amounts to produce hexamethylene diamine (HMDA). The HMDA pathway includes glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate aminotransferase or 3-oxopimeloyl-CoA ligase. Melate aminating oxidoreductase, 3-aminopimelate kinase, 5-aminopimeloylphosphonate reductase, 3-amino-7-oxoheptanoate 2,3-aminomutase, 2-amino-7-oxoheptano ate 7-aminotransferase, 2-amino-7-oxoheptanoate 7-aminating oxidoreductase, or homolysine decarboxylase (see Examples XXIV and XXVI). step A/B/J/M/N/P/Q/S in Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate kinase; 5-aminopimelate phosphonate reductase ; 3-amino-7-oxoheptanoate 2,3-aminomutase; 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and homo Encodes lysine decarboxylase.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in an amount sufficient to produce hexamethylene diamine (HMDA). The HMDA pathway includes glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate aminotransferase, and 3-oxopimelate aminotransferase. Melate aminating oxidoreductase, 3-aminopimelate CoA transferase, 3-aminopimelate CoA ligase, 5-aminopimeloyl-CoA reductase (aldehyde formation), 3-amino-7-oxoheptanoate 2,3-aminomutase, 2-amino -7-oxoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoate aminating oxidoreductase or homolysine decarboxylase (Examples XXIV and XXVI) See steps A/B/J/K/L/P/Q/S in Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimelate-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate-CoA transferase or 3-aminopimelate-CoA ligase; 5-aminopimeloyl -CoA reductase (aldehyde formation); 3-amino-7-oxoheptanoate 2,3-aminomutase; 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate It encodes an aminating oxidoreductase; and a homolysine decarboxylase.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in an amount sufficient to produce hexamethylene diamine (HMDA). The HMDA pathway includes glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate aminotransferase, and 3-oxopimeloyl-CoA hydrolase. Melate aminating oxidoreductase, 3-aminopimelate reductase, 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate 7-aminating oxidoreductase, 3,7 -Providing a non-naturally occurring microbial organism comprising diaminoheptanoate 2,3-aminomutase, or homolysine decarboxylase (see Examples XXIV and XXVI; Steps A/B/J of Figure 21) /O/Z/R/S). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate amination oxidoreductase; 3-aminopimelate reductase; 3-amino-7-oxo It encodes heptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; 3,7-diaminoheptanoate 2,3-aminomutase; and homolysine decarboxylase.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in sufficient amounts to produce hexamethylene diamine (HMDA). The HMDA pathway includes glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate aminotransferase, and 3-oxopimeloyl-CoA hydrolase. Melate aminating oxidoreductase, 3-aminopimelate CoA transferase, 3-aminopimelate CoA ligase, 5-aminopimeloyl-CoA reductase (aldehyde formation), 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7 - provides a non-naturally occurring microbial organism comprising an oxoheptanoate aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see Examples XXIV and XXVI). See steps A/B/J/K/L/Z/R/S in Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimelate-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate-CoA transferase or 3-aminopimelate-CoA ligase; 5-aminopimelate- CoA-reductase (aldehyde formation); 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate aminating oxidoreductase; 3,7-diaminoheptanoate 2,3- It encodes aminomutase; and homolysine decarboxylase.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in an amount sufficient to produce hexamethylene diamine (HMDA). The HMDA pathway includes glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate aminotransferase, and 3-oxopimelate aminotransferase. Melate amination oxidoreductase, 3-aminopimelate kinase, 5-aminopimeloylphosphonate reductase, 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate amino oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomutase, or homolysine decarboxylase (see Examples XXIV and XXVI; FIG. steps A/B/J/M/N/Z/R/S). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate kinase; 5-aminopimelate phosphonate reductase ;3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate aminating oxidoreductase;3,7-diaminoheptanoate 2,3-aminomutase; Encodes carboxylase.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in an amount sufficient to produce hexamethylene diamine (HMDA). The HMDA pathway includes glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate aminotransferase, and 3-oxopimelate aminotransferase. Melate aminating oxidoreductase, 3-aminopimelate 2,3-aminomutase, 2-aminopimelate reductase, 2-amino-7-oxoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoate Provide a non-naturally occurring microbial organism comprising an aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/J/T/W/Q/ of Figure 21). S). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate 2,3-aminomutase; 2-aminopimelate encodes rate reductase; 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and homolysine decarboxylase.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in an amount sufficient to produce hexamethylene diamine (HMDA). The HMDA pathway includes glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate aminotransferase, and 3-oxopimelate aminotransferase. Melate aminating oxidoreductase, 3-aminopimelate 2,3-aminomutase, 2-aminopimelate kinase, 6-aminopimeloylphosphonate reductase, 2-amino-7-oxoheptanoate 7-aminotransferase, 2 - provides a non-naturally occurring microbial organism comprising an amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; Steps A/B of Figure 21). /J/T/U/X/Q/S). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate 2,3-aminomutase; 2-aminopimelate encodes rate kinase; 6-aminopimeloylphosphonate reductase; 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and homolysine decarboxylase .

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in an amount sufficient to produce hexamethylene diamine (HMDA). The HMDA pathway includes glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate aminotransferase, and 3-oxopimelate aminotransferase. Melate aminating oxidoreductase, 3-aminopimelate 2,3-aminomutase, 2-aminopimelate CoA transferase, 2-aminopimelate CoA ligase, 6-aminopimeloyl-CoA reductase (aldehyde formation), 2-amino-7-oxoheptanoate Provided are non-naturally occurring microbial organisms comprising a 7-aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine decarboxylase (see Examples XXIV and XXVI; FIG. 21 steps A/B/J/T/V/Y/Q/S). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimelate-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate 2,3-aminomutase; 2-aminopimelate CoA transferase or 2-aminopimelate-CoA ligase; 6-aminopimeloyl-CoA reductase (aldehyde formation); 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and homo Encodes lysine decarboxylase.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in an amount sufficient to produce hexamethylene diamine (HMDA). The HMDA pathway includes 2-oxo-4-hydroxy-7-aminoheptanoate aldolase, 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase, 2-oxo-7-aminohepta-3 -enoate reductase, 2-oxo-7-aminoheptanoate aminotransferase, 2-oxo-7-aminoheptanoate aminotransferase amination oxidoreductase, homolysine decarboxylase, 2-oxo-7-aminoheptanoate Provide a non-naturally occurring microbial organism comprising a decarboxylase, 6-aminohexal aminotransferase, or 6-aminohexal aminating oxidoreductase (see Examples XXIV and XXVI; steps A-G of Figure 22). ). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising 2-oxo-4-hydroxy-7-aminoheptanoate. Aldolase; 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; 2-oxo-7-aminohepta-3-enoate reductase; 2-oxo-7-aminoheptanoate aminotransferase or 2-oxo-7 -encodes aminoheptanoate aminating oxidoreductase; and homolysine decarboxylase. In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising 2-oxo-4-hydroxy-7-aminoheptanoate. aldolase; 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; 2-oxo-7-aminohepta-3-enoate reductase; 2-oxo-7-aminoheptanoate decarboxylase; and 6-aminohexanal Encodes an aminotransferase or 6-aminohexanalaminating oxidoreductase.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in sufficient amounts to produce hexamethylene diamine (HMDA). The HMDA pathway includes 6-aminocaproate reductase, 6-aminocaproate semialdehyde aminotransferase, 6-aminocaproate semialdehyde oxidoreductase (amination), 6-aminocaproate N-acetyltransferase, Naturally occurring, including 6-acetamidohexanoate reductase, 6-acetamidohexanal aminotransferase, 6-acetamidohexanal oxidoreductase (amination), 6-acetamidohexanamine N-acetyltransferase, or acetamidohexanamine hydrolase (amide) (See Example XXVII; Steps O/C or D/P/G/H of Figure 24 and Example XXXI). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: 6-aminocaproate reductase; and 6-aminocaproate semi- Encodes aldehyde aminotransferase or 6-aminocaproic acid semialdehyde oxidoreductase (amination). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: 6-aminocaproate N-acetyltransferase; 6-acetamide Encodes hexanoate reductase; 6-acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase (amination); and 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide). The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in sufficient amounts to produce hexamethylene diamine (HMDA). The HMDA pathway includes 2-amino-7-oxosabarate ketoacid decarboxylase, 2-amino-7-oxoheptanoate decarboxylase, 6-aminohexanalaminating oxidoreductase, 6-aminohexanalamino Transferase, 2-amino-7-oxoheptanoate decarboxylase, homolysine decarboxylase, 2-amino-7-oxosabarate amino acid decarboxylase, 2-oxo-7-aminoheptanoate aminating oxidoreductase, 2- Oxo-7-aminoheptanoate aminotransferase, 2-amino-7-oxosabarate aminating oxidoreductase, 2-amino-7-oxosabarate aminotransferase, 2,7-diaminosabarate decarboxylase, 2-amino -7-oxoheptanoate aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase (see Examples XXIV and XXVI; FIG. 26 steps A/B/C/G/H/I/J/K/L/M). In further embodiments, the microbial organism contains at least one exogenous enzyme encoding a 2-amino-7-oxosubarate pathway enzyme expressed in sufficient amount to produce 2-amino-7-oxosabarate. It has a 2-amino-7-oxosabarate pathway with nucleic acids, and the 2-amino-7-oxosabarate pathway is 2-amino-5-hydroxy-7-oxosabarate aldolase, 2-amino-5- hydroxy-7-oxosubarate dehydratase, or 2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; Steps A/B/C of Figure 27).

In another embodiment of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising 2-amino-7-oxosabarate aminating oxidoreductase. or 2-amino-7-oxosubarate aminotransferase; 2,7-diaminosubarate decarboxylase; and homolysine decarboxylase (see Examples XXIV and XXVI; steps K/L/ of FIG. 26) H). In another embodiment of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: 2-amino-7-oxosubarate amino acid decarboxylase; 2-oxo-7-aminoheptanoate aminating oxidoreductase or 2-oxo-7-aminoheptanoate aminotransferase; and homolysine decarboxylase (see Examples XXIV and XXVI; FIG. 26) Step I/J/H). In another embodiment of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: 2-amino-7-oxosubarate amino acid decarboxylase; 2-oxo-7-aminoheptanoate decarboxylase; and 6-aminohexanalaminating oxidoreductase or 6-aminohexanal aminotransferase (see Examples XXIV and XXVI; Steps I/G in Figure 26). /C). In another embodiment of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising 2-amino-7-oxosabarate ketoacid decarboxylase. ; 2-amino-7-oxoheptanoate decarboxylase; and 6-aminohexanalaminating oxidoreductase or 6-aminohexanal aminotransferase (see Examples XXIV and XXVI; Step A/ of Figure 26). B/C). In another embodiment of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising 2-amino-7-oxosabarate ketoacid decarboxylase. ; 2-amino-7-oxoheptanoate aminating oxidoreductase or 2-amino-7-oxoheptanoate aminotransferase; and homolysine decarboxylase (see Examples XXIV and XXVI; FIG. 26 steps A/M/H). In a further aspect of each of the above embodiments, the microbial organism encodes a 2-amino-7-oxosabalate pathway enzyme expressed in an amount sufficient to produce 2-amino-7-oxosabalate. the 2-amino-7-oxosabarate pathway has a second set of exogenous nucleic acids that 2-amino-5-hydroxy-7-oxosubarate dehydratase; and 2-amino-5-en-7-oxosabarate reductase (see Examples XXV and XXVI; steps of FIG. 27). A/B/C). The present invention further provides for a non-naturally occurring microbial organism having an LA pathway comprising at least one exogenous nucleic acid encoding an LA pathway enzyme expressed in sufficient quantity to produce levulinic acid (LA). wherein the LA pathway comprises 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA/acyl-CoA transferase, 3-oxoadipyl-CoA synthase, 3-oxoadipyl-CoA hydrolase, or 3-oxoadipyl-CoA decarboxylase. Provide a non-naturally occurring microbial organism (see Example XXIX; steps A/E/F/G/AA of Figure 25). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding LA pathway enzymes, the set comprising: 3-oxoadipyl-CoA thiolase; 3-oxoadipyl-CoA/acyl -CoA transferase, 3-oxoadipyl-CoA synthase, or 3-oxoadipyl-CoA hydrolase; and 3-oxoadipate decarboxylase.

The non-naturally occurring microbial organisms disclosed herein can have, for example, a 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway; As disclosed herein, it includes at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product. Thus, a non-naturally occurring microbial organism can contain at least one exogenous nucleic acid encoding a polypeptide, the polypeptide being , and enzymes or proteins that convert substrates and products of the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway, such as those shown in 20-27.

For example, a non-naturally occurring microbial organism can have an adipate pathway; a microbial organism can include succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA; CoA; 3-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA; 5-carboxy-2-pentenoyl-CoA to adipyl-CoA; adipyl-CoA to adipate conversion from a substrate to a product. (See Figure 2). Furthermore, the non-naturally occurring microbial organism can have an adipate pathway, the microbial organism converting succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA; 3- oxoadipyl 3-oxoadipate to 3-hydroxyadipate; 3-hydroxyadipate to hex-2-enedioate (also referred to herein as 5-carboxy-2-pentanoate); hex-2-enedioate to adipate; Contains at least one exogenous nucleic acid encoding a polypeptide that undergoes conversion into a product (see Figure 3). Additionally, the non-naturally occurring microbial organism can have a 6-aminocaproic acid pathway, and the microbial organism is selected from adipyl-CoA to adipate semialdehyde; and adipate semialdehyde to 6-aminocaproate. Contains at least one exogenous nucleic acid encoding a polypeptide that performs the conversion of substrate to product (see Figure 8). Additionally, non-naturally occurring microbial organisms can have a caprolactam pathway, and the microbial organisms can convert adipyl-CoA to adipate semialdehyde; adipate semialdehyde to 6-aminocaproate; and 6-aminocaproate. contains at least one exogenous nucleic acid encoding a polypeptide that performs the conversion of a substrate selected from caprolactam to a product. Additionally, non-naturally occurring microbial organisms can have an adipate pathway, and microbial organisms can include alpha-ketoadipate to alpha-ketoadipyl-CoA; alpha-ketoadipyl-CoA to 2-hydroxyadipyl-CoA; - Polymers for the conversion of substrates to products selected from hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA; 5-carboxy-2-pentenoyl-CoA to adipyl-CoA; and adipyl-CoA to adipate. Contains at least one exogenous nucleic acid encoding a peptide (see Figure 9). In addition, non-naturally occurring microbial organisms can have an adipate pathway, and microbial organisms can convert alpha-ketoadipate to 2-hydroxyadipate; 2-hydroxyadipate to 2-hydroxyadipyl-CoA; 2-hydroxy A polypeptide that performs the conversion of a substrate to a product selected from adipyl-CoA to 5-carboxy-2-pentenoyl-CoA; 5-carboxy-2-pentenoyl-CoA to adipyl-CoA; and adipyl-CoA to adipate. Contains at least one encoding exogenous nucleic acid (Figure 9).

In addition, non-naturally occurring microbial organisms can have a 6-aminocaproyl-CoA pathway, and microbial organisms can contain 4-aminobutyryl-CoA and acetyl-CoA to 3-oxo-6-aminohexanoyl- CoA;3-oxo-6-aminohexanoyl-CoA to 3-hydroxy-6-aminohexanoyl-CoA;3-hydroxy-6-aminohexanoyl-CoA to 6-aminohex-2-enoyl-CoA;6 Contains at least one exogenous nucleic acid encoding a polypeptide that performs the conversion of a substrate to a product selected from -aminohex-2-enoyl-CoA to 6-aminocaproyl-CoA (Figure 11). Further substrates and products of such pathways are 6-aminocaproyl-CoA to 6-aminocaproate; 6-aminocaproyl-CoA to caprolactam; or 6-aminocaproyl-CoA to 6-aminocaproate. ate semialdehyde, and 6-aminocaproate semialdehyde to hexamethylene diamine (Figure 11). Non-naturally occurring microbial organisms can also have a 6-aminocaproic acid pathway, which allows microbial organisms to convert from 4-aminobutyryl-CoA and acetyl-CoA to 3-oxo-6-aminohexanoyl-CoA;3- Oxo-6-aminohexanoyl-CoA to 3-oxo-6-aminohexanoate; 3-oxo-6-aminohexanoate to 3-hydroxy-6-aminohexanoate; 3-hydroxy-6-amino at least one exogenous agent encoding a polypeptide that performs a substrate-to-product conversion selected from hexanoate to 6-aminohex-2-enoate; and 6-aminohex-2-enoate to 6-aminocaproate; (Figure 11). Further substrates and products of such pathways are 6-aminocaproate to caprolactam or 6-aminocaproate to 6-aminocaproyl-CoA, 6-aminocaproyl-CoA to 6-aminocaproate semi aldehydes, and 6-aminocaproate semialdehyde to hexamethylene diamine (Figure 11).

Additionally, non-naturally occurring microbial organisms can have a 6-aminocaproic acid pathway, and microbial organisms can convert pyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane-1,7-dioate; -Hydroxy-2-oxoheptane-1,7-dioate (HODH) to 2-oxohept-4-ene-1,7-dioate (OHED); 2-oxohept-4-ene-1,7-dioate (OHED) selected from 2-oxoheptane-1,7-dioate (2-OHD); 2-oxoheptane-1,7-dioate (2-OHD) from adipate semialdehyde; and adipate semialdehyde from 6-aminocaproate (Figure 12). Non-naturally occurring microbial organisms can alternatively have the 6-aminocaproic acid pathway, and microbial organisms can convert pyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane-1,7-dioate; 4-Hydroxy-2-oxoheptane-1,7-dioate (HODH) to 2-oxohept-4-ene-1,7-dioate (OHED); 2-oxohept-4-ene-1,7-dioate (OHED) ) from 6-oxohex-4-enoate (6-OHE); 6-oxohex-4-enoate (6-OHE) from adipate semialdehyde; and adipate semialdehyde from 6-aminocaproate. (Figure 12). Non-naturally occurring microbial organisms can alternatively have the 6-aminocaproic acid pathway, and microbial organisms can convert pyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane-1,7-dioate; 4-Hydroxy-2-oxoheptane-1,7-dioate (HODH) to 2-oxohept-4-ene-1,7-dioate (OHED); 2-oxohept-4-ene-1,7-dioate (OHED) ) to 2-aminohept-4-ene-1,7-dioate (2-AHE); 2-aminohept-4-ene-1,7-dioate (2-AHE) to 2-aminoheptane-1,7-dioate (2-AHD); and at least one exogenous agent encoding a polypeptide that performs a substrate-to-product conversion selected from 2-aminoheptane-1,7-dioate (2-AHD) to 6-aminocaproate; (Figure 12). Non-naturally occurring microbial organisms can alternatively have the 6-aminocaproic acid pathway, and microbial organisms can convert pyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane-1,7-dioate; 4-Hydroxy-2-oxoheptane-1,7-dioate (HODH) to 2-oxohept-4-ene-1,7-dioate (OHED); 2-oxohept-4-ene-1,7-dioate (OHED) ) to 2-oxoheptane-1,7-dioate (2-OHD); 2-oxoheptane-1,7-dioate (2-OHD) to 2-aminoheptane-1,7-dioate (2-AHD); and at least one exogenous nucleic acid encoding a polypeptide that performs a substrate-to-product conversion selected from 2-aminoheptane-1,7-dioate (2-AHD) to 6-aminocaproate. (Figure 12). Non-naturally occurring microbial organisms can alternatively have the 6-aminocaproic acid pathway, and microbial organisms can convert pyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane-1,7-dioate; 4-Hydroxy-2-oxoheptane-1,7-dioate (HODH) to 3-hydroxyadipyl-CoA;3-hydroxyadipyl-CoA to 2,3-dehydroadipyl-CoA;2,3-dehydroazi at least one exogenous polypeptide encoding a substrate-to-product conversion selected from pyr-CoA to adipyl-CoA; adipyl-CoA to adipate semialdehyde; and adipate semialdehyde to 6-aminocaproate. Contains nucleic acids (Figure 12). Non-naturally occurring microbial organisms can alternatively have the 6-aminocaproic acid pathway, and microbial organisms can convert pyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane-1,7-dioate; 4-Hydroxy-2-oxoheptane-1,7-dioate (HODH) to 2-oxohept-4-ene-1,7-dioate (OHED); 2-oxohept-4-ene-1,7-(OHED) from 2,3-dehydroadipyl-CoA; from 2,3-dehydroadipyl-CoA to adipyl-CoA; from adipyl-CoA to adipate semialdehyde; and from adipate semialdehyde to 6-aminocaproate. Contains at least one exogenous nucleic acid encoding a polypeptide that undergoes conversion into a product (Figure 12). Non-naturally occurring microbial organisms can alternatively have the 6-aminocaproic acid pathway, and microbial organisms can convert pyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane-1,7-dioate; 4-Hydroxy-2-oxoheptane-1,7-dioate (HODH) to 2-oxohept-4-ene-1,7-dioate (OHED); 2-oxohept-4-ene-1,7-dioate (OHED) ) to 2-oxoheptane-1,7-dioate (2-OHD); 2-oxoheptane-1,7-dioate (2-OHD) to adipyl-CoA; adipyl-CoA to adipate semialdehyde; and adipate semialdehyde 6-aminocaproate (FIG. 12).

Additionally, non-naturally occurring microbial organisms can have a 6-aminocaproic acid pathway; -Oxo-6-amino-pimeloyl-CoA to 3-hydroxy-6-amino-pimeloyl-CoA; 3-hydroxy-6-amino-pimeloyl-CoA to 6-amino-7-carboxy-hept-2-enoyl-CoA from a substrate selected from; 6-amino-7-carboxy-hept-2-enoyl-CoA to 6-aminopimeloyl-CoA; 6-aminopimeloyl-CoA to 2-aminopimelate; and 2-aminopimelate to 6-aminocaproate. Contains at least one exogenous nucleic acid encoding a polypeptide that undergoes conversion into a product (Figure 20). Non-naturally occurring microbial organisms can alternatively have a 6-aminocaproic acid pathway, and microbial organisms can convert glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; at least one exogenous nucleic acid encoding a polypeptide that performs a substrate-to-product conversion selected from oxopimelate to 3-aminopimelate; 3-aminopimelate to 2-aminopimelate; and 2-aminopimelate to 6-aminocaproate. Contains (Figure 21). Non-naturally occurring microbial organisms can alternatively have a 6-aminocaproic acid pathway; microbial organisms can produce homolysine to 6-aminohexanamide; and 6-aminohexanamide to 6-aminocaproate. Contains at least one exogenous nucleic acid encoding a polypeptide that performs the conversion of the selected substrate to product (Figure 23). A non-naturally occurring microbial organism can alternatively have a 6-aminocaproic acid pathway, and the microbial organism can convert adipate to adipate semialdehyde; adipyl phospate to adipyl phosphate; and adipyl phosphate to adipyl phosphate to semialdehyde. Contains at least one exogenous nucleic acid encoding a polypeptide that performs the conversion of the selected substrate to product (Figure 25).

Additionally, non-naturally occurring microbial organisms can have a 6-aminocaproic acid pathway, and microbial organisms can convert 2-amino-7-oxosabarate to 2-amino-7-oxoheptanoate; Amino-7-oxoheptanoate to 6-aminohexanal; 6-aminohexanal to 6-aminocaproate; 2-amino-7-oxosabarate to 2-amino-7-oxoheptanoate; 2-amino -7-oxoheptanoate to 6-aminohexanal; 2-amino-7-oxoheptanoate to 2-aminopimelate; and 2-aminopimelate to 6-aminocaproate. (Figure 26). The non-naturally occurring microbial organism may further have a 2-amino-7-oxosabarate pathway, in which the microbial organism converts glutamic acid-5-semialdehyde to 2-amino-5-hydroxy-7-oxosabarate. 2-amino-5-hydroxy-7-oxosabarate to 2-amino-5-ene-7-oxosabarate; and 2-amino-5-ene-7-oxosabarate to 2-amino-7 - Contains at least one exogenous nucleic acid encoding a polypeptide that performs the conversion of a substrate to a product selected from oxosubarates (Figure 27). Additionally, non-naturally occurring microbial organisms can have hexamethylene diamine (HMDA) pathways, and microbial organisms can convert 6-aminocaproate to [(6-aminohexanoyl)oxy]phosphonate (6- AHOP); [(6-aminohexanoyl)oxy]phosphonate (6-AHOP) to 6-aminocaproic acid semialdehyde; and 6-aminocaproic semialdehyde to hexamethylene diamine. Contains at least one exogenous nucleic acid encoding a polypeptide (Figure 13). A non-naturally occurring microbial organism can alternatively have an HMDA pathway, in which the microbial organism converts 6-aminocaproate to [(6-aminohexanoyl)oxy]phosphonate (6-AHOP); [(6-aminohexanoyl)oxy]phosphonate (6-AHOP) to 6-aminocaproyl-CoA; 6-aminocaproyl-CoA to 6-aminocaproic acid semialdehyde; and 6-aminocaproic acid semialdehyde to hexamethylene Contains at least one exogenous nucleic acid encoding a polypeptide that performs the conversion of a substrate selected from a diamine to a product (Figure 13). A non-naturally occurring microbial organism can alternatively have a HMDA pathway, in which the microbial organism produces 6-aminocaproate to 6-aminocaproyl-CoA; 6-aminocaproyl-CoA to 6 -aminocaproic acid semialdehyde; and 6-aminocaproic acid semialdehyde to hexamethylene diamine. A non-naturally occurring microbial organism can alternatively have an HMDA pathway, in which the microbial organism converts 6-aminocaproate to 6-acetamidohexanoate; 6-acetamidohexanoate to [(6 -acetamidohexanoyl)oxy]phosphonate (6-AAHOP); [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) to 6-acetamidohexanal; 6-acetamidohexanal to 6-acetamidohexanamine; and 6- Contains at least one exogenous nucleic acid encoding a polypeptide that performs a substrate to product conversion selected from acetamidohexanamine to hexamethylene diamine (FIG. 13). A non-naturally occurring microbial organism can alternatively have an HMDA pathway, and the microbial organism can convert 6-aminocaproate to 6-acetamidohexanoate; 6-acetamidohexanoate to 6-acetamide Conversion of a substrate to a product selected from hexanoyl-CoA; 6-acetamidohexanoyl-CoA to 6-acetamidohexanal; 6-acetamidohexanal to 6-acetamidohexanamine; and 6-acetamidohexanamine to hexamethylenediamine. (Figure 13). A non-naturally occurring microbial organism can alternatively have an HMDA pathway, in which the microbial organism converts 6-aminocaproate to 6-acetamidohexanoate; 6-acetamidohexanoate to [(6 -acetamidohexanoyl)oxy]phosphonate (6-AAHOP); [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) to 6-acetamidohexanoyl-CoA; 6-acetamidohexanoyl-CoA to 6-acetamide containing at least one exogenous nucleic acid encoding a polypeptide that performs a substrate-to-product conversion selected from hexanal; 6-acetamidohexanal to 6-acetamidohexanamine; and 6-acetamidohexanamine to hexamethylenediamine. (Figure 13).

Additionally, non-naturally occurring microbial organisms can have hexamethylene diamine (HMDA) pathways; ;3-oxo-6-amino-pimeloyl-CoA to 3-hydroxy-6-amino-pimeloyl-CoA;3-hydroxy-6-amino-pimeloyl-CoA to 6-amino-7-carboxy-hept-2-enoyl -CoA;6-amino-7-carboxy-hept-2-enoyl-CoA to 6-aminopimeloyl-CoA;6-aminopimeloyl-CoA to 2-amino-7-oxoheptanoate;-amino-7-oxoheptano and homolysine to HMDA (FIG. 20). Non-naturally occurring microbial organisms can alternatively have the HMDA pathway, and the microbial organisms can be glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3 -oxo-1-carboxyheptanal; 3-oxo-1-carboxyheptanal to 3-oxo-7-aminoheptanoate; 3-oxo-7-aminoheptanoate to 3,7-diaminoheptanoate; Contains at least one exogenous nucleic acid encoding a polypeptide that performs a substrate-to-product conversion selected from 3,7-diaminoheptanoate to homolysine; and homolysine to HMDA (FIG. 21). A non-naturally occurring microbial organism can alternatively have the HMDA pathway, and the microbial organism can be glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 5 -oxopimeloylphosphonate;5-oxopimeloylphosphonate to 3-oxo-1-carboxyheptanal;3-oxo-1-carboxyheptanal to 3-oxo-7-aminoheptanoate;3-oxo- At least one polypeptide encoding a substrate-to-product conversion selected from 7-aminoheptanoate to 3,7-diaminoheptanoate; 3,7-diaminoheptanoate to homolysine; and homolysine to HMDA. (Figure 21). A non-naturally occurring microbial organism can alternatively have the HMDA pathway, and the microbial organism can be glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 5 -oxopimeloyl-CoA; 5-oxopimeloyl-CoA to 3-oxo-1-carboxyheptanal; 3-oxo-1-carboxyheptanal to 3-oxo-7-aminoheptanoate; 3-oxo-7-aminohepta at least one exogenous polypeptide encoding a substrate-to-product conversion selected from noate to 3,7-diaminoheptanoate; 3,7-diaminoheptanoate to homolysine; and homolysine to HMDA. Contains nucleic acids (Figure 21). Non-naturally occurring microbial organisms can alternatively have the HMDA pathway, and the microbial organisms can be glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3 -Oxo-1-carboxyheptanal; 3-oxo-1-carboxyheptanal to 3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to 3,7-diaminoheptanoate; Contains at least one exogenous nucleic acid encoding a polypeptide that performs a substrate-to-product conversion selected from 3,7-diaminoheptanoate to homolysine; and homolysine to HMDA (FIG. 21). A non-naturally occurring microbial organism can alternatively have the HMDA pathway, and the microbial organism can be glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 5 -oxopimeloyl-CoA; 5-oxopimeloyl-CoA to 3-oxo-1-carboxyheptanal; 3-oxo-1-carboxyheptanal to 3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate at least one exogenous polypeptide encoding a substrate-to-product conversion selected from noate to 3,7-diaminoheptanoate; 3,7-diaminoheptanoate to homolysine; and homolysine to HMDA. Contains nucleic acids (Figure 21). A non-naturally occurring microbial organism can alternatively have the HMDA pathway, and the microbial organism can be glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 5 -oxopimeloylphosphonate;5-oxopimeloylphosphonate to 3-oxo-1-carboxyheptanal;3-oxo-1-carboxyheptanal to 3-amino-7-oxoheptanoate;3-amino- 7-oxoheptanoate to 3,7-diaminoheptanoate; 3, 7-diaminoheptanoate to homolysine; and homolysine to HMDA; and homolysine to HMDA. Non-naturally occurring microbial organisms can alternatively have the HMDA pathway, and the microbial organisms can be glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3 -aminopimelate; 3-aminopimelate to 3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to 2-amino-7-oxo(axo)heptanoate; 2-amino-7-oxo(axo) ) heptanoate to homolysine; and homolysine to HMDA (FIG. 21). Non-naturally occurring microbial organisms can alternatively have the HMDA pathway, and the microbial organisms can be glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3 -Aminopimelate; 3-aminopimelate to 5-aminopimeloylphosphonate; 5-aminopimeloylphosphonate to 3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to 2-amino-7 - at least one exogenous nucleic acid encoding a polypeptide that performs a substrate-to-product conversion selected from oxo(axo)heptanoate; 2-amino-7-oxo(axo)heptanoate to homolysine; and homolysine to HMDA; Contains (Figure 21). A non-naturally occurring microbial organism can alternatively have the HMDA pathway, and the microbial organism can be glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 5 -aminopimeloyl-CoA;5-aminopimeloyl-CoA to 3-amino-7-oxoheptanoate;3-amino-7-oxoheptanoate to 2-amino-7-oxo(axo)heptanoate;2-amino-7 -oxo (axo) heptanoate to homolysine; and homolysine to HMDA. Non-naturally occurring microbial organisms can alternatively have the HMDA pathway, and the microbial organisms can be glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3 -aminopimelate; 3-aminopimelate to 3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to 3,7-diaminoheptanoate; 3,7-diaminoheptanoate to homolysine; Contains at least one exogenous nucleic acid encoding a polypeptide that performs the conversion of a substrate to a product selected from homolysine to HMDA (Figure 21). Non-naturally occurring microbial organisms can alternatively have the HMDA pathway, and the microbial organisms can be glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3 -Aminopimelate; 3-aminopimelate to 5-aminopimeloyl-CoA; 5-aminopimeloyl-CoA to 3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to 3,7-diaminoheptanoate; Contains at least one exogenous nucleic acid encoding a polypeptide that performs a substrate-to-product conversion selected from 3,7-diaminoheptanoate to homolysine; and homolysine to HMDA (FIG. 21). Non-naturally occurring microbial organisms can alternatively have the HMDA pathway, and the microbial organisms can be glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3 -Aminopimelate; 3-aminopimelate to 5-aminopimeloylphosphonate; 5-aminopimeloylphosphonate to 3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to 3,7-diamino heptanoate;3, 7-diaminoheptanoate to homolysine; and homolysine to HMDA; and homolysine to HMDA. Non-naturally occurring microbial organisms can alternatively have the HMDA pathway, and the microbial organisms can be glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3 - aminopimelate; 3-aminopimelate to 2-aminopimelate; 2-aminopimelate to 2-amino-7-oxoheptanoate; 2-amino-7-oxoheptanoate to homolysine; and homolysine to HMDA. (Figure 21). Non-naturally occurring microbial organisms can alternatively have the HMDA pathway, and the microbial organisms can be glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3 -aminopimelate; 3-aminopimelate to 2-aminopimelate; 2-aminopimelate to 6-aminopimeloylphosphonate; 6-aminopimeloylphosphonate to 2-amino-7-oxoheptanoate; 2-amino-7-oxoheptanoate Contains at least one exogenous nucleic acid encoding a polypeptide that performs a substrate-to-product conversion selected from noate to homolysine; and homolysine to HMDA (FIG. 21). Non-naturally occurring microbial organisms can alternatively have the HMDA pathway, and the microbial organisms can be glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3 -Aminopimelate; 3-aminopimelate to 2-aminopimelate; 2-aminopimelate to 6-aminopimeloyl-CoA; 6-aminopimeloyl-CoA to 2-amino-7-oxoheptanoate; 2-amino-7-oxoheptanoate to homolysine and at least one exogenous nucleic acid encoding a polypeptide that performs a substrate-to-product conversion selected from homolysine to HMDA (FIG. 21). Non-naturally occurring microbial organisms can alternatively have the HMDA pathway, and microbial organisms can convert pyruvate and 4-aminobutanal to 2-oxo-4-hydroxy 7-aminoheptanoate; -4-hydroxy 7-aminoheptanoate to 2-oxo-7-aminohepta-3-enoate; 2-oxo-7-aminohepta-3-enoate to 2-oxo-7-aminoheptanoate; 2-oxo- Contains at least one exogenous nucleic acid encoding a polypeptide that performs a substrate-to-product conversion selected from 7-aminoheptanoate to homolysine; and homolysine to HMDA (FIG. 22). Non-naturally occurring microbial organisms can alternatively have the HMDA pathway, and microbial organisms can convert pyruvate and 4-aminobutanal to 2-oxo-4-hydroxy 7-aminoheptanoate; -4-hydroxy 7-aminoheptanoate to 2-oxo-7-aminohepta-3-enoate; 2-oxo-7-aminohepta-3-enoate to 2-oxo-7-aminoheptanoate; 2-oxo- Contains at least one exogenous nucleic acid encoding a polypeptide that performs a substrate-to-product conversion selected from 7-aminoheptanoate to 6-aminohexanal; and 6-aminohexanal to HMDA (Figure 22) . A non-naturally occurring microbial organism can alternatively have an HMDA pathway, and the microbial organism is selected from 6-aminocaproate to 6-aminocaproic acid semialdehyde; and 6-aminocaproic acid semialdehyde to HMDA (Figure 24). Non-naturally occurring microbial organisms can alternatively have the HMDA pathway, and microbial organisms can convert 6-aminocaproate to 6-acetamidohexanoate; 6-acetamidohexanoate to 6-acetamidohexanal contains at least one exogenous nucleic acid encoding a polypeptide that performs a substrate-to-product conversion selected from; 6-acetamidohexanal to 6-acetamidohexanamine; 6-acetamidohexanamine to HMDA (Figure 24) . Non-naturally occurring microbial organisms can alternatively have the HMDA pathway, and microbial organisms can convert 2-amino-7-oxosabarate to 2-amino-7-oxoheptanoate; 7-oxoheptanoate to 6-aminohexanal; 6-aminohexanal to HMDA; 2-amino-7-oxosabarate to 2-oxo-7-aminoheptanoate; 2-amino-7-oxoheptanoate homolysine from; HMDA from homolysine; homolysine from 2-oxo-7-aminoheptanoate; 6-aminohexanal from 2-oxo-7-aminoheptanoate; 2,7- from 2-amino-7-oxosabarate diaminosubarate; and 2,7-diaminosubarate to homolysine (FIG. 26). The non-naturally occurring microbial organism may further have a 2-amino-7-oxosabarate pathway, in which the microbial organism converts glutamic acid-5-semialdehyde to 2-amino-5-hydroxy-7-oxosabarate. 2-amino-5-hydroxy-7-oxosabarate to 2-amino-5-ene-7-oxosabarate; and 2-amino-5-ene-7-oxosabarate to 2-amino-7 - Contains at least one exogenous nucleic acid encoding a polypeptide that performs the conversion of a substrate to a product selected from oxosubarates (Figure 27). Contains at least one exogenous nucleic acid encoding a polypeptide that performs a substrate to product conversion selected from 7-diaminosubarate to homolysine (Figure 26). The non-naturally occurring microbial organism may further have a 2-amino-7-oxosabarate pathway, in which the microbial organism converts glutamic acid-5-semialdehyde to 2-amino-5-hydroxy-7-oxosabarate. rate; 2-amino-5-hydroxy-7-oxosabarate to 2-amino-5-ene-7-oxosabarate; and 2-amino-5-ene-7-oxosabarate to 2-amino-7 - Contains at least one exogenous nucleic acid encoding a polypeptide that performs the conversion of a substrate to a product selected from oxosubarates (Figure 27). Contains at least one exogenous nucleic acid encoding a polypeptide that performs a substrate to product conversion selected from 7-diaminosubarate to homolysine (Figure 26). The non-naturally occurring microbial organism may further have a 2-amino-7-oxosabarate pathway, in which the microbial organism converts glutamic acid-5-semialdehyde to 2-amino-5-hydroxy-7-oxosabarate. 2-amino-5-hydroxy-7-oxosabarate to 2-amino-5-ene-7-oxosabarate; and 2-amino-5-ene-7-oxosabarate to 2-amino-7 - Contains at least one exogenous nucleic acid encoding a polypeptide that performs the conversion of a substrate to a product selected from oxosubarates (Figure 27).

Additionally, non-naturally occurring microbial organisms can have a levulinic acid pathway; microbial organisms can convert succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA; 3-oxoadipyl-CoA to 3-oxoadipate; and at least one exogenous nucleic acid encoding a polypeptide that performs a substrate to product conversion selected from 3-oxoadipate to levulinic acid. It is understood that any of the pathways disclosed herein that produce intermediates for one pathway can be used to produce intermediates for other pathways, if desired. For example, as disclosed herein, the alpha-ketoadipate to adipate pathway shown in Figure 9 produces the intermediate adipyl-CoA, which is also represented in Figure 10. It is also an intermediate in the pathway. Therefore, an alternative route involves adipyl-CoA from alpha-ketoadipate, which can be converted to adipate, 6-aminocaproate, caprolactam, or hexamethylene diamine, as depicted in Figure 10. It is understood that this is possible. Any of the routes disclosed herein that produce the desired intermediate can be used in combination with any other routes disclosed herein so long as the desired product is produced. is understood. For example, the non-naturally occurring microbial organisms disclosed herein may include 2-AHD decarboxylase (Step I of Figure 12) and 6-acetamidohexanoate kinase (Step E of Figure 13) or alternatively 2-oxohept-4-ene-1,7-dioate (OHED) decarboxylase (Step F of Figure 12), adipate semialdehyde aminotransferase (Step E of Figure 12), and 6-acetamidohexanoyl-CoA oxidoreductase. (Step J of Figure 13) or alternatively 5-carboxy-2pentenoyl-CoA reductase (Step D of Figure 10), adipyl-CoA dehydrogenase (Step O of Figure 12), and 6-aminocaproyl-CoA oxidoreductase ( Step N of Figure 13) or alternatively 2-amino-7-oxoheptanoate aminotransferase (Step G of Figure 20) and 3,7-diaminoheptanoate 2,3-aminomutase (Step R of Figure 21). ) or alternatively 6-aminocaproate reductase (step O of Figure 24) and 6-aminohex-2-enoate reductase (step J of Figure 11) or alternatively adipate reductase (step X of Figure 25) and at least one nucleic acid encoding a 6-aminocaproic acid pathway enzyme and at least one nucleic acid encoding a hexamethylenediamine pathway enzyme, such as 6-acetamidohexanoate reductase (Step P of Figure 24). can.

In a further embodiment, the present invention provides a non-naturally occurring microbial organism having a 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway, wherein the non-naturally occurring microbial organism is herein at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product, selected from any of the substrates or products disclosed in Figures 1-14 and 20-27. include. Those skilled in the art will appreciate that any of the substrate-product pairs disclosed herein are suitable for producing the desired product and for which the appropriate activity is available for the conversion of the substrate to the product. It will be understood that this can be readily determined by one of ordinary skill in the art based on the teachings herein. Accordingly, the present invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, the enzyme or protein shown in Figures 1-14 and 20-27. Provided are non-naturally occurring microbial organisms that convert substrates and products of the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathways, such as any of the following.

Although generally described herein as microbial organisms containing the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway, the present invention provides A non-naturally occurring microorganism comprising at least one exogenous nucleic acid encoding a 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway enzyme expressed in an amount sufficient to produce an acid pathway intermediate. It is understood that the organism is further provided. For example, as disclosed herein, the 6-aminocaproic acid, caprolactam, hexamethylene diamine, or levulinic acid pathways are illustrated in Figures 1-14 and 20-27. Therefore, in addition to microbial organisms containing the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway that produce 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid, the present invention further provides: A non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway enzyme, wherein the microbial organism , hexamethylene diamine, or a levulinic acid pathway intermediate, such as any of the intermediates shown in FIGS. 1-14 and 20-27.

Any of the routes disclosed herein, including those described in the Examples and those illustrated in the figures, including the routes of Figures 1-14 and 20-27, can be modified as desired. It is understood that microbial organisms can be utilized to generate non-naturally occurring microbial organisms that produce pathway intermediates or products. As disclosed herein, such microbial organisms that produce intermediates can be used in combination with other microbial organisms that express downstream pathway enzymes to produce the desired products. I can do it. However, it is understood that non-naturally occurring microbial organisms that produce 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway intermediates can be utilized to produce the intermediates as desired products. Ru.

The present invention relates generally to metabolic reactions, reactants, or products thereof, or particularly to one or more nucleic acids or genes encoding enzymes associated with or catalyzing the metabolic reactions, reactants, or products described above. It is described in Unless explicitly stated otherwise herein, those skilled in the art will understand that references to a reaction also refer to the reactants and products of the reaction. Similarly, unless explicitly stated otherwise herein, references to reactants or products also refer to reactions, and references to any of these metabolic components also refer to the above-mentioned reactions, reactions. It also refers to the gene(s) encoding the enzyme that catalyzes the product. Similarly, given the well-known fields of metabolic biochemistry, enzymology, and genomics, references herein to genes or encoding nucleic acids refer to the corresponding encoding enzymes and the reactions they catalyze, as well as reactions. It can also refer to reactants and products.

The non-naturally occurring microbial organisms of the invention may incorporate expressible nucleic acids encoding one or more enzymes involved in the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthetic pathway. can be produced by Depending on the host microbial organism selected for biosynthesis, nucleic acids for some or all of the particular 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthetic pathways may be expressed. can. For example, if the chosen host is deficient in one or more enzymes for the desired biosynthetic pathway, the expressible nucleic acids for the deficient enzyme(s) can be found in the host for subsequent exogenous expression. will be introduced in Alternatively, if the chosen host exhibits endogenous expression of some pathway genes but is deficient in others, the encoding nucleic acid may contain 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulin. A deficient enzyme(s) is required to accomplish acid biosynthesis. Accordingly, the non-naturally occurring microbial organisms of the present invention can be produced by introducing exogenous enzymatic activity to obtain the desired biosynthetic pathway or the desired biosynthetic pathway can be produced using one or more endogenous enzymes. can be obtained by introducing one or more exogenous enzymatic activities that produce the desired product, such as 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid, together with a specific enzyme.

Depending on the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthetic pathway components of the host microbial organism chosen, the non-naturally occurring microbial organisms of the invention can contain at least one exogenously expressed 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway encoding nucleic acids and any encoding nucleic acids for one or more adipate, 6-aminocaproic acid, or caprolactam biosynthetic pathways. For example, 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthesis can be established in a host deficient in pathway enzymes through exogenous expression of the corresponding encoding nucleic acids. Exogenous expression of all enzymes in the pathway can be included in a host deficient in all enzymes of the 6-aminocaproic acid, caprolactam, hexamethylene diamine, or levulinic acid pathway, but the host is deficient in at least one of the pathway enzymes. It is understood that all enzymes of the pathway can be expressed even when containing .

For example, exogenous expression of all enzymes in the pathway for the production of adipate includes succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy -2-pentenoyl-CoA reductase, and adipyl-CoA synthetase or phosphotransadipyrase/adipate kinase or adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase, etc., can be included in the host organism. In particular, the host organism is capable of producing the adipate pathway enzymes, succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl -Can contain CoA synthetase. Alternatively, the host organism can contain adipate pathway enzymes, succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and phosphotransadipyrase/adipate kinase. In addition, the host organism is capable of producing adipate pathway enzymes, succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl -CoA: Can contain acetyl-CoA transferase. In addition, the host organism is capable of producing adipate pathway enzymes, succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl -Can contain CoA hydrolase.

In the case of 6-aminocaproic acid producing microbial organisms, exogenous expression of all enzymes in the pathway for the production of 6-aminocaproic acid can be either CoA-dependent aldehyde dehydrogenases and transaminases or CoA-dependent aldehyde dehydrogenases. It can be included in the host organism, such as dehydrogenase and 6-aminocaproate dehydrogenase. For caprolactam-producing microbial organisms, exogenous expression of all enzymes in the pathway for the production of caprolactam is essential, such as CoA-dependent aldehyde dehydrogenase, transaminase or 6-aminocaproate dehydrogenase, and amidohydrolase. can be included in the host organism as such. In another example, exogenous expression of all enzymes in the pathway for the production of 6-aminocaproic acid (6-ACA) includes HODH aldolase; OHED hydratase; OHED reductase; 2-OHD decarboxylase; aminotransferase or adipate semialdehyde oxidoreductase (amination) or alternatively HODH aldolase; OHED hydratase; OHED decarboxylase; 6-OHE reductase; and adipate semialdehyde aminotransferase or adipate semialdehyde oxidoreductase (amination) or alternative HODH aldolase; OHED hydratase; OHED aminotransferase or OHED oxidoreductase (amination); 2-AHE reductase; and 2-AHD decarboxylase or alternatively HODH aldolase; OHED hydratase; OHED reductase; 2-OHD aminotransferase or 2 -OHD oxidoreductase (amination); and 2-AHD decarboxylase or alternatively HODH aldolase; HODH formate lyase and pyruvate formate lyase activating enzyme or HODH dehydrogenase; 3-hydroxyadipyl-CoA dehydratase; 2,3- dehydroadipyl-CoA reductase; adipyl-CoA dehydrogenase; and adipate semialdehyde aminotransferase or adipate semialdehyde oxidoreductase (amination) or alternatively HODH aldolase; OHED hydratase; OHED formate lyase and pyruvate formate lyase activating enzyme or 2- such as OHD formate lyase and pyruvate formate lyase activating enzyme or 2-OHD dehydrogenase; adipyl-CoA dehydrogenase; and adipate semialdehyde aminotransferase or adipate semialdehyde oxidoreductase (amination). can. In further embodiments, all of the 6-ACA pathways described above can include succinate semialdehyde dehydrogenase, alpha-ketoglutarate decarboxylase, or phosphoenolpyruvate (PEP) carboxykinase. In other examples, exogenous expression of all enzymes in the pathway for the production of 6-aminocaproic acid (6-ACA) includes: glutamyl-CoA transferase or glutamyl-CoA ligase; beta-ketothiolase; 3-oxo-6 -aminopimeloyl-CoA oxidoreductase; 3-hydroxy-6-aminopimeloyl-CoA dehydratase; 6-amino-7-carboxyhept-2-enoyl-CoA reductase; 6-aminopimeloyl-CoA reductase (aldehyde formation); Melate decarboxylase or glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate amino oxidoreductase; 3-aminopimelate 2,3-aminomutase; and 2-aminopimelate decarboxylase.

In another example, exogenous expression of all enzymes in the pathway for the production of hexamethylene diamine includes: 6-aminocaproate kinase; 6-AHOP oxidoreductase; and 6-aminocaproate semialdehyde oxidoreductase (amino 6-aminocaproic acid semialdehyde aminotransferase; or 6-aminocaproate kinase; 6-AHOP acyltransferase; 6-aminocaproyl-CoA oxidoreductase; and 6-aminocaproic acid semialdehyde oxidoreductase (amination) or 6-aminocaproate semialdehyde aminotransferase, or 6-aminocaproate CoA transferase or 6-aminocaproate CoA ligase; 6-aminocaproyl-CoA oxidoreductase; ) or 6-aminocaproate semialdehyde aminotransferase, or 6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate kinase; 6-AAHOP oxidoreductase; 6-acetamidohexanal aminotransferase or 6-acetamidohexanal oxide reductase (amination); and 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide); or 6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate CoA transferase or 6- Acetamidohexanoate CoA ligase; 6-acetamidohexanoyl-CoA oxidoreductase; 6-acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase (amination); and 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexane Amine hydrolase (amide), or 6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate kinase; 6-AAHOP oxidoreductase; 6-acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase (amination); and 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide). In other examples, exogenous expression of all enzymes in the pathway for the production of hexamethylene diamine includes: glutamyl-CoA transferase or ligase; beta-ketothiolase; 3-oxo-6-aminopimeloyl-CoA oxidoreductase; Hydroxy-6-aminopimeloyl-CoA dehydratase; 6-amino-7-carboxyhept-2-enoyl-CoA reductase; 6-aminopimeloyl-CoA reductase (aldehyde formation); 2-amino-7-oxoheptanoate aminotransferase or amino oxidoreductase; and homolysine decarboxylase, or glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate reductase; -1-carboxyheptanal 7-aminotransferase or 3-oxo-1-carboxyheptanal 7-aminated oxidoreductase; 3-oxo-7-aminoheptanoate 3-aminotransferase or 3-oxo-7-aminoheptase Noate 3-amination oxidoreductase; 3,7-diaminoheptanoate 2,3-aminomutase; and homolysine decarboxylase, or glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA Transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate kinase; 5-oxopimeloylphosphonate reductase; 3-oxo-1-carboxyheptanal 7-aminotransferase or 3-oxo-1-carboxyheptanal 7 -Aminated oxidoreductase; 3-oxo-7-aminoheptanoate 3-aminotransferase or 3-oxo-7-aminoheptanoate 3-aminated oxidoreductase; 3,7-diaminoheptanoate 2,3- aminomutase; and homolysine decarboxylase, or glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate-CoA transferase or 3-oxopimelate-CoA ligase; 5-oxopimeloyl-CoA reductase (aldehyde formation); 3-oxo-1-carboxyheptanal 7-aminotransferase or 3-oxo-1-carboxyheptanal 7-amination oxidoreductase; 3-oxo-7-aminoheptano ate 3-aminotransferase or 3-oxo-7-aminoheptanoate 3-aminated oxidoreductase; 3,7-diaminoheptanoate 2,3-aminomutase; and homolysine decarboxylase, or glutaryl-CoA beta- Ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate reductase; 3-oxo-1-carboxyheptanal 3-aminotransferase or 3-oxo-1 -carboxyheptanal 3-aminated oxidoreductase; 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminated oxidoreductase; 3,7-diaminoheptano ate 2,3-aminomutase; and homolysine decarboxylase, or glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate kinase ;5-oxopimeloylphosphonate reductase;3-oxo-1-carboxyheptanal 3-aminotransferase or 3-oxo-1-carboxyheptanal 3-aminated oxidoreductase;3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminated oxidoreductase; 3,7-diaminoheptanoate 2,

Depending on the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthetic pathway components of the host microbial organism chosen, the non-naturally occurring microbial organisms of the invention can contain at least one exogenously expressed 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway encoding nucleic acids and any encoding nucleic acids for one or more 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthetic pathways. . For example, 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid. In a host deficient in all enzymes or proteins of the 6-aminocaproic acid, caprolactam, hexamethylene diamine, or levulinic acid pathway, exogenous expression of all enzymes or proteins in the pathway can be included, but not limited to the host deficient in the pathway enzymes. It is understood that all enzymes or proteins of the pathway can be expressed, even if they contain at least one of the enzymes or proteins. For example, as disclosed herein, exogenous expression of all enzymes or proteins in the pathway for the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid can be included.

In view of the teachings and guidance provided herein, those skilled in the art will appreciate that the number of encoding nucleic acids for introduction in expressible form is at least as large as the adipate, 6-aminocaprone of the selected host microbial organism. It will be appreciated that this would be comparable to acid, caprolactam, hexamethylene diamine, or levulinic acid pathway deficiencies. Therefore, the non-naturally occurring microbial organism of the present invention comprises at least 1, 2, 3, 4, It can have any number of 5, 6, 7, 8, 9, 10, 11, or 12 nucleic acids. In some embodiments, the non-naturally occurring microbial organism also promotes or optimizes 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthesis or has other beneficial functions for the host microbial organism. Other genetic modifications can be included to provide for. Other such single functionalities include, for example, succinyl-CoA and/or acetyl-CoA for adipate synthesis or 6-aminocaproic acid or caprolactam synthesis, including the adipate pathway enzymes disclosed herein. adipyl-CoA or adipate or 6-aminocaproate in the case of pyruvate and succinic semialdehyde, glutamic acid, glutaryl-CoA, homolysine, or 2-amino-7-oxosabarate or hexamethylene diamine in the case of the synthesis. 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulin, such as 6-aminocaproate, glutamic acid, glutaryl-CoA, pyruvate and 4-aminobutanal, or 2-amino-7-oxosabarate, etc. can include increasing the synthesis of one or more acid pathway precursors.

In general, the host microbial organism may be modified as a naturally produced molecule or genetically engineered to provide de novo production of desired precursors or increased production of precursors naturally produced by the host microbial organism. The selected product is selected to produce 6-aminocaproic acid, caprolactam, hexamethylenediamine, or a precursor of the levulinic acid pathway. As disclosed herein, host organisms can be genetically engineered to increase production of precursors. Additionally, microbial organisms genetically engineered to produce the desired precursors can be used as host organisms to express enzymes or proteins of the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathways. can be further genetically engineered to

In some embodiments, the non-naturally occurring microbial organisms of the invention are produced from a host that contains the enzymatic capacity to synthesize 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. In this particular embodiment, for example, to drive a 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway reaction toward 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid production, It may be useful to increase the synthesis or accumulation of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway products. Increased synthesis or accumulation can be achieved, for example, by overexpression of a nucleic acid encoding one or more of the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway enzymes described above. Overexpression of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway enzyme(s) can be achieved, for example, through exogenous expression of endogenous gene(s) or exogenous expression of heterologous gene(s). can occur through the expression of Therefore, naturally occurring organisms encode, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthetic pathway enzymes. . can be easily produced. Additionally, non-naturally occurring organisms can be generated by mutagenesis of endogenous genes resulting in increased activity of enzymes in the 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthetic pathways.

A particularly useful embodiment utilizes exogenous expression of the encoding nucleic acid. Exogenous expression provides the host and application with the ability to tailor expression and/or regulatory elements to achieve desired expression levels that are controlled by the user. However, endogenous expression is also exploited in other embodiments, such as by removing negative regulatory effectors or inducing the promoter of a gene when linked to an inducible promoter or other regulatory element. be able to. Thus, endogenous genes with naturally occurring inducible promoters can be upregulated by providing an appropriate inducing agent or the regulatory regions of endogenous genes can be upregulated to incorporate inducible regulatory elements into the gene. can be manipulated, thereby allowing regulation of increased expression of endogenous genes when desired. Similarly, inducible promoters can be included as regulatory elements for exogenous genes introduced into non-naturally occurring microbial organisms.

The present invention further provides a non-naturally occurring microbial organism comprising one or more gene disruptions, such as those disclosed in Example XXX and Tables 14-16, wherein the organism comprises 6-ACA, 6-ACA, Produces adipate and/or HMDA. If the gene disruption reduces the activity of the enzyme, such that the gene disruption confers increased production of adipate, 6-ACA, and/or HMDA in non-naturally occurring organisms, the disruption , and/or occur in genes encoding enzymes that link the production of HMDA to the growth of the organism. Accordingly, the present invention provides a non-naturally occurring microbial organism comprising one or more gene disruptions, the one or more gene disruptions occurring in a gene encoding a protein or enzyme, the one or more gene disruptions comprising: Non-naturally occurring microbial organisms are provided that provide increased production of adipate, 6-ACA, and/or HMDA in an organism. As disclosed herein, such organisms, in addition to gene disruptions such as those exemplified in Example XXX and Tables 14-16, may contain adipate, 6-ACA, and/or Contains the pathway for the production of HMDA.

It is understood that in the methods of the invention any one or more exogenous nucleic acids can be introduced into a microbial organism to produce a non-naturally occurring microbial organism of the invention.

Nucleic acids can be introduced to provide the microbial organism with a 6-aminocaproic acid, caprolactam, hexamethylene diamine, or levulinic acid biosynthetic pathway, for example. Alternatively, the encoding nucleic acid encodes an intermediate with biosynthetic capacity to catalyze some of the reactions required to confer 6-aminocaproic acid, caprolactam, hexamethylene diamine, or levulinic acid biosynthetic capacity. It may also be introduced to produce microbial organisms. For example, a non-naturally occurring microbial organism having a 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthetic pathway can contain at least two exogenous nucleic acids encoding the desired enzymes. In the case of adipate production, the at least two exogenous nucleic acids are succinyl-CoA:acetyl-CoA acyltransferase and 3-hydroxyacyl-CoA dehydrogenase, or succinyl-CoA:acetyl-CoA acyltransferase and 3-hydroxyadipyl -Encodes enzymes such as CoA dehydratase, or 3-hydroxyadipyl-CoA and 5-carboxy-2-pentenoyl-CoA reductase, or 3-hydroxyacyl-CoA and adipyl-CoA synthetase, and other similar combinations. can do. In the case of caprolactam production, the at least two exogenous nucleic acids encode enzymes such as a CoA-dependent aldehyde dehydrogenase and transaminase or a CoA-dependent aldehyde dehydrogenase and amidohydrolase or a combination of a transaminase and an amidohydrolase. I can do it. In the case of 6-aminocaproic acid production, the at least two exogenous nucleic acids are 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) aldolase and 2-oxohept-4-ene-1,7- dioate (OHED) hydratase, or 2-oxohept-4-ene-1,7-dioate (OHED) hydratase and 2-aminoheptane-1,7-dioate (2-AHD) decarboxylase, 3-hydroxyadipyl-CoA Enzymes such as dehydratase and adipyl-CoA dehydrogenase, glutamyl-CoA transferase and 6-aminopimeloyl-CoA hydrolase, or a combination of glutaryl-CoA beta-ketothiolase and 3-aminopimelate 2,3-aminomutase, etc. can be encoded. In the case of hexamethylenediamine production, the at least two exogenous nucleic acids are 6-aminocaproate kinase and [(6-aminohexanoyl)oxy]phosphonate (6-AHOP) oxidoreductase, or 6-acetamidohexanoyl Noatekinase and [(6-acetamidohexanoyl)oxy]

Phosphonate (6-AAHOP) oxidoreductase, 6-aminocaproate N-acetyltransferase and 6-acetamidohexanoyl-CoA oxidoreductase, 3-hydroxy-6-aminopimeloyl-CoA dehydratase and 2-amino-7-oxoheptano Enzymes such as ate aminotransferase or a combination of 3-oxopimeloyl-CoA ligase and homolysine decarboxylase can be encoded. It is therefore understood that any combination of two or more enzymes of a biosynthetic pathway can be included in the non-naturally occurring microbial organisms of the invention.

Similarly, any combination of three or more enzymes of a biosynthetic pathway, for example in the case of adipate production, is an enzyme, as long as the combination of enzymes of the desired biosynthetic pathway results in the production of the corresponding desired product. succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyadipyl-CoA dehydratase; or succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, and 5-carboxy -2-pentenoyl-CoA reductase; or succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase and adipyl-CoA synthetase; or 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase and It is understood that adipyl-CoA:acetyl-CoA transferase, as well as other combinations, can be included in the non-naturally occurring microbial organisms of the invention as desired. In the case of 6-aminocaproic acid production, the at least three exogenous nucleic acids are 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) aldolase, 2-oxohept-4-ene-1,7- dioate (OHED) hydratase and 2-oxoheptane-1,7-dioate (2-OHD) decarboxylase, or 2-oxohept-4-ene-1,7-dioate (OHED) hydratase, 2-aminohept-4-ene -1,7-dioate (2-AHE) reductase and 2-aminoheptane-1,7-dioate (2-AHD) decarboxylase, or 3-hydroxyadipyl-CoA dehydratase, 2,3-dehydroadipyl-CoA reductase and adipyl-CoA dehydrogenase, or 6-amino-7-carboxyhept-2-enoyl-CoA reductase, 6-aminopimeloyl-CoA hydrolase and 2-aminopimelate decarboxylase, or glutaryl-CoA beta-ketothiolase, 3- Enzymes such as aminating oxidoreductase and 2-aminopimelate decarboxylase, or a combination of 3-oxoadipyl-CoA thiolase, 5-carboxy-2-pentenoate reductase and adipate reductase, and the like can be encoded. In the case of hexamethylene diamine production, the at least three exogenous nucleic acids are 6-aminocaproate kinase, [(6-aminohexanoyl)oxy]phosphonate (6-AHOP) oxidoreductase and 6-aminocaproate semi- Aldehyde aminotransferase, or 6-aminocaproate N-acetyltransferase, 6-acetamidohexanoate kinase and [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) oxidoreductase, or 6-aminocaproate N-acetyltransferase, [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) acyltransferase and 6-acetamidohexanoyl-CoA oxidoreductase, or 3-oxo-6-aminopimeloyl-CoA oxidoreductase, 3-hydroxy -6-aminopimeloyl-CoA dehydratase and homolysine decarboxylase, or 2-oxo-4-hydroxy-7-aminoheptanoate aldolase, 2-oxo-7-aminohept-3-enoate reductase and homolysine decarboxylase, or Enzymes such as 6-acetamidohexanoate reductase, 6-acetamidohexanal aminotransferase and a combination of 6-acetamidohexanamine N-acetyltransferase, etc. can be encoded. Similarly, any combination of four or more enzymes of the biosynthetic pathways disclosed herein can be used as desired, so long as the combination of enzymes of the desired biosynthetic pathway results in the production of the corresponding desired product. can be included in the non-naturally occurring microbial organisms of the present invention.

In addition to the biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid described herein, the non-naturally occurring microbial organisms and methods of the present invention also produce products by other routes. They can also be utilized in a wide variety of combinations with each other and with other microbial organisms and methods well known in the art to accomplish the synthesis. For example, some alternatives to producing 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid other than using 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid producing strains include adipate, 6 - through the addition of other microbial organisms that can convert aminocaproic acid, or caprolactam pathway intermediates, to 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. Certain such procedures include, for example, fermentation of microbial organisms that produce 6-aminocaproic acid, caprolactam, hexamethylene diamine, or levulinic acid pathway intermediates. The 6-aminocaproic acid, caprolactam, hexamethylene diamine, or levulinic acid pathway intermediate then converts the 6-aminocaproic acid, caprolactam, hexamethylene diamine, or levulinic acid pathway intermediate into 6-aminocaproic acid, caprolactam, hexamethylene diamine , or can be used as a substrate for a second microbial organism that converts it to levulinic acid. 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid pathway intermediates can be added directly to other cultures of a second organism or 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid The original culture of the pathway intermediate producing strain can be cleared of these microbial organisms, e.g. by cell separation, and subsequent addition of a second organism to the fermentation broth then involves an intermediate purification step. It can be used to produce the final product without any

In other embodiments, the non-naturally occurring microbial organisms and methods of the invention utilize a variety of sub-pathways to achieve the biosynthesis of, for example, 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. It can be assembled at In these embodiments, the biosynthetic pathway for the desired product of the invention can be separated into different microbial organisms, and the different microbial organisms can be co-cultured to produce the final product. . In such a biosynthetic scheme, the product of a first microbial organism becomes a substrate for a second microbial organism until the final product is synthesized. For example, the biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylene diamine, or levulinic acid constructs microbial organisms containing biosynthetic pathways for the conversion of one pathway intermediate to another pathway intermediate or product. This can be achieved by Alternatively, 6-aminocaproic acid, caprolactam, hexamethylene diamine, or levulinic acid can also be produced biosynthetically from microbial organisms through co-culture or co-fermentation using the two organisms in the same vessel. , the first microbial organism produces the 6-aminocaproic acid, caprolactam, hexamethylene diamine, or levulinic acid intermediate, and the second microbial organism produces the intermediate 6-aminocaproic acid, caprolactam, hexamethylene Converts to diamine or levulinic acid.

Given the teachings and guidance provided herein, one of ordinary skill in the art will recognize that other microbial organisms, sub-routes and In addition to co-cultivation of other non-naturally occurring microbial organisms and combinations of other chemical and/or biochemical procedures well known in the art, a wide variety of combinations and permutations are contemplated by the present invention. It will be understood that non-naturally occurring microbial organisms and methods exist.

Similarly, host organisms can be selected based on the desired characteristics for the introduction of one or more gene disruptions to increase production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. will be understood by those skilled in the art. Therefore, if a genetic modification is to be introduced into a host organism to disrupt a gene, any homologs, orthologs, or paralogs that catalyze similar but not identical metabolic reactions are It is understood that the desired metabolic reaction can be sufficiently disrupted to ensure that the desired metabolic reaction is sufficiently disrupted. Those skilled in the art will appreciate that the actual genes that are disrupted in a given organism may differ between organisms, as certain differences exist in the metabolic networks between different organisms. Probably. However, given the teachings and guidance provided herein, one skilled in the art will also be able to determine which species of interest would increase 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthesis. It will be appreciated that the methods of the invention can be applied to any suitable host microorganism to identify the cognate metabolic variations required to construct an organism. In certain embodiments, increased production couples 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthesis to growth of the organism, as disclosed herein, if desired. , 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid can be unavoidably linked to the growth of the organism.

Sources encoding nucleic acids for 6-aminocaproic acid, caprolactam, hexamethylene diamine, or levulinic acid pathway enzymes can include, for example, any species in which the encoding gene product is capable of catalyzing the reactions described above. Such species include, but are not limited to, prokaryotes and eukaryotes, including bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plants, insects, animals, and mammals, including humans. Including both. Exemplary species for such sources are, for example, Escherichia coli, E. coli strain K12, E. coli C, E. coli W, Pseudomonas sp., Pseudomonas knackmussii, Pseudomonas sp. strain B13, Pseudomonas putida. (Pseudomonas putida), Pseudomonas fluorescens (Pseudomonas fluorescens), Pseudomonas stutzeri (Pseudomonas stutzeri), Pseudomonas mendocina (Pseudomonas mendocina), Rhodopseudomonas palustris, Mycobacterium tuberculosis, Vibrio cholera, Helicobacter pylori, Klebsiella pneumoniae, Serratia proteamaculans, Streptomyces species 2065, Pseudomonas aeruginosa, Pseudomonas aeruginosa PAO1, Ralstonia eutropha, Ralstonia eutropha H16, Clostridium acetobutylicum, Euglena gracilis, Treponema Treponema denticola, Clostridium kluyveri, Homo sapiens, Rattus norvegicus), Acinetobacter sp. ADP1, Acinetobacter sp. M-1, Streptomyces coelicolor, Eubacterium barkeri, Peptostreptococcus asaccharolyticus, Clostridium botulinum ( Clostridium botulinum), Clostridium botulinum strain A3, Clostridium tyrobutyricum, Clostridium pasteurianum, Clostridium thermoaceticum (Moorella thermoaceticum), Moorella thermoaceticum ( Moorella thermoacetica), Acinetobacter calcoaceticus, mouse (Mus musculus), wild boar (Sus scrofa), Flavobacterium species, Arthrobacter aurescens, Penicillium chrysogenum, Aspergillus niger, Aspergillus nidulans, Bacillus subtilis, Saccharomyces cerevisiae, Zymomonas mobilis, Mannheimia succiniciproducens, Clostridium ljungdahlii, Clostridium carboxidivorans, Geobacillus stearothermophilus (Geobacillus stearothermophilus), Agrobacterium tumefaciens (Geobacillus stearothermophilus) tumefaciens), Achromobacter denitrificans, Arabidopsis thaliana, Haemophilus influenzae, Acidaminococcus fermentans, Clostridium sp. M62/1, Fusobacterium nucleatum (Fusobacterium nucleatum), Bos taurus, Zoogloea ramigera, Rhodobacter sphaeroides, Clostridium beijerinckii, Metallosphaera sedula, Thermoanaerobacter sp., Thermoanaeroides Bacter brockii (Thermoanaerobacter brockii), Acinetobacter bailey (Acinetobacter baylyi), Porphyromonas gingivalis, Leuconostoc mesenteroides, Sulfolobus tokodaii, Sulfolobus tokodaii7, Sulfolobus solfataricus, Sulfolobus solfataricus Sulfolobus acidocaldarius, Salmonella typhimurium, Salmonella enterica, Thermotoga maritima, Halobacterium salinarum, Bacillus cereus, Clostridium difficile, Alkaliphilus metalliregiens metalliredigens), Thermoanaerobacter tengcongensis, Saccharomyces kluyveri, Helicobacter pylori, Corynebacterium glutamicum, Clostridium saccharoperbutylacetonicum ( Clostridium saccharoperbutylacetonicum), Pseudomonas chlororaphis, Streptomyces clavuligerus, Campylobacter jejuni, Thermus thermophilus, Pelotomaculum thermopropionicum , Bacteroides capirosus capillosus), Anaerotruncus colihominis, Natranaerobius thermophilius, Archaeoglobus fulgidus, Archaeoglobus fulgidus DSM 4304, Haloarcula marismortui, Pyrobaculum aerophii Lamb (Pyrobaculum) aerophilum), Pyrobaculum aerophilum strain IM2, tobacco (Nicotiana tabacum), peppermint (Menthe piperita), loblolly pine (Pinus taeda), barley (Hordeum vulgare), corn (Zea mays), Rhodococcus opacus (Rhodococcus opacus), Capriavidus necator (Cupriavidus necator), Bradyrhizobium japonicum (Bradyrhizobium japonicum), Bradyrhizobium japonicum USDA110, Ascarius suum, butyrate-producing bacteria L2-50, Bacillus megaterium, Methanococcus maripaludis, Methanosarcina mazei ), Methanosarcina mazei, Methanosarcina barkeri, Methanocaldococcus jannaschii, Caenorhabditis elegans, Leishmaniamajor, Methyl. Methylomicrobium alcaliphilum 20Z, Chromohalobacter salexigens, Archaeglobus fulgidus), Chlamydomonas reinhardtii, trichomonas vaginalis G3, Trypanosoma brucei, Mycoplana ramosa, Micrococcus luteus, Acetobacter pasteurians ), Kluyveromyces lactis, Mesorhizobium loti, Lactococcus lactis, Lysinibacillus sphaericus, Candida boidinii, Candida albicans ( Candida albicans) SC5314, Burkholderia ambiphalia ambifaria) AMMD, roundworm, Acinetobacter baumanii, Acinetobacter calcoaceticus, Burkholderia pymatum, Candida albicans, Clostridium subterminale, Cupriavidus taiwanensis), Flavobacterium lutescens, Lachancea kluyveri, Lactobacillus sp. 30a, Leptospira interrogans, Moorella thermoacetica, Myxococcusxanthus, Nicotiana glutinosa (Nicotiana glutinosa), Nocardia iowensis (species NRRL 5646), Pseudomonas reinekei) MT1, Ralstonia eutropha JMP134, Ralstonia metallidurans, Rhodococcus jostii, Schizosaccharomyces pombe, Selenomonas ruminantium, Streptomyces clavuligerus ), Syntrophus aciditrophicus, Vibrio parahaemolyticus, Vibrio vulnificus vulnificus), and other exemplary species disclosed herein or available as source organisms for the corresponding genes (see Examples). However, to date, the complete genome sequences available are over 550 species, including 395 microbial genomes and various yeast, fungi, plant, and mammalian genomes (over half of these are (available in public databases), essential 6-aminocaproic acid for one or more genes in closely or distantly related species, including, for example, homologs, orthologs, paralogs, and non-orthologous gene substitutions of known genes. The identification of genes encoding , caprolactam, hexamethylenediamine, or levulinic acid biosynthetic activity, as well as the exchange of genetic modifications between organisms, is routine and well known in the art. Thus, the metabolic variations that enable the biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid described herein for certain organisms such as E. coli are similar to prokaryotes and It can be easily applied to other microorganisms, including eukaryotes. Given the teachings and guidance provided herein, those skilled in the art will appreciate that the metabolic variations illustrated in one organism are equally applicable to other organisms.

In some cases, such as when 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid biosynthetic pathways exist in unrelated species, 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid Acid biosynthesis can be induced in a host species by, for example, exogenous expression of paralog(s) from unrelated species that catalyze similar but not identical metabolic reactions and replace the reactions described above. can give.

One of ordinary skill in the art will appreciate that the actual gene utilization between different organisms may differ because certain differences in metabolic networks exist between different organisms. However, given the teachings and guidance provided herein, one skilled in the art will also know that certain microbial organisms of interest would synthesize 6-aminocaproic acid, caprolactam, hexamethylene diamine, or levulinic acid. It should be understood that the teachings and methods of the present invention can be applied to all microbial organisms, using similar metabolic variations to those exemplified herein to build the body. Dew.

The host microbial organism can be selected from, for example, bacteria, yeast, fungi, or any of a variety of other microorganisms applicable to fermentation processes, and non-naturally occurring microbial organisms can be obtained therefrom. . Exemplary bacteria include Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannhemia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium Bacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum plantarum), Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida. Exemplary yeasts or fungi include S. cerevisiae, fission yeast, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris , Rhizopus arrhizus, Rhizopus oryzae, and the like. For example, E. coli is a particularly useful host organism because it is a well-characterized microbial organism amenable to genetic engineering. Other particularly useful host organisms include yeasts such as Saccharomyces cerevisiae and the like. It is understood that any suitable microbial host organism can be used to introduce metabolic and/or genetic modifications to produce the desired product.

Methods for constructing hosts that produce non-naturally occurring 6-aminocaproic acid, caprolactam, hexamethylene diamine, or levulinic acid and testing their expression levels can be described, for example, by methods well known in the art. This can be carried out by different methods of detection and detection. Such methods are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al. , Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999).

Exogenous nucleic acid sequences involved in the pathway for the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid can be synthesized by conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transduction. They can be stably or transiently introduced into host cells using techniques well known in the art, including, but not limited to, transformation. For exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the eukaryotic nucleic acid gene or cDNA can be removed, if desired, prior to transformation into a prokaryotic host cell. , an N-terminal mitochondrial signal or other targeting signals. For example, removal of the mitochondrial leader sequence led to increased expression in E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338(2005). For exogenous expression in yeast or other eukaryotic cells, the gene can be expressed in the cytosol without the addition of a leader sequence or can be directed to mitochondria or other organelles or host cells. can be directed for secretion by the addition of suitable targeting sequences, such as mitochondrial targeting signals or secretion signals suitable for. It is therefore understood that appropriate modifications to a nucleic acid sequence to remove or include a target sequence can be incorporated into an exogenous nucleic acid sequence to confer desirable properties. Additionally, genes can be subjected to codon optimization using techniques well known in the art to achieve optimization of protein expression.

The expression vector(s) comprises one or more 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulin, as exemplified herein, operably linked to expression control sequences functional in the host organism. Can be constructed to include nucleic acids encoding acid biosynthetic pathways. Expression vectors applicable for use in microbial host organisms of the invention include, for example, plasmids, phage vectors, viral vectors, containing operable vectors and selection sequences or markers for stable integration into the host chromosome. , episomes, and artificial chromosomes. Additionally, the expression vector can include one or more selectable marker genes and appropriate expression control sequences. For example, selectable marker genes can also be included that provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or provide critical nutrients not present in the culture medium. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, as are well known in the art. If two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or into separate expression vectors. For single vector expression, the encoding nucleic acid can be operably linked to one common expression control sequence or to different expression control sequences, such as one inducible promoter and one constitutive promoter. can be combined with Transformation of exogenous nucleic acid sequences involved in metabolic or synthetic pathways can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blot or polymerase chain reaction (PCR) amplification of mRNA or immunoblotting for the expression of a gene product or the expression of an introduced nucleic acid sequence or its corresponding gene product. including other suitable analytical methods for testing. It is understood by those skilled in the art that exogenous nucleic acids are expressed in amounts sufficient to produce the desired product, and expression levels are well known in the art and disclosed herein. It is further understood that methods can be used and optimized to obtain sufficient expression.

Directed evolution is one approach that involves the introduction of mutations that target specific genes to improve and/or change the properties of an enzyme. Improved and/or altered enzymes can be identified through embodiment screening assays that allow for the identification of useful variants. Particularly useful screening methods are those with large numbers of enzyme variants (e.g. &gt;10⁴) includes a sensitive high-throughput assay that allows for automated screening. Iterative rounds of mutagenesis and screening are typically performed to identify enzymes with optimized properties. The greater the number of variants screened, the greater the chance of identifying an ideally suited variant. Computational algorithms that can help identify areas of genes for mutagenesis have also been developed and can significantly reduce the number of enzyme variants that need to be generated and screened. .

A number of directed evolution techniques have been developed (for review, see Hibbert et al., Biomol. Eng 22:11-19 (2005); Huisman and Lalonde, In Biocatalysis in the Pharmaceutical and Biotechnology Industries). Patel (ed.), CRC Press; Otten and Quax. et al., Biomol. Eng 22:1-9 (2005); and Sen et al., Appl Biochem.Biotechnol 143 212-223 (2007)), these methods have been successfully applied to improve a wide range of properties across many enzyme classes.

Characteristics of enzymes that have been improved and/or changed by directed evolution techniques include, for example, selectivity/specificity (for conversion of non-natural substrates); temperature stability (for intense high temperature processing); pH stability (low or for bioprocessing under high pH conditions); substrate or product tolerance (so that high product titers can be achieved); binding (K_m) (broadens substrate binding to include non-natural substrates); inhibition (K_i) (to remove inhibition by product, substrate, or key intermediates); activity (kcat) (increase enzyme reaction rate to achieve desired flux); expression level (protein yield and overall pathway oxygen stability (for operation of air-sensitive enzymes under aerobic conditions); and anaerobic activity (for operation of aerobic enzymes in the absence of oxygen).

The following exemplary methods have been developed for gene mutagenesis and diversification to target desired properties of specific enzymes. Any of these can be used to alter/optimize the activity of the decarboxylase enzyme.

EpPCR (Pritchard et al., J Theor. Biol 234:497-509 (2005))²⁺Random point mutations are introduced by reducing the fidelity of the DNA polymerase in the PCR reaction, by adding ions, by biasing the dNTP concentration, or by other conditional variations. A five-step cloning process to limit mutagenesis to the target gene of interest is: 1) error-prone PCR amplification of the gene of interest; 2) digestion with restriction enzymes; 3) gel purification of the desired DNA fragment; 4) ligation into a vector; 5) transformation of the genetic variant into a suitable host and screening of the library for improved performance. This method can simultaneously generate multiple mutations in a single gene, which can be useful. Many mutants can be generated by EpPCR, so high-throughput screening assays or selection methods (especially using robotics) are useful to identify those with desirable characteristics.

Error-prone Rolling Circle Amplification (epRCA) (Fujii et al., Nucleic Acids Res 32:el45 (2004); and Fujii et al., Nat. Protoc. 1:2493-2497 (2006)) A circular plasmid is used as a template and a random 6 base length with an exonuclease-resistant thiophosphate linkage on the last two nucleotides is used to amplify the plasmid, within which the plasmid is tandemly It has many of the same elements as epPCR, except that it is followed by transformation into cells that recircularize with repeats. Mn²⁺By adjusting the concentration, the mutation rate can be varied somewhat. This technique uses a simple error-prone single-step method to generate complete copies of plasmids with 3-4 mutations/kbp. No restriction enzyme digestion or specific primers are required. Additionally, this method is typically available as a kit.

DNA or family shuffling (Stemmer, Proc Natl Acad Sci USA 91: 10747-10751 (1994); and Stemmer, Nature 370:389-391 (1994)) is typically used to generate libraries of chimeric genes. involves digestion of two or more mutant genes with a nuclease such as Dnase I or EndoV to generate a pool of random fragments that is reconstituted by cycles of annealing and extension in the presence of DNA polymerase. . The fragments prime each other and recombination occurs when one copy primes the other (template switch). This method can be used with &gt;1kbp DNA sequences. In addition to mutant recombinants generated by fragment reconstitution, this method introduces point mutations in the extension step at a rate similar to error-prone PCR. This method can be used to remove deleterious, random, neutral mutations that may confer antigenicity.

Staggered Extension (StEP) (Zhao et al., Nat. Biotechnol 16:258-261 (1998)) involves template priming, followed by denaturation and a very short period of annealing/extension (~5 seconds). A repeated cycle of two-step PCR follows. The growing fragments anneal to different templates and are further elongated, and this is repeated until a full-length sequence is created. Template switching means that most resulting fragments have multiple parents.

The combination of low fidelity polymerases (Taq and Mutazyme) reduces error prone bias due to opposing mutation spectra.

In Random Priming Recombination (RPR), random sequence primers are used to generate many short DNA fragments that are complementary to different segments of the template. (Shao et al., Nucleic Acids Res 26:681-683 (1998)) Point mutations result from base misincorporation and mispriming via epPCR. Short DNA fragments are primed with each other based on homology, recombined, and reassembled into full length through repeated thermocycling. Removal of the template before this step ensures fewer parental recombinants. This method, like most others, can be performed with multiple iterations to evolve distinct properties. This technique avoids sequence bias, is gene length independent, and requires little parental DNA for application.

In heteroduplex recombination, linearized plasmid DNA is used to form a heteroduplex that is repaired by mismatch repair. (Volkov et al., Nucleic Acids Res 27:e18(1999); and Volkov et al., Methods Enzymol. 328:456-463(2000)). The mismatch repair step is at least somewhat mutagenic. Heteroduplexes deform more efficiently than linear homoduplexes. This method is suitable for large genes and whole operons.

Random Chimeragenesis on Transient Templates (RACHITT) (Coco et al., Nat. Biotechnol 19:354-359 (2001)) Utilizing I fragmentation and size fractionation. Homologous fragments hybridize to complementary ssDNA scaffolds in the absence of polymerase. Any overlapping non-hybridized fragment ends are excised by the exonuclease. Gaps between the fragments are filled and then ligated, resulting in a pool of full-length diverse strands hybridized to the scaffold (containing U to prevent amplification). The scaffold is then disrupted and replaced by new strands complementary to the diverse strands by PCR amplification. The method involves one strand (scaffold) derived from only one parent; the priming fragment is derived from the other gene; selection is made to exclude the parental scaffold. Therefore, no reannealing with the parent fragment occurs. Overlapping fragments are excised with an exonuclease. In other cases, this is conceptually similar to DNA shuffling and StEP. Therefore, there should be no populations with the same parents, very few inert bodies, and no non-shuffled parents. This technique has the advantage that fewer or no parental genes are created and more cross-over types can occur compared to standard DNA shuffling.

Recombined Extension on Truncated templates (RETT) involves template switching of unidirectionally growing strands from primers in the presence of unidirectional ssDNA fragments used as a pool of templates. (Lee et al., J. Molec. Catalysis 26: 119-129(2003)). DNA endonucleases are not used. Unidirectional ssDNA is created by DNA polymerase using random primers or by sequential deletions by exonucleases. Unidirectional ssDNA is only a template, not a primer. Random priming and exonucleases do not introduce sequence biases that correspond to DNA shuffling/RACHITT enzymatic cleavage. RETT can be easier to optimize than StEP because it uses regular PCR conditions instead of very short extensions. Recombination occurs as a component of the PCR step - not direct shuffling. This method can also be more random than StEP due to the lack of resting periods.

In Degenerate Oligonucleotide Gene Shuffling (DOGS), degenerate primers are used to control recombination between molecules; (Bergquist and Gibbs, Methods Mol. Biol 352:191 -204 (2007); Bergquist et al., Biomol. Eng 22:63-72 (2005); Gibbs et al., Gene 271:13-20 (2001)). This can be used to control the tendency of other methods such as DNA shuffling to regenerate parental genes. This method can be combined with random mutagenesis (epPCR) of selected gene segments. This can be a good way to block reconfiguration of the parent array. Endonuclease is not required. By adjusting the input concentration of the segments produced, one can bias toward the desired backbone. This method allows DNA shuffling from unrelated parents without restriction enzyme digests and allows for the selection of random mutagenesis methods.

Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY) generates combinatorial libraries with single base pair deletions of genes or gene fragments of interest (Ostermeier et al., Proc Natl. Acad Sci USA 96:3562-3567 (1999); and Ostermeier et al., Nat. Biotechnol 17: 1205-1209 (1999)). The cutting moieties are introduced into two different parts of the gene in opposite directions. These are ligated together and the fusion cloned. This technique does not require homology between the two parental genes. When ITCHY is combined with DNA shuffling, this system is called SCRATCHY (see below). The main advantage of both is that homology between the parental genes is not required; for example, functional fusions between E. coli and human genes have been created via ITCHY. When the ITCHY library is created, all possible crossover types are captured.

Thio-Incremental Truncation for the Creation of Hybrid Enzymes (THIO-ITCHY) is similar to ITCHY except that phosphorothioate dNTPs are used to generate the truncation ( Lutz et al., Nucleic Acids Res 29:E16 (2001)). Compared to ITCHY, THIO-ITCHY can be easier to optimize and offers more reproducibility and flexibility.

SCRATCHY combines two methods for recombining genes, ITCHY and DNA shuffling (Lutz et al., Proc Natl Acad Sci USA 98:11248-11253 (2001)). SCRATCHY combines the best features of ITCHY and DNA shuffling. First, ITCHY is used to generate a comprehensive set of fusions between gene fragments in a DNA homology-independent manner. This artificial family is then subjected to a DNA shuffling step to increase the number of cross-types. Computational predictions can be used in optimization. SCRATCHY is more effective than DNA shuffling when sequence identity is less than 80%.

In Random Drift Mutagenesis (RNDM), mutations are created via epPCR followed by screening/selection for those that retain usable activity (Bergquist et al., Biomol. Eng 22:63-72(2005)). These are then used in DOGS to generate recombinants using fusions between active mutants or between an active mutant and some other desired parent. Designed to facilitate the isolation of neutral mutations, the aim is to screen for retained catalytic activity, regardless of whether this activity is higher or lower than the original gene. RNDM can be used in high-throughput assays if the screen can detect activity above background. RNDM was used as a pre-step to DOGS in generating diversity. This technique requires activity before shuffling or other subsequent steps; neutral drift libraries are shown to yield higher/more immediate improvements in activity from smaller libraries. Although published using epPCR, this can be applied to other large-scale mutagenesis methods.

Sequence Saturation Mutagenesis (SeSaM) is a random mutagenesis method that uses 1) random incorporation of phosphothioate nucleotides and truncations to generate pools of random length fragments; This pool is used as a template and 2) extended in the presence of a "uniform" base such as inosine; 3) replication of the inosine-containing complement results in random base incorporation and consequent mutagenesis. (Wong et al., Biotechnol J3:74-82 (2008); Wong et al., Nucleic Acids Res 32:e26 (2004); and Wong et al., Anal.Biochem. 341:187-189 (2005)). Using this technique, it may be possible to generate large libraries of mutants within 2-3 days using a simple method. This technique is non-specific compared to the mutational bias of DNA polymerases. This difference in approach makes this technology complementary (or alternative) to epPCR.

In synthetic shuffling, overlapping oligonucleotides are designed to encode "all the genetic diversity in the target," allowing for very high diversity of shuffled progeny (Ness et al. Nat.Biotechnol 20: 1251-1255(2002)). With this technique, the pieces that are to be shuffled can be designed. This helps increase the resulting diversity of progeny. Sequence/codon biases can be designed such that more distantly related sequences recombine at a rate similar to that observed with more closely related sequences. Furthermore, the technique does not require physically having the template gene.

Nucleotide Exchange and Excision Technology NexT utilizes a combination of dUTP incorporation followed by treatment with uracil DNA glycosylase and then piperidine to perform endpoint DNA fragmentation (Muller et al. , Nucleic Acids Res 33:ell7(2005)). Genes are reconstituted using internal PCR primer extension with a proofreading polymerase. The size for shuffling can be directly controlled using various dUPT::dTTP ratios. This is an endpoint reaction using a simple method for uracil incorporation and cleavage. Other nucleotide analogs such as 8-oxo-guanine and the like can be used with this method. Furthermore, the technique works well with very short fragments (86 bp) and has low error rates. The chemical cutting of DNA used in this technique rarely results in unshuffled clones.

In Sequence Homology-Independent Protein Recombination (SHIPREC), linkers are used to facilitate fusion between two distantly/unrelated genes. Nuclease treatment is used to generate a series of chimeras between two genes. These fusions result in a library of single cross-over hybrids (Sieber et al., Nat. Biotechnol 19:456-460 (2001)). This produces a limited type of shuffling and separate processes are required for mutagenesis. Furthermore, since homology is not required, this technique can generate libraries of chimeras having various portions of each of two unrelated parental genes. SHIPREC was tested using the heme-binding domain of bacterial CP450 fused to the N-terminal region of mammalian CP450; this resulted in mammalian activity in the more soluble enzyme.

In Gene Site Saturation Mutagenesis(TM) (GSSM(TM)), the starting material is a supercoiled dsDNA plasmid containing the insert and a degenerate 2 at the desired site of mutation. (Kretz et al., Methods Enzymol. 388:3-11 (2004)). Primers carrying the mutation of interest anneal to the same sequence on opposite strands of DNA.

The mutation is typically in the middle of the primer, flanked on each side by a precise sequence of about 20 nucleotides. The sequences in the primers are NNN or NNK (coding) and MNN (non-coding) (N=all 4, K=G, T, M=A, C). After extension, DpnI is used to digest the dam-methylated DNA to eliminate the wild-type template. This technique searches for all possible amino acid substitutions (ie, one codon) at a given locus. The technique facilitates the generation of all possible substitutions at a single site free of nonsense codons, resulting in equivalent to nearly equivalent representation of most possible alleles. This technique does not require prior knowledge of the structure, mechanism, or domain of the target enzyme. If followed by shuffling or gene rearrangement, this technique generates a diverse library of recombinants containing all possible combinations of single-site up-mutations. The utility of this combination of techniques has been demonstrated for the successful evolution of over 50 different enzymes, as well as for more than one property in a given enzyme.

Combinatorial Cassette Mutagenesis (CCM) involves the use of short oligonucleotide cassettes to exchange restricted regions with many possible amino acid sequence changes (Reidhaar-Olson et al., Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al., Science 241:53-57 (1988)). Using this technique, simultaneous substitutions at two or three sites are possible. Furthermore, the method tests the multiplicity of possible sequence changes within a restricted range of sites. This technique was used to investigate the information content of the lambda repressor DNA-binding domain.

Combinatorial Multiple Cassette Mutagenesis (CMCM) is essentially similar to CCM, except that it is utilized as part of a larger program: 1) 2) ID Hotspots and Hot Regions The use of epPCR at high mutation rates, then 3) extension with CMCM to cover defined regions of protein sequence space (Reetz, MT, S. Wilensek, D. Zha, and KE Jaeger, 2001, "Combinatorial "Directed Evolution of an Enantioselective Enzyme through Combinatorial Multiple-Cassette Mutagenesis.", Angew.Chem.Int.Ed Engl. 40:3589-3591.) As with CCM, this method can test virtually all possible changes on the target site. When used in conjunction with random mutations and methods for generating shuffled genes, it provides an excellent means of generating a variety of shuffled proteins. This approach was successful in increasing the enantioselectivity of the enzyme by 51-fold.

In mutator strain technology, the conditional ts mutator plasmid allows for a 20-4000-fold increase in the frequency of random, natural mutations during selection, and eliminates deleterious mutations when selection is not required. block accumulation (Selifonova et al., Appl Environ Microbiol 67:3645-3649 (2001)). This technology is based on the plasmid-derived mutD5 gene, which encodes a mutant subunit of DNA polymerase III. This subunit binds endogenous DNA polymerase III and impairs the proofreading ability of polymerase III in any strain carrying the plasmid.

A broad spectrum of base substitutions and frameshift mutations occur. In order of effective use, the mutator plasmid should be removed once the desired phenotype is achieved; this is achieved through a temperature-sensitive origin of replication, which Enables curing. It is noted that mutator strains have been investigated for quite some time (see, eg, Low et al., J. Mol. Biol. 260:359-3680 (1996)). Very high spontaneous mutation rates are observed with this technique. Conditional properties minimize unwanted background mutations. This technique can be combined with adaptive evolution to enhance mutagenesis rates and achieve desired phenotypes more rapidly.

"Look-Through Mutagenesis (LTM) is a multidimensional mutagenesis method that evaluates and optimizes combinatorial mutations of selected amino acids." (Rajpal et al., Proc Natl Acad Sci USA 102:8466-8471(2005)). Rather than saturate each site with all possible amino acid changes, a set of nine is chosen to cover the range of amino acid R group chemistries. Fewer changes per site allows multiple sites to be subjected to this type of mutagenesis. &gt;800-fold increase in binding affinity for low nanomolar to picomolar antibodies was achieved through this method. This method is a reasonable approach to minimizing the number of random combinations and increases the ability to find improved traits by significantly reducing the number of clones that will be screened. can be increased. This has been applied in antibody engineering, particularly to increase binding affinity and/or reduce dissociation. The technique can be combined with screening or selection.

Gene rearrangement is a DNA shuffling method that can be applied to multiple genes at once or to the creation of many libraries of chimeras (multiple mutations) of a single gene (supplied by Verenium) Tunable GeneReassembly(TM) (TGR(TM) Technology supplied).

Typically, this technique is used in conjunction with ultra-high throughput screening to determine the sequence space represented for the desired improvement. This technique allows for multiple genetic recombinations independent of homology. The exact number and location of crossover events can be predetermined using fragments designed through bioinformatics analysis. This technique leads to very high levels of diversity with virtually no parental gene rearrangement and with low levels of inactive genes. In combination with GSSM, a wide range of mutations can be tested for improved activity. The method allows for "blending" and "tweaking" of DNA shuffling, for example codon usage can be optimized.

In silico Protein Design Automation (PDA) fixes a structurally determined protein backbone with a specific fold and for amino acid substitutions that can stabilize the fold and overall protein energy properties. (Hayes et al., Proc Natl Acad Sci USA99:15926-15931 (2002)). This technique is used in in silico structure-based entropy prediction to search for structural tolerance towards protein amino acid variations.

Statistical mechanics is applied to calculate the binding interactions at each location. The tolerance of a structure toward amino acid substitutions is an indicator of binding. Ultimately, this technique is designed to obtain desired modifications of protein properties while maintaining the integrity of structural features. The method computationally evaluates a large number of possible sequence variants and allows their filtering (10⁵⁰). The selection of sequence variants to test relates to the most favorable thermodynamically based predictions. Ostensibly, only stability or properties associated with stability can be efficiently studied using this technique. The method has been successfully used to genetically engineer several therapeutic proteins, particularly immunoglobulins. In silico prediction avoids testing too many potential variants. Predictions based on existing three-dimensional structures appear to be more likely to be successful than those based on hypothesized structures. This technique can easily predict multiple simultaneous mutations and allows their targeted screening, which, due to the exponential growth in numbers, sometimes precludes purely experimental techniques. It is not possible to use it.

Iterative Saturation Mutagenesis (ISM) uses: 1) structural/functional information to select promising sites for enzyme improvement; 2) using Stratagene QuikChange (or other suitable means) saturation mutagenesis at selected sites; 3) screening/selection for desired properties; and 4) continued rework and iteration at other sites with improved clones (Reetz et al. Nat. Protoc. 2:891-903 (2007); and Reetz et al., Angew. Chem. Int. Ed Engl. 45:7745-7751 (2006)). This is a proven methodology, which ensures that all possible substitutions at a given position are made for screening/selection.

Any of the aforementioned methods of mutagenesis can be used alone or in any combination. Additionally, any one or a combination of directed evolution methods can be used with adaptive evolution techniques.

The present invention further provides methods for producing desired intermediates or products such as adipate, 6-aminocaproic acid, caprolactam, hexamethylene diamine, levulinic acid, and the like. For example, a method for producing adipate can include culturing a non-naturally occurring microbial organism having an adipate pathway, the pathway producing adipate in an amount sufficient to produce adipate. at least one exogenous nucleic acid encoding an adipate pathway enzyme that is expressed under conditions and for a period sufficient for the purpose of CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA synthetase or phosphotransadipyrase/adipate kinase or adipyl-CoA:acetyl-CoA transferase or adipyl-CoA Contains hydrolase. Further, a method for producing adipate can include culturing a non-naturally occurring microbial organism having an adipate pathway, the pathway producing adipate in an amount sufficient to produce adipate. at least one exogenous nucleic acid encoding an adipate pathway enzyme that is expressed under conditions and for a period sufficient for the purpose of succinyl-CoA:acetyl-CoA acyltransferase; transferase, 3-oxoadipate reductase, 3-hydroxyadipate dehydratase, and 2-enoate reductase.

Additionally, a method for producing 6-aminocaproic acid can include culturing a non-naturally occurring microbial organism having a 6-aminocaproic acid pathway, wherein the pathway is capable of producing 6-aminocaproic acid. 6-aminocaproic acid comprising at least one exogenous nucleic acid encoding a 6-aminocaproic acid pathway enzyme that is expressed in a sufficient amount under conditions and for a period sufficient to produce 6-aminocaproic acid; The pathway includes CoA-dependent aldehyde dehydrogenase and transaminase or 6-aminocaproate dehydrogenase. Further, a method for producing caprolactam can include culturing a non-naturally occurring microbial organism having a caprolactam pathway, the pathway producing caprolactam in an amount sufficient to produce caprolactam. at least one exogenous nucleic acid encoding a caprolactam pathway enzyme that is expressed under conditions and for a period sufficient for the purpose of Contains dehydrogenase and amidohydrolase.

The present invention provides for the production of 6-aminocaproic acid (6-ACA) in a non-naturally occurring microbial organism having the 6-ACA pathway described herein under conditions and for a sufficient period of time to produce 6-aminocaproic acid (6-ACA). Further provided is a method for producing 6-ACA by culturing. In one aspect, the 6-ACA pathway includes HODH aldolase; OHED hydratase; OHED reductase; 2-OHD decarboxylase; and adipate semialdehyde aminotransferase or adipate semialdehyde oxidoreductase (amination). In other embodiments, the 6-ACA pathway includes HODH aldolase; OHED hydratase; OHED decarboxylase; 6-OHE reductase; and adipate semialdehyde aminotransferase or adipate semialdehyde oxidoreductase (amination). In other embodiments, the 6-ACA pathway includes HODH aldolase; OHED hydratase; OHED aminotransferase or OHED oxidoreductase (amination); 2-AHE reductase; and 2-AHD decarboxylase. In other embodiments, the 6-ACA pathway includes HODH aldolase; OHED hydratase; OHED reductase; 2-OHD aminotransferase or 2-OHD oxidoreductase (amination); and 2-AHD decarboxylase. In other embodiments, the 6-ACA pathway comprises: HODH aldolase; HODH formate lyase and pyruvate formate lyase activating enzymes or HODH dehydrogenase; 3-hydroxyadipyl-CoA dehydratase; 2,3-dehydroadipyl-CoA reductase; adipyl -CoA dehydrogenase; and adipate semialdehyde aminotransferase or adipate semialdehyde oxidoreductase (amination). In other embodiments, the 6-ACA pathway comprises HODH aldolase; OHED hydratase; OHED formate lyase and pyruvate formate lyase activating enzymes or OHED dehydrogenase; 2,3-dehydroadipyl-CoA reductase; adipyl-CoA dehydrogenase; Contains semialdehyde aminotransferase or adipate semialdehyde oxidoreductase (amination). In other embodiments, the 6-ACA pathway comprises HODH aldolase; OHED hydratase; OHED reductase; 2-OHD formate lyase and pyruvate formate lyase activating enzymes or 2-OHD dehydrogenase; adipyl-CoA dehydrogenase; and adipate semialdehyde aminotransferase. or adipate semialdehyde oxidoreductase (amination). In further embodiments, the 6-ACA pathway described above can include succinate semialdehyde dehydrogenase, alpha-ketoglutarate decarboxylase, or phosphoenolpyruvate (PEP) carboxykinase.

The present invention provides methods for producing hexamethylene diamine (HMDA) by culturing a non-naturally occurring microbial organism having the HMDA pathway described herein under conditions and for a sufficient period of time to produce hexamethylene diamine (HMDA). Further provided are methods for producing HMDA. In one embodiment, the HMDA pathway includes 6-aminocaproate kinase; 6-AHOP oxidoreductase; and 6-aminocaproate semialdehyde oxidoreductase (amination) or 6-aminocaproate semialdehyde aminotransferase. In other embodiments, the HMDA pathway comprises: 6-aminocaproate kinase; 6-AHOP acyltransferase; 6-aminocaproyl-CoA oxidoreductase; and 6-aminocaproic acid semialdehyde oxidoreductase (amination) or 6-aminocaproic Contains acid semialdehyde aminotransferase. In other embodiments, the HMDA pathway comprises 6-aminocaproate CoA transferase or 6-aminocaproate CoA ligase; 6-aminocaproyl-CoA oxidoreductase; and 6-aminocaproate semialdehyde oxidoreductase (amination) or 6-aminocaproic acid semialdehyde aminotransferase. In other embodiments, the HMDA pathway comprises 6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate kinase; 6-AAHOP oxidoreductase; 6-acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase ); and 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide). In other embodiments, the HMDA pathway comprises: 6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate CoA transferase or 6-acetamidohexanoate CoA ligase; 6-acetamidohexanoyl-CoA oxidoreductase; Contains acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase (amination); and 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide). In other embodiments, the HMDA pathway comprises 6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate kinase; 6-AAHOP oxidoreductase; 6-acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase ); and 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide).

Further, a method for producing adipate can include culturing a non-naturally occurring microbial organism having an adipate pathway, the pathway producing adipate in an amount sufficient to produce adipate. at least one exogenous nucleic acid encoding an adipate pathway enzyme that is expressed under conditions and for a period sufficient to provide for alpha-ketoadipyl-CoA synthetase, phosphotransketoadipyrase/alpha -ketoadipate kinase, or alpha-ketoadipyl-CoA: acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydrogenase; 2-hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase, phosphotransadipyrase/adipate kinase, adipyl-CoA:acetyl-CoA transferase, or adipyl-CoA hydrolase. Further, a method for producing adipate can include culturing a non-naturally occurring microbial organism having an adipate pathway, the pathway producing adipate in an amount sufficient to produce adipate. at least one exogenous nucleic acid encoding an adipate pathway enzyme that is expressed under conditions and for a period sufficient to provide an adipate pathway enzyme; 2-hydroxyadipate dehydrogenase; 2-hydroxyadipyl-CoA synthetase; Phosphotranshydroxyadipyrase/2-hydroxyadipate kinase, or 2-hydroxyadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl- Includes CoA synthetase, phosphotransadipyrase/adipate kinase, adipyl-CoA:acetyl-CoA transferase, or adipyl-CoA hydrolase.

As disclosed herein, the invention also includes at least one exogenous nucleic acid encoding a 6-aminocaproic acid pathway enzyme expressed in sufficient amounts to produce 6-aminocaproic acid. A method for producing 6-aminocaproic acid by culturing a non-naturally occurring microbial organism having a 6-aminocaproic acid pathway, the 6-aminocaproic acid pathway comprising 3-oxo-6-aminohexanoyl -CoA thiolase; 3-oxo-6-aminohexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoA dehydratase; 6-aminohex-2-enoyl-CoA reductase; and 6-aminocaproyl-CoA /acyl-CoA transferase, 6-aminocaproyl-CoA synthase, or 6-aminocaproyl-CoA hydrolase (see Examples XII and XIII; steps A/B/ of FIG. 11). C/D/K/L/M). The present invention further relates to a naturally occurring 6-aminocaproic acid pathway comprising at least one exogenous nucleic acid encoding a 6-aminocaproic acid pathway enzyme expressed in sufficient amounts to produce 6-aminocaproic acid. 3-oxo-6-aminohexanoyl-CoA thiolase; aminohexanoyl-CoA/acyl-CoA transferase, 3-oxo-6-aminohexanoyl-CoA synthase, or 3-oxo-6-aminohexanoyl-CoA hydrolase; 3-oxo-6-aminohexanoate reductase; 3-hydroxy-6-aminohexanoate dehydratase; and 6-aminohex-2-enoate reductase (see Examples XII and XIV; steps A/E/F of FIG. /G/H/I/J).

In other embodiments, the present invention provides a non-naturally occurring microbial organism having a caprolactam pathway comprising at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in an amount sufficient to produce caprolactam. Provided is a method for producing caprolactam by culturing See Examples XII and XV; step K/L in FIG. 11). In such methods, caprolactam can be produced by spontaneous cyclization of 6-aminocaproyl-CoA to caprolactam (see Example XII; step Q of Figure 11). The present invention also provides non-naturally occurring microbial organisms having a hexamethylenediamine pathway comprising at least one exogenous nucleic acid encoding a hexamethylenediamine pathway enzyme expressed in sufficient amounts to produce hexamethylenediamine. 6-aminocaproyl-CoA/acyl-CoA transferase or 6-aminocaproyl-CoA synthase; 6-aminocaproyl-CoA reductase (aldehyde formation); and hexamethylenediamine Also provided are non-naturally occurring microbial organisms containing transaminases or hexamethylene diamine dehydrogenases (see Examples XII and XVI; steps K/L/N/O/P of Figure 11).

In still other embodiments, the present invention provides a non-naturally occurring microbial organism having a caprolactam pathway comprising at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in sufficient amount to produce caprolactam. 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA reductase; hydroxy-6-aminohexanoyl-CoA dehydratase; and 6-aminohex-2-enoyl-CoA reductase (see Examples XII and XVII; steps A/B/C of FIG. 11). /D). In such methods, caprolactam can (see Example XII; step Q of Figure 11). Cultivating a non-naturally occurring microbial organism having a hexamethylene diamine pathway comprising at least one exogenous nucleic acid encoding a hexamethylene diamine pathway enzyme expressed in an amount sufficient to produce hexamethylene diamine. 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA reductase; 3-hydroxy -6-aminohexanoyl-CoA dehydratase; 6-aminohex-2-enoyl-CoA reductase; 6-aminocaproyl-CoA reductase (aldehyde formation); and hexamethylenediamine transaminase or hexamethylenediamine dehydrogenase. Also provided (see Examples XII and XVIII; steps A/B/C/D/N/O/P of Figure 11).

In another embodiment, the invention provides a method for producing 6-ACA by culturing a non-naturally occurring microbial organism that has a 6-aminocaproic acid (6-ACA) pathway, the method comprising: The body contains at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in sufficient quantities to produce 6-ACA, the 6-ACA pathway being succinic semialdehyde dehydrogenase, alpha- Ketoglutarate decarboxylase, phosphoenolpyruvate (PEP) carboxykinase, 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) aldolase, 2-oxohept-4-ene-1,7-dioate (OHED) ) hydratase, 2-oxohept-4-ene-1,7-dioate (OHED) reductase, 2-oxoheptane-1,7-dioate (2-OHD) decarboxylase, adipate semialdehyde aminotransferase, adipate semialdehyde oxidoreductase (amination), 2-oxohept-4-ene-1,7-dioate (OHED) decarboxylase, 6-oxohex-4-enoate (6-OHE) reductase, 2-oxoheptane-1,7-dioate (2 -OHD) aminotransferase, 2-oxoheptane-1,7-dioate (2-OHD) oxidoreductase (amination), 2-aminoheptane-1,7-dioate (2-AHD) decarboxylase, 2-oxoheptane- 4-ene-1,7-dioate (OHED) aminotransferase, 2-oxohept-4-ene-1,7-dioate (OHED) oxidoreductase (amination), 2-aminohept-4-ene-1,7- dioate (2-AHE) reductase, 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) formate lyase, 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) dehydrogenase, 3-hydroxy Adipyl-CoA dehydratase, 2,3-dehydroadipyl-CoA reductase, adipyl-CoA dehydrogenase, 2-oxohept-4-ene-1,7-dioate (OHED) formate lyase, 2-oxohept-4-ene-1 ,7-dioate (OHED) dehydrogenase, 2-oxoheptane-1,7-dioate (2-OHD) formate lyase, 2-oxoheptane-1,7-dioate (2-OHD) dehydrogenase, or pyruvate formate lyase activity (See Examples XIX and XXI; Steps A-Q of Figure 12).

In another embodiment, the invention provides a method for producing 6-ACA by culturing a non-naturally occurring microbial organism that has a 6-aminocaproic acid (6-ACA) pathway, the method comprising: Provided are methods, wherein the body comprises at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in an amount sufficient to produce 6-ACA. In one aspect, the 6-ACA pathway comprises HODH aldolase; OHED hydratase; OHED reductase; 2-OHD decarboxylase; and adipate semialdehyde aminotransferase or adipate semialdehyde oxidoreductase (amination) (see Examples XIX and XXI See steps A/B/C/D/E in Figure 12). In other aspects of the invention, the 6-ACA pathway comprises HODH aldolase; OHED hydratase; OHED decarboxylase; 6-OHE reductase; and adipate semialdehyde aminotransferase or adipate semialdehyde oxidoreductase (amination) (Example See XIX and XXI; steps A/B/F/G/E of Figure 12). In other embodiments of the invention, the 6-ACA pathway includes HODH aldolase; OHED hydratase; OHED aminotransferase or OHED oxidoreductase (amination); 2-AHE reductase; and 2-AHD decarboxylase (Examples XIX and See XXI; steps A/B/J/D/I in Figure 12). In other embodiments of the invention, the 6-ACA pathway comprises HODH aldolase; OHED hydratase; OHED reductase; 2-OHD aminotransferase or 2-OHD oxidoreductase (amination); and 2-AHD decarboxylase (Example See XIX and XXI; steps A/B/C/H/I in Figure 12). In other aspects of the invention, the 6-ACA pathway comprises: HODH aldolase; HODH formate lyase and pyruvate formate lyase activating enzymes or HODH dehydrogenase; 3-hydroxyadipyl-CoA dehydratase; 2,3-dehydroadipyl-CoA reductase; adipyl-CoA dehydrogenase; and adipate semialdehyde aminotransferase or adipate semialdehyde oxidoreductase (amination) (see Examples XIX and XXI; steps A/L/M/N/O/ of Figure 12). E). The 6-ACA pathway involves HODH aldolase; OHED hydratase; OHED formate lyase and pyruvate formate lyase activating enzymes or OHED dehydrogenase; 2,3-dehydroadipyl-CoA reductase; adipyl-CoA dehydrogenase; and adipate semialdehyde aminotransferase or adipate semialdehyde oxidoreductase (amination) (see Examples XIX and XXI; steps A/B/P/N/O/E of Figure 12). In other embodiments of the invention, the 6-ACA pathway comprises HODH aldolase; OHED hydratase; OHED reductase; 2-OHD formate lyase and pyruvate formate lyase activating enzymes or 2-OHD dehydrogenase; adipyl-CoA dehydrogenase; aldehyde aminotransferase or adipate semialdehyde oxidoreductase (amination) (see Examples XIX and XXI; steps A/B/C/Q/O/E of Figure 12). In further embodiments, the 6-ACA pathway described above can include succinate semialdehyde dehydrogenase, alpha-ketoglutarate decarboxylase, or phosphoenolpyruvate (PEP) carboxykinase.

In other embodiments, the invention provides 6-aminocaproic acid (6-ACA) comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in sufficient amounts to produce 6-ACA. A method for producing 6-ACA by culturing a non-naturally occurring microbial organism having an ACA pathway, the 6-ACA pathway comprising glutamyl-CoA transferase, glutamyl-CoA ligase, beta-ketothiolase, 3-oxo-6-aminopimeloyl-CoA oxidoreductase, 3-hydroxy-6-aminopimeloyl-CoA dehydratase, 6-amino-7-carboxyhept-2-enoyl-CoA reductase, 6-aminopimeloyl-CoA reductase (aldehyde formation), or 2-aminopimelate decarboxylase (see Examples XXV and XXVI; steps A/B/C/D/E/I/J of Figure 20). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, the set comprising glutamyl-CoA transferase or glutamyl-CoA ligase; beta- Ketothiolase; 3-oxo-6-aminopimeloyl-CoA oxidoreductase; 3-hydroxy-6-aminopimeloyl-CoA dehydratase; 6-amino-7-carboxyhept-2-enoyl-CoA reductase; 6-aminopimeloyl-CoA reductase (aldehyde formation ); and encodes 2-aminopimelate decarboxylase.

In other embodiments, the invention provides 6-aminocaproic acid (6-ACA) comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in sufficient amounts to produce 6-ACA. A method for producing 6-ACA by culturing a non-naturally occurring microbial organism having an ACA pathway, the 6-ACA pathway comprising glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, 3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, 3-aminopimelate 2,3-aminomutase, or 2-aminopimelate (See Examples XXV and XXVI; Steps A/B/J/T/AA of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate 2,3-aminomutase; and 2- Encodes aminopimelate decarboxylase.

In other embodiments, the invention provides 6-aminocaproic acid (6-ACA) comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in sufficient amounts to produce 6-ACA. Provided is a method for producing 6-ACA by culturing a non-naturally occurring microbial organism having an ACA pathway, the 6-ACA pathway comprising homolysine 2-monooxygenase. See Examples XXV and XXVI; step A of Figure 23). In a further embodiment, the 6-ACA pathway involves hydrolysis of the 6-aminohexanamide product with a dilute acid or base to convert the 6-aminohexanamide to 6-aminocaproate (see Example XXV). step B in Figure 23).

In other embodiments, the invention provides 6-aminocaproic acid (6-ACA) comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in sufficient amounts to produce 6-ACA. A method for producing 6-ACA by culturing a non-naturally occurring microbial organism having an ACA pathway, the 6-ACA pathway comprising adipate reductase, adipate kinase, or adipyl phosphate reductase. A method is provided (see Example XXVIII; steps X/Y/Z of Figure 25). In further embodiments, the 6-aminocaproic acid (6-ACA) pathway includes adipate reductase. In other further embodiments, the 6-ACA pathway includes adipate kinase and adipyl phosphate reductase. In other embodiments, the microbial organism having a 6-ACA pathway described above further comprises an adipate pathway, a caprolactam pathway, and/or a hexamethylenediamine pathway as described herein (see Example XXVIII; Steps A to W in Figure 25).

In other embodiments, the invention provides 6-aminocaproic acid (6-ACA) comprising at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in sufficient amounts to produce 6-ACA. A method for producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally occurring microbial organism having an ACA pathway, the 6-ACA pathway being a 2-amino-7-oxosabaproic acid. rate ketoacid decarboxylase, 2-amino-7-oxoheptanoate decarboxylase, 2-amino-7-oxoheptanoate oxidoreductase, 2-aminopimelate decarboxylase, 6-aminohexanal oxidoreductase, 2- Amino-7-oxoheptanoate decarboxylase, or 2-amino-7-oxosabarate amino acid decarboxylase (see Examples XXV and XXVI; steps A/B/ of FIG. D/E/F/G/I).

In further embodiments, the microbial organism contains at least one exogenous enzyme encoding a 2-amino-7-oxosubarate pathway enzyme expressed in sufficient amount to produce 2-amino-7-oxosabarate. It has a 2-amino-7-oxosabarate pathway with nucleic acids, and the 2-amino-7-oxosabarate pathway is 2-amino-5-hydroxy-7-oxosabarate aldolase, 2-amino-5- hydroxy-7-oxosubarate dehydratase, or 2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; Steps A/B/C of Figure 27).

In another embodiment of the invention, the invention provides a method for producing a naturally occurring enzyme that has a 6-aminocaproic acid (6-ACA) pathway, comprising a set of exogenous nucleic acids encoding 6-aminocaproic acid (6-ACA) pathway enzymes. A method for producing 6-ACA by culturing non-existent microbial organisms, the set comprising 2-amino-7-oxosabarate ketoacid decarboxylase; 2-amino-7-oxohepta and 2-aminopimelate decarboxylase (see Example XXV; Steps A/D/E of Figure 26). In another embodiment of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, the set comprising 2-amino-7-oxosabarate ketoacid. encodes decarboxylase; 2-amino-7-oxoheptanoate decarboxylase; and 6-aminohexanal oxidoreductase (see Example XXV; steps A/B/F of Figure 26). In another embodiment of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, the set comprising a 2-amino-7-oxosabalate amino acid encodes carboxylase; 2-amino-7-oxoheptanoate decarboxylase; and 6-aminohexanal oxidoreductase (see Example XXV; Steps I/G/F of Figure 26). In a further aspect of each of the above embodiments, the microbial organism encodes a 2-amino-7-oxosabalate pathway enzyme expressed in an amount sufficient to produce 2-amino-7-oxosabalate. the 2-amino-7-oxosabarate pathway has a second set of exogenous nucleic acids that 2-amino-5-hydroxy-7-oxosabarate dehydratase; and 2-amino-5-en-7-oxosabarate reductase (see Examples XXV and XXVI; steps of FIG. 27). A/B/C).

In another embodiment, the invention provides a method for producing HMDA by culturing a non-naturally occurring microbial organism that has a hexamethylenediamine (HMDA) pathway, the microbial organism producing HMDA. the HMDA pathway comprises at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in sufficient amounts to produce 6-aminocaproate kinase, [(6-aminohexanoyl)oxy]phosphonate; (6-AHOP) oxidoreductase, 6-aminocaproate semialdehyde aminotransferase, 6-aminocaproate semialdehyde oxidoreductase (amination), 6-aminocaproate N-acetyltransferase, 6-acetamidohexanoate kinase, [ (6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) oxidoreductase, 6-acetamidohexanal aminotransferase, 6-acetamidohexanal oxidoreductase (amination), 6-acetamidohexanamine N-acetyltransferase, 6-acetamidohexane Amine hydrolase (amide), 6-acetamidohexanoate CoA transferase, 6-acetamidohexanoate CoA ligase, 6-acetamidohexanoyl-CoA oxidoreductase, [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) Provided are methods comprising acyltransferases, [(6-aminohexanoyl)oxy]phosphonate (6-AHOP) acyltransferases, 6-aminocaproate CoA transferases, and 6-aminocaproate CoA ligases (Example XX and XXI; steps A-N of Figure 13).

In another embodiment, the invention provides a method for producing HMDA by culturing a non-naturally occurring microbial organism that has a hexamethylenediamine (HMDA) pathway, the microbial organism producing HMDA. The method comprises at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in sufficient amounts to produce a HMDA pathway enzyme. In one aspect, the HMDA pathway comprises 6-aminocaproate kinase; 6-AHOP oxidoreductase; and 6-aminocaproate semialdehyde oxidoreductase (amination) or 6-aminocaproate semialdehyde aminotransferase (Example XX and XXI; steps A/B/C of Figure 13). In other aspects of the invention, the HMDA pathway comprises: 6-aminocaproate kinase; 6-AHOP acyltransferase; 6-aminocaproyl-CoA oxidoreductase; and 6-aminocaproate semialdehyde oxidoreductase (amination) or 6-aminocaproic acid semialdehyde aminotransferase (see Examples XX and XXI; steps A/L/N/C of Figure 13). In other aspects of the invention, the HMDA pathway comprises 6-aminocaproate CoA transferase or 6-aminocaproate CoA ligase; 6-aminocaproyl-CoA oxidoreductase; and 6-aminocaproate semialdehyde oxidoreductase ( amination) or 6-aminocaproic acid semialdehyde aminotransferase (see Examples XX and XXI; steps M/N/C of Figure 13). In other aspects of the invention, the HMDA pathway comprises 6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate kinase; 6-AAHOP oxidoreductase; 6-acetamidohexanal aminotransferase or 6-acetamidohexanoate kinase; (amination); and 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide) (see Examples XX and XXI; steps D/E/F/G/ of Figure 13). H). In other aspects of the invention, the HMDA pathway comprises: 6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate CoA transferase or 6-acetamidohexanoate CoA ligase; 6-acetamidohexanoyl-CoA oxidoreductase ; 6-acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase (amination); and 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide) (see Examples XX and XXI). step D/I/J/G/H in Figure 13). In other aspects of the invention, the HMDA pathway comprises 6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate kinase; 6-AAHOP oxidoreductase; 6-acetamidohexanal aminotransferase or 6-acetamidohexanoate kinase; (amination); and 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide) (see Examples XX and XXI; steps D/E/K/J/ of Figure 13). G).

In other embodiments, the invention provides methods for producing hexamethylene diamine (HMDA) having a naturally occurring A method for producing HMDA by culturing non-existent microbial organisms, the HMDA pathway comprising: glutamyl-CoA transferase, glutamyl-CoA ligase, beta-ketothiolase, 3-oxo-6-aminopimeloyl-CoA oxide. Reductase, 3-hydroxy-6-aminopimeloyl-CoA dehydratase, 6-amino-7-carboxyhept-2-enoyl-CoA reductase, 6-aminopimeloyl-CoA reductase (aldehyde formation), 2-amino-7-oxoheptanoate (See Examples XXIV and XXVI; Steps A-H of Figure 20). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutamyl-CoA transferase or ligase; beta-ketothiolase; -6-aminopimeloyl-CoA oxidoreductase; 3-hydroxy-6-aminopimeloyl-CoA dehydratase; 6-amino-7-carboxyhept-2-enoyl-CoA reductase; 6-aminopimeloyl-CoA reductase (aldehyde formation); 2-amino -7-oxoheptanoate aminotransferase or aminating oxidoreductase; and homolysine decarboxylase.

In other embodiments, the invention provides methods for producing hexamethylene diamine (HMDA) having a naturally occurring A method for producing HMDA by culturing non-existent microbial organisms, the HMDA pathway comprising glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate reductase, 3-oxo-1-carboxyheptanal aminotransferase, 3-oxo-1-carboxyheptanal aminating oxidoreductase, 3-oxo-7-aminoheptanoate 3- Aminotransferase, 3-oxo-7-aminoheptanoate 3-aminated oxidoreductase, 3-oxopimelate kinase, 5-oxopimeloylphosphonate reductase, 3-oxopimelate CoA transferase, 3-oxopimelate ligase , 5-oxopimelate-CoA reductase (aldehyde formation), 3-oxopimelate aminotransferase, 3-oxopimelate amination oxidoreductase, 3-aminopimelate-CoA transferase, 3-aminopimelate ligase, 5-aminopimelate- CoA reductase (aldehyde formation), 3-aminopimelate kinase, 5-aminopimeloylphosphonate reductase, 3-aminopimelate reductase, 3-amino-7-oxoheptanoate 2,3-aminomutase, 2 -Amino-7-oxoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoate aminating oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomutase, homolysine decarboxylase, 3 -Aminopimelate 2,3-aminomutase, 2-aminopimelate kinase, 2-aminopimelate CoA transferase, 2-aminopimelate CoA ligase, 2-aminopimelate reductase, 6-aminopimelate phosphonate reductase, 6-aminopimelate- (Examples XXIV and XXVI See Figure 21).

In other embodiments, the invention provides methods for producing hexamethylene diamine (HMDA) having a naturally occurring A method for producing HMDA by culturing non-existent microbial organisms, the HMDA pathway comprising glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate reductase, 3-oxo-1-carboxyheptanal 7-aminotransferase, 3-oxo-1-carboxyheptanal 7-amination oxidoreductase, 3-oxo-7-aminohepta Noate 3-aminotransferase, 3-oxo-7-aminoheptanoate 3-aminated oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomutase, or homolysine decarboxylase. (See Examples XXIV and XXVI; Steps A/B/C/D/E/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate reductase; 3-oxo-1-carboxyheptanal 7-aminotransferase or 3-oxo-1-carboxyheptanal 7-amination Oxidoreductase; 3-oxo-7-aminoheptanoate 3-aminotransferase or 3-oxo-7-aminoheptanoate 3-amination oxidoreductase; 3,7-diaminoheptanoate 2,3-aminomutase; and encodes homolysine decarboxylase.

In other embodiments, the invention provides methods for producing hexamethylene diamine (HMDA) having a naturally occurring A method for producing HMDA by culturing non-existent microbial organisms, the HMDA pathway comprising glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate kinase; 5-oxopimeloylphosphonate reductase, 3-oxo-1-carboxyheptanal 7-aminotransferase, 3-oxo-1-carboxyheptanal 7-aminated oxidoreductase , 3-oxo-7-aminoheptanoate 3-aminotransferase, 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomutase, or homo (See Examples XXIV and XXVI; Steps A/B/F/G/D/E/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate kinase; 5-oxopimeloylphosphonate reductase; 3-oxo-1-carboxyheptanal 7-aminotransferase or 3-oxo- 1-Carboxyheptanal 7-aminated oxidoreductase; 3-oxo-7-aminoheptanoate 3-aminotransferase or 3-oxo-7-aminoheptanoate 3-aminated oxidoreductase; 3,7-diaminohepta It encodes noate 2,3-aminomutase; and homolysine decarboxylase.

In other embodiments, the invention provides methods for producing hexamethylene diamine (HMDA) having a naturally occurring A method for producing HMDA by culturing non-existent microbial organisms, the HMDA pathway comprising glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate-CoA transferase, 3-oxopimelate-CoA ligase, 5-oxopimeloyl-CoA reductase (aldehyde formation), 3-oxo-1-carboxyheptanal 7-aminotransferase, 3-oxo-1-carboxyheptanal 7-aminated oxidoreductase, 3-oxo-7-aminoheptanoate 3-aminotransferase, 3-oxo-7-aminoheptanoate 3-aminated oxidoreductase, 3,7-diaminoheptanoate 2,3 - aminomutase, or homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/H/I/D/E/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate-CoA transferase or 3-oxopimelate-CoA ligase; 5-oxopimeloyl-CoA reductase (aldehyde formation); 3-oxo-1-carboxyheptanal 7-amino transferase or 3-oxo-1-carboxyheptanal 7-amination oxidoreductase; 3-oxo-7-aminoheptanoate 3-aminotransferase or 3-oxo-7-aminoheptanoate 3-amination oxidoreductase; Encodes 3,7-diaminoheptanoate 2,3-aminomutase; and homolysine decarboxylase.

In other embodiments, the invention provides methods for producing hexamethylene diamine (HMDA) having a naturally occurring A method for producing HMDA by culturing non-existent microbial organisms, the HMDA pathway comprising glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate reductase, 3-oxo-1-carboxyheptanal 3-aminotransferase, 3-oxo-1-carboxyheptanal 3-amination oxidoreductase, 3-amino-7-oxohepta Noate 7-aminotransferase, 3-amino-7-oxoheptanoate 7-aminating oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomutase, or homolysine decarboxylase. (See Examples XXIV and XXVI; Steps A/B/C/AB/Z/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate reductase; 3-oxo-1-carboxyheptanal 3-aminotransferase or 3-oxo-1-carboxyheptanal 3-amination oxidoreductase; 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; 3,7-diaminoheptanoate 2,3-aminomutase; and Encodes homolysine decarboxylase.

In other embodiments, the invention provides methods for producing hexamethylene diamine (HMDA) having a naturally occurring A method for producing HMDA by culturing non-existent microbial organisms, the HMDA pathway comprising glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate kinase, 5-oxopimeloylphosphonate reductase, 3-oxo-1-carboxyheptanal 3-aminotransferase, 3-oxo-1-carboxyheptanal 3-aminated oxidoreductase , 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate 7-aminating oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomutase, or homo (See Examples XXIV and XXVI; Steps A/B/H/I/AB/Z/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate kinase; 5-oxopimeloylphosphonate reductase; 3-oxo-1-carboxyheptanal 3-aminotransferase or 3-oxo- 1-Carboxyheptanal 3-aminated oxidoreductase; 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminated oxidoreductase; 3,7-diaminohepta It encodes noate 2,3-aminomutase; and homolysine decarboxylase.

In other embodiments, the invention provides methods for producing hexamethylene diamine (HMDA) having a naturally occurring A method for producing HMDA by culturing non-existent microbial organisms, the HMDA pathway comprising glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate-CoA transferase or 3-oxopimelate-CoA ligase, 5-oxopimeloyl-CoA reductase (aldehyde formation), 3-oxo-1-carboxyheptanal 3-aminotransferase, 3-oxo-1-carboxyheptanal 3-aminated oxidoreductase, 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate 7-aminated oxidoreductase, 3,7-diaminoheptanoate 2,3 - aminomutase, or homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/F/G/AB/Z/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate-CoA transferase or 3-oxopimelate-CoA ligase; 5-oxopimeloyl-CoA reductase (aldehyde formation); 3-oxo-1-carboxyheptanal 3-amino transferase or 3-oxo-1-carboxyheptanal 3-amination oxidoreductase; 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-amination oxidoreductase; Encodes 3,7-diaminoheptanoate 2,3-aminomutase; and homolysine decarboxylase.

In other embodiments, the invention provides methods for producing hexamethylene diamine (HMDA) having a naturally occurring A method for producing HMDA by culturing non-existent microbial organisms, the HMDA pathway comprising glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase, 3-aminopimelate reductase, 3-amino-7-oxoheptanoate 2,3-aminomutase, 2 - amino-7-oxoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoate aminating oxidoreductase, or homolysine decarboxylase (see Examples XXIV and XXVI) step A/B//J/O/P/Q/S in Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate reductase; 3-amino-7-oxohepta It encodes noate 2,3-aminomutase; 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate 7-amination oxidoreductase; and homolysine decarboxylase.

In other embodiments, the invention provides methods for producing hexamethylene diamine (HMDA) having a naturally occurring A method for producing HMDA by culturing non-existent microbial organisms, the HMDA pathway comprising glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase, 3-aminopimelate kinase, 5-aminopimeloylphosphonate reductase, 3-amino-7-oxoheptano 2,3-aminomutase, 2-amino-7-oxoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoate aminating oxidoreductase, or homolysine decarboxylase. (See Examples XXIV and XXVI; Steps A/B/J/M/N/P/Q/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate kinase; 5-aminopimelate phosphonate reductase ; 3-amino-7-oxoheptanoate 2,3-aminomutase; 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and homo Encodes lysine decarboxylase.

In other embodiments, the invention provides methods for producing hexamethylene diamine (HMDA) having a naturally occurring A method for producing HMDA by culturing non-existent microbial organisms, the HMDA pathway comprising glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, 3-aminopimelate CoA transferase, 3-aminopimelate CoA ligase, 5-aminopimeloyl-CoA reductase (aldehyde formation), 3-amino -7-oxoheptanoate 2,3-aminomutase, 2-amino-7-oxoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoate amination oxidoreductase or homolysine decarboxylase , provides a method (see Examples XXIV and XXVI; steps A/B/J/K/L/P/Q/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimelate-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate-CoA transferase or 3-aminopimelate-CoA ligase; 5-aminopimeloyl -CoA reductase (aldehyde formation); 3-amino-7-oxoheptanoate 2,3-aminomutase; 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate It encodes 7-aminated oxidoreductase; and homolysine decarboxylase.

In other embodiments, the invention provides methods for producing hexamethylene diamine (HMDA) having a naturally occurring A method for producing HMDA by culturing non-existent microbial organisms, the HMDA pathway comprising glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, 3-aminopimelate reductase, 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino -7-oxoheptanoate 7-amination oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomutase, or homolysine decarboxylase (see Examples XXIV and XXVI step A/B/J/O/Z/R/S in Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate amination oxidoreductase; 3-aminopimelate reductase; 3-amino-7-oxo It encodes heptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; 3,7-diaminoheptanoate 2,3-aminomutase; and homolysine decarboxylase.

In other embodiments, the invention provides methods for producing hexamethylene diamine (HMDA) having a naturally occurring A method for producing HMDA by culturing non-existent microbial organisms, the HMDA pathway comprising glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, 3-aminopimelate CoA transferase, 3-aminopimelate CoA ligase, 5-aminopimeloyl-CoA reductase (aldehyde formation), 3-amino -7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate aminating oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomutase, or homolysine decarboxylase, (See Examples XXIV and XXVI; Steps A/B/J/K/L/Z/R/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimelate-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate-CoA transferase or 3-aminopimelate-CoA ligase; 5-aminopimelate- CoA-reductase (aldehyde formation); 3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate aminating oxidoreductase; 3,7-diaminoheptanoate 2,3- It encodes aminomutase; and homolysine decarboxylase.

In other embodiments, the invention provides methods for producing hexamethylene diamine (HMDA) having a naturally occurring A method for producing HMDA by culturing non-existent microbial organisms, the HMDA pathway comprising glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, 3-aminopimelate kinase, 5-aminopimeloylphosphonate reductase, 3-amino-7-oxoheptano ate 7-aminotransferase, 3-amino-7-oxoheptanoate aminating oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomutase, or homolysine decarboxylase. See Examples XXIV and XXVI; steps A/B/J/M/N/Z/R/S in Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate kinase; 5-aminopimelate phosphonate reductase ;3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate aminating oxidoreductase;3,7-diaminoheptanoate 2,3-aminomutase; Encodes carboxylase.

In other embodiments, the invention provides methods for producing hexamethylene diamine (HMDA) having a naturally occurring A method for producing HMDA by culturing non-existent microbial organisms, the HMDA pathway comprising glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, 3-aminopimelate 2,3-aminomutase, 2-aminopimelate reductase, 2-amino-7-oxohepta Noate 7-aminotransferase, 2-amino-7-oxoheptanoate aminating oxidoreductase, or homolysine decarboxylase (see Examples XXIV and XXVI; Step A of Figure 21). /B/J/T/W/Q/S). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate 2,3-aminomutase; 2-aminopimelate encodes rate reductase; 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and homolysine decarboxylase.

In other embodiments, the invention provides methods for producing hexamethylene diamine (HMDA) having a naturally occurring A method for producing HMDA by culturing non-existent microbial organisms, the HMDA pathway comprising glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, 3-aminopimelate 2,3-aminomutase, 2-aminopimelate kinase, 6-aminopimelate phosphonate reductase , 2-amino-7-oxoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoate aminating oxidoreductase, or homolysine decarboxylase (Examples XXIV and XXVI). See steps A/B/J/T/U/X/Q/S in Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimeloyl-CoA transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate 2,3-aminomutase; 2-aminopimelate encodes rate kinase; 6-aminopimeloylphosphonate reductase; 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and homolysine decarboxylase .

In other embodiments, the invention provides methods for producing hexamethylene diamine (HMDA) having a naturally occurring A method for producing HMDA by culturing non-existent microbial organisms, the HMDA pathway comprising glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl -CoA ligase, 3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, 3-aminopimelate 2,3-aminomutase, 2-aminopimelate CoA transferase, 2-aminopimelate CoA ligase, 6-aminopimeloyl- CoA reductase (aldehyde formation), 2-amino-7-oxoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoate aminating oxidoreductase, or homolysine decarboxylase ( See Examples XXIV and XXVI; steps A/B/J/T/V/Y/Q/S of Figure 21). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase; 3-oxopimelate-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate 2,3-aminomutase; 2-aminopimelate CoA transferase or 2-aminopimelate-CoA ligase; 6-aminopimeloyl-CoA reductase (aldehyde formation); 2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase; and homo Encodes lysine decarboxylase.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in an amount sufficient to produce hexamethylene diamine (HMDA). A method for producing hexamethylene diamine (HMDA) by culturing the body, the HMDA pathway is a method for producing hexamethylene diamine (HMDA) by culturing -7-aminoheptanoate dehydratase, 2-oxo-7-aminoheptanoate reductase, 2-oxo-7-aminoheptanoate aminotransferase, 2-oxo-7-aminoheptanoate aminotransferase amination oxidoreductase, homolysine decarboxylase, 2-oxo-7-aminoheptanoate decarboxylase, 6-aminohexanal aminotransferase, or 6-aminohexanal aminating oxidoreductase (Examples XXIV and XXVI) See steps A-G in Figure 22). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising 2-oxo-4-hydroxy-7-aminoheptanoate. Aldolase; 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; 2-oxo-7-aminohepta-3-enoate reductase; 2-oxo-7-aminoheptanoate aminotransferase or 2-oxo-7 -encodes aminoheptanoate aminating oxidoreductase; and homolysine decarboxylase. In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising 2-oxo-4-hydroxy-7-aminoheptanoate. aldolase; 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; 2-oxo-7-aminohepta-3-enoate reductase; 2-oxo-7-aminoheptanoate decarboxylase; and 6-aminohexanal Encodes an aminotransferase or 6-aminohexanalaminating oxidoreductase.

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in sufficient amounts to produce hexamethylene diamine (HMDA). A method for producing hexamethylene diamine (HMDA) by culturing the body, the HMDA pathway comprises 6-aminocaproate reductase, 6-aminocaproic acid semialdehyde aminotransferase, 6-aminocaproic acid semialdehyde Oxidoreductase (amination), 6-aminocaproate N-acetyltransferase, 6-acetamidohexanoate reductase, 6-acetamidohexanal aminotransferase, 6-acetamidohexanal oxidoreductase (amination), 6-acetamidohexanamine N -acetyltransferase, or acetamidohexanamine hydrolase (amide) (see Example XXVII; steps O/C or D/P/G/H of Figure 24). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: 6-aminocaproate reductase; and 6-aminocaproate semi- Encodes aldehyde aminotransferase or 6-aminocaproic acid semialdehyde oxidoreductase (amination).

In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: 6-aminocaproate N-acetyltransferase; 6-acetamide Encodes hexanoate reductase; 6-acetamidohexanal aminotransferase or 6-acetamidohexanal oxidoreductase (amination); and 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide).

The present invention further provides for a non-naturally occurring microbial organism having an HMDA pathway comprising at least one exogenous nucleic acid encoding an HMDA pathway enzyme expressed in an amount sufficient to produce hexamethylene diamine (HMDA). A method for producing hexamethylene diamine (HMDA) by culturing a human body, the HMDA pathway is a 2-amino-7-oxosabarate keto acid decarboxylase, 2-amino-7-oxoheptano ate decarboxylase, 6-aminohexanal aminating oxidoreductase, 6-aminohexal aminotransferase, 2-amino-7-oxoheptanoate aminotransferase, 2-amino-7-oxoheptanoate aminating oxidoreductase, 2- Oxo-7-aminoheptanoate decarboxylase, homolysine decarboxylase, 2-amino-7-oxosabarate amino acid decarboxylase, 2-oxo-7-aminoheptanoate amination oxidoreductase, 2-oxo-7- Provided are methods comprising aminoheptanoate aminotransferase, 2-amino-7-oxosubarate aminating oxidoreductase, 2-amino-7-oxosubarate aminotransferase, or 2,7-diaminosubarate decarboxylase. (See Examples XXIV and XXVI; Steps A/B/C/G/H/I/J/K/L/M of Figure 26). In further embodiments, the microbial organism contains at least one exogenous enzyme encoding a 2-amino-7-oxosubarate pathway enzyme expressed in sufficient amount to produce 2-amino-7-oxosabarate. It has a 2-amino-7-oxosabarate pathway with nucleic acids, and the 2-amino-7-oxosabarate pathway is 2-amino-5-hydroxy-7-oxosabarate aldolase, 2-amino-5- hydroxy-7-oxosubarate dehydratase, or 2-amino-5-ene-7-oxosabarate reductase (see Examples XXV and XXVI; Steps A/B/C of Figure 27).

In another embodiment, the present invention provides hexamethylene diamine (HMDA) pathway enzymes by culturing a non-naturally occurring microbial organism with an HMDA pathway that includes a set of exogenous nucleic acids encoding hexamethylene diamine (HMDA) pathway enzymes. 2-amino-7-oxosabarate aminating oxidoreductase or 2-amino-7-oxosabarate aminotransferase; 2,7-diaminosabarate decarboxylase. ; and homolysine decarboxylase (see Examples XXIV and XXVI; steps K/L/H of Figure 26). In another embodiment of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: 2-amino-7-oxosubarate amino acid decarboxylase; 2-oxo-7-aminoheptanoate aminating oxidoreductase or 2-oxo-7-aminoheptanoate aminotransferase; and homolysine decarboxylase (see Examples XXIV and XXVI; FIG. 26) Step I/J/H). In another embodiment of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising: 2-amino-7-oxosubarate amino acid decarboxylase; 2-oxo-7-aminoheptanoate decarboxylase; and 6-aminohexanalaminating oxidoreductase or 6-aminohexanal aminotransferase (see Examples XXIV and XXVI; Steps I/G in Figure 26). /C). In another embodiment of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising 2-amino-7-oxosabarate ketoacid decarboxylase. ; 2-amino-7-oxoheptanoate decarboxylase; and 6-aminohexanalaminating oxidoreductase or 6-aminohexanalaminotransferase (see Examples XXIV and XXVI; Step A/ of Figure 26). B/C). In another embodiment of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set comprising 2-amino-7-oxosabarate ketoacid decarboxylase. ; 2-amino-7-oxoheptanoate aminating oxidoreductase or 2-amino-7-oxoheptanoate aminotransferase; and homolysine decarboxylase (see Examples XXIV and XXVI; FIG. 26 steps A/M/H). In a further aspect of each of the above embodiments, the microbial organism encodes a 2-amino-7-oxosabalate pathway enzyme expressed in an amount sufficient to produce 2-amino-7-oxosabalate. the 2-amino-7-oxosabarate pathway has a second set of exogenous nucleic acids that 2-amino-5-hydroxy-7-oxosabarate dehydratase; and 2-amino-5-en-7-oxosabarate reductase (see Examples XXV and XXVI; steps of FIG. 27). A/B/C).

The invention further provides for a non-naturally occurring microbial organism having an LA pathway comprising at least one exogenous nucleic acid encoding an LA pathway enzyme expressed in sufficient amounts to produce levulinic acid (LA). A method for producing hexamethylene diamine (HMDA) by culturing , 3-oxoadipyl-CoA hydrolase, or 3-oxoadipate decarboxylase (see Example XXIX; steps A/E/F/G/AA of Figure 25). In another aspect of the invention, the non-naturally occurring microbial organism comprises a set of exogenous nucleic acids encoding LA pathway enzymes, the set comprising: 3-oxoadipyl-CoA thiolase; 3-oxoadipyl-CoA/acyl -CoA transferase, 3-oxoadipyl-CoA synthase, or 3-oxoadipyl-CoA hydrolase; and 3-oxoadipate decarboxylase.

The present invention relates to non-natural microbial organisms in which the production of adipate, 6-ACA, and/or HMDA is increased by disruption of one or more genes to increase the production of adipate, 6-ACA, and/or HMDA. Further provided are methods for producing. Such gene disruptions include those exemplified in Example XXX and Tables 14-16 herein.

The present invention involves culturing a non-naturally occurring microbial organism containing one or more gene disruptions that increase the production of adipate, 6-ACA, and/or HMDA. Further provided are methods for producing HMDA. Disruption occurs when genetic disruption reduces the activity of the enzyme such that it confers stable, growth-associated production of adipate, 6-ACA, and/or HMDA in the non-natural microbial organism. -Can occur in genes encoding essential enzymes that link ACA and/or HMDA production to microbial growth.

In some embodiments, gene disruption can include complete gene deletion. Methods for gene disruption are well known to those skilled in the art and are described herein (see Example XXX). In some embodiments, other methods for disrupting a gene include, for example, by deletion of oligonucleotides, frameshifting by addition, or mutations that render the gene inoperable. Those skilled in the art will recognize the advantages of gene deletion; however, due to stability, it may revert a non-naturally occurring organism to a phenotype expressing a previously disrupted gene. In particular, the gene disruption is selected from the gene sets listed in Tables 14-16.

Suitable purifications and/or assays to test for the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid can be performed using well-known methods. Suitable replicates, such as triplicate cultures, can be grown for each genetically engineered strain to be tested. For example, product and byproduct formation in genetically engineered production hosts can be monitored.

Final products and intermediates as well as other organic compounds can be analyzed using HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass Spectroscopy), and LC-MS (Liquid Chromatography-Mass Spectroscopy or other suitable analytical methods using routine procedures well known in the art. Product release in fermentation broth can also be tested with culture supernatants. By-products and residual glucose can be detected using e.g. refractive index detectors for glucose and alcohols and UV detectors for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779 (2005)) or other suitable assays and detection methods well known in the art. Individual enzyme activity from exogenous DNA sequences can also be assayed using methods well known in the art.

6-Aminocaproic acid, caprolactam, hexamethylene diamine, or levulinic acid can be separated from other components in the culture using a variety of methods well known in the art. Such separation methods include, for example, extraction procedures and continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography. chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration. All of the above methods are well known in the art.

Any of the non-naturally occurring microbial organisms described herein can be cultured to produce and/or secrete the biosynthetic products of the invention. For example, a 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid producing strain can be cultured for biosynthetic production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid.

For the production of 6-aminocaproic acid, caprolactam, hexamethylene diamine, or levulinic acid, the recombinant strain is cultured in a medium with a carbon source and other essential nutrients. It is sometimes desirable, and can be highly desirable, to maintain anaerobic conditions in the fermenter to reduce the cost of the overall process. Such conditions can be obtained, for example, by first sparging the medium with nitrogen and then sealing the flask using a septum and crimp cap.

For strains whose growth is not observed anaerobically, microaerobic or substantially anaerobic conditions can be applied by punching small holes in the septum, limiting ventilation. Exemplary anaerobic conditions have been previously described and are well known in the art. Exemplary aerobic and anaerobic conditions are described, for example, in US Patent Application Publication No. 2009/0047719 (No. 11/891,602), filed August 10, 2007. Fermentation can be performed in a batch, fed-batch, or continuous manner as disclosed herein.

If desired, the pH of the medium is adjusted to the desired pH, particularly by the addition of a base or acid, such as NaOH or other bases, to a pH of about 7, as necessary to maintain the culture medium at the desired pH. As such, it can be maintained at a neutral pH. Growth rate can be determined by measuring optical density using a spectrophotometer (600 nm) and glucose uptake rate can be determined by monitoring carbon source depletion over a period of time.

The growth medium can also include any carbohydrate source that can, for example, provide a non-naturally occurring carbon source to the microorganism. Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, starch, and the like. Other sources of carbohydrates include, for example, renewable feedstocks and biomass. Exemplary types of biomass that can be used as a feedstock in the methods of the invention include cellulosic biomass, hemicellulose biomass, and lignin feedstock or portions of feedstocks. Such biomass feedstocks contain carbohydrate substrates useful as carbon sources, such as, for example, glucose, xylose, arabinose, galactose, mannose, fructose, starch, and the like. In view of the teachings and guidance provided herein, those skilled in the art will appreciate that renewable feedstocks and biomass other than those exemplified above are 6-aminocaproic acid, caprolactam, hexamethylene diamine, or levulin. It will be appreciated that the microbial organisms of the invention can be used to culture for the production of acids.

In addition to renewable feedstocks such as those exemplified above, the 6-aminocaproic acid, caprolactam, hexamethylene diamine or levulinic acid microbial organisms of the present invention are modified for growth with syngas as its carbon source. You can also. In this particular embodiment, one or more proteins or enzymes are expressed in a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid producing organism to facilitate metabolism to utilize syngas or other gaseous carbon sources. Provide a route.

Synthesis gas, also known as syngas or producer gas, is the primary product of the gasification of carbonaceous materials such as coal and biomass materials, including agricultural crops and residues. Syngas is mainly H₂and CO and can be obtained from the gasification of any organic feedstock, including but not limited to coal, oil, natural gas, biomass, and waste organic matter. Gasification is generally carried out under high fuel-to-oxygen ratios.

Mainly H₂and CO, but syngas has a smaller amount of CO₂and other gases. Synthesis gas therefore contains CO and even CO₂Provides cost-effective gaseous carbon such as

The Wood-Ljungdahl pathway includes CO and H₂catalyzes the conversion of acetyl-CoA and other products such as acetate. Organisms capable of utilizing CO and syngas generally produce CO2 through the same basic set of enzymes and transformations encompassed by the Wood-Ljungdahl pathway.₂and CO₂/H₂It also has the ability to utilize mixtures. CO caused by microorganisms₂H to acetate₂Dependent transformations were recognized long before it became clear that CO could also be used by the same organism and required the same pathway. Many acetogens are CO₂has been shown to grow in the presence of acetate and produce compounds such as acetate as long as hydrogen is present to supply the necessary reducing equivalents (e.g., Drake, Acetogenesis, pp. 3-60 (See Chapman and Hall, New York, (1994)). This can be summarized by the following equation.

2CO₂ +4H₂ + nADP + nPiCH₃COOH+2H₂O + nATP Therefore, non-naturally occurring microorganisms with the Wood-Ljungdahl pathway use CO2 to produce acetyl-CoA and other desired products.₂and H₂Mixtures of can also be used.

The Wood-Ljungdahl pathway is well known in the art and consists of 12 reactions that can be divided into two branches: (1) methyl branch and (2) carbonyl branch. The methyl branch converts syngas to methyl-tetrahydrofolate (methyl-THF), whereas the carbonyl branch converts methyl-THF to acetyl-CoA. The reaction at the methyl branch is catalyzed in sequence by the following enzymes: ferredoxin oxidoreductase, formate dehydrogenase, formyltetrahydrofolate synthetase, methenyltetrahydrofolate cyclodehydratase, methylenetetrahydrofolate dehydrogenase, and methylenetetrahydrofolate reductase. The reaction at the carbonyl branch is catalyzed in order by the following enzymes or proteins: cobalamide corrinoid/iron-sulfur protein, methyltransferase, carbon monoxide dehydrogenase, acetyl-CoA synthase, acetyl-CoA synthase disulfide reductase and hydrogenase. Enzymes include methyltetrahydrofolate:corrinoid protein methyltransferase (e.g. AcsE), corrinoid iron-sulfur protein, nickel-protein assembly protein (e.g. AcsF), ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase and nickel-protein assembly protein. It can also be referred to as a protein (eg CooC). With the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to produce a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway, one skilled in the art can at least: It will be appreciated that the same technical design can be carried out for introducing nucleic acids encoding Wood-Ljungdahl enzymes or proteins that are not present in the host organism. Therefore, introducing one or more encoding nucleic acids into a microbial organism of the invention, such that the modified organism contains the complete Wood-Ljungdahl pathway, will confer the ability to utilize syngas.

Additionally, the reducing (reverse) tricarboxylic acid cycle linked to carbon monoxide dehydrogenase and/or hydrogenase activity is₂and/or H₂It can also be used for the conversion of acetyl-CoA and other products such as acetate. Organisms capable of fixing carbon via the reductive TCA pathway include the following enzymes: ATP citrate lyase, citrate lyase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate:ferredoxin oxidoreductase, One or more of succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase, fumarase, malate dehydrogenase, NAD(P)H:ferredoxin oxidoreductase, carbon monoxide dehydrogenase, and hydrogenase can be utilized. Specifically, CO and/or H are removed by carbon monoxide dehydrogenase and hydrogenase.₂CO via the reductive TCA cycle using reducing equivalents extracted from₂is fixed in acetyl-CoA or acetate. Acetate can be converted to acetyl-CoA by enzymes such as acetyl-CoA transferase, acetate kinase/phosphotransacetylase, and acetyl-CoA synthetase. Acetyl-CoA is converted to p-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate precursors, glyceraldehyde-3-phosphate, phosphoenol by pyruvate:ferredoxin oxidoreductase and gluconeogenic enzymes. It can be converted to pyruvate and pyruvate. The teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to produce the p-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway. Accordingly, one skilled in the art will understand that the same technical design can be practiced, at least with respect to introducing a nucleic acid encoding a reducing TCA pathway enzyme or protein that is not present in the host organism. Therefore, introducing one or more encoding nucleic acids into a microbial organism of the invention, such that the modified organism contains a complete reductive TCA pathway, will confer the ability to utilize syngas.

From the teachings and guidance provided herein, one skilled in the art will appreciate that non-naturally occurring microbial organisms can be produced that secrete the biosynthetic compounds of the present invention when grown on carbon sources such as carbohydrates. Probably. Such compounds include, for example, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, and any of the intermediate metabolites in the 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway. All that is required is to manipulate one or more of the required enzymatic activities to produce a desired product containing, for example, some or all of the 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathways. It is only necessary to accomplish the biosynthesis of the compound or intermediate. Therefore, the present invention produces and/or secretes 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid when grown on carbohydrates; Non-naturally occurring microbial organisms that produce and/or secrete any of the intermediate metabolites of the levulinic acid pathway are provided. For example, an adipate-producing microbial organism can optionally initiate synthesis from intermediates such as 3-oxoadipyl-CoA, 3-hydroxyadipyl-CoA, 5-carboxy-2-pentenoyl-CoA or adipyl-CoA. (See Figure 2). In addition, adipate-producing microbial organisms can initiate synthesis from intermediates such as 3-oxoadipyl-CoA, 3-oxoadipate, 3-hydroxyadipate or hex-2-enedioate (see Figure 3). sea bream). The 6-aminocaproic acid producing microbial organisms of the invention can initiate synthesis from intermediates such as adipate semialdehyde (see Figure 8). The caprolactam-producing microbial organisms of the invention can optionally initiate synthesis from intermediates such as adipate semialdehyde or 6-aminocaproic acid (see Figure 8).

The non-naturally occurring microbial organisms of the invention contain 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid pathway enzymes in amounts sufficient to produce 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. Constructed using methods well known in the art, as exemplified herein, to exogenously express at least one encoding nucleic acid. It is understood that the microbial organisms of the invention are cultured under conditions sufficient to produce 6-aminocaproic acid, caprolactam, hexamethylene diamine or levulinic acid. With the teachings and guidance provided herein, the non-naturally occurring microbial organisms of the present invention can produce 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid that produces intracellular concentrations of about 0.1 to 200 mM or more. biosynthesis can be achieved. Generally, the intracellular concentration of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid is about 3-150mM, especially about 5-125mM, especially about 8-100mM, and more than about 10mM, 20mM, 50mM or 80mM. include. Intracellular concentrations between and above each of these exemplary ranges can also be achieved from the non-naturally occurring microbial organisms of the present invention.

In some embodiments, the culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions. Exemplary anaerobic conditions have been previously described and are well known in the art. Exemplary anaerobic conditions for fermentation processing are described herein, eg, in US Patent Publication No. 2009/0047719, filed Aug. 10, 2007. Any of these conditions, as well as other anaerobic conditions well known in the art, can be employed for non-naturally occurring microbial organisms. Under such anaerobic conditions, 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid producers are present at intracellular concentrations of 5-10 mM or higher, as well as all other concentrations exemplified herein. 6-Aminocaproic acid, caprolactam, hexamethylene diamine or levulinic acid can be synthesized. Although the above description refers to intracellular concentrations, microbial organisms producing 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid may contain 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid intracellularly. and/or secrete the product into the culture medium.

Culture conditions can include, for example, liquid culture methods, as well as fermentation and other large-scale culture methods. As described herein, particularly useful yields of biosynthetic products of the invention can be obtained under anaerobic or substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achieving biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylene diamine or levulinic acid includes anaerobic culture or fermentation conditions. In certain embodiments, the non-naturally occurring microbial organisms of the invention can be maintained, cultured, or fermented under anaerobic or substantially anaerobic conditions. Briefly, anaerobic conditions refer to an environment devoid of oxygen. Substantially anaerobic conditions include, for example, culturing, batch fermentation or continuous fermentation where the concentration of dissolved oxygen in the medium is maintained between 0% and 10% of saturation. Substantially anaerobic conditions also include growing or quiescent cells in liquid media or solid agar inside a closed chamber maintained in an atmosphere of less than 1% oxygen. Increase the proportion of oxygen, e.g., N to the culture₂/CO₂The mixture can be maintained by sparging with one or more other suitable non-oxygen gases.

The culture conditions described herein can be scaled up and grown continuously for the production of 6-aminocaproic acid, caprolactam, hexamethylene diamine or levulinic acid. Exemplary growth methods include, for example, fed batch fermentation and batch separation, batch fed fermentation and continuous separation, or continuous fermentation and continuous separation. All of these methods are well known in the art. Fermentation methods are particularly useful for the biosynthetic production of commercial quantities of 6-aminocaproic acid, caprolactam, hexamethylene diamine or levulinic acid. In general, and as with discontinuous culture methods, continuous and/or near continuous production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid maintains and/or nearly maintains growth in log phase. culturing the non-naturally occurring 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid producing organisms of the present invention in nutrients and media sufficient to . Continuous culturing under such conditions can include, for example, 1, 2, 3, 4, 5, 6, or 7 or more days. Continuous culture can also include periods of 1 week, 2, 3, 4 or 5 weeks or more, up to several months. Alternatively, the organisms of the invention can be cultured for several hours if appropriate for a particular application. It should be understood that continuous and/or near continuous culture conditions can also include all time intervals between these exemplary time periods. Furthermore, it is understood that the time for culturing the microbial organisms of the present invention corresponds to a period of time sufficient to produce a sufficient amount of product for the desired purpose.

Fermentation methods are well known in the art. Briefly, fermentation for the biosynthetic production of 6-aminocaproic acid, caprolactam, hexamethylene diamine or levulinic acid can be carried out by, for example, fed batch fermentation and batch separation, fed batch fermentation and continuous separation, or continuous fermentation and continuous separation. It can be used for. Examples of batch and continuous fermentation methods are well known in the art.

In addition to the above fermentation method using the 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid producers of the invention for the continuous production of substantial amounts of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. The 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid producers are then subjected to simultaneous chemical synthesis treatments, for example, to convert the products into other compounds, or, if desired, the products are transferred to fermentation cultures. The product can be separated into other compounds by subsequent chemical transformations. As described herein, an intermediate in the adipate pathway that utilizes 3-oxoadipate, i.e., hexa-2-enedioate, can be converted to adipate by, for example, platinum-catalyzed chemical hydrogenation. (See Example III).

As described herein, exemplary growth conditions for achieving biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid include adding an osmoprotectant to the culture conditions. include. In certain embodiments, the non-naturally occurring microbial organisms of the invention can be maintained, cultured or fermented as described above in the presence of an osmoprotectant. Briefly, an osmoprotectant refers to a compound that acts as an osmolyte and helps the microbial organisms described herein to withstand osmotic stress. Osmoprotectants include, but are not limited to, betaines, amino acids, and the sugar trehalose.

Non-limiting examples thereof are glycine betaine, praline betaine, dimethyltetin, dimethylsulfonioproprionate, 3-dimethylsulfonio-2-methylproprionate, pipecolic acid, dimethylsulfonioacetate, choline, L- Carnitine and ectoine. In one embodiment, the osmoprotectant is glycine betaine. It will be appreciated by those skilled in the art that the amount and type of osmoprotectant suitable for protecting the microbial organisms described herein from osmotic stress will depend on the microbial organism used. For example, as described in Example XXII, E. coli in the presence of different amounts of 6-aminocaproic acid grows well in the presence of 2mM glycine betaine. The amount of the osmoprotectant in the culture conditions is, for example, about 0.1 mM or less, about 0.5 mM or less, about 1.0 mM or less, about 1.5 mM or less, about 2.0 mM or less, about 2.5 mM or less, about 3.0 mM or less, about 5.0 It can be less than or equal to about 7.0 mM, less than or equal to about 10 mM, less than or equal to about 50 mM, less than or equal to about 100 mM, or less than or equal to about 500 mM.

Metabolite modeling can be used to optimize growth conditions to generate better producers. Modeling can be used to design gene knockouts that further optimize pathway utilization (e.g., U.S. Patent Publications Nos. 2002/0012939; 2003/0224363; 2004/0072723, 2003/0059792, 2002/0168654 and 2004/0009466 and US Pat. No. 7,127,379). Modeling analyzes allow reliable prediction of the impact on cell growth of shifting metabolism towards more efficient production of 6-aminocaproic acid, caprolactam, hexamethylene diamine or levulinic acid.

One computational method for identifying and designing metabolic changes that favor the biosynthesis of desired products is the OptKnock computational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657(2003)). OptKnock is a metabolic modeling and simulation program that suggests gene deletion strategies that result in genetically stable microorganisms that overproduce target products. Specifically, the framework interrogates the complete metabolic and/or biochemical networks of microorganisms to suggest genetic manipulations that make desired biochemicals essential byproducts of cell growth. By coupling biochemical production with cell growth through strategically placed gene deletions or other functional gene disruptions, growth selection pressures exerted on engineered strains after extended periods of time in bioreactors Increased performance results from forced growth-linked biochemical production. Finally, once the gene deletion is constructed, the genes selected by OptKnock will be completely removed from the genome, so there is a negligible chance that the engineered strains will revert to their wild-type state. . Therefore, this computational approach can be used to identify alternative routes leading to the biosynthesis of desired products or to use non-naturally occurring microbial organisms for further optimization of the biosynthesis of desired products. Can be used in conjunction with

The concept of growth-coupled biochemical production can be visualized in the context of the range of biochemical production of typical metabolic networks calculated using in silico models. These ranges are determined by fixing the limiting substrate uptake rates to their experimental measurements and calculating the maximum and minimum biochemical production rates at each achievable growth level. Although exceptions exist, typically the production of desired biochemicals competes directly with biomass formation for intracellular resources. Therefore, increased biochemical production rates necessarily result in submaximal growth rates. Knockouts suggested by OptKnock are designed to limit the acceptable solution boundaries that alter metabolic behavior from the wild-type strain. The actual solution bounds for a given strain will expand or contract as the substrate uptake rate increases or decreases, but each experimental point should fall within the calculated solution bounds. Plots such as these make it possible to visualize how close strains are to their performance limits, or in other words, how much room there is for improvement. The OptKnock framework is already capable of identifying promising gene deletion strategies for biochemical overproduction (Burgard et al., Biotechnol Bioeng, 84(6):647-657 (2003); Pharkya et al., Biotechnol Bioeng , 84(7):887-899 (2003)), establish a systematic framework that necessarily encompasses future improvements in metabolic and regulatory modeling frameworks.

Briefly, OptKnock is a term used herein to refer to computational methods and systems for modeling cellular metabolism. The OptKnock program relates to a framework of models and methods that incorporate specific conditions into flow balance analysis (FBA) models. These conditions include, for example, qualitative kinetic information, qualitative regulatory information, and/or DNA microarray experimental data. OptKnock also solves various metabolic problems by, for example, fixing the flow boundaries derived via flow balance models and subsequently exploring the performance limits of metabolic networks in the presence of gene additions or deletions. calculate. The OptKnock computational framework enables the construction of model formulations that enable effective interrogation of the performance limits of metabolic networks, and provides methods for solving mixed-integer linear programming problems that arise. The metabolic modeling and simulation method, referred to herein as OptKnock, is described, for example, in U.S. Patent Publication No. 2002/0168654, filed on January 10, 2002, and International Patent No. PCT, filed on January 10, 2002. /US02/00660 and US Patent Application No. 2009/0047719, filed August 10, 2007.

Another computational method for identifying and designing metabolic changes that favor biosynthetic production of products is a metabolic modeling and simulation system named SimPheny®. This calculation method and system is described, for example, in U.S. Patent Publication No. 2003/0233218, filed on June 14, 2002, and International Patent Application No. PCT/US03/18838, filed on June 13, 2003. Are listed. SimPheny® generates network models in silico to simulate the flow of mass, energy, or charge due to chemical reactions in biological systems, defining a solution space that includes all possible functions of chemical reactions in the system. This is a computational system that can be used to determine the range of activity that is permissible for biological systems. This approach is called condition-based modeling because the solution space is determined by conditions such as the known stoichiometry of the reactions involved, as well as reaction thermodynamics and volumetric conditions associated with maximum flux through the reaction. . The space defined by these conditions can be interrogated to measure the phenotypic function and behavior of biological systems or biochemical components. For example, Schilling et al., J. Theor. Biol. 203:229-248 (2000); convex as described in Schilling et al., Biotech. Bioeng. 71:286-306 (2000) and Schilling et al., Biotech. Analytical methods such as parsing, linear programming and polar path calculations can be used to measure such phenotypic functions.

As described above, one condition-based method used in calculation programs applicable to the present invention is flow balance analysis. Flow balance analysis is based on flow balancing at steady state conditions and can be performed, for example, as described in Varma and Palsson, Biotech. Bioeng. 12:994-998 (1994). Applying the flow balance approach to reaction networks, e.g. adipocyte metabolism as described in Fell and Small, J. Biochem. 138:781-786 (1986), Majewski and Domach, Biotech. Bioeng. 35:732- 738 (1990), or systemic characteristics of ethanol secretion by yeast, as described in Vanrolleghem et al., Biotech. Prog. 12:434-448 (1996). simulated or predicted. Also using this approach, Edwards and Palsson, Proc. Natl. Acad. Sci. 97:5528-5533 (2000), Edwards and Palsson, J.Bio. Chem. 274:17410-17416 (1999) and Edwards et al., Nature Biotech. 19:125-130 (2001). The growth of Saccharomyces cerevisiae on various single carbon sources as well as the metabolic growth of H. influenzae can be predicted or simulated.

Once the solution space is established, it can be analyzed to find possible solutions under various conditions. This computational approach is consistent with biological reality, as biological systems are flexible and the same result can be reached in many different ways. Biological systems are designed through evolutionary mechanisms constrained by the fundamental conditions that all living systems must face. Condition-based modeling strategies therefore encompass these general realities. Furthermore, by fixing conditions, further constraints can be sequentially imposed on the network model, thereby reducing the size of the solution space and thereby increasing the accuracy with which physiological performance or phenotype can be predicted.

These computational approaches are consistent with biological reality because biological systems are flexible and the same result can be reached in many different ways. Biological systems are designed through evolutionary mechanisms constrained by the fundamental conditions that all living systems must face. Condition-based modeling strategies therefore encompass these general realities. Furthermore, by fixing conditions, further constraints can be sequentially imposed on the network model, thereby reducing the size of the solution space and thereby increasing the accuracy with which physiological performance or phenotype can be predicted.

With the teachings and guidance provided herein, those skilled in the art will be able to apply various computational frameworks for metabolic modeling and simulation to design and implement the biosynthesis of desired compounds in host microbial organisms. becomes possible. Such metabolic modeling and simulation methods include, for example, the computational systems exemplified above as SimPheny® and OptKnock. To illustrate the invention, several methods are described herein with respect to the OptKnock computational framework for modeling and simulation. Those skilled in the art will know how to apply the identification, design and implementation of metabolic changes using OptKnock to any of such other metabolic modeling and simulation computational frameworks and methods that are well known in the art. You will understand.

The ability of a cell or organism to forcefully couple growth to the production of biochemical products can be illustrated in the context of the biochemical production range of typical metabolic networks calculated using in silico models. These ranges are determined by fixing the limiting substrate uptake rates to their experimentally measured values and calculating the maximum and minimum biochemical production rates at each achievable growth level. Production of desired biochemicals directly competes with biomass formation for intracellular resources. Under these circumstances, increased biochemical production rates will inevitably result in submaximal growth rates. Knockouts suggested by the metabolic modeling and simulation programs described above, such as OptKnock, are designed to restrict the acceptable solution boundaries that alter metabolic behavior from the wild-type strain. Although the actual solution boundary for a given strain will expand or contract as the substrate uptake rate increases or decreases, each experimental point lies within the calculated solution boundary. Plots like these allow us to accurately predict how close engineered strains are to their performance limits, which also gives an indication of how much room there is for improvement. Make it.

The OptKnock mathematical framework is illustrated herein to define genetic deletions that lead to growth-associated biochemical production (see Example XXX). The method is based on condition-based metabolic modeling that narrows the range of possible phenotypes that a cell system can display by sequentially imposing prevailing physicochemical conditions (Price et al., Nat Rev Microbiol , 2:886-97 (2004)).

As described above, condition-based models and simulations are well known in the art and generally facilitate optimization of specific cellular goals governed by network stoichiometry to estimate suggests a fluid distribution of

Briefly, it is quantified as the collective reaction flux for a steady-state metabolic network containing a set of metabolites N={1,...,N} and a set of metabolic reactions M={1,...,M}. The maximization of the cell goal is expressed mathematically as follows.

<math num="1"><img file="JP7370366B2_D0001.tif" /></math>[In the formula, S_ijis the stoichiometric coefficient of metabolite i in reaction j, and ν_jis the flow of reaction j and ν_{substrate _uptake}represents the predicted or observed uptake rate of the limiting substrate, and ν_{atp main}is the non-growth related ATP maintenance requirement. ]. Vector ν includes both internal and external flow. In this investigation, the cellular target is often assumed to be the drain of biosynthetic precursors in the ratio required for biomass formation (Neidhardt, FC et al., 2nd edition, 1996, Washington, DC: ASM Press.2 v. (xx, 2822, lxxvi)). Flow is generally reported per gDW·hr (dry weight (g) x time), such that biomass formation is expressed in gDW·hr or the amount (g) of biomass produced per 1/hr. be done.

Modeling of gene deletions and reaction exclusion initially employs the insertion of binary variables into a condition-based approach framework (Burgard et al., Biotechnol Bioeng, 74:364-375 (2001), Burgard et al., Biotechnol Prog, 17:791-797(2001)). These binary variables,<math num="2"><img file="JP7370366B2_D0002.tif" /></math>takes the value 1 if reaction j is active, and 0 if it is not active. The following conditions,<math num="3"><img file="JP7370366B2_D0003.tif" /></math>is the variable y_jThe reaction flux ν only if is equal to 0_jis set to 0. Alternatively, y_jis equal to 1, then ν_jis the lower limit<math num="4"><img file="JP7370366B2_D0004.tif" /></math>and upper limit<math num="5"><img file="JP7370366B2_D0005.tif" /></math>Freely take any value between . here,<math num="6"><img file="JP7370366B2_D0006.tif" /></math>are specified by minimizing and maximizing, respectively, all reaction fluxes subject to the above network conditions (Mahadevan et al., Metab Eng, 5:264-76 (2003)).

Optimal gene/reaction knockout is achieved by combining the set of active reactions (y_j=1) by solving a bilevel optimization problem. Schematically, this bilevel optimization problem is shown in Figure 2. Mathematically, this bilevel optimization problem can be expressed as the following bilevel mixed integer optimization problem.

<math num="7"><img file="JP7370366B2_D0007.tif" /></math>[In the formula, ν_chemicalis the production of the desired target product, e.g., adipate, 6-ACA and/or HMDA, or other biochemical product, and K is the number of tolerable knockouts. ]. Setting K equal to 0 produces the maximum biomass solution for the complete network, and setting K equal to 1 ensures that the resulting network has the maximum overproduction of a single gene when considering its maximum biomass yield. /reaction knockout(y_j=0) is specified. The final conditions ensure that the resulting network meets a minimum biomass yield. A more detailed explanation of model formulations and solutions is provided in Burgard et al., Biotechnol Bioeng, 84:647-57 (2003). CPLEX 8.0 accessed via GAMS, Brooke et al., GAMS Development Corporation (1998), modeling the environment on an IBM RS6000-270 workstation for problems involving hundreds of binary variables, GAMS: The Solver Manuals.2003 :GAMS Development Corporation can be used to solve in order of minutes and hours. The OptKnock framework is already capable of identifying promising gene deletion strategies for biochemical overproduction (Burgard et al., Biotechnol Bioeng, 84:647-57 (2003), Pharkya et al., Biotechnol Bioeng, 84:887-899 (2003)), establishing a systematic framework that necessarily encompasses future improvements in metabolic and regulatory modeling frameworks.

The above method results in the destruction of one set of metabolic reactions. Elimination or metabolic modification of each reaction within the set can result in the desired product as a necessary product throughout the growth phase of the organism. Since the reactions are known, the solution to the bilevel OptKnock problem will also give one or more relevant genes encoding one or more enzymes that catalyze each reaction in the set of reactions. Identification of a set of reactions and their corresponding genes encoding the enzymes involved in each reaction is generally accomplished through association of the reactions with a reaction database that has relationships between enzymes and encoding genes. It is an automated process.

Once identified, the set of reactions to be disrupted to achieve production of the desired product is carried out in the target cell or organism by functional disruption of at least one gene encoding each metabolic reaction in the set. be done. One particularly useful means of achieving functional disruption of a reaction set is through deletion of each encoding gene. However, in some cases, other genetic abnormalities, including for example displacements, deletions of regulatory regions such as promoters or cis-binding sites for regulatory elements, or truncation of the coding sequence at any of a number of locations disrupt the response. It may be beneficial to let the These latter abnormalities, resulting in less than complete deletion of a gene set, may be useful, for example, when rapid assessment of product linkage is desired or when gene conversion is unlikely to occur.

Integer cuts and A called optimization method can be implemented. The method works by iteratively solving the OptKnock problem exemplified above by introducing an additional condition called an integer cut at each iteration. The integer cut condition effectively prevents the solution method from selecting the exact same set of reactions identified in any previous iteration that forces product biosynthesis to be coupled to growth. For example, if a previously identified growth-linked metabolic modification specifies reactions 1, 2, and 3 for destruction, the following condition prevents the same reactions from being considered simultaneously in the next solution. Integer cut methods are well known in the art and have been described, for example, in Burgard et al., Biotechnol. Prog. 17:791-797 (2001). As with all the methods described herein for the OptKnock computational framework and their combination for metabolic modeling and simulation, integer cut methods to reduce redundancy in iterative computational analyzes are also available, such as in SimPheny ( It can be applied with other computational frameworks well known in the art, including (trademark).

The methods illustrated herein include forcing the production of a target biochemical product to the growth of a cell or organism engineered to retain the identified genetic alteration. It allows for the construction of cells and organisms that biosynthetically produce the desired product. The computational methods described herein therefore enable the identification and implementation of metabolic modifications identified by in silico methods selected from OptKnock or SimPheny®. A set of metabolic modifications can include, for example, addition of one or more biosynthetic pathway enzymes and/or functional disruption of one or more metabolic reactions, including disruption by, for example, gene deletion.

As described above, the OptKnock approach demonstrates that subjecting mutant microbial networks to long-term growth selection can cause them to evolve toward their computationally predicted maximal growth phenotype. It was developed as a premise. In other words, the approach exploits the ability of organisms to self-optimize under selective pressure. The OptKnock framework enables exhaustive enumeration of gene deletion combinations that couple biochemical production and cell growth based on network stoichiometry. Identifying optimal gene/reaction knockouts requires solving a bilevel optimization problem in which the set of active reactions is selected such that the optimal growth solution to the resulting network overproduces the biochemical of interest. (Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)).

For example, U.S. Patent Publications No. 2002/0012939, 2003/0224363, 2004/0029149, 2004/0072723, 2003/0059792, 2002/0168654, An in silico stoichiometric model of E. coli metabolism can be employed to identify essential genes for the metabolic pathways described in 2004/0009466 and US Pat. No. 7,127,379.

As disclosed herein, the OptKnock mathematical framework can be applied to define genetic deletions that result in growth-coupled production of desired products. Furthermore, the solution to the bilevel OptKnock problem gives only one set of deletions. An optimization technique called integer cut can be performed to enumerate all meaningful solutions, ie, all sets of knockouts that result in growth-coupled production formation. This requires solving the OptKnock problem iteratively, as described above, by introducing an additional condition called an integer cut at each iteration.

Given the teachings and guidance provided herein, those skilled in the art will understand that to disrupt an enzymatic reaction, the catalytic activity of one or more enzymes involved in the reaction is disrupted. Disruption can occur by a wide variety of means including, for example, deletion of the encoding gene or introduction of genetic mutations in one or more encoding gene sequences. The encoding genes targeted for disruption may be one, several or all of the genes encoding enzymes involved in catalytic activity. For example, if a single enzyme is responsible for the target catalytic activity, disruption may occur by genetic mutations that reduce or abolish the catalytic activity of the encoded gene product. Similarly, if a single enzyme is a multimer, including heteromers, disruption may occur by genetic mutations that reduce or abolish the function of one or all subunits of the encoded gene product. Disruption of activity can be achieved by loss of binding activity of one or more subunits to form an active complex, disruption of a catalytic subunit of a multimeric complex, or both. Other functions of multimeric protein association and activity can also be targeted to disrupt the metabolic reactions of the invention. Such other features are well known to those skilled in the art. Additionally, some or all of the functions of a single polypeptide or multimeric complex can be modified according to the invention to reduce or destroy the catalytic activity of one or more enzymes involved in the reactions or metabolic modifications of the invention. It can be destroyed by Similarly, some or all of the enzymes involved in the reactions or metabolic modifications of the invention can be disrupted so long as the targeted reaction is reduced or eliminated.

With the teachings and guidance provided herein, one skilled in the art will be able to reduce or eliminate responses encoded by common genes, and/or reduce or eliminate responses encoded by common genes, and/or It will also be appreciated that enzymatic reactions can be disrupted by orthologs. Reducing both the common gene and all orthologs can completely abolish the catalytic activity of the target reaction. However, disrupting either the common gene or one or more orthologs can reduce the catalytic activity of the target reaction sufficient to promote the coupling of growth to product biosynthesis. Common genes encoding catalytic activities for a wide variety of metabolic modifications, as well as their orthologs, are both exemplified herein.

Those skilled in the art will be able to carry out the disruption of some or all of the genes encoding the enzymes of the target metabolic reactions in the methods of the invention to achieve growth-related product production in the non-naturally occurring microbial organisms of the invention. You will understand that it can be incorporated into Exemplary disruptions that result in increased production of adipate, 6-ACA and/or HMDA are described in Example XXX and Tables 14-16.

Employing the method exemplified above, the method of the invention couples the production of a desired product to the growth of, for example, a cell or organism engineered to carry an identified genetic variation. This allows for the construction of cells and organisms that increase the production of desired products. As disclosed herein, metabolic mutations have been identified that link the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid to growth of an organism. Microbial organism strains constructed with identified metabolic mutations produce higher levels of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid during exponential growth phase compared to those in the absence of metabolic mutations. do. These strains can be advantageously used for the commercial production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid in a continuous fermentation process without being exposed to the previously described negative selective pressures. . Although exemplified herein as metabolic mutations, in particular one or more gene disruptions resulting in growth-associated production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, 6- It is understood that any genetic disruption that increases aminocaproic acid, caprolactam, hexamethylene diamine or levulinic acid can be introduced into the host microbial organism.

The methods of the invention therefore provide a set of metabolic alterations identified by in silico methods such as OptKnock. A set of metabolic modifications can include, for example, functional disruption of one or more metabolic reactions, including disruption by gene deletion. For the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, metabolic modifications can be selected from the set of metabolic modifications shown in Tables 14-16 (see Example XXX).

Also provided are methods of producing non-naturally occurring microbial organisms with stable growth-related production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. The method involves identifying in silico a set of metabolic modifications that increase the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, e.g. during exponential growth; Genetically modifying an organism to include a set of metabolic modifications that increase production of methylene diamine or levulinic acid; and culturing the genetically modified organism. Optionally, culturing can include adaptively evolving the genetically modified organism under conditions requiring production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid. can. The methods of the invention are applicable to bacteria, yeasts and fungi, as well as a wide variety of other cells and microorganisms disclosed herein.

Accordingly, the present invention provides non-naturally occurring microbial organisms containing one or more gene disruptions that result in increased production of 6-aminocaproic acid, caprolactam, hexamethylene diamine or levulinic acid. In one embodiment, the one or more gene disruptions result in growth-associated production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, such as stabilization of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. growth-related production. In another embodiment, such one or more gene disruptions reduce the activity of the respective one or more encoded enzymes.

A non-naturally occurring microbial organism can have one or more gene disruptions included in the metabolic modifications shown in Tables 14-16. As disclosed herein, one or more gene disruptions can be deletions. Such non-naturally occurring microbial organisms of the invention include bacteria, yeasts, fungi, or any of a wide variety of other microorganisms applicable to the fermentation methods disclosed herein.

Accordingly, the present invention provides a non-naturally occurring microbial organism comprising one or more gene disruptions, wherein the one or more gene disruptions occur in a gene encoding a protein or enzyme; , provides a non-naturally occurring microbial organism that results in increased production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid in an organism. The production of 6-aminocaproic acid, caprolactam, hexamethylene diamine or levulinic acid can be growth-related or non-growth-related. In certain embodiments, the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid can be forced to be coupled to the growth of an organism, as disclosed herein.

The present invention provides genetic mutations such as gene disruptions that increase the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid, e.g. Provided are non-naturally occurring microbial organisms having Production of the product can be forced into the exponential growth phase of the microorganism by, for example, genetically mutating the metabolic pathways of the cell, as disclosed herein. Genetic variation can increase the production of a desired product or even make a desired product an essential product of the growing season. A set of metabolic mutations or transformations that result in increased production and increased levels of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid biosynthesis are illustrated in Tables 14-16 (see Example XXX). ). Each mutation in the set corresponds to an essential metabolic reaction that is to be functionally disrupted. Functional disruption of all reactions within each set can result in increased production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid by the engineered strain throughout the growth phase. The reactions corresponding to the listed mutations can be found in Tables 14-16 (see Example XXX) and one or more genes encoding enzymes or proteins carrying out the reactions can be found in Tables 14-16. It is shown.

For example, for each strain exemplified in Tables 14 to 16, metabolic mutations that can be produced for the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine, or levulinic acid are shown in each column. These mutations include functional disruption of the reactions shown in Tables 14-16. Each of these non-naturally occurring mutations results in increased production, e.g., through the exponential growth phase of the microbial organism, compared to strains not containing such metabolic mutations, under appropriate culture conditions, and 6- resulting in increased levels of production of aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid. Suitable conditions include, for example, those disclosed herein, including conditions such as availability of particular carbon sources or reactants and/or adaptive evolution.

It is understood that modifications that do not substantially affect the activity of the various embodiments of the invention are also provided within the definition of the invention set forth herein. Accordingly, the following examples are intended to illustrate the invention and not to limit it.

Example I (Reverse Adipate Degradation Pathway) This example describes an exemplary adipate synthesis route via the reverse adipate decomposition pathway.

Organisms such as Penicillium chrysogenum have the ability to naturally degrade adipate (Thykaer et al., Metab. Eng. 4:151-158. (2002)). The mechanism is similar to fatty acid oxidation (see Figure 1). The first step in adipate degradation is an ATP-dependent reaction that activates adipate with CoA. The second reaction is catalyzed by dehydrogenase, which forms 5-carboxy-2-pentenoyl-CoA from adipyl-CoA. Through peroxisomal adipate degradation, the dehydrogenase enzyme contains FAD, which accepts electrons and then transports them directly to oxygen. The catalase enzyme produces H formed by the reduction of oxygen₂O₂dissipate. In mitochondrial fatty acid oxidation, FAD from dehydrogenase transports electrons directly into the electron transport chain. Multifunctional fatty acid oxidizing proteins in eukaryotes such as Saccharomyces cerevisiae and Penicillium chrysogenum carry out the following hydratase and dehydrogenase steps. The final step is an acyltransferase that splits 3-oxoadipyl-CoA into acetyl-CoA and succinyl-CoA.

A highly efficient route for the production of adipate is achieved by genetically mutating microorganisms such that similar enzymatic reactions are adopted for adipate synthesis from succinyl-CoA and acetyl-CoA (Fig. 2). To do this successfully, express the appropriate genes and adjust their expression so that high acetyl-CoA, succinyl-CoA and/or redox (e.g. NADH/NAD+) ratios direct metabolic flux into this pathway. It is necessary to change the culture conditions to direct adipate synthesis rather than degradation via . The strong parallelism with butyrate formation in Clostridium (Kanehisa and Goto, Nucl. Acids Res. 28:27-30 (2000)) suggests that each step in the adipate synthesis pathway is a reaction-directed one governed by the concentration of the metabolites involved. This confirms that it is thermodynamically realizable due to its properties. The final step of forming adipate from adipyl-CoA can occur via a synthetase, phosphotransadipyrase/kinase, transferase or hydrolase mechanism.

The maximum theoretical yield of adipate using this route was calculated in the presence and absence of an external electron acceptor such as oxygen. These calculations show that the route converts glucose into adipate and CO with a molar yield of 92%.₂has been shown to be able to be efficiently converted under anaerobic conditions (Table 1). Adipate production using this pathway does not require oxygen uptake, as NAD+ can be regenerated in two hydrogenase steps to form 3-hydroxyadipyl-CoA and adipyl-CoA (see Figure 2). . Furthermore, assuming a synthetase, phosphotransadipyrase/kinase or transferase mechanism for the final conversion step, up to 1.55 moles of ATP are formed per mole of glucose consumed at the maximum theoretical yield of adipate, so that The route is energetically favorable. Assuming that phosphoenolpyruvate carboxykinase (PPCK) functions in the direction of ATP production toward the formation of oxaloacetate, it is possible to increase the ATP yield to a level where 2.47 moles of ATP are produced per mole of glucose. can. Maximum ATP yield calculations were then performed assuming that the conversion of adipyl-CoA to adipate was the hydrolysis step. This reduces the maximum ATP yield at maximum adipate production to 0.85 and 1.77 moles of ATP per mole of glucose consumed, respectively, assuming that PPCK is irreversible or reversible, respectively. However, these ATP yields are sufficient for cell growth, maintenance and production.

Table 1: Maximum theoretical yield of adipate and associated ATP yield per mole of glucose using the reverse degradation pathway assuming the final step in the pathway is a synthetase, phosphotransadipyrase/kinase or transferase.<tables><img file="JP7370366B2_D0008.tif" /></tables>

Successful manipulation of this pathway requires identifying a suitable set of enzymes with sufficient activity and specificity. This requires identifying a suitable set of enzymes, cloning their corresponding genes into the production host, optimizing fermentation conditions, and assaying for product formation after fermentation. shall be. To engineer a production host for the production of adipate, one or more exogenous DNA sequences are expressed in a suitable host microorganism. In addition, microorganisms can functionally delete endogenous genes. These modifications enable the production of adipate using renewable feedstocks.

Several biochemically characterized candidate genes encoding enzymes that catalyze each step of the reverse adipate degradation pathway in the production host are described below. Although described using E. coli as the host organism for engineering the pathway, essentially any suitable host organism can be used. Specific mention is made of genes unique to E. coli, as well as genes in other organisms that, when properly cloned and expressed, can be adapted to catalyze appropriate transformations.

Referring to Figure 2, Step 1 involves succinyl-CoA:acetyl-CoA acyltransferase (β-ketothiolase). The first step in the pathway combines acetyl-CoA and succinyl-CoA to form 3-oxoadipyl-CoA. pcaF of Pseudomonas strain B13 (Kaschabek et al., J. Bacteriol. 184:207-215 (2002)), phaD of Pseudomonas putida U (Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)) ), paaE of Pseudomonas fluorescens ST (Di Gennaro et al., Arch. Microbiol. 188:117-125 (2007)) and paaJ of E. coli (Nogales et al., Microbiol. 153:357-365 (2007)) catalyzes the conversion of 3-oxoadipyl-CoA to succinyl-CoA and acetyl-CoA during the degradation of aromatic compounds such as phenylacetate and styrene. do. Because β-ketothiolase enzymes catalyze reversible conversions, these enzymes can be employed in the first step in adipate synthesis shown in Figure 2. For example, the ketothiolase phaA from Ralstonia eutropha combines two molecules of acetyl-CoA to form acetoacetyl-CoA (Sato et al., J. Biosci. Bioengineer. 103:38-44 (2007)). Similarly, β-ketothiolase (bktB) was reported to catalyze the condensation of acetyl-CoA and propionyl-CoA to form β-ketovaleryl-CoA in Ralstonia eutropha (Slater et al., J. Bacteriol.180:1979-1987(1998)). A further candidate is found in Burkholderia ambifaria AMMD. Protein sequences for the above gene products are well known in the art and can be obtained in public databases such as GenBank using the GI numbers and/or GenBank identifiers below.

<tables><img file="JP7370366B2_D0009.tif" /></tables>

These exemplary sequences can be used to identify homologue proteins in GenBank or other databases through sequence similarity searches (eg, BLASTp). The resulting homologue proteins and their corresponding gene sequences provide additional exogenous DNA sequences for transformation into E. coli or other suitable host microorganisms to generate production hosts.

For example, the following GI numbers and/or GenBank identifiers can be used to find the ortholog of paaJ from E. coli K12.

<tables><img file="JP7370366B2_D0010.tif" /></tables>

Examples of orthologs of pcaF from Pseudomonas nachmussi can be found using the GI numbers and/or GenBank identifiers below.

<tables><img file="JP7370366B2_D0011.tif" /></tables>

Additional prototypic candidate genes for the ketothiolase step include atoB (Sato et al., J. Biosci. Bioengineer. 103:38-44 (2007)), which can catalyze the reversible condensation of two acetyl-CoA molecules; Its homolog yqeF is mentioned. Candidate non-primitive genes include phaA (Sato et al., supra, 2007) and bktB (Slater et al., J. Bacteriol. 180:1979-1987 (1998)) from Ralstonia eutropha, and two genes from Clostridium acetobutylicum. Ketothiolases, thiA and thiB (Winzer et al., J. Mol. Microbiol. Biotechnol. 2:531-541 (2000)). The protein sequence for each of these exemplary gene products can be found using the GI numbers and/or GenBank identifiers below.

<tables><img file="JP7370366B2_D0012.tif" /></tables>

In this exemplary pathway, it is less desirable to use the thiolase-encoding genes fadA and fadB, which are genes in the E. coli fatty acid degradation pathway. These genes form a complex that encodes for multiple activities, most of which are undesirable for this pathway.

Referring to Figure 2, step 2 includes 3-hydroxyacyl-CoA dehydrogenase. The second step in the pathway involves the reduction of 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA. phaC of Pseudomonas putida U (Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)) and paaC of Pseudomonas fluorescens ST (Di Gennaro et al., Arch. Microbiol. 188:117-125) (2007)) catalyzes the reverse reaction, oxidation of 3-hydroxyadipyl-CoA to form 3-oxoadipyl-CoA, through the catabolism of phenylacetate or styrene. Since the reactions catalyzed by such dehydrogenases are reversible, these genes represent candidates for carrying out the second step of adipate synthesis shown in Figure 2. A similar transformation is carried out by the gene product of Clostridium acetobutylicum hbd (Atsumi et al., Metab. Eng. (epub Sep. 14, 2007);Boynton et al., J. Bacteriol. 178:3015-3024(1996)). This enzyme converts acetoacetyl-CoA to 3-hydroxybutyryl-CoA. The closeness of paaH E. coli to other genes in the phenylacetate-degrading operon (Nogales et al., Microbiol. 153:357-365 (2007)) and the inability of the paaH mutant to grow on phenylacetate (Ismail et al., Eur. J Biochem. 270:3047-3054 (2003)), it is estimated that the E. coli paaH gene encodes 3-hydroxyacyl-CoA dehydrogenase. The protein sequence for each of these exemplary gene products can be found using the GI numbers and/or GenBank identifiers below.

<tables><img file="JP7370366B2_D0013.tif" /></tables>

Referring to Figure 2, step 3 includes 3-hydroxyadipyl-CoA dehydratase. The gene product of crt from Clostridium acetobutylicum catalyzes the dehydration of 3-hydroxybutyryl-CoA to crotonyl-CoA (see Figure 2) (Atsumi et al., supra, 2007; Boynton et al., J Bacteriol. 178:3015-3024(1996)). Homologues of this gene are strong candidates for carrying out the third step in the adipate synthesis pathway illustrated in FIG. In addition, genes known to catalyze double bond hydroxylation in enoyl-CoA compounds are further candidates due to the reversibility of such enzymatic conversions. For example, the enoyl-CoA hydratases phaA and phaB of Pseudomonas putida appear to carry out double bond hydroxylation through phenylacetate catabolism (Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)), thus making it a further candidate for introduction into E. coli. Deletion of these genes prevents phenylacetate degradation in Pseudomonas putida. paaA and paaB from Pseudomonas fluorescens catalyze similar transformations (Olivera et al., supra, 1998). Finally, some E. coli genes are maoC (Park and Lee, J. Bacteriol. 185:5391-5397(2003)), paaF (Ismail et al., Eur. J. Biochem. 270:3047-3054(2003); Park and Lee, Biotechnol. Bioeng. 86:681-686(2004);Park and Lee, Appl. Biochem. Biotechnol. 113-116:335-346 (2004)) and paaG (Ismail et al., supra, 2003; Park and Lee, supra, 2004; Park and Lee, supra, 2004). It has been shown. The protein sequence for each of these exemplary gene products can be found using the GI numbers and/or GenBank identifiers below.

<tables><img file="JP7370366B2_D0014.tif" /></tables>

Alternatively, beta-oxidation genes are candidates for the first three steps in adipate synthesis. Candidate genes for the proposed adipate synthesis pathway also include the native fatty acid oxidation genes of E. coli and their homologues in other organisms. The E. coli genes fadA and fadB encode a multienzyme complex that exhibits ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, and enoyl-CoA hydratase activities (Yang et al., Biochem. 30:6788-6795 (1991); Yang et al., J. Biol. Chem. 265:10424-10429(1990); Yang et al., J. Biol. Chem. 266:16255(1991); Nakahigashi and Inokuchi, Nucl. Acids Res. 18:4937(1990)). These activities are mechanistically similar to the first three transformations shown in Figure 2. The fadI and fadJ genes encode similar functions and are necessarily expressed only anaerobically (Campbell et al., Mol. Microbiol. 47:793-805(2003)). These gene products necessarily convert succinyl-CoA and acetyl-CoA to 5-carboxy-2-pentenoyl-CoA as presented in Figure 2, but also convert short, medium and long chains. It acts to break down fatty acyl-CoA compounds into acetyl-CoA. However, ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase enzymes are well known to catalyze reversible conversions. Directed evolution and related approaches can also be applied to tune the substrate specificity of E. coli's native beta-oxidation machinery. These enzymes or their homologues can therefore be applied to adipate production. If native genes act to degrade adipate or its precursors in vivo, appropriate genetic modifications are made to weaken or eliminate these functions. However, the production of poly[(R)-3-hydroxybutyrate] in E. coli involves activating fadB by knocking out the negative regulator fadR and expressing the non-native ketothiolase phaA from Ralstonia eutropha. It may not be necessary, as methods have been described for (Sato et al., J. Biosci. Bioeng. 103:38-44 (2007)). This study shows that the gene product of beta-oxidases, specifically fadB, which encodes both 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase activities, produces longer-chain molecules from acetyl-CoA precursors. clearly demonstrated that it can function as part of a pathway for The protein sequence for each of these exemplary gene products can be found using the GI numbers and/or GenBank identifiers below.

<tables><img file="JP7370366B2_D0015.tif" /></tables>

Referring to Figure 2, step 4 includes 5-carboxy-2-pentenoyl-CoA reductase. The ketothiolase, dehydrogenase and enoyl-CoA hydratase steps are generally reversible, whereas the enoyl-CoA reductase step is almost always oxidative and irreversible under physiological conditions (Hoffmeister et al., J. Biol. Chem. 280:4329-4338(2005)). FadE catalyzes this likely irreversible conversion in E. coli (Campbell and Cronan, J. Bacteriol. 184:3759-3764 (2002)). The pathway requires enzymes that can reduce 2-enoyl-CoA intermediates, rather than enzymes such as FadE that simply oxidize acyl-CoA to 2-enoyl-CoA compounds. It has also been suggested that E. coli necessarily possesses the enzyme for the reduction of enoyl-CoA (Mizugaki et al., J. Biochem. 92:1649-1654 (1982); Nishimaki et al., J. Biochem. 95:1315-1321 (1984)), the E. coli gene possessing this function has not been biochemically characterized.

One candidate gene for the enoyl-CoA reductase step is the butyryl-of crotonyl-CoA, a reaction similar in mechanism to the desired reduction of 5-carboxy-2-pentanoyl-CoA to adipyl-CoA in the adipate synthesis pathway. It is the gene product of bcd from Clostridium acetobutylicum that necessarily catalyzes the reduction to CoA (Atsumi et al., supra, 2007; Boynton et al., J. Bacteriol. 178:3015-3024 (1996)). The activity of this enzyme can be enhanced by expressing bcd in conjunction with expression of the Clostridium acetobutylicum etfAB gene, which encodes an electron-transporting flavoprotein. A further candidate for the enoyl-CoA reductase step is the mitochondrial enoyl-CoA reductase from Euglena gracilis (Hoffmeister et al., J. Biol. Chem. 280:4329-4338(2005)). Following removal of the mitochondrial targeting leader sequence, a construct derived from this sequence was cloned into E. coli to yield the active enzyme (Hoffmeister et al., supra, 2005). This approach to expressing eukaryotic genes, particularly genes with leader sequences capable of targeting the gene product to specific subcellular compartments in prokaryotes, is well known to those skilled in the art. A close homolog of this gene from the prokaryotic Treponema denticola, TDE0597, is the third enoyl-CoA reductase cloned and expressed in E. coli (Tucci and Martin, FEBS Lett. 581:1561-1566 (2007)). The protein sequence for each of these exemplary gene products can be found using the GI numbers and/or GenBank identifiers below.

<tables><img file="JP7370366B2_D0016.tif" /></tables>

Referring to FIG. 2, step 5 includes adipyl-CoA synthetase (also referred to as adipate-CoA ligase), phosphotransadipyrase/adipate kinase, adipyl-CoA:acetyl-CoA transferase, or adipyl-CoA hydrolase. From an energy standpoint, it is desirable to catalyze the final step in the adipate synthesis pathway by an enzyme or enzyme pair capable of protecting the ATP equivalent stored in the thioester bond of adipyl-CoA. The products of the E. coli sucC and sucD genes or their homologs could potentially catalyze the final conversion shown in FIG. 2 if they exhibit activity towards adipyl-CoA. The sucCD gene naturally forms a succinyl-CoA synthetase complex that catalyzes the formation of succinyl-CoA from succinate with simultaneous consumption of one ATP, a reversible reaction in vivo (Buck et al., Biochem. 24:6245-6252(1985)). Due to the structural similarity of succinate and adipate, ie, both are linear dicarboxylic acids, it is reasonable to expect some activity of the sucCD enzyme towards adipyl-CoA. Here, AMP and PPi are used as cofactors to produce ATP production of adipate from adipyl-CoA, where enzymes exhibiting adipyl-CoA ligase activity act in opposite physiological directions as shown in Figure 1. can be done equally well. Exemplary CoA-ligases include rat dicarboxylate CoA ligase, whose sequence has not yet been characterized (Vamecq et al., Biochem. J. 230:683-693 (1985)), two features from Penicillium chrysogenum Either of the attached phenylacetate-CoA ligases (Lamas-Maceiras et al., Biochem. J. 395, 147-155 (2005); Wang et al., Biochem. Biophy. Res. Commun. 360:453-458 (2007)), phenylacetate-CoA ligase from Pseudomonas putida (Martinez-Bianco et al., J. Biol. Chem. 265:7084-7090 (1990)) and 6-carboxyhexane from Bacillus subtilis. Noate-CoA ligase (Bower et al., J. Bacteriol. 178:4122-4130 (1996)). The protein sequence for each of these exemplary gene products can be found using the GI numbers and/or GenBank identifiers below.

<tables><img file="JP7370366B2_D0017.tif" /></tables>

Another option to use phosphotransadipyrase/adipate kinase is to use the buk1, buk2 and ptb gene products from Clostridium acetobutylicum (Walter et al., Gene 134:107-111 (1993); Huang et al., J. Mol. Microbiol. Biotechnol. 2:33-38 (2000)) or their homologs. The ptb gene encodes an enzyme that can convert butyryl-CoA to butyryl-phosphate. The converted butyryl-phosphate is then converted to butyrate via one of the buk gene products while ATP is produced. A similar set of transformations can be performed by the buk1, buk2 and ptb gene products, namely the conversion of adipyl-CoA to adipyl-phosphate and the subsequent conversion of adipyl-phosphate to adipate. The protein sequence for each of these exemplary gene products can be found using the GI numbers and/or GenBank identifiers below.

<tables><img file="JP7370366B2_D0018.tif" /></tables>

Alternatively, acetyltransferases capable of transferring the CoA group from adipyl-CoA to acetate can be applied. Similar conversions are catalyzed by the clostridium kluyveri cat1, cat2, and cat3 gene products, which were shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA acetyltransferase activities, respectively (Sohling and Gottschalk, J. Bacteriol. 178:871-880(1996); Seedorf et al., Proc. Natl. Acad. Sci. USA 105:2128-2133(2008)). The protein sequence for each of these exemplary gene products can be found using the GI numbers and/or GenBank identifiers below.

<tables><img file="JP7370366B2_D0019.tif" /></tables>

Finally, although undesirable from an energy point of view, the conversion of adipyl-CoA to adipate can also be carried out by means of acyl-CoA hydrolases or likewise thioesterases.

The top E. coli gene candidate shows high similarity to human acot8, a dicarboxylic acid acetyltransferase with activity towards adipyl-CoA (Westin et al., J. Biol. Chem. 280:38125-38132 (2005)) tesB (Naggert et al., J. Biol. Chem. 266:11044-11050 (1991)). This activity has also been characterized in rat liver (Deana, Biochem. Int. 26:767-773 (1992)). The protein sequence for each of these exemplary gene products can be found using the GI numbers and/or GenBank identifiers below.

<tables><img file="JP7370366B2_D0020.tif" /></tables>

Other protist candidate genes include tesA (Bonner and Bloch, J. Biol. Chem. 247:3123-3133(1972)), ybgC (Kuznetsova et al., FEMS Microbiol. Rev. 29:263-279(2005); Zhuang et al., FEBS Lett. 516:161-163(2002)), paal (Song et al., J. Biol. Chem. 281:11028-11038(2006)) and ybdB (Leduc et al., J. Bacteriol. 189:7112-7126 (2007)). The protein sequence for each of these exemplary gene products can be found using the GI numbers and/or GenBank identifiers below.

<tables><img file="JP7370366B2_D0021.tif" /></tables>

The above description depicts an exemplary adipate pathway via the reverse adipate decomposition pathway.

Example II (Production of an adipate-producing microbial organism with a reverse degradation pathway) This example describes the production of a microbial organism capable of producing adipate using the reverse degradation pathway.

Use E. coli as the target organism to manipulate the reverse adipate degradation pathway shown in Figure 2. E. coli provides a good host for producing non-naturally occurring microorganisms capable of producing adipate. Escherichia coli is amenable to genetic manipulation and can effectively produce various products such as ethanol, acetic acid, formic acid, lactic acid and succinic acid under anaerobic or microaerobic conditions. Are known.

To generate E. coli strains engineered to produce adipate, nucleic acids encoding enzymes utilized in the reverse degradation pathway are expressed in E. coli using well-known molecular biology techniques (e.g., Sambrook, supra. , 2001; see Ausubel, supra, 1999). In particular, paaJ (NP_415915.1), paaH (NP_415913.1) and maoC (NP_415905) encode succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase and 3-hydroxyadipyl-CoA dehydratase activities, respectively. .1) Clone the gene into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the bcd (NP_349317.1), etfAB (349315.1 and 349316.1) and sucCD (NP_415256.1 and AAC73823.1) genes encoding 5-carboxy-2-pentenoyl-CoA reductase and adipyl-CoA synthetase activities, respectively, were transferred to PA1. Clone into the pZA33 vector (Expressys, Ruelzheim, Germany) under the /lacO promoter. The two sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for adipate synthesis via the reverse degradation pathway.

The resulting genetically engineered organisms are cultured in glucose-containing media according to procedures well known in the art (see, eg, Sambrook et al., supra, 2001). Confirm expression of reverse degradation pathway genes using methods well known in the art for measuring polypeptide expression or enzymatic activity, including, for example, Northern blots, PCR amplification of mRNA, and immunoblots. do. Confirm the enzymatic activity of the expressed enzyme using assays specific for the individual activity. The ability of the engineered E. coli strain to produce adipate is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have functional adipate synthesis pathways are further enhanced by optimization for efficient utilization of the pathway. Briefly, engineered strains are evaluated to determine whether any of the exogenous genes are expressed at rate-limiting levels. For example, introduction of additional gene copy numbers increases expression for any enzymes expressed at low levels that can limit flux through the pathway.

Utilize metabolic modeling to optimize growth conditions to generate better producers.

Additionally, modeling is used to design gene knockouts that further optimize pathway utilization (e.g., U.S. Patent Publications Nos. 2002/0012939; 2003/0224363; 2004/0072723, 2003/0059792, 2002/0168654 and 2004/0009466 and US Pat. No. 7,127,379). Modeling analysis allows reliable prediction of the effects on cell growth of shifting metabolism towards more efficient production of adipate. One modeling method is the bilevel optimization approach OptKnock, which is applied to select gene knockouts that collectively result in better production of adipate (Burgard et al., Biotechnol. Bioengineer. 84:647-657(2003)). Adaptive evolution can also be used, for example, to generate better producers of acetyl-CoA and succinyl-CoA intermediates or adipate products. Adaptive evolution is carried out to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on these results, a series of subsequent modeling, genetic manipulation and adaptive evolution can be applied to the adipate producers to further increase production.

For large-scale production of adipate, the reverse pathway-containing organism is cultivated in a fermentor using a medium known in the art to support the growth of the organism under anaerobic conditions. . Fermentation is carried out in batch, fed batch or continuous mode. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel. For example, the flask can be sealed with a septum and crimp cap. Microaerophilic conditions can also be utilized by providing small holes in the membrane for limited ventilation. H₂S.O.₄Maintain the pH of the medium at a pH of approximately 7 by adding an acid such as Growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and glucose uptake rate is determined by monitoring carbon source depletion over time. By-products such as undesirable alcohols, organic acids and residual glucose can be detected using, for example, Aminex® series HPLC columns (e.g. HPX- 87 series) (BioRad, Hercules, Calif.) (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

This example describes the production of an adipate-producing microbial organism using the reverse degradation pathway.

Example III (Adipate Synthesis via 3-oxoadipate) This example describes an exemplary adipate synthesis route via 3-oxoadipate.

A further route from that described in Examples I and II using acetyl-CoA and succinyl-CoA as precursors for adipate formation and via the metabolic intermediate 3-oxoadipate is shown in FIG. The first two transformations in this pathway are the two final steps in the decomposition pathway for aromatic and chloroaromatic compounds that act in opposite directions (Kaschabek et al., J. Bacteriol. 184:207-215 (2002) ; Nogales et al., Microbiol. 153:357-365 (2007); Ismail et al., Eur. J. Biochem. 270:3047-3054 (2003)). Specifically, the first step forms 3-oxoadipyl-CoA by condensation of succinyl- and acetyl-CoA. The second step forms 3-oxoadipate and is reported to be reversible in Pseudomonas sp. strain B13 (Kaschabek et al., J. Bacteriol. 184:207-215 (2002)).

The subsequent steps are reduction of 3-oxoadipate to 3-hydroxyadipate (conversion of keto group to hydroxyl group), dehydration of 3-hydroxyadipate to obtain hexa-2-enedioate, and forming adipate. Includes reduction of hexa-2-enedioate. These steps in the pathway are similar to the conversion of oxaloacetate to succinate via the reductive TCA cycle (see Figure 4). This supports that these steps of the pathway are thermodynamically favorable, subject to the presence of appropriate metabolite concentrations. The final reduction step can be carried out biochemically or by employing a chemical catalyst to convert hexa-2-enedioate to adipate. Chemical hydrogenation can be carried out using activated carbon supported Pt catalysts as described in (Niu et al., Biotechnol. Prog. 18:201-211 (2002)).

The maximum theoretical yield of adipate using this route is 0.92 moles per mole of glucose consumed, and no oxygen is required to achieve these yields (see Table 2). The energy mechanism involved is identical to that of the reverse adipate pathway. Theoretically, up to 1.55 moles of ATP are formed per mole of glucose utilized via this pathway. Assuming that phosphoenolpyruvate kinase (PPCK) acts in the direction of ATP production, the ATP yield increases to about 2.47 mol. Interestingly, using chemical hydrogenation in the last step and assuming 100% catalyst efficiency, the product yield can be increased to 1 mole of adipate per mole of glucose consumed. In this scenario, up to 1.95 moles of ATP could theoretically be formed, assuming no reversal function of PPCK.

Table 2: Maximum theoretical adipate yield and associated ATP yield per mole of glucose using the 3-oxoadipate route.<tables><img file="JP7370366B2_D0022.tif" /></tables>

Successful manipulation of this pathway involves identifying a suitable set of enzymes with sufficient activity and specificity. This requires identifying a suitable set of enzymes, cloning their corresponding genes into the production host, optimizing fermentation conditions, and assaying for product formation after fermentation. To engineer a production host for the production of adipate, one or more exogenous DNA sequences can be expressed in the host microorganism.

In addition, the host microorganism can be functionally deleted from endogenous genes. These modifications enable the production of adipate using renewable feedstocks.

Several biochemically characterized candidate genes capable of encoding enzymes that catalyze each step of the 3-oxoadipate pathway for adipate synthesis are described below. Although this method has been described for E. coli, one skilled in the art can apply these teachings to any other suitable host organism. Specifically, genes unique to E. coli, as well as genes in other organisms that, when properly cloned and expressed, can be applied to catalyze appropriate transformations are listed below.

Referring to Figure 3, Step 1 involves succinyl-CoA:acetyl-CoA acyltransferase (β-ketothiolase). Gene candidates for this enzyme are shown above (Figure 2, Step 1).

Referring to Figure 3, step 2 includes 3-oxoadipyl-CoA transferase. In this step, 3-oxoadipate is formed by transferring the CoA group from 3-oxoadipyl-CoA to succinate. This activity is reported for the two-unit enzyme encoded by Pseudomonas pcaI and pcaJ (Kaschabek et al., J. Bacteriol. 184:207-215 (2002)). This enzyme catalyzes a reversible conversion. Exemplary gene product protein sequences for subunit A of this complex can be found using the GI numbers and/or GenBank identifiers below.

<tables><img file="JP7370366B2_D0023.tif" /></tables>

Exemplary gene product protein sequences for subunit B of this complex can be found using the GI numbers and/or GenBank identifiers below.

<tables><img file="JP7370366B2_D0024.tif" /></tables>

Referring to Figure 3, step 3 includes 3-oxoadipate reductase. E. coli has several candidate alcohol dehydrogenases, two with similar functions are malate dehydrogenase (mdh) and lactate dehydrogenase (ldhA). Although these two enzymes have not been shown to have broad substrate specificity in E. coli, lactate dehydrogenase from Ralstonia eutropha is capable of producing lactate, 2-oxobutyrate, 2-oxopentanoate and 2-oxoglutamate. It has been shown to exhibit high activity against substrates of various chain lengths, such as esters (Steinbuchel and Schlegel, Eur. J. Biochem. 130:329-334 (1983)). A further non-native enzyme candidate for this step is mitochondrial 3-hydroxybutyrate dehydrogenase (bdh) from the human heart, which has been cloned and characterized (Marks et al., J. Biol. Chem. 267:15459-15463(1992)). Of particular interest is that this enzyme is a dehydrogenase that acts on 3-hydroxy acids. Since dehydrogenases are typically reversible, this gene product or its homologue reduces a 3-oxo acid, e.g. 3-oxoadipate, to the corresponding 3-hydroxy acid, e.g. 3-hydroxyadipate. It is estimated that it will be possible to do so. The protein sequence for each of these exemplary gene products can be found using the GI numbers and/or GenBank identifiers below.

<tables><img file="JP7370366B2_D0025.tif" /></tables>

Referring to Figure 3, step 4 includes 3-hydroxyadipate dehydratase. In this reaction, 3-hydroxyadipate is dehydrated to hexa-2-enedioate.

Most dehydratases catalyze the α,β removal of water, although direct evidence for this enzymatic conversion has not been identified. This involves activation of the α-hydrogen by an electron-withdrawing carbonyl, carboxylate or CoA-thiol ester group and removal of the hydroxyl group from the β position (Martins et al., Proc. Natl. Acad .Sci. USA 101 :15645-15649(2004);Buckel and Golding,. FEMS Microbiol. Rev. 22:523-541(1998)). Protein sequences for exemplary gene products can be found using the GI numbers and/or GenBank identifiers below.

<tables><img file="JP7370366B2_D0026.tif" /></tables>

Other good candidates to perform this function are serine dehydratases. These enzymes catalyze a very similar conversion in the removal of ammonia from serine, which is required in this dehydration step. Protein sequences for exemplary gene products can be found using the GI numbers and/or GenBank identifiers below.

<tables><img file="JP7370366B2_D0027.tif" /></tables>

Non-native gene candidates for this transformation were also identified. For example, multisubunit L-serine dehydratase from Peptostreptococcus asaccharolyticus was shown to complement E. coli strains lacking L-serine dehydratase activity (Hofmeister et al., J. Bacteriol. 179: 4937-4941(1997)). Furthermore, the putative 2-(hydroxymethyl)glutarate dehydratase encoded by the gene hmd of Eubacterium barkeri is directed against both α- and β-subunits of [4Fe-4S]-containing bacterial serine dehydratases. (Alhapel et al., Proc. Natl. Acad. Sci. USA 103:12341-12346 (2006)). Protein sequences for exemplary gene products can be found using the GI numbers and/or GenBank identifiers below.

<tables><img file="JP7370366B2_D0028.tif" /></tables>

Referring to Figure 3, step 5 includes 2-enoate reductase. The final step in the 3-oxoadipate pathway is the reduction of the double bond of hexa-3-enedioate to form the adipate. Biochemically, this conversion is catalyzed by 2-enoate reductase (EC 1.3.1.31), which is known to catalyze the NADH-dependent reduction of a wide variety of α,β-unsaturated carboxylic acids and aldehydes. (Rohdich et al., J. Biol. Chem. 276:5779-5787 (2001)). This enzyme is encoded by enr of several Clostridium species, including Clostridium tyrobutylicum and Clostridium thermoaceticum (now called Moorella thermoaceticum) (Giesel and Simon, Arch. Microbiol. 135:51- 57(1983)). In the recently published genome sequence of Saccharomyces kluyveri, nine coding sequences for enoate reductase were reported, one of which was characterized (Seedorf et al., Proc. Natl. Acad. Sci. USA 105:2128-2133(2008)). The enr genes from both Clostridium tyrobutylicum and Clostridium thermoaceticum have been cloned and sequenced and show 59% identity to each other. The former gene was also found to have approximately 75% similarity to the characterized Saccharomyces kluybergi gene (Giesel and Simon, Arch. Microbiol. 135:51-57 (1983)). Based on these sequence results, enr was reported to be highly similar to E. coli dienoyl-CoA reductase (fadH) (Rohdich et al., J. Biol. Chem. 276:5779-5787(2001)). Therefore, several gene candidates exist for catalyzing the last step in the 3-oxoadipate pathway and are listed below. The C. thermoaceticum enr gene has also been expressed in E. coli in an enzymatically active form (Rohdich et al., supra, 2001). Protein sequences for exemplary gene products can be found using the GI numbers and/or GenBank identifiers below.

<tables><img file="JP7370366B2_D0029.tif" /></tables>

The above description depicts an exemplary adipate synthesis route via the 3-oxoadipate pathway.

Example IV (Production of an adipate-producing microbial organism having a 3-oxoadipate pathway) This example describes the production of a microbial organism capable of producing adipate using the 3-oxoadipate pathway. do.

E. coli is used as the target organism to engineer the 3-oxoadipate pathway shown in Figure 3. E. coli provides a good host for producing non-naturally occurring microorganisms capable of producing adipate. E. coli is amenable to genetic manipulation and is capable of effectively producing various products such as ethanol, acetic acid, formic acid, lactic acid and succinic acid under anaerobic or microaerobic conditions. Are known.

To generate E. coli strains engineered to produce adipate, nucleic acids encoding enzymes utilized in the 3-oxoadipate pathway are expressed in E. coli using well-known molecular biology techniques (e.g., Sambrook, (see Ausubel, supra, 1999). In particular, paaJ (NP_415915.1), pcaIJ (AAN69545.1 and NP_746082.1) and bdh (AAA58352), which encode succinyl-CoA:acetyl-CoA acyltransferase, 3-oxoadipyl-CoA transferase and 3-oxoadipate reductase activities, respectively. .1) Clone the gene into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the acnA (P25516.3) and enr (ACA54153.1) genes encoding 3-hydroxyadipate dehydratase and 2-enoate reductase activities, respectively, were cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. do. The two sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for adipate synthesis via the 3-oxoadipate pathway.

The resulting genetically engineered organisms are cultured in glucose-containing media according to procedures well known in the art (see, eg, Sambrook et al., supra, 2001). The 3-oxo Confirm expression of adipate pathway genes. Confirm the enzymatic activity of the expressed enzyme using assays specific for the individual activity. The ability of the engineered E. coli strain to produce adipate is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have functional adipate synthesis pathways are further enhanced by optimization for efficient utilization of the pathway. Briefly, engineered strains are evaluated to determine whether any of the exogenous genes are expressed at rate-limiting levels. For example, introduction of additional gene copy numbers increases expression for any enzymes expressed at low levels that can limit flux through the pathway.

Utilize metabolic modeling to optimize growth conditions to generate better producers.

Additionally, modeling is used to design gene knockouts that further optimize pathway utilization (e.g., U.S. Patent Publications Nos. 2002/0012939; 2003/0224363; 2004/0072723, 2003/0059792, 2002/0168654 and 2004/0009466 and US Pat. No. 7,127,379). Modeling analysis allows reliable prediction of the effects on cell growth of shifting metabolism towards more efficient production of adipate. One modeling method is the bilevel optimization approach OptKnock, which is applied to select gene knockouts that collectively result in better production of adipate (Burgard et al., Biotechnol. Bioengineer. 84:647-657(2003)). Adaptive evolution can also be used, for example, to generate better producers of acetyl-CoA and succinyl-CoA intermediates or adipate products. Adaptive evolution is carried out to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on these results, a series of subsequent modeling, genetic manipulation and adaptive evolution can be applied to the adipate producers to further increase production.

For large-scale production of adipate, 3-oxoadipate pathway-containing organisms are cultured in fermentors using media known in the art to support growth of the organisms under anaerobic conditions. do. Fermentation is carried out in batch, fed batch or continuous mode. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel. For example, the flask can be sealed with a septum and crimp cap. Microaerophilic conditions can also be utilized by providing small holes in the membrane for limited ventilation. H₂S.O.₄Maintain the pH of the medium at a pH of approximately 7 by adding an acid such as Growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and glucose uptake rate is determined by monitoring carbon source depletion over time. By-products such as undesirable alcohols, organic acids and residual glucose can be detected using, for example, Aminex® series HPLC columns (e.g. HPX- 87 series) (BioRad) (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

This example describes the production of an adipate-producing microbial organism containing the 3-oxide adipate pathway.

(Example V) (Adipate synthesis via cis, cis-muconic acid) In this example, a previously described adipate synthesis route is described (Niu et al., Biotechnol. Prog. 18(2):p. 201- 11. 2002; see Frost et al., US Pat. No. 5,487,987, issued January 30, 1996).

Adipate synthesis via an integrated biological and chemical transformation method has been previously described (Niu et al., Biotechnol. Prog. 18:201-211 (2002)) and is illustrated in FIG. This method is further described in US Pat. No. 5,487,987. Adipate synthesis via this route requires the introduction into E. coli of three heterologous genes capable of converting dehydroshikimate to cis, cis-muconic acid (Niu et al., supra, 2002). A final chemical hydrogenation step forms adipic acid. In this step, pre-treated fermentation broth containing 150 mM cis, cis-muconate was mixed with 10% platinum (Pt) on activated carbon. The hydrogenation reaction was carried out at 250 degrees with stirring at a hydrogen pressure of 3400 KPa for 2.5 hours. The calculated adipate yields are shown in Table 3, assuming that either enzymatic or chemical catalytic steps are utilized to convert cis, cis-muconate to adipate. Under aerobic conditions, 85% molar yields of adipate can be obtained when chemical reactions are employed for hydrogenation, and 75% molar yields are obtained when NADH-based hydrogenases are used.

Table 3: Maximum theoretical yield of adipate per mole of glucose using the cis, cis-muconic acid pathway<tables><img file="JP7370366B2_D0030.tif" /></tables>

Although this is an exemplary method, this method has drawbacks compared to other methods, such as those described in Examples I-IV. For example, the first limitation of this method is the low theoretical yield compared to the reverse adipate decomposition and 3-oxoadipate routes. A second limitation is that the ATP yield of this pathway is extremely low. A third limitation of this pathway is that it requires a supply of oxygen to the bioreactor and involves dioxygenases, eliminating the option of anaerobic fermentation.

The above description depicts an exemplary adipate synthesis route via the cis, cis-muconic acid pathway.

Example VI Adipate Synthesis via Alpha-Ketoadipate This example describes an exemplary adipate synthesis route via the alpha-ketoadipate route.

Alpha-ketoadipate is a known intermediate for lysine biosynthesis in Saccharomyces cerevisiae, and this information was used to identify additional pathways for adipic acid biosynthesis (see Figure 6). The conversion of alpha-ketoglutarate to alpha-ketoadipate is catalyzed by homocitrate synthase, homoaconitase and homoisocitrate dehydrogenase, as indicated by the dotted arrows in FIG. The conversion of alpha-ketoadipate to alpha-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, an enzyme reported to be found in rat and human placenta (Suda et al., Arch. Biochem. Biophys. 176:610-620 (1976); Suda et al., Biochem. Biophys. Res. Commun. 77:586-591(1977)). Subsequent steps include a dehydratase to convert alpha-hydroxyadipate to hexa-2-enedioate and then reduce it to adipic acid. This last step is enzymatically catalyzed or is described in Example II. It can be done through a chemical reaction. Genes encoding enzymes for the alpha-ketoadipate pathway are identified as described in Examples I-IV.

The adipate yields associated with this route are shown in Table 4. two COs through the conversion of acetyl-CoA to adipate₂Only 67% of glucose can be converted to adipate because molecules are lost. This is reflected in the molar yield for this route under aerobic conditions.

Without oxygen uptake, yields are further reduced. Additionally, the maximum ATP yield under anaerobic conditions is extremely low, forcing engineered organisms to utilize additional substrates to form energy for cell growth and maintenance under such conditions. .

Table 4: Maximum theoretical adipate yield and associated ATP yield per mole of glucose using the alpha-ketoadipate route.<tables><img file="JP7370366B2_D0031.tif" /></tables>

The above description illustrates an exemplary adipate synthesis route via the alpha-ketoadipate pathway.

Example VII (Adipate Synthesis via Lysine Degradation) This example describes an exemplary adipate synthesis route via the lysine degradation pathway.

Two additional routes for adipate synthesis rely on lysine degradation to form adipate. One pathway (the one found in Saccharomyces cerevisiae and not unique to E. coli) starts from alpha-ketoglutarate to form lysine; the other pathway (unique to E. coli) starts from alpha-ketoglutarate to form lysine. Use aspartate as a starting point for biosynthesis. Figure 7 shows adipate formation from lysine. The maximum theoretical yields for adipate, both in the presence and absence of oxygen, using the E. coli stoichiometry model are shown in Tables 5 and 6 for alpha-ketoglutarate and aspartate, respectively, as starting points for lysine. . The maximum ATP yields associated with these theoretical yields were also calculated. They are shown in the same table. These yields are low compared to other routes described in Examples I-IV. Genes encoding enzymes for the alpha-ketoadipate pathway are identified as described in Examples I-IV.

Table 5: Maximum theoretical yield of adipate per mole of glucose assuming a lysine biosynthetic pathway starting from alpha-ketoglutarate and concomitant ATP yield.<tables><img file="JP7370366B2_D0032.tif" /></tables>Table 6: Maximum theoretical yield of adipate per mole of glucose assuming a lysine biosynthetic pathway starting from aspartate and concomitant ATP yield.<tables><img file="JP7370366B2_D0033.tif" /></tables>

The above description illustrates an exemplary adipate synthesis route via the lysine degradation pathway.

Example VIII (Production of caprolactam and 6-aminocaproic acid via the adipyl-CoA pathway) This example describes an exemplary caprolactam and/or 6-aminocaproic acid synthesis route via the adipyl-CoA pathway.

An exemplary route for forming caprolactam and/or 6-aminocaproic acid using adipyl-CoA as a precursor is shown in FIG. The pathway includes a CoA-dependent aldehyde dehydrogenase that can reduce adipyl-CoA to adipate semialdehyde, and a transaminase or 6-aminocaproate dehydrogenase that can convert this molecule to 6-aminocaproic acid. The final step of converting 6-aminocaproate to caprolactam can be carried out via amidohydrolase or via chemical conversion (Guit et al., published March 7, 2002). Buijs et al., U.S. Pat. No. 6,353,100; Wolters et al., U.S. Pat. No. 5,700,934, issued Dec. 23, 1997; Agterberg et al., U.S. Pat. No. 6,660,857, issued Dec. 9, 2003). Assuming that the reverse adipate decomposition pathway was complemented with the reaction scheme shown in Figure 8, the maximum theoretical yield of caprolactam was calculated to be 0.8 moles per mole of glucose consumed (see Table 7). . The route is energetically favorable since up to 0.78 moles of ATP are formed per mole of glucose consumed at the maximum theoretical yield of caprolactam. Assuming that phosphoenolpyruvate carboxykinase (PPCK) functions in the direction of ATP production towards the formation of oxaloacetate, it is possible to further improve the ATP yield to a level where 1.63 moles of ATP are produced for every mole of glucose. can.

Because the final amidohydrolase step is energetically and redox-neutral, the product and ATP molar yields associated with the production of 6-aminocaproic acid are comparable to those associated with the production of caprolactam. Therefore, alternatively, microorganisms and associated fermentation processes can be envisaged in which 6-aminocaproic acid is formed instead of caprolactam followed by further unit processing to dehydrate/cyclize 6-aminocaproic acid to caprolactam.

Table 7: Maximum theoretical caprolactam yield and concomitant ATP yield per mole of glucose assuming that the reverse fatty acid degradation pathway is complemented with the reaction scheme of Figure 8.<tables><img file="JP7370366B2_D0034.tif" /></tables>

Successful manipulation of this pathway requires identifying a suitable set of enzymes with sufficient activity and specificity. This requires identifying a suitable set of enzymes, cloning their corresponding genes into the production host, optimizing fermentation conditions, and assaying for product formation after fermentation. . To engineer a production host for the production of 6-aminocaproic acid or caprolactam, one or more exogenous DNA sequences can be expressed in the host microorganism. In addition, microorganisms can functionally delete endogenous genes. These modifications allow the production of 6-aminocaproate or caprolactam using renewable feedstocks.

Several biochemically characterized candidate genes that can encode enzymes that catalyze each step of the caprolactam formation pathway described in Figure 8 are described below. Although described for E. coli, one skilled in the art can apply these teachings to any other suitable host organism. Specifically, the genes shown are either native to E. coli or genes in other organisms that, when properly cloned and expressed, can be applied to catalyze the appropriate transformations.

Referring to Figure 8, Step 1 involves CoA-dependent aldehyde dehydrogenase. An exemplary gene encoding an enzyme to catalyze the reduction of an acyl-CoA to its corresponding aldehyde includes Acinetobacter calcoaceticus acrl, which encodes fatty acyl-CoA reductase (Reiser and Somerville, J. Bacteriol 179:2969-2975 (1997)), Acinetobacter sp. The sucD gene from Clostridium kluyveri (Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996)) can be mentioned.

<tables><img file="JP7370366B2_D0035.tif" /></tables>

Referring to Figure 8, step 2 includes transaminases. The second step in the pathway is the conversion of the 6-aldehyde to an amine. It is likely that this conversion can be accomplished by gamma-aminobutyrate transaminase (GABA transaminase), a native enzyme encoded by gabT that transfers an amino group from glutamic acid to the terminal aldehyde of succinyl semialdehyde (Bartch et al., J. Bacteriol. 172:7035-7042(1990)). The puuE gene product catalyzes another 4-aminobutyrate transaminase in E. coli (Kurihara et al., J. Biol. Chem. 280:4602-4608 (2005)). GABA transaminase in mouse, Pseudomonas fluorescens, and wild boar was shown to react with 6-aminocaproic acid (Cooper, Methods Enzymol. 113:80-82 (1985); Scott and Jakoby, J. Biol. Chem. 234:932-936(1959)). Protein sequences for exemplary gene products can be found using the GI numbers and/or GenBank identifiers below.

<tables><img file="JP7370366B2_D0036.tif" /></tables>

Referring to FIG. 8, step 2 can alternatively include 6-aminocaproate dehydrogenase comprising reductive amination of adipate semialdehyde to form 6-aminocaproate. This conversion can be accomplished by lysine-6-dehydrogenase, which necessarily converts L-lysine to 2-aminoadipate-6-semialdehyde. Exemplary enzymes include Geobacillus stearothermophilus (Heydari et al., Appl. Environ. Microbiol. 70(2):937-942 (2004)) and Agrobacterium tumefaciens (Hashimoto et al., J. Biochem. (Tokyo)). , 106(1):76-80(1989); Misono et al., J. Biochem. (Tokyo), 105(6):1002-1008(1989)) and Achromobacter denitrificans (Ruldeekulthamrong et al., BMB Reports 790-795 (2008)).

<tables><img file="JP7370366B2_D0037.tif" /></tables>

Referring to Figure 8, step 3 includes amidohydrolase. The final step in caprolactam synthesis is the cyclization of 6-aminocaproic acid. This conversion, although not enzymatically characterized, is very similar to the cyclization of lysine by D-lysine lactamase from Cryptococcus laurentii (EC 3.5.2.11) (Fukumura et al., FEBS Lett. 89:298- 300(1978)). However, the protein and nucleotide sequences of this enzyme are currently unknown and lysine lactamase activity has not previously been demonstrated in other organisms.

A plasmid contained in some strains of Pseudomonas species isolated from soil confers the ability to grow on caprolactam as the sole carbon source (Boronin et al., FEMS Microbiol. Lett. 22:167-170 (1984) ). However, no associated gene or protein sequences have been associated with this function to date.

The most closely related candidate enzyme with available sequence information is the 6-aminohexanoate cyclic dimeric hydrolase, which has been characterized in Pseudomonas and Flavobacterium species. The nylB gene product was cloned from Pseudomonas sp. NK87 and expressed in E. coli (Kanagawa et al., J. Gen. Microbiol. 139:787-795 (1993)). The substrate specificity of the enzyme was tested in Flavobacterium sp. K172 and was shown to react with higher oligomers of 6-aminohexanoate, but not with caprolactam (Kinoshita et al., Eur. J. Biochem. 116:547-551 (1981)). The reversibility and ability of the 6-aminohexanoate dimeric hydrolase in other organisms to react in the desired direction with the desired substrate can be further tested. Protein sequences for exemplary gene products can be found using the GI numbers and/or GenBank identifiers below.

<tables><img file="JP7370366B2_D0038.tif" /></tables>

The above description illustrates an exemplary route for producing caprolactam and/or 6-aminocaproic acid via the adipyl-CoA pathway.

Example IX (Production of a 6-aminocaproate or caprolactam producing microbial organism with a 3-oxoadipate pathway) In this example, the reverse degradation pathway was used to produce adipate and the intracellular adipate to 6 - Describes the production of microbial organisms capable of converting aminocaproate and/or caprolactam.

E. coli is used as the target organism to engineer the genes necessary for adipate, 6-aminocaproate and/or caprolactam synthesis (see Figures 2 and 8). E. coli provides a good host for producing non-naturally occurring microorganisms capable of producing adipate, 6-aminocaproate and/or caprolactam. E. coli is amenable to genetic manipulation and is capable of effectively producing various products such as ethanol, acetic acid, formic acid, lactic acid and succinic acid under anaerobic or microaerobic conditions. Are known.

Nucleic acids encoding enzymes utilized in the reverse adipate degradation pathway and the 6-aminocaproate or caprolactam synthesis pathway to produce E. coli strains engineered to produce 6-aminocaproate and/or caprolactam. is expressed in E. coli using well-known molecular biology techniques (see, eg, Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, paaJ (NP_415915.1), paaH (NP_415913.1) and maoC (NP_415905) encode succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase and 3-hydroxyadipyl-CoA dehydratase activities, respectively. .1) Clone the gene into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the bcd (NP_349317.1), etfAB (349315.1 and 349316.1) and sucCD (NP_415256.1 and AAC73823.1) genes encoding 5-carboxy-2-pentenoyl-CoA reductase and adipyl-CoA synthetase activities, respectively, were transferred to PA1. Clone into the pZA33 vector (Expressys, Ruelzheim, Germany) under the /lacO promoter. Finally, a third compatible acrl (YP_047869.1), gabT (NP_417148.1) and nylB (AAA24929.1) genes encoding CoA-dependent aldehyde dehydrogenase, transaminase and amidohydrolase activities were placed under the PA1/lacO promoter. Clone into plasmid pZS23. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with the kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for 6-aminocaproate and/or caprolactam synthesis.

The resulting genetically engineered organisms are cultured in glucose-containing media according to procedures well known in the art (see, eg, Sambrook et al., supra, 2001). 6-aminocaproate and caprolactam using methods well known in the art for measuring polypeptide expression or enzymatic activity, including, for example, Northern blots, PCR amplification of mRNA and immunoblots, etc. Confirm the expression of the synthetic gene. Confirm the enzymatic activity of the expressed enzyme using assays specific for the individual activity. Confirm the ability of engineered E. coli strains to produce 6-aminocaproate and/or caprolactam using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS) do.

Microbial strains engineered to have functional pathways for the synthesis of 6-aminocaproate and/or caprolactam are further enhanced by optimization for efficient utilization of the pathway. Briefly, engineered strains are evaluated to determine whether any of the exogenous genes are expressed at rate-limiting levels. For example, introduction of additional gene copy numbers increases expression for any enzymes expressed at low levels that can limit flux through the pathway.

Utilize metabolic modeling to optimize growth conditions to generate better producers.

Additionally, modeling is used to design gene knockouts that further optimize pathway utilization (e.g., U.S. Patent Publications Nos. 2002/0012939; 2003/0224363; 2004/0072723, 2003/0059792, 2002/0168654 and 2004/0009466 and US Pat. No. 7,127,379). The modeling analysis allows reliable prediction of the effects on cell growth of shifting metabolism towards more efficient production of 6-aminocaproate and/or caprolactam. One modeling method is the bilevel optimization approach OptKnock, which is applied to select gene knockouts that collectively result in better production of 6-aminocaproate and/or caprolactam (Burgard et al., Biotechnol Bioengineer. 84:647-657(2003)). Adaptive evolution can also be used, for example, to generate better producers of the products acetyl-CoA and succinyl-CoA intermediates. Adaptive evolution is carried out to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on those results, a series of subsequent modeling, genetic manipulation and adaptive evolution can be applied to the 6-aminocaproate and/or caprolactam producers to further increase production.

For large-scale production of 6-aminocaproate and/or caprolactam, the organisms are fermented using media known in the art to support the growth of the organisms under anaerobic conditions. Culture in a tank. Fermentation is carried out in batch, fed batch or continuous mode. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel. For example, the flask can be sealed with a septum and crimp cap. Microaerophilic conditions can also be utilized by providing small holes in the membrane for limited ventilation. H₂S.O.₄Maintain the pH of the medium at a pH of approximately 7 by adding an acid such as Growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and glucose uptake rate is determined by monitoring carbon source depletion over time. By-products such as undesirable alcohols, organic acids and residual glucose can be detected using, for example, Aminex® series HPLC columns (e.g. HPX- 87 series) (BioRad) (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

Example Describe about.

As described in Example VI, alpha-ketoadipate is a known intermediate in lysine biosynthesis that can be formed from alpha-ketoglutarate via homocitrate synthase, homoaconitase and homoisocitrate dehydrogenase. It is the body.

Alpha-ketoadipate can be converted to 2-hydroxyadipyl-CoA by two routes shown in Figure 9. Subsequently, 2-hydroxyadipyl-CoA can be dehydrated and reduced to adipyl-CoA, which can then be converted to adipate as shown in Figure 9.

The maximum yield of adipate from glucose via these routes is 0.67 mol/mol.

The conversion of alpha-ketoadipate to 2-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, an enzyme reported to be found in rat and human placenta (Suda et al., Arch. Biochem. Biophys. 176:610-620 (1976); Suda et al., Biochem. Biophys. Res. Commun. 77:586-591 (1977)). Alternatively, enzymes capable of reducing alpha-ketoglutarate to 2-hydroxyglutarate may also exhibit activity on alpha-ketoadipates that are one carbon atom longer. One such enzyme that possesses alpha-ketoglutarate reductase activity is serA of Escherichia coli (Zhao and Winkler, J. Bacteriol. 178(1)232-9 (1996)). Additional exemplary enzymes can be found in Arabidopsis thaliana (Ho et al., J. Biol. Chem. 274(1):397-402 (1999)) and Haemophilus influenzae.

<tables><img file="JP7370366B2_D0039.tif" /></tables>

Referring to FIG. 9, it is likely that 2-hydroxyadipate can be converted to 2-hydroxyadipyl-CoA by the synthetases, transferases, phosphotransadipyrases and kinases described in Example I. Alternatively, enzymes with 2-hydroxyglutarate CoA-transferase or glutaconate CoA-transferase activity are likely to be suitable for transferring the CoA moiety to 2-hydroxyadipate. One example of such an enzyme is encoded by the gctA and gctB genes of Acidaminococcus fermentans (Buckel et al., Eur. J. Biochem. 118(2):315-321 (1981); Mack et al. , Eur. J. Biochem.226(1):41-51(1994)). Similarly, as shown in Figure 9, synthetase, transferase or phosphotransadipyrase and kinase activity are required to convert alpha-ketoadipate to alpha-ketoadipyl-CoA. Conversion of alpha-ketoadipyl-CoA to 2-hydroxyadipyl-CoA can be performed by an alpha-hydroxyacyl-CoA dehydrogenase enzyme. Similar activity was reported in propionate-adapted E. coli cells, the extract of which catalyzes the oxidation of lactyl-CoA to form pyruvyl-CoA (Megraw et al., J. Bacteriol. 90(4): 984-988 (1965) ). Additional hydroxyacyl-CoA dehydrogenases are described in Example I.

<tables><img file="JP7370366B2_D0040.tif" /></tables>

Dehydration of 2-hydroxyadipyl-CoA to form 5-carboxy-2-pentenoyl-CoA can be carried out by 2-hydroxyacyl-CoA dehydratase. The 2-hydroxyglutaryl-CoA dehydratase system has been characterized in Acidaminococcus fermentans and requires both hgdA and hgdB subunits, as well as the activator protein hgdC for optimal activity. (Dutscho et al., Eur. J. Biochem. 181(3):741-746(1989); Locher et al., J. Mol. Biol. 307(1):297-308; Muller and Buckel, Eur. J. Biochem. 230(2):698-704(2001); Schweiger et al., Eur. J. Biochem. 169(2):441-448(1987)). This enzyme system is similar in mechanism to the lactoyl-CoA dehydratase from Clostridium propionicum (Hofmeister and Buckel, Eur. J. Biochem. 206(2):547-552(1992); Kuchta and Abeles, J. Biol. Chem. 260(24):13181-13189(1985)). Homologues of hgdA, hgdB and hgdC exist in several organisms.

<tables><img file="JP7370366B2_D0041.tif" /></tables>

The conversion of 5-carboxy-2-pentenoyl-CoA to adipate is carried out by the enzyme described in Example I.

The above description illustrates an exemplary adipate synthesis route via the 2-hydroxyadipyl-CoA pathway.

EXAMPLE Describe the generation of living organisms.

E. coli is used as the target organism to engineer the genes required for adipate synthesis (see Figure 9). E. coli provides a good host for producing non-naturally occurring microorganisms capable of producing adipate. E. coli is amenable to genetic manipulation and is capable of effectively producing various products such as ethanol, acetic acid, formic acid, lactic acid and succinic acid under anaerobic or microaerobic conditions. Are known.

To generate E. coli strains engineered to produce adipate, nucleic acids encoding enzymes utilized in the 2-hydroxyadipyl-CoA to adipate pathway were introduced into E. coli using well-known molecular biology techniques. (see, eg, Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the serA (NP_417388.1), gctA (Q59111) and gctB (Q59112) genes encoding 2-hydroxyadipate dehydrogenase and 2-hydroxyadipyl-CoA:acetyl-CoA transferase activities, respectively, were transferred to pZE13 under the PA1/lacO promoter. Cloning into vector (Expressys, Ruelzheim, Germany). In addition, the hgdA (P11569), hgdB (P11570) and hgdC (P11568) genes, each encoding 2-hydroxyadipyl-CoA dehydratase activity, are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. Additionally, the bcd (NP_349317.1), etfAB (349315.1 and 349316.1), and sucCD (NP_415256.1 and AAC73823.1) genes encoding 5-carboxy-2-pentenoyl-CoA reductase and adipyl-CoA synthetase activities were transferred to PA1/ Clone into the third compatible plasmid pZS23 under the lacO promoter. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with the kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for adipate synthesis.

The resulting genetically engineered organisms are cultured in glucose-containing media according to procedures well known in the art (see, eg, Sambrook et al., supra, 2001). 2-hydroxy for adipate synthesis using methods well known in the art for measuring polypeptide expression or enzymatic activity, including, for example, Northern blots, PCR amplification of mRNA and immunoblots, etc. Confirm expression of adipyl-CoA pathway genes. Confirm the enzymatic activity of the expressed enzyme using assays specific for the individual activity. The ability of the engineered E. coli strain to produce adipate is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional adipate synthesis pathway are further enhanced by optimization for efficient utilization of the pathway. Briefly, engineered strains are evaluated to determine whether any of the exogenous genes are expressed at rate-limiting levels. For example, introduction of additional gene copy numbers increases expression for any enzymes expressed at low levels that can limit flux through the pathway.

Utilize metabolic modeling to optimize growth conditions to generate better producers.

Additionally, modeling is used to design gene knockouts that further optimize pathway utilization (e.g., U.S. Patent Publications Nos. 2002/0012939; 2003/0224363; 2004/0072723, 2003/0059792, 2002/0168654 and 2004/0009466 and US Pat. No. 7,127,379). Modeling analysis allows reliable prediction of the effects on cell growth of shifting metabolism towards more efficient production of adipate. One modeling method is the bilevel optimization approach OptKnock, which is applied to select gene knockouts that collectively result in better production of adipate (Burgard et al., Biotechnol. Bioengineer. 84:647-657) 2003)). Adaptive evolution can also be used, for example, to generate better producers of alpha-ketoadipate intermediates or adipate products. Adaptive evolution is carried out to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058(2004); Alper et al., Science 314:1565-1568(2006)). Based on those results, a series of subsequent modeling, genetic manipulation and adaptive evolution can be applied to the adipate producers to further increase production.

For large-scale production of adipate, 2-hydroxyadipyl-CoA pathway-containing organisms are fermented using media known in the art to support the growth of the organisms under anaerobic conditions. Cultivate at Fermentation is carried out in batch, fed batch or continuous mode. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel. For example, the flask can be sealed with a septum and crimp cap. Microaerophilic conditions can also be utilized by providing small holes in the membrane for limited ventilation. H₂S.O.₄Maintain the pH of the medium at a pH of approximately 7 by adding an acid such as Growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and glucose uptake rate is determined by monitoring carbon source depletion over time. By-products such as undesirable alcohols, organic acids and residual glucose can be detected using, for example, Aminex® series HPLC columns (e.g. HPX- 87 series) (BioRad) (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

This example describes the production of an adipate-producing microbial organism containing the 2-hydroxyadipyl-CoA pathway.

EXAMPLE .

Various routes leading to the production of caprolactam, hexamethylene diamine (HMDA) or 6-aminocaproate from common central metabolites are described below. The first described pathway activates 6-aminocaproate to 6-aminocaproyl-CoA by a transferase or synthase enzyme (Figure 10, step Q or R), followed by 6-aminocaproyl-CoA. Requires spontaneous cyclization of CoA to form caprolactam (Figure 10, Step T). The second described pathway activates 6-aminocaproate to 6-aminocaproyl-CoA (Figure 10, Step Q or R), followed by reduction (Figure 10, Step U) and amination. (Figure 10, step V or W) to form the HMDA. Alternatively, 6-aminocaproic acid can be activated to 6-aminocaproyl-phosphate instead of 6-aminocaproyl-CoA. 6-Aminocaproyl-phosphate can spontaneously cyclize to form caprolactam. Alternatively, 6-aminocaproyl-phosphate can be reduced to 6-aminocaproate semialdehyde, which can then be converted to HMDA as shown in FIGS. 10 and 11. In both cases, the amination reaction must be carried out relatively quickly to minimize spontaneous formation of the 6-aminocaproate semialdehyde cyclic imine. Coupling or supporting the participating enzymes on a scaffold is a potentially attractive option for efficiently deriving the 6-aminocaproate semialdehyde intermediate from the reductase enzyme to the aminating enzyme.

Another option to minimize or even eliminate the formation of cyclic imines or caprolactam through the conversion of 6-aminocaproic acid to HMDA is to convert functional groups (e.g. acetyl, succinyl) to 6-aminocaproic acid. amine group to protect it from cyclization. This is similar to ornithine formation from L-glutamic acid in E. coli.

Specifically, glutamic acid is first converted to N-acetyl-L-glutamic acid by N-acetylglutamic acid synthase. N-acetyl-L-glutamic acid is then activated to N-acetylglutamyl-phosphate, which is reduced and transaminated to form N-acetyl-L-ornithine. The acetyl group is then removed from N-acetyl-L-ornithine by N-acetyl-L-ornithine deacetylase to form L-ornithine. Formation of glutamic acid-5-phosphate from glutamic acid followed by reduction to glutamic acid-5-semialdehyde results in (S)-1-pyrroline-5, a cyclic imine that is formed spontaneously from glutamic acid-5-semialdehyde. - Such a route is necessary because carboxylates are formed. When HMDA is formed from 6-aminocaproic acid, the steps are acetylation of 6-aminocaproic acid to acetyl-6-aminocaproic acid, activation of the carboxylic acid group with CoA or phosphate groups, reduction, amination and desorption. Acetylation may be included.

Note that 6-aminocaproate can be formed from a variety of starting molecules. For example, the carbon backbone of 6-aminocaproate can be derived from succinyl-CoA and acetyl-CoA, as shown in FIG. 10 and also described in FIGS. 2, 3, and 8. Alternatively, 6-aminocaproate can be derived from alpha-ketoadipate, converting alpha-ketoadipate to adipyl-CoA (see Figure 9) and adipyl-CoA as shown in Figure 10. It is converted to 6-aminocaproate as follows.

Figure 11 shows two further metabolic routes to 6-aminocaproate or 6-aminocapropyl-CoA starting from 4-aminobutyryl-CoA and acetyl-CoA. The first route condenses 4-aminobutyryl-CoA and acetyl-CoA to form 3-oxo-6-aminohexanoyl-CoA (step A), followed by reduction (step B), dehydration (step C) and reduction (Step D) to form 6-aminocaproyl-CoA. 6-Aminocaproyl-CoA can be converted to 6-aminocaproate by transferase (Step K), synthase (Step L) or hydrolase (Step M) enzymes. Alternatively, 6-aminocaproyl-CoA is converted to caprolactam by spontaneous cyclization (step Q) or to HMDA by its reduction (step N) and amination (step O or P). be able to. The second pathway, described in Figure 11, condenses 4-aminobutyryl-CoA and acetyl-CoA to form 3-oxo-6-aminohexanoyl-CoA (step A), which is then transferred by transferase ( Step E) requires conversion to 3-oxo-6-aminohexanoate by synthase (Step F) or hydrolase (Step G). The 3-oxo-6-aminohexanoate is then reduced (Step H), dehydrated (Step I) and reduced (Step J) to form 6-aminocaproate.

The starting molecule 4-aminobutyryl-CoA can be formed from a variety of common central metabolites. For example, glutamic acid can be decarboxylated to 4-aminobutyrate, which is then activated to 4-aminobutyryl-CoA by a CoA-transferase or synthase. Alternatively, succinic semialdehyde formed by reduction of succinyl-CoA or decarboxylation of alpha-ketoglutarate can be transaminated to 4-aminobutyrate prior to activation by CoA-transferase or synthase. 4-aminobutyryl-CoA can be formed. It is noted that 4-aminobutyryl-CoA and some of the intermediates in the 4-aminobutyryl-CoA to 6-aminocaproyl-CoA pathway can spontaneously cyclize to their corresponding lactams. Therefore, the addition of protective functional groups to some terminal amine groups of 4-aminobutyryl-CoA and/or amino-CoA intermediates can be used to minimize the formation of undesirable cyclic by-products. In this case, the same general set of transformations shown in Figure 11 applies, but two additional steps can be added to the pathway, for example an acetylase and a deacetylase.

All transformations shown in Figures 10-11 fall into the 12 general transformation categories shown in Table 8. Some biochemically characterized candidate genes in each category are described below. Specifically, genes that, when cloned and expressed, can be applied to catalyze the appropriate transformations in Figures 10-11 are mentioned.

Table 8. Enzyme types for converting succinyl-CoA, acetyl-CoA and/or 4-aminobutyryl-CoA to 6-aminocaproate, caprolactam and/or hexamethylenediamine.

The first three numbers of each label correspond to the first three enzyme commission number numbers representing common types of conversions independent of substrate specificity.

<tables><img file="JP7370366B2_D0042.tif" /></tables>

1.1.1.a Oxidoreductase. The four transformations shown in Figures 10 and 11 require oxidoreductases to convert ketone functional groups to hydroxyl groups. Step B in both Figures 10 and 11 involves converting 3-oxoacyl-CoA to 3-hydroxyacyl-CoA.

Step H in both Figures 1 and 2 involves converting the 3-oxoacid to a 3-hydroxy acid.

3-oxoacyl-CoAs, such as 3-oxoadipyl-CoA and 3-oxo-6-aminohexanoyl-CoA, and 3-hydroxy, such as 3-hydroxyadipyl-CoA and 3-hydroxy-6-aminohexanoyl-CoA, respectively. Exemplary enzymes that can be converted to acyl-CoA molecules include enzymes whose natural physiological role is fatty acid beta-oxidation or phenylacetate catabolism. For example, two fatty acid oxidation complex subunits in E. coli encoded by fadB and fadJ function as 3-hydroxyacyl-CoA dehydrogenases (Binstock et al., Methods Enzymol. 71:403-411 (1981)). Also, phaC in Pseudomonas putida U (Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)) and paaC in Pseudomonas fluorescens ST (Di Gennaro et al., Arch. Microbiol. 188:177-125 (2007)) catalyzed the reverse reaction of step B in Figure 10, i.e., the oxidation of 3-hydroxyadipyl-CoA to form 3-oxoadipyl-CoA, through the catabolism of phenylacetate or styrene. do. Note that reactions catalyzed by such enzymes are reversible. In addition, in E. coli paaH is close to other genes in the phenylacetate degradation operon (Nogales et al., Microbiology 153:357-365 (2007)) and paaH mutants are unable to grow on phenylacetate (Ismail et al., Eur.J Biochem. 270:3047-3054 (2003)), the E. coli paaH gene is presumed to encode 3-hydroxyacyl-CoA dehydrogenase.

<tables><img file="JP7370366B2_D0043.tif" /></tables>

Additional exemplary oxidoreductases capable of converting 3-oxoacyl-CoA molecules to their corresponding 3-hydroxyacyl-CoA molecules include 3-hydroxybutyryl-CoA dehydrogenase. The enzyme from Clostridium acetobutylicum encoded by hbd has been cloned and functionally expressed in E. coli (Youngleson et al., J. Bacteriol. 171:6800-6807 (1989)). Additional gene candidates include Hbd1 (C-terminal region) and Hbd2 (N-terminal region) in Clostridium kluyveri (Hillmer et al., FEBS Lett. 21:351-354 (1972)) and HSD17B10 in cattle (Wakil et al., J. Biol. Chem. 207:631-638 (1954)). Another gene candidate demonstrated to reduce acetoacetyl-CoA to 3-hydroxybutyryl-CoA is phbB from Zooglea lamigera (Ploux et al., Eur. J. Biochem. 174:177-182 (1988)) and phaB from Rhodobacter sphaeroides (Alber et al., Mol. Microbiol 61:297-309 (2006)). The former gene candidate is NADPH dependent, its nucleotide sequence was determined (Peoples et al., Mol. Microbiol 3:349-357 (1989)), and the gene was expressed in E. coli. Substrate specificity tests for the gene led to the conclusion that it can accept 3-oxopropionyl-CoA as substrate in addition to acetoacetyl-CoA (Ploux et al., supra).

<tables><img file="JP7370366B2_D0044.tif" /></tables>

Several similar enzymes were found in other species of Clostridium and Mettalospaera cedula (Berg et al., Science 318:1782-1786 (2007)).

<tables><img file="JP7370366B2_D0045.tif" /></tables>

Various alcohol dehydrogenases convert 3-oxoadipate to 3-hydroxyadipate (Step H, Figure 10) or 3-oxo-6-aminohexanoate to 3-hydroxy-6-aminohexanoate. (Step H, Figure 11). Two such enzymes capable of converting oxoacids to hydroxy acids are encoded by the malate dehydrogenase (mdh) and lactate dehydrogenase (ldhA) genes in E. coli. In addition, lactate dehydrogenase from Ralstonia eutropha has been shown to exhibit high activity towards substrates of various chain lengths such as lactate, 2-oxobutyrate, 2-oxopentanoate and 2-oxoglutarate. (Steinbuchel et al., Eur. J. Biochem. 130:329-334 (1983)). The conversion of alpha-ketoadipate to alpha-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, an enzyme reported to be found in rat and human placenta (Suda et al., Arch. Biochem. Biophys. 176:610-620 (1976); Suda et al., Biochem. Biophys. Res. Commun. 77:586-591 (1977)). A further candidate for these steps is mitochondrial 3-hydroxybutyrate dehydrogenase (bdh) from the human heart, which has been cloned and characterized (Marks et al., J. Biol. Chem. 267:15459-15463 (1992)). . This enzyme is a dehydrogenase that acts on 3-hydroxy acids. Other exemplary alcohol dehydrogenases are Clostridium beijerinckii (Ismaiel et al., J. Bacteriol. 175:5097-5105 (1993)) and Thermoanaerobacter brockii ((Lamed et al., Biochem. J. 195:183-190 (1981) ); converting acetone to isopropanol as shown in Peretz et al., Biochemistry 28:6549-6555 (1989)).

<tables><img file="JP7370366B2_D0046.tif" /></tables>

1.2.1.b Oxidoreductase (acyl-CoA to aldehyde). Conversion of adipyl-CoA to adipate semialdehyde (Step N, Figure 10) and conversion of 6-aminocaproyl-CoA to 6-aminocaproate semialdehyde (Step U, Figure 10; Step N, Figure 11) requires an acyl-CoA dehydrogenase capable of reducing acyl-CoA to its corresponding aldehyde. Exemplary genes encoding such enzymes are Acinetobacter calcoaceticus acr1, which encodes fatty acyl-CoA reductase (Reiser et al., J. Bacteriology 179:2969-2975 (1997)), Acinetobacter sp. M-1 Fatty acyl-CoA reductase (Ishige et al., Appl. Environ. Microbiol. 68:1192-1195 (2002)), and CoA and NADP-dependent succinic semialdehyde dehydrogenase encoded by the sucD gene in Clostridium kluyveri (Sohling et al., J. Bacteriol. 178:871-880 (1996)). Porphyromonas gingivalis sucD is another succinic semialdehyde dehydrogenase (Takahashi et al., J. Bacteriol. 182:4704-4710 (2000)). The enzyme that acylates acetaldehyde dehydrogenase in Pseudomonas species encoded by bphG is yet another candidate as it has been demonstrated to oxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde and formaldehyde (Powlowski et al. , J Bacteriol. 175:377-385(1993)). The enzyme encoded by adhE in Leuconostoc mesenteroides was shown to oxidize the branched-chain compound isobutyraldehyde to isobutyryl-CoA, in addition to reducing acetyl-CoA to ethanol (Kazahaya et al., J .Gen. Appl. Microbiol. 18:43-55 (1972); Koo et al., Biotechnol Lett. 27:505-510 (2005)).

<tables><img file="JP7370366B2_D0047.tif" /></tables>

A further enzyme type that converts acyl-CoA to its corresponding aldehyde is malonyl-CoA reductase, which converts malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is the key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle in thermoacidophilic archaea (Berg et al., supra; Thauer RK, Science 318:1732-1733 (2007 )). The enzyme utilizes NADPII as a cofactor and has been characterized in Metallospaera and Sulfolobus species (Alber et al., J. Bacteriol. 188:8551-8559 (2006); Hugler et al., J. Bacteriol. 184:2404-2410(2002)). The enzyme is encoded by Msed_0709 in Metallospaera cedula (Alber et al., supra; Berg et al., supra). The gene encoding malonyl-CoA reductase from Sulfolobus tokodaiii was cloned and expressed heterologously in E. coli (Albert et al., supra). This enzyme was also shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde (WO 2007/141208). The aldehyde dehydrogenase functionality of these enzymes is similar to bifunctional dehydrogenases from green non-sulfur bacteria, but there is little sequence similarity. Both malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate-semialdehyde dehydrogenase, an enzyme that catalyzes the reduction and simultaneous dephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde. Additional gene candidates can be found by sequence homology with proteins in other organisms, including Sulfolobus solfataricus and Sulfolobus acidocaldarius, and are shown below. Another candidate for a CoA-acylated aldehyde dehydrogenase is the ald gene from Clostridium beijerincki (Toth et al., Appl Environ Microbiol 65:4973-4980 (1999)). This enzyme was reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to eutE, which encodes the acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth et al., supra).

<tables><img file="JP7370366B2_D0048.tif" /></tables>

1.3.1.a Oxidoreductase acting on CH-CH donors. Referring to Figure 10, Step D refers to the conversion of 5-carboxy-2-pentenoyl-CoA to adipyl-CoA by 5-carboxy-2-pentenoyl-CoA reductase. Referring to Figure 11, Step D refers to the conversion of 6-aminohex-2-enoyl-CoA to 6-aminocaproyl-CoA. Enoyl-CoA reductase enzyme is a suitable enzyme for both conversions. One exemplary enoyl-CoA reductase is the gene product of bcd from Clostridium acetobutylicum, which necessarily catalyzes the reduction of crotonyl-CoA to butyryl-CoA (Boynton et al., J Bacteriol. 178:3015- 3024(1996); Atsumi et al., Metab. Eng. 2008 10(6):305-311(2008)(Epub Sep. 14, 2007). The activity of this enzyme can be increased by expressing bcd along with expression of the Clostridium acetobutylicum etfAB gene, which encodes an electron-transporting flavoprotein. A further candidate for the enoyl-CoA reductase step is mitochondrial enoyl-CoA reductase from Euglena gracilis (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). A construct derived from this sequence was cloned into E. coli after removal of its mitochondrial targeting leader sequence to yield the active enzyme (Hoffmeister et al., supra). This approach to expressing eukaryotic genes, especially genes with leader sequences capable of directing the gene product to specific subcellular compartments in prokaryotes, is well known to those skilled in the art.

A close homolog of this gene from the prokaryotic Treponema denticola, TDE0597, is the third enoyl-CoA reductase cloned and expressed in E. coli (Tucci et al., FEBS Letters 581:1561-1566 (2007)).

<tables><img file="JP7370366B2_D0049.tif" /></tables>

Step J of Figures 10 and 11 requires the 2-enoate reductase enzyme. 2-enoate reductase (EC 1.3.1.31) is known to catalyze the NAD(P)H-dependent reduction of a wide variety of α,β-unsaturated carboxylic acids and aldehydes (Rohdich et al., J. Biol.Chem. 276:5779-5787(2001)). 2-enoate reductase is an enzyme in several species of Clostridium, including Clostridium tyrobutylicum and Clostridium thermoaceticum (now called Moorella thermoaceticum) (Giesel et al., Arch. Microbiol. 135:51-57 (1983)) (Rohdich et al., supra). In the published genome sequence of Saccharomyces kluyberg, nine coding sequences for enoate reductase were reported, one of which was characterized (Seedorf et al., Proc. Natl. Acad. Sci. USA 105:2128-2133(2008)). The enr genes from both Clostridium tyrobutylicum and Clostridium thermoaceticum have been cloned and sequenced and show 59% identity to each other. The former gene was also found to have approximately 75% similarity to the characterized Saccharomyces kluybergi genes (Giesel et al., supra). Based on these sequence results, enr was reported to be highly similar to E. coli dienoyl CoA reductase (fadH) (Rohdich et al., supra). The C. thermoaceticum enr gene has also been expressed in an enzymatically active form in E. coli (Rohdich et al., supra).

<tables><img file="JP7370366B2_D0050.tif" /></tables>

1.4.1.a Oxidoreductase that acts on amino acids. Figure 10 shows two reductive aminations. Specifically, Step P of Figure 10 involves the conversion of adipate semialdehyde to 6-aminocaproate, and Step W of Figure 10 involves the conversion of 6-aminocaproate semialdehyde to hexamethylene diamine. It takes. The latter transformation is also required in step P of FIG.

Most oxidoreductases acting on amino acids catalyze the oxidative deamination of alpha-amino acids with NAD+ or NADP+ acceptors, but the reaction is typically reversible. Exemplary oxidoreductases that act on amino acids include glutamate dehydrogenase (deamination) encoded by gdhA, leucine dehydrogenase (deamination) encoded by ldh, and aspartate dehydrogenase (deamination) encoded by nadX. ). gdhA gene products from E. coli (McPherson et al., Nucleic. Acids Res. 11:5257-5266 (1983); Korber et al., J. Mol. Biol. 234:1270-1273 (1993)), gdh from Thermotoga maritima (Kort et al., Extremophiles 1:52-60(1997); Lebbink et al., J. Mol. Biol. 280:287-296(1998); Lebbink et al., J. Mol. Biol. 289:357-369 (1999)) and gdhA1 from Halobacterium salinarum (Ingoldsby et al., Gene. 349:237-244 (2005)), which reversibly interconverts glutamate to 2-oxoglutarate and ammonia. catalyze, and each has an advantageous effect on NADP(H), NAD(H), or both. The ldh gene from Bacillus cereus encodes the LeuDH protein, which has a wide range of substrates including leucine, isoleucine, valine and 2-aminobutanoate (Stoyan et al., J. Biotechnol 54:77-80 (1997); Ansorge et al. , Biotechnol Bioeng. 68:557-562(2000)). The nadX gene from Thermotoga maritima, which encodes for aspartate dehydrogenase, is involved in the biosynthesis of NAD (Yang et al., J. Biol. Chem. 278:8804-8808 (2003)).

<tables><img file="JP7370366B2_D0051.tif" /></tables>

Lysine 6-dehydrogenase (deamination), encoded by the lysDH gene, catalyzes the oxidative deamination of the ε-amino group of L-lysine to form 2-aminoadipate-6-semialdehyde, which is then non-enzymatically cyclized to form Δ¹-piperidine-6-carboxylate (Misono et al., J. Bacteriol. 150:398-401 (1982)). Exemplary enzymes include Geobacillus stearothermophilus (Heydari et al., Appl Environ. Microbiol 70:937-942 (2004)), Agrobacterium tumefaciens (Hashimoto et al., J Biochem 106:76-80 (1989); Misono et al. , supra) and Achromobacter denitrificans (Ruldeekulthamrong et al., BMB.Rep. 41:790-795 (2008)). Such enzymes are particularly good candidates for converting adipate semialdehyde to 6-aminocaproate due to the structural similarities between adipate semialdehyde and 2-aminoadipate-6-semialdehyde.

<tables><img file="JP7370366B2_D0052.tif" /></tables>

2.3.1.b Acyltransferases. Referring to Figure 10, Step A comprises 3-oxoadipyl-CoA thiolase, or similarly succinyl-CoA:acetyl-CoA acyltransferase (β-ketothiolase). pcaF in Pseudomonas strain B13 (Kaschabek et al., J. Bacteriol. 184:207-215 (2002)), phaD in Pseudomonas putida U (Olivera et al., supra), paaE in Pseudomonas fluorescens ST (Di Gennaro et al., supra) and from E. coli. paaJ (Nogales et al., supra) catalyzes the conversion of 3-oxoadipyl-CoA to succinyl-CoA and acetyl-CoA through the decomposition of aromatic compounds such as phenylacetate or styrene. Since β-ketothiolase enzymes catalyze reversible conversions, these enzymes can be employed in the synthesis of 3-oxoadipyl-CoA. For example, the ketothiolase phaA from Ralstonia eutropha combines two molecules of acetyl-CoA to form acetoacetyl-CoA (Sato et al., J Biosci Bioeng 103:38-44 (2007)). Similarly, β-ketothiolase (bktB) was reported to catalyze the condensation of acetyl-CoA and propionyl-CoA to form β-ketovaleryl-CoA in Ralstonia eutropha (Slater et al., J. Bacteriol. 180:1979-1987(1998)). In addition to the probability of possessing 3-oxoadipyl-CoA thiolase activity, in their native form or when properly manipulated, all such enzymes condense 4-aminobutyryl-CoA and acetyl-CoA. to form 3-oxo-6-aminohexanoyl-CoA (Step A, Figure 11).

<tables><img file="JP7370366B2_D0053.tif" /></tables>

2-amino-4-oxopentanoate (AKP) thiolase or AKP thiolase (AKPT) enzymes are further candidates for carrying out step A in FIGS. 10 and 11. AKPT is a pyridoxalphosphate-dependent enzyme that participates in ornithine degradation in Clostridium sticklandii (Jeng et al., Biochemistry 13:2898-2903 (1974); Kenklies et al., Microbiology 145:819-826 (1999)). The gene cluster encoding the alpha and beta subunits (or-2 (ortA) and or-3 (ortB)) of AKPT was recently identified and the biochemical properties of the enzyme were characterized (Fonknechten et al., J. Bacteriol. . In Press (2009)). The enzyme is capable of acting in both directions and naturally reacts with the D-isomer of alanine. Although AKPT from Clostridium sticklandii has been characterized, its protein sequence has not yet been published. Enzymes with a high degree of sequence homology are found in Clostridium difficile, Alcalophilus metalliregigens QYF, Thermoanaerobacter sp.

<tables><img file="JP7370366B2_D0054.tif" /></tables>

2.6.1.a Aminotransferases. Step O of Figures 10 and 11 and Step V of Figure 10 require transamination of a 6-aldehyde to an amine. These conversions can be catalyzed by gamma-aminobutyrate transaminases (GABA transaminases). One E. coli GABA transaminase, encoded by gabT, transfers an amino group from glutamic acid to the terminal aldehyde of succinyl semialdehyde (Bartsch et al., J. Bacteriol. 172:7035-7042 (1990)). The puuE gene product catalyzes another 4-aminobutyrate transaminase in E. coli (Kurihara et al., J. Biol. Chem. 280:4602-4608 (2005)).

GABA transaminase in mouse, Pseudomonas fluorescens and wild boar was shown to react with 6-aminocaproic acid (Cooper, Methods Enzymol. 113:80-82 (1985); Scott et al., J. Biol. Chem. 234 :932-936(1959)).

<tables><img file="JP7370366B2_D0055.tif" /></tables>

Additional enzyme candidates include putrescine aminotransferase or other diamine aminotransferases. Such enzymes are particularly well suited for carrying out the conversion of 6-aminocaproate semialdehyde to hexamethylene diamine. E. coli putrescine aminotransferase is encoded by the ygjG gene, and the purified enzyme was also capable of transaminating cadaverine and spermidine (Samsonova et al., BMCMicrobiol 3:2 (2003)). In addition, activity of this enzyme toward amino receptors other than 1,7-diaminoheptane and 2-oxoglutarate (e.g., pyruvate, 2-oxobutanoate) has been reported (Samsonova et al., supra; Kim, KH, J Biol Chem 239:783-786(1964)). Putrescine aminotransferase, which has higher activity towards pyruvate as an amino receptor than alpha-ketoglutarate, is the spuC gene of Pseudomonas aeruginosa (Lu et al., J Bacterial 184:3765-3773 (2002)).

<tables><img file="JP7370366B2_D0056.tif" /></tables>

Additional candidate enzymes include beta-alanine/alpha-ketoglutarate aminotransferase, which generates malonate semialdehyde from beta-alanine (WO 08027742). The gene product of SkPYD4 in Saccharomyces kluyveri was also shown to preferentially use beta-alanine as the amino group donor (Andersen et al., FEBSJ. 274:1804-1817(2007)). SkUGA1 encodes a homologue of Saccharomyces cerevisiae GABA aminotransferase (Ramos et al., Eur. (Andersen et al., supra). 3-Amino-2-methylpropionate transaminase catalyzes the conversion of methylmalonate semialdehyde to 3-amino-2-methylpropionate. This enzyme has been characterized in rat and boar and is encoded by Abat (Tamaki et al., Methods Enzymol, 324:376-389 (2000)).

<tables><img file="JP7370366B2_D0057.tif" /></tables>

2.8.3.a Coenzyme A transferase. CoA transferase catalyzes a reversible reaction that involves the transfer of a CoA moiety from one molecule to another. For example, step E of Figure 10 is catalyzed by 3-oxoadipyl-CoA transferase. In this step, 3-oxoadipate is formed by transfer of a CoA group from 3-oxoadipyl-CoA to succinate, acetate or another CoA acceptor. Step E of Figure 11 requires transfer of the CoA moiety from another 3-oxoacyl-CoA to 3-oxo-6-aminohexanoyl-CoA. One candidate enzyme for these steps is the two-unit enzyme encoded by pcaI and pcaJ in Pseudomonas that was shown to have 3-oxoadipyl-CoA/succinate transferase activity (Kaschabek et al., supra). ). A similar enzyme based on homology is Acinetobacter species ADP1 (Kowalchuk et al., Gene 146:23-30 (1994)) and Streptomyces coelicolor. Additional exemplary succinyl-CoA:3:oxoacid-CoA transferases include H. pylori (Corthesy-Theulaz et al., J. Biol. Chem. 272:25659-25667 (1997)) and Bacillus subtilis (Stols et al., Protein. Expr. .Purif. 53:396-403 (2007)).

<tables><img file="JP7370366B2_D0058.tif" /></tables>

The 3-oxoacyl-CoA transferase that can utilize acetate as a CoA receptor is the acetoacetyl-CoA transferase encoded by the E. coli atoA (alpha subunit) and atoD (beta subunit) genes (Vanderwinkel et al., Biochem. .Biophys.Res Commun. 33:902-908(1968);Korolev et al., Acta Crystallogr.D Biol Crystallogr. 58:2116-2121(2002)). This enzyme has a wide variety of branched and linear forms, including isobutyrate (Matthies et al., Appl EnvironMicrobiol 58:1435-1439 (1992)), valerate (Vanderwinkel et al., supra), and butanoate (Vanderwinkel et al., supra). It was shown to transfer the CoA moiety from the acyl-CoA substrate to the acetate. Similar enzymes include Corynebacterium glutamicum ATCC 13032 (Duncan et al., Appl Environ Microbiol 68:5186-5190 (2002)), Clostridium acetobutylicum (Cary et al., Appl Environ Microbiol 56:1576-1583 (1990)) and Clostridium - Present in Saccharoperbutylacetonicum (Kosaka et al., Biosci.Biotechnol Biochem.71:58-68 (2007)).

<tables><img file="JP7370366B2_D0059.tif" /></tables>

The enzymes also have the desired activity towards adipyl-CoA and adipate (Figure 10, Step K) or 6-aminocaproate and 6-aminocaproyl-CoA (Figure 10, Step Q; Figure 2, Step K). can demonstrate. However, further exemplary transferase candidates are provided by the clostridium kluyveri cat1, cat2 and cat3 gene products, which have been shown to exert succinyl-CoA, 4-hydroxybutyryl-CoA and butyryl-CoA transferase activities, respectively. catalyzed (Seedorf et al., supra; Sohling et al., Eur. J Biochem. 212:121-127 (1993); Sohling et al., J Bacteriol. 178:871-880 (1996)).

<tables><img file="JP7370366B2_D0060.tif" /></tables>

The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from the anaerobic bacterium Acidaminococcus fermentans reacts with the diacids glutaconyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS Lett. 405:209 -212(1997)). The genes encoding this enzyme are gctA and gctB. This enzyme has small but detectable activity against other CoA derivatives including glutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckel et al., Eur. J. Biochem. 118:315-321(1981)). This enzyme was cloned and expressed in E. coli (Mack et al., Eur. J. Biochem. 226:41-51 (1994)).

<tables><img file="JP7370366B2_D0061.tif" /></tables>

3.1.2.a Thiol ester hydrolase (CoA specificity). Some eukaryotic acetyl-CoA hydrolases have broad substrate specificity and therefore include 3-oxoadipyl-CoA, adipyl-CoA, 3-oxo-6-aminohexanoyl-CoA or 6-aminocaproyl-CoA. (steps G and M in Figures 10 and 11). For example, enzymes from rat brain (Robinson et al., Biochem. Biophys. Res. Commun. 71:959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA and malonyl-CoA.

<tables><img file="JP7370366B2_D0062.tif" /></tables>

Additional hydrolase enzymes include 3-hydroxyisobutyryl-CoA hydrolase, which has been shown to efficiently catalyze the conversion of 3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate during valine degradation. (Shimomura et al., J Biol Chem. 269:14248-14253 (1994)). Genes encoding this enzyme include hibch of rat (Shimomura et al., supra; Shimomura et al., Methods Enzymol. 324:229-240 (2000)) and Homo sapiens (Shimomura et al., supra). Candidate genes based on sequence homology include hibch from Saccharomyces cerevisiae and BC_2292 from Bacillus cereus.

<tables><img file="JP7370366B2_D0063.tif" /></tables>

Yet another candidate hydrolase is the human dicarboxylic acid thioesterase acot8 (Westin et al., J.Biol.Chem. 280 :38125-38132 (2005)) as well as the near E. coli homolog tesB (Naggert et al., J Biol Chem 266:11044-11050 (1991)), which can also hydrolyze a wide range of CoA thiol esters. A similar enzyme was also characterized in rat liver (Deana R., Biochem Int 26:767-773 (1992)).

<tables><img file="JP7370366B2_D0064.tif" /></tables>

Other promising E. coli thiol ester hydrolases include tesA (Bonner et al., J Biol Chem 247:3123-3133 (1972)), ybgC (Kuznetsova et al., FEMS Microbiol Rev 29:263-279 (2005); Zhuang et al. FEBS Lett 516:161-163(2002)), paaI (Song et al., J Biol Chem 281:11028-11038(2006)) and ybdB (Leduc et al., J Bacteriol 189:7112-7126(2007)) gene products can be mentioned.

<tables><img file="JP7370366B2_D0065.tif" /></tables>

6.3.1.a/6.3.2.a Amide synthase/peptide synthase. Direct conversion of 6-aminocaproate to caprolactam (Step S, Figure 10; Step R, Figure 11) requires the formation of an intramolecular peptide bond. The ribosome, which assembles amino acids into proteins during translation, is nature's most abundant peptide bond-forming catalyst. Nonribosomal peptide synthetases are messenger mRNA-free peptide bond-forming catalysts (Schwarzer et al., Nat Prod. Rep. 20:275-287 (2003)). Additional enzymes capable of forming peptide bonds include acyl-CoA synthetase from Pseudomonas chlororaphis (Abe et al., J Biol Chem 283:11312-11321 (2008)), gamma-glutamylputrescine synthetase from E. coli (Kurihara et al., J Biol Chem 283:19981-19990 (2008)) and beta-lactam synthetase from Streptomyces clavuligeras (Bachmann et al., Proc Natl Acad Sci USA 95:9082-9086(1998);Bachmann et al., Biochemistry 39:11187-11193(2000);Miller et al., Nat Struct.Biol 8:684-689(2001);Miller et al., Proc Natl Acad Sci USA 99:14752- 14757 (2002); Tahlan et al., Antimicrob. Agents. Chemother. 48:930-939 (2004)).

<tables><img file="JP7370366B2_D0066.tif" /></tables>

4.2.1.a Hydrolyase. Most dehydratases catalyze the α,β-removal of water. This involves activation of the α-hydrogen by an electron-withdrawing carbonyl, carboxylate or CoA-thiol ester group and removal of the hydroxyl group from the β position. Enzymes that exhibit activity toward substrates with electron-withdrawing carboxylate groups dehydrate 3-hydroxyadipate (Figure 10, Step I) or 3-hydroxy-6-aminohexanoate (Figure 11, Step I) is an excellent candidate for

For example, fumarase enzymes necessarily catalyze the reversible dehydration of malate to fumarate. E. coli has three fumarases that are regulated by growth conditions: FumA, FumB and FumC. FumB is oxygen sensitive and active only under anaerobic conditions. FumA is active under microaerobic conditions and FumC is an enzyme active only in aerobic growth (Tseng et al., J Bacteriol 183:461-467 (2001); Woods et al., Biochim Biophys Acta 954:14 -26 (1988); Guest et al., J Gen Microbiol 131:2971-2984 (1985)). Additional enzyme candidates include Campylobacter jejuni (Smith et al., Int. )) and rat (Kobayashi et al., J Biochem. 89:1923-1931 (1981)). Similar enzymes with high sequence homology include fum1 from Arabidopsis and fumC from Corynebacterium glutamicum. MmcBC fumarase from Perotomaculum thermopropionicum is another type of fumarase with two subunits (Shimoyama et al., FEMS Microbiol Lett 270:207-213 (2007)).

<tables><img file="JP7370366B2_D0067.tif" /></tables>

Two additional dehydratase candidates were enzymes investigated for their role in nicotinate catabolism in Eubacterium barkeri (formerly Clostridium barkeri) (Alhapel et al., Proc Natl Acad Sci U SA 103:12341-6 (2006)). 2-(hydroxymethyl)glutarate dehydratase and dimethylmalate hydratase. 2-(Hydroxymethyl)glutarate dehydrase is a [4Fe-4S]-containing enzyme that dehydrates 2-(hydroxymethyl)glutarate to 2-methylene-glutarate. This enzyme is encoded by hmd in Eubacterium barkeri (Alhapel et al., supra). Similar enzymes with high sequence homology are found in Bacteroides capirosus, Anaerotrunchus coryhominis and Natranaerobius thermophilus. These enzymes are homologous to the alpha and beta subunits of [4Fe-4S]-containing bacterial serine dehydratases (eg, the enzymes encoded by tdcG, sdhB and sdaA).

<tables><img file="JP7370366B2_D0068.tif" /></tables>

Dimethylmalate hydratase (EC 4.2.1.85) is a reversible Fe hydratase in the aconitase family that dehydrates dimethylmaeate to form (2R,3S)-2,3-dimethylmalate.²⁺It is a dependent and oxygen-sensitive enzyme. This enzyme is encoded by dmdAB in Eubacterium barkeri (Alhapel et al., supra; Kollmann-Koch et al., Hoppe Seylers. Z. Physiol Chem. 365:847-857 (1984)).

<tables><img file="JP7370366B2_D0069.tif" /></tables>

A further enzyme candidate is 2-methylmalate dehydratase, also called citramalate hydrolyase, which is a reversible hydrolyase that catalyzes the alpha, beta removal of water from citramalate to form mesaconate. This enzyme was purified and characterized in Clostridium tetanomorphum (Wang et al., J Biol. Chem. 244:2516-2526 (1969)). The activity of this enzyme has also been detected in some bacteria in the genera Citrobacter and Morganella in the context of the glutamate degradation VI pathway (Kato et al., Arch. Microbiol 168:457-463 (1997)). The gene encoding this enzyme has not been identified in any organism to date.

Enzymes that exhibit activity against substrates with an electron-withdrawing CoA-thiol ester group adjacent to the α-hydrogen are either 3-hydroxyadipyl-CoA (Figure 10, Step C) or 3-hydroxy-6-aminohexanoyl - is an excellent candidate for dehydrating CoA (Figure 11, Step C). The enoyl-CoA hydratases phaA and phaB of Pseudomonas putida are thought to carry out double bond hydroxylation through phenylacetate catabolism (Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998) ). paaA and paaB from Pseudomonas fluorescens catalyze similar transformations (Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)). Finally, some E. coli genes are known as maoC (Park et al., J Bacteriol. 185:5391-5397(2003)), paaF(Ismail et al., supra; Park et al., Appl.Biochem.Biotechnol 113-116:335-346(2004); Park et al., Biotechnol Bioeng 86:681-686(2004) ) and paaG (Ismail et al., supra; Park et al., Appl.Biochem.Biotechnol 113-116:335-346 (2004); Park et al., Biotechnol Bioeng 86:681-686 (2004)) was shown to exhibit enoyl-CoA hydratase function. The crotonase enzyme is a further candidate for dehydrating the necessary 3-hydroxyacyl-CoA molecules shown in FIGS. 10 and 11. These enzymes are required for n-butanol formation in several organisms, especially Clostridia species, and 3-hydroxypropionate/4-in thermophilic acidic archaea of the genera Sulphorobus, Acidianus, and Metallospaera. Also includes one step of the hydroxybutyrate cycle. Exemplary genes encoding crotonase enzymes are Clostridium acetobutylicum (Boynton et al., supra), Clostridium kluyveri (Hillmer et al., FEBS Lett. 21:351-354 (1972)) and Metallospaera cedula (Berg et al., supra), but the sequence of the latter gene is unknown. Enoyl-CoA hydratases, involved in fatty acid beta-oxidation and/or metabolism of various amino acids, can also catalyze the hydration of crotonyl-CoA, forming 3-hydroxybutyryl-CoA (Roberts et al., Arch. Microbiol. 117:99-108 (1978); Agnihotri et al., Bioorg.Med.Chem. 11:9-20 (2003); Conrad et al., J Bacteriol. 118:103-111 (1974)).

<tables><img file="JP7370366B2_D0070.tif" /></tables>

6.2.1.a Acid thiol ligase. Steps F, L, and R of Figure 10 and steps F and L of Figure 11 require an acid-thiol ligase or synthetase function (ligase, synthetase, and synthase are used interchangeably herein and refer to the same enzyme class). ). An exemplary gene encoding an enzyme likely to perform these conversions includes the sucCD gene of E. coli, which naturally forms the succinyl-CoA synthetase complex. This enzyme complex naturally catalyzes the formation of succinyl-CoA from succinate with simultaneous consumption of one ATP, a reaction that is reversible in vivo (Buck et al., Biochem. 24:6245-6252 (1985)). Due to the structural similarity of succinate and adipate, ie, both are linear dicarboxylic acids, it is reasonable to expect some activity of the sucCD enzyme towards adipyl-CoA.

<tables><img file="JP7370366B2_D0071.tif" /></tables>

Further exemplary CoA-ligases include rat dicarboxylate CoA ligase, whose sequence has not yet been characterized (Vamecq et al., Biochemical Journal 230:683-693 (1985)), two characterized from Penicillium chrysogenum phenylacetate-CoA ligase (Lamas-Maceiras et al., Biochem. J. 395, 147-155 (2005); Wang et al., Biochem. Biophy. Res. Commun. 360(2):453-458 (2007) )), phenylacetate-CoA ligase from Pseudomonas putida (Martinez-Bianco et al., J. Biol. Chem. 265:7084-7090 (1990)) and 6-carboxyhexanoate-CoA ligase from Bacillus subtilis (Bower et al., J. Bacteriol. 178(14):4122-4130(1996)). Additional candidate enzymes include mouse (Hasegawa et al., Biochim Biophys Acta 1779:414-419 (2008)) and Homo sapiens (Ohgami et al., Biochem Pharmacol. 65:989-994 (2003)).

<tables><img file="JP7370366B2_D0072.tif" /></tables>

ADP-forming acetyl-CoA synthetase (ACD, EC6.2.1.13) is another candidate enzyme that couples the conversion of acyl-CoA esters to their corresponding acids with the simultaneous synthesis of ATP. Several enzymes with broad substrate specificity have been described in the literature. ACD I from Archaeoglobus fulgidus, encoded by AF1211, contains a wide variety of compounds including acetyl-CoA, propionyl-CoA, butyryl-CoA, acetate, propionate, butyrate, isobutyrate, isovalerate, succinate, fumarate, phenylacetate, indole acetate. It has been shown to act on linear and branched substrates (Musfeldt et al., J Bacteriol 184:636-644 (2002)). An enzyme from Haloarchula marismortui (noted as succinyl-CoA synthetase) was shown to accept propionate, butyrate and branched chain acids (isovalerate and isobutyrate) as substrates and to act in the forward and reverse directions. (Brasen et al., Arch Microbiol 182:277-287(2004)). The ACD encoded by the hyperthermophilic crenarchaeon, PAE3250 from Pyrobaculum aerophyllum, is the most of all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA. It exhibited a wide substrate range (Brasen et al., supra). Enzymes from Archaeoglobus fulgidus, Haloarchula marismortii, and Pyrobaculum aerophyllum have all been cloned, functionally expressed, and characterized in E. coli (Musfeldt et al., supra; Brasen et al., supra).

<tables><img file="JP7370366B2_D0073.tif" /></tables>

Yet another option is to employ a set of enzymes with net ligase or synthetase activity. For example, the phosphotransadipyrase and adipate kinase enzymes are catalyzed by the buk1, buk2 and ptb gene products from Clostridium acetobutylicum (Walter et al., Gene 134:107-111 (1993); Huang et al., J. Mol. Microbiol. Biotechnol. 2:33-38(2000)). The ptb gene encodes an enzyme that can convert butyryl-CoA to butyryl-phosphate, which is then converted to butyrate via one of the buk gene products simultaneously with the production of ATP.

<tables><img file="JP7370366B2_D0074.tif" /></tables>

Spontaneous cyclization without the need for enzymes. 6-Aminocaproyl-CoA spontaneously cyclizes to caprolactam and does not require a dedicated enzyme for this step. A similar spontaneous cyclization is observed with 4-aminobutyryl-CoA to form pyrrolidinone (Ohsugi et al., J BiolChem 256:7642-7651 (1981)).

EXAMPLE The production of microbial organisms capable of producing 6-aminocaproic acid from aminobutyryl-CoA is described.

E. coli is used as the target organism to engineer the 6-aminocaproic acid pathway shown in Figure 11, starting from acetyl-CoA and 4-aminobutyryl-CoA. E. coli provides a good host for producing non-naturally occurring microorganisms capable of producing 6-aminocaproic acid. E. coli is amenable to genetic manipulation and is capable of effectively producing various products such as ethanol, acetic acid, formic acid, lactic acid and succinic acid under anaerobic or microaerobic conditions. Are known.

To generate E. coli strains engineered to produce 6-aminocaproic acid, nucleic acids encoding the necessary enzymes are expressed in E. coli using well-known molecular biology techniques (e.g., Sambrook, supra, 2001 ; see Ausubel, supra, 1999). In particular, paaJ (NP_415915.1) encoding 3-oxo-6-aminohexanoyl-CoA thiolase, 3-oxo-6-aminohexanoyl-CoA reductase, and 3-hydroxy-6-aminohexanoyl-CoA dehydratase activities, respectively. ), paaH (NP_415913.1) and maoC (NP_415905.1) genes are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition, bcd (NP_349317.1), etfAB (NP_349315.1 and NP_349316.1) and acot8 (CAA15502) genes encoding 6-aminohex-2-enoyl-CoA reductase and 6-aminocaproyl-CoA hydrolase activities. is cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter.

Finally, the sucD (NP_904963.1), gabT (NP_417148.1) and cat2 (P38942.2) genes encode succinyl-CoA reductase (aldehyde formation), GABA transaminase and 4-aminobutyryl-CoA/acyl-CoA transferase activities. into a third compatible plasmid pZS23 under the PA1/lacO promoter to increase the availability of 4-aminobutyryl-CoA. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with the kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for 6-aminocaproic acid synthesis.

The resulting genetically engineered organisms are cultured in glucose-containing media according to procedures well known in the art (see, eg, Sambrook et al., supra, 2001). Expression of the 6-aminocaproic acid synthesis gene using methods well known in the art for measuring polypeptide expression or enzymatic activity, including, for example, Northern blots, PCR amplification of mRNA and immunoblots, etc. Check. Confirm the enzymatic activity of the expressed enzyme using assays specific for the individual activity. The ability of the engineered E. coli strain to produce 6-aminocaproic acid is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional 6-aminocaproic acid synthesis pathway are further enhanced by optimization for efficient utilization of the pathway. Briefly, engineered strains are evaluated to determine whether any of the exogenous genes are expressed at rate-limiting levels. For example, introduction of additional gene copy numbers increases expression for any enzymes expressed at low levels that can limit flux through the pathway.

Utilize metabolic modeling to optimize growth conditions to generate better producers.

Additionally, modeling is used to design gene knockouts that further optimize pathway utilization (e.g., U.S. Patent Publications Nos. 2002/0012939; 2003/0224363; 2004/0072723, 2003/0059792, 2002/0168654 and 2004/0009466 and US Pat. No. 7,127,379). The modeling analysis allows reliable prediction of the impact on cell growth of shifting metabolism towards more efficient production of 6-aminocaproic acid. One modeling method is the bilevel optimization approach OptKnock, which is applied to select gene knockouts that collectively result in better production of 6-aminocaproic acid (Burgard et al., Biotechnol. Bioengineer. 84:647-657(2003)). Adaptive evolution can also be used, for example, to generate better producers of the acetyl-CoA and succinyl-CoA intermediates of the 6-aminocaproic acid product. Adaptive evolution is carried out to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on these results, a series of subsequent modeling, genetic manipulation and adaptive evolution can be applied to the 6-aminocaproic acid producer to further increase production.

For large scale production of 6-aminocaproic acid, the organisms are cultured in fermentors using media known in the art to support the growth of the organisms under anaerobic conditions.

Fermentation is carried out in batch, fed batch or continuous mode. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel. For example, the flask can be sealed with a septum and crimp cap. Microaerophilic conditions can also be utilized by providing small holes in the membrane for limited ventilation. H₂S.O.₄Maintain the pH of the medium at a pH of approximately 7 by adding an acid such as Growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and glucose uptake rate is determined by monitoring carbon source depletion over time. By-products such as undesirable alcohols, organic acids and residual glucose can be detected using, for example, Aminex® series HPLC columns (e.g. HPX- 87 series) (BioRad, Hercules, Calif.) (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

EXAMPLE The production of microbial organisms capable of producing 6-aminocaproic acid from aminobutyryl-CoA is described.

E. coli is used as the target organism to engineer the 6-aminocaproic acid pathway shown in Figure 11, starting from acetyl-CoA and 4-aminobutyryl-CoA. E. coli provides a good host for producing non-naturally occurring microorganisms capable of producing 6-aminocaproic acid. E. coli is amenable to genetic manipulation and is capable of effectively producing various products such as ethanol, acetic acid, formic acid, lactic acid and succinic acid under anaerobic or microaerobic conditions. Are known.

To generate E. coli strains engineered to produce 6-aminocaproic acid, nucleic acids encoding the necessary enzymes are expressed in E. coli using well-known molecular biology techniques (e.g., Sambrook, supra, 2001 ; see Ausubel, supra, 1999). In particular, paaJ( NP_415915.1), pcaIJ (AAN69545.1 and NP_746082.1) and bdh (AAA58352.1) genes are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition, enr (CAA76083.1) and hmd (ABC88407.1) genes encoding 6-aminohex-2-enoate reductase and 3-hydroxy-6-aminohexanoate dehydratase activities were generated under the PA1/lacO promoter. Cloning into pZA33 vector (Expressys, Ruelzheim, Germany). Finally, the sucD (NP_904963.1), gabT (NP_417148.1) and cat2 (P38942.2) genes encode succinyl-CoA reductase (aldehyde formation), GABA transaminase and 4-aminobutyryl-CoA/acyl-CoA transferase activities. into a third compatible plasmid pZS23 under the PA1/lacO promoter to increase the availability of 4-aminobutyryl-CoA. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with the kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for 6-aminocaproic acid synthesis.

The resulting genetically engineered organisms are cultured in glucose-containing media according to procedures well known in the art (see, eg, Sambrook et al., supra, 2001). Expression of the 6-aminocaproic acid synthesis gene using methods well known in the art for measuring polypeptide expression or enzymatic activity, including, for example, Northern blots, PCR amplification of mRNA and immunoblots, etc. Check. Confirm the enzymatic activity of the expressed enzyme using assays specific for the individual activity. The ability of the engineered E. coli strain to produce 6-aminocaproic acid is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional 6-aminocaproic acid synthesis pathway are further enhanced by optimization for efficient utilization of the pathway. Briefly, engineered strains are evaluated to determine whether any of the exogenous genes are expressed at rate-limiting levels. For example, introduction of additional gene copy numbers increases expression for any enzymes expressed at low levels that can limit flux through the pathway.

Utilize metabolic modeling to optimize growth conditions to generate better producers.

Additionally, modeling is used to design gene knockouts that further optimize pathway utilization (e.g., U.S. Patent Publications Nos. 2002/0012939; 2003/0224363; 2004/0072723, 2003/0059792, 2002/0168654 and 2004/0009466 and US Pat. No. 7,127,379). The modeling analysis allows reliable prediction of the impact on cell growth of shifting metabolism towards more efficient production of 6-aminocaproic acid. One modeling method is the bilevel optimization approach OptKnock, which is applied to select gene knockouts that collectively result in better production of 6-aminocaproic acid (Burgard et al., Biotechnol. Bioengineer. 84:647-657(2003)). Adaptive evolution can also be used, for example, to generate better producers of the acetyl-CoA and succinyl-CoA intermediates of the 6-aminocaproic acid product. Adaptive evolution is carried out to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on these results, a series of subsequent modeling, genetic manipulation and adaptive evolution can be applied to the 6-aminocaproic acid producer to further increase production.

For large scale production of 6-aminocaproic acid, the organisms are cultured in fermentors using media known in the art to support the growth of the organisms under anaerobic conditions.

Fermentation is carried out in batch, fed batch or continuous mode. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel. For example, the flask can be sealed with a septum and crimp cap. Microaerophilic conditions can also be utilized by providing small holes in the membrane for limited ventilation. H₂S.O.₄Maintain the pH of the medium at a pH of approximately 7 by adding an acid such as Growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and glucose uptake rate is determined by monitoring carbon source depletion over time. By-products such as undesirable alcohols, organic acids and residual glucose can be detected using, for example, Aminex® series HPLC columns (e.g. HPX- 87 series) (BioRad, Hercules, Calif.) (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

EXAMPLE The production of microbial organisms capable of

E. coli is used as the target organism to engineer the caprolactam pathway shown in Figure 10, starting from acetyl-CoA and succinyl-CoA. E. coli provides a good host for producing non-naturally occurring microorganisms capable of producing caprolactam. E. coli is amenable to genetic manipulation and is capable of effectively producing various products such as ethanol, acetic acid, formic acid, lactic acid and succinic acid under anaerobic or microaerobic conditions. Are known.

To generate an E. coli strain engineered to produce caprolactam, nucleic acids encoding the necessary enzymes are expressed in E. coli using well-known molecular biology techniques (e.g., Sambrook, supra, 2001; Ausubel, et al. (see supra, 1999). In particular, the paaJ (NP_415915.1), paaH (NP_415913.1) and maoC (NP_415905.1) genes encode 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA reductase and 3-hydroxyadipyl-CoA dehydratase activities, respectively. is cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the bcd (NP_349317.1) and etfAB (NP_349315.1 and NP_349316.1) genes encoding 5-carboxy-2-pentenoyl-CoA reductase activity were transferred under the PA1/lacO promoter into the pZA33 vector (Expressys, Ruelzheim, Germany). to be cloned. Finally, acrI (YP_047869.1), gabT (NP_417148.1) and bioW (NP_390902.2) encode adipyl-CoA reductase (aldehyde formation), 6-aminocaproic acid transaminase and 6-aminocaproyl-CoA synthase activities. The gene is cloned into a third compatible plasmid pZS23 under the PA1/lacO promoter. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with the kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for caprolactam synthesis.

The resulting genetically engineered organisms are cultured in glucose-containing media according to procedures well known in the art (see, eg, Sambrook et al., supra, 2001). Confirm expression of the caprolactam synthesis gene using methods well known in the art for measuring polypeptide expression or enzymatic activity, including, for example, Northern blots, PCR amplification of mRNA, immunoblots, etc. . Confirm the enzymatic activity of the expressed enzyme using assays specific for the individual activity. The ability of the engineered E. coli strain to produce caprolactam is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional caprolactam synthesis pathway are further enhanced by optimization for efficient utilization of the pathway. Briefly, engineered strains are evaluated to determine whether any of the exogenous genes are expressed at rate-limiting levels. For example, introduction of additional gene copy numbers increases expression for any enzymes expressed at low levels that can limit flux through the pathway.

Utilize metabolic modeling to optimize growth conditions to generate better producers.

Additionally, modeling is used to design gene knockouts that further optimize pathway utilization (e.g., U.S. Patent Publications Nos. 2002/0012939; 2003/0224363; 2004/0072723, 2003/0059792, 2002/0168654 and 2004/0009466 and US Pat. No. 7,127,379). Modeling analysis allows reliable prediction of the effects on cell growth of shifting metabolism towards more efficient production of caprolactam. One modeling method is the bilevel optimization approach OptKnock, which is applied to select gene knockouts that collectively result in better production of caprolactam (Burgard et al., Biotechnol. Bioengineer. 84:647-657(2003)). Adaptive evolution can also be used, for example, to generate better producers of the acetyl-CoA and succinyl-CoA intermediates of the caprolactam product. Adaptive evolution is carried out to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on those results, a series of subsequent modeling, genetic manipulation and adaptive evolution can be applied to caprolactam producers to further increase production.

For large-scale production of caprolactam, the organisms are cultured in fermentors using media known in the art to support the growth of the organisms under anaerobic conditions. Fermentation is carried out in batch, fed batch or continuous mode. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel. For example, the flask can be sealed with a septum and crimp cap. Microaerophilic conditions can also be utilized by providing small holes in the membrane for limited ventilation. H₂S.O.₄Maintain the pH of the medium at a pH of approximately 7 by adding an acid such as Growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and glucose uptake rate is determined by monitoring carbon source depletion over time.

By-products such as undesirable alcohols, organic acids and residual glucose can be detected using, for example, Aminex® series HPLC columns (e.g. HPX- 87 series) (BioRad, Hercules, Calif.) (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

EXAMPLE The production of microbial organisms capable of producing methylene diamine is described.

E. coli is used as the target organism to engineer the hexamethylene diamine pathway shown in Figure 10, starting from acetyl-CoA and succinyl-CoA. E. coli provides a good host for producing non-naturally occurring microorganisms capable of producing hexamethylene diamine. E. coli is amenable to genetic manipulation and is capable of effectively producing various products such as ethanol, acetic acid, formic acid, lactic acid and succinic acid under anaerobic or microaerobic conditions. Are known.

To generate an E. coli strain engineered to produce hexamethylene diamine, nucleic acids encoding the necessary enzymes are expressed in E. coli using well-known molecular biology techniques (e.g., Sambrook, supra, 2001; (see Ausubel, supra, 1999). In particular, the paaJ (NP_415915.1), paaH (NP_415913.1) and maoC (NP_415905.1) genes encode 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA reductase and 3-hydroxyadipyl-CoA dehydratase activities, respectively. is cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the bcd (NP_349317.1) and etfAB (NP_349315.1 and NP_349316.1) genes encoding 5-carboxy-2-pentenoyl-CoA reductase activity were transferred under the PA1/lacO promoter into the pZA33 vector (Expressys, Ruelzheim, Germany). to be cloned. Finally, acrI (YP_047869 .1), cloning the gabT (NP_417148.1), bioW (NP_390902.2) and ygjG (NP_417544) genes into the third compatible plasmid pZS23 under the PA1/lacO promoter. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with the kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for hexamethylene diamine synthesis.

The resulting genetically engineered organisms are cultured in glucose-containing media according to procedures well known in the art (see, eg, Sambrook et al., supra, 2001). Expression of the hexamethylenediamine synthesis gene was determined using methods well known in the art for measuring polypeptide expression or enzymatic activity, including, for example, Northern blots, PCR amplification of mRNA and immunoblots. confirm. Confirm the enzymatic activity of the expressed enzyme using assays specific for the individual activity. The ability of the engineered E. coli strain to produce hexamethylene diamine is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

A microbial strain engineered to have a functional hexamethylenediamine synthesis pathway is further enhanced by optimization for efficient utilization of the pathway. Briefly, engineered strains are evaluated to determine whether any of the exogenous genes are expressed at rate-limiting levels. For example, introduction of additional gene copy numbers increases expression for any enzymes expressed at low levels that can limit flux through the pathway.

Utilize metabolic modeling to optimize growth conditions to generate better producers.

Additionally, modeling is used to design gene knockouts that further optimize pathway utilization (e.g., U.S. Patent Publications Nos. 2002/0012939; 2003/0224363; 2004/0072723, 2003/0059792, 2002/0168654 and 2004/0009466 and US Pat. No. 7,127,379). The modeling analysis allows reliable prediction of the effect on cell growth of shifting metabolism towards more efficient production of hexamethylene diamine. One modeling method is the bilevel optimization approach OptKnock, which is applied to select gene knockouts that collectively result in better production of hexamethylenediamine (Burgard et al., Biotechnol. Bioengineer. 84:647-657(2003)). Adaptive evolution can also be used, for example, to generate better producers of the acetyl-CoA and succinyl-CoA intermediates of the hexamethylene diamine product. Adaptive evolution is carried out to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on these results, a series of subsequent modeling, genetic manipulation and adaptive evolution can be applied to hexamethylene diamine producers to further increase production.

For large scale production of hexamethylene diamine, the organisms are cultured in fermentors using media known in the art to support the growth of the organisms under anaerobic conditions. Fermentation is carried out in batch, fed batch or continuous mode. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel. For example, the flask can be sealed with a septum and crimp cap. Microaerophilic conditions can also be utilized by providing small holes in the membrane for limited ventilation. H₂S.O.₄Maintain the pH of the medium at a pH of approximately 7 by adding an acid such as Growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and glucose uptake rate is determined by monitoring carbon source depletion over time. By-products such as undesirable alcohols, organic acids and residual glucose can be detected using, for example, Aminex® series HPLC columns (e.g. HPX- 87 series) (BioRad, Hercules, Calif.) (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

EXAMPLE The production of microbial organisms capable of producing caprolactam from aminobutyryl-CoA is described.

E. coli is used as the target organism to engineer the caprolactam pathway shown in Figure 11, starting from acetyl-CoA and 4-aminobutyryl-CoA. E. coli provides a good host for producing non-naturally occurring microorganisms capable of producing caprolactam. E. coli is amenable to genetic manipulation and is capable of effectively producing various products such as ethanol, acetic acid, formic acid, lactic acid and succinic acid under anaerobic or microaerobic conditions. Are known.

To generate an E. coli strain engineered to produce caprolactam, nucleic acids encoding the necessary enzymes are expressed in E. coli using well-known molecular biology techniques (e.g., Sambrook, supra, 2001; Ausubel, et al. (see supra, 1999). In particular, paaJ (NP_415915.1) encoding 3-oxo-6-aminohexanoyl-CoA thiolase, 3-oxo-6-aminohexanoyl-CoA reductase, and 3-hydroxy-6-aminohexanoyl-CoA dehydratase activities, respectively. ), paaH (NP_415913.1) and maoC (NP_415905.1) genes are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the bcd (NP_349317.1) and etfAB (NP_349315.1 and NP_349316.1) genes encoding 6-aminohex-2-enoyl-CoA reductase activity were transferred under the PA1/lacO promoter into the pZA33 vector (Expressys, Ruelzheim, Germany). ). Finally, the sucD (NP_904963.1), gabT (NP_417148.1) and cat2 (P38942.2) genes encode succinyl-CoA reductase (aldehyde formation), GABA transaminase and 4-aminobutyryl-CoA/acyl-CoA transferase activities. into a third compatible plasmid pZS23 under the PA1/lacO promoter to increase the availability of 4-aminobutyryl-CoA. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with the kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for caprolactam synthesis.

The resulting genetically engineered organisms are cultured in glucose-containing media according to procedures well known in the art (see, eg, Sambrook et al., supra, 2001). Confirm expression of the caprolactam synthesis gene using methods well known in the art for measuring polypeptide expression or enzymatic activity, including, for example, Northern blots, PCR amplification of mRNA, immunoblots, etc. . Confirm the enzymatic activity of the expressed enzyme using assays specific for the individual activity. The ability of the engineered E. coli strain to produce caprolactam is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional caprolactam synthesis pathway are further enhanced by optimization for efficient utilization of the pathway. Briefly, engineered strains are evaluated to determine whether any of the exogenous genes are expressed at rate-limiting levels. For example, introduction of additional gene copy numbers increases expression for any enzymes expressed at low levels that can limit flux through the pathway.

Utilize metabolic modeling to optimize growth conditions to generate better producers.

Additionally, modeling is used to design gene knockouts that further optimize pathway utilization (e.g., U.S. Patent Publications Nos. 2002/0012939; 2003/0224363; 2004/0072723, 2003/0059792, 2002/0168654 and 2004/0009466 and US Pat. No. 7,127,379). Modeling analysis allows reliable prediction of the effects on cell growth of shifting metabolism towards more efficient production of caprolactam. One modeling method is the bilevel optimization approach OptKnock, which is applied to select gene knockouts that collectively result in better production of caprolactam (Burgard et al., Biotechnol. Bioengineer. 84:647-657(2003)). Adaptive evolution can also be used, for example, to generate better producers of the acetyl-CoA and succinyl-CoA intermediates of the caprolactam product. Adaptive evolution is carried out to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on those results, a series of subsequent modeling, genetic manipulation and adaptive evolution can be applied to caprolactam producers to further increase production.

For large-scale production of caprolactam, the organisms are cultured in fermentors using media known in the art to support the growth of the organisms under anaerobic conditions. Fermentation is carried out in batch, fed batch or continuous mode. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel. For example, the flask can be sealed with a septum and crimp cap. Microaerophilic conditions can also be utilized by providing small holes in the membrane for limited ventilation. H₂S.O.₄Maintain the pH of the medium at a pH of approximately 7 by adding an acid such as Growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and glucose uptake rate is determined by monitoring carbon source depletion over time.

By-products such as undesirable alcohols, organic acids and residual glucose can be detected using, for example, Aminex® series HPLC columns (e.g. HPX- 87 series) (BioRad, Hercules, Calif.) (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

EXAMPLE The production of microbial organisms capable of producing hexamethylene diamine from 4-aminobutyryl-CoA is described.

E. coli is used as the target organism to engineer the hexamethylene diamine pathway shown in Figure XVII, starting from acetyl-CoA and 4-aminobutyryl-CoA. E. coli provides a good host for producing non-naturally occurring microorganisms capable of producing hexamethylene diamine. E. coli is amenable to genetic manipulation and is capable of effectively producing various products such as ethanol, acetic acid, formic acid, lactic acid and succinic acid under anaerobic or microaerobic conditions. Are known.

To generate an E. coli strain engineered to produce hexamethylene diamine, nucleic acids encoding the necessary enzymes are expressed in E. coli using well-known molecular biology techniques (e.g., Sambrook, supra, 2001; (see Ausubel, supra, 1999). In particular, paaJ (NP_415915.1) encoding 3-oxo-6-aminohexanoyl-CoA thiolase, 3-oxo-6-aminohexanoyl-CoA reductase, and 3-hydroxy-6-aminohexanoyl-CoA dehydratase activities, respectively. ), paaH (NP_415913.1) and maoC (NP_415905.1) genes are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition, 6-aminohex-2-enoyl-CoA reductase, bcd (NP_349317.1), etfAB (NP_349315.1 and NP_349316. 1) Clone the acrI (YP_047869.1) and ygjG (NP_417544) genes into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. Finally, the sucD (NP_904963.1), gabT (NP_417148.1) and cat2 (P38942.2) genes encode succinyl-CoA reductase (aldehyde formation), GABA transaminase and 4-aminobutyryl-CoA/acyl-CoA transferase activities. into a third compatible plasmid pZS23 under the PA1/lacO promoter to increase the availability of 4-aminobutyryl-CoA. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with the kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for hexamethylene diamine synthesis.

The resulting genetically engineered organisms are cultured in glucose-containing media according to procedures well known in the art (see, eg, Sambrook et al., supra, 2001). Expression of the hexamethylenediamine synthesis gene was determined using methods well known in the art for measuring polypeptide expression or enzymatic activity, including, for example, Northern blots, PCR amplification of mRNA and immunoblots. confirm. Confirm the enzymatic activity of the expressed enzyme using assays specific for the individual activity. The ability of the engineered E. coli strain to produce hexamethylene diamine is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

A microbial strain engineered to have a functional hexamethylenediamine synthesis pathway is further enhanced by optimization for efficient utilization of the pathway. Briefly, engineered strains are evaluated to determine whether any of the exogenous genes are expressed at rate-limiting levels. For example, introduction of additional gene copy numbers increases expression for any enzymes expressed at low levels that can limit flux through the pathway.

Utilize metabolic modeling to optimize growth conditions to generate better producers.

Additionally, modeling is used to design gene knockouts that further optimize pathway utilization (e.g., U.S. Patent Publications Nos. 2002/0012939; 2003/0224363; 2004/0072723, 2003/0059792, 2002/0168654 and 2004/0009466 and US Pat. No. 7,127,379) . The modeling analysis allows reliable prediction of the effect on cell growth of shifting metabolism towards more efficient production of hexamethylene diamine. One modeling method is the bilevel optimization approach OptKnock, which is applied to select gene knockouts that collectively result in better production of hexamethylenediamine (Burgard et al., Biotechnol. Bioengineer. 84:647-657(2003)). Adaptive evolution can also be used, for example, to generate better producers of the acetyl-CoA and succinyl-CoA intermediates of the hexamethylene diamine product. Adaptive evolution is carried out to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on these results, a series of subsequent modeling, genetic manipulation and adaptive evolution can be applied to hexamethylene diamine producers to further increase production.

For large scale production of hexamethylene diamine, the organisms are cultured in fermentors using media known in the art to support the growth of the organisms under anaerobic conditions. Fermentation is carried out in batch, fed batch or continuous mode. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel. For example, the flask can be sealed with a septum and crimp cap. Microaerophilic conditions can also be utilized by providing small holes in the membrane for limited ventilation. H₂S.O.₄Maintain the pH of the medium at a pH of approximately 7 by adding an acid such as Growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and glucose uptake rate is determined by monitoring carbon source depletion over time. By-products such as undesirable alcohols, organic acids and residual glucose can be detected using, for example, Aminex® series HPLC columns (e.g. HPX- 87 series) (BioRad, Hercules, Calif.) (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

Example XIX (Production route for 6-aminocaproate from succinic semialdehyde and pyruvate) This example describes an exemplary production route for 6-aminocaproate.

A novel route for producing 6-aminocaproate (6-ACA) and related products is described herein. These routes synthesize 6-ACA from succinic semialdehyde and pyruvate using the enzymes aldolase and hydratase from the 4-hydroxyphenylacetic acid degradation pathway. Candidate enzymes and the risks associated with their implementation are discussed in Example XXI below.

The present invention relates, in part, to non-naturally occurring microorganisms that express genes encoding enzymes that catalyze the production of 6-ACA. Successful genetic engineering of these pathways involves identifying a suitable set of enzymes with sufficient activity and specificity, cloning their corresponding genes into the production host, and disseminating these enzymes in the production host. There is a need to optimize gene expression, optimize fermentation conditions, and assay for post-fermentation product formation.

6-Aminocaproate and derivatives are produced from succinic semialdehyde and pyruvate in a minimum of five enzymatic steps. In the first step of the entire pathway, pyruvate and succinic semialdehyde are linked by 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) aldolase. The product of this reaction, HODH, is then dehydrated by 2-oxohept-4-ene-1,7-dioate (OHED) hydratase to form OHED. In subsequent steps, the OHED is transaminated, decarboxylated, or reduced, as shown in FIG. 12.

In one route, the alkene of OHED is reduced by OHED reductase to form the 2-keto acid 2-oxoheptane-1,7-dioate (2-OHD) (Figure 12, Step C). 2-OHD is then converted to adipate semialdehyde by keto acid decarboxylase (Figure 12, Step D). In the final step, the aldehyde of adipate semialdehyde is converted to an amine by an aminotransferase or aminating oxidoreductase (Figure 12, Step E).

In a similar route, the 2-keto group of 2-OHD undergoes transamination by an aminotransferase or aminating oxidoreductase (Figure 12, Step H) to form 2-aminoheptane-1,7-dioate (2-AHD ) is formed. This product is then decarboxylated by 2-AHD decarboxylase to form 6-aminocaproate (Figure 12, Step I).

In an alternative pathway, OHED is first decarboxylated by OHED decarboxylase (Figure 12, Step F) to form 6-oxo-4-hexenoic acid (6-OHE). The alkenal group of 6-OHE is reduced to adipate semialdehyde by oxidoreductase (Figure 12, Step G).

Adipate semialdehyde is then converted to 6-aminocaproate by an aminotransferase or aminating oxidoreductase (Figure 12, Step E).

Yet another route requires an aminotransferase or aminating oxidoreductase that converts OHED to 2-amino-4 hepten-1,7-dioate (2-AHE) (Figure 12, Step J). The alkene of 2-AHE is then reduced by an alkene oxidoreductase (Figure 12, Step K). The product of this reaction, 2-AHD, is then decarboxylated by amino acid decarboxylase (Figure 12, Step I) to form 6-aminocaproate.

In yet another route, HODH is converted to 3-hydroxyadipyl-CoA by either HODH dehydrogenase or HODH formate lyase (Figure 12, Step L). 3-hydroxyadipyl-CoA is then dehydrated and reduced to form adipyl-CoA (Figure 12, steps M, N). Adipyl-CoA is reduced and deacylated to form adipate semialdehyde (Figure 12, Step O), which is then converted to 6-aminocaproate by an aminotransferase or aminating oxidoreductase (Figure 12, Step O). 12, Step E).

In a similar route, HODH is first converted to OHED as described above (Figure 12, Step B). OHED is then converted to 2,3-dehydroadipyl-CoA by dehydrogenase or OHED formate lyase (Figure 12, Step P). 2,3-dihydroadipyl-CoA is then reduced to adipyl-CoA (Figure 12, Step N), which is converted to 6-aminocaproate via adipate semialdehyde as described above. (Figure 12, steps O, E).

In the final route, HODH is converted to 2-OHD via steps B and C as described above. 2-OHD is converted to adipyl-CoA by 2-OHD formate lyase or dehydrogenase (Figure 12, Step Q), and adipyl-CoA is then reduced by a CoA-dependent aldehyde dehydrogenase (Figure 12, Step O). The product adipate semialdehyde is converted to 6-aminocaproate by an aminotransferase or aminating oxidoreductase (Figure 12, Step E).

The route detailed in Figure 12 allows a maximum theoretical 6-ACA yield of 0.8 moles of 6-ACA per mole of glucose utilized to be achieved. Energy yields are also advantageous, with maximum product yields of up to 1.6 moles of ATP per mole of glucose utilized. Yields were calculated using the following assumptions: 1) phosphoenolpyruvate (PEP) carboxykinase can operate in the direction of ATP generation, 2) NH4 and 6-ACA enter the cell by proton countertransport. and 3) succinic semialdehyde is formed from alpha-ketoglutarate and/or succinyl-CoA. Succinic semialdehyde dehydrogenase is a NAD(P)H and CoA-dependent aldehyde dehydrogenase that converts succinyl-CoA to succinic semialdehyde. Succinic semialdehyde is formed from alpha-ketoglutarate by two enzymes: alpha-ketoglutarate decarboxylase and 4-aminobutyrate transamylase.

Example XX (Production route for hexamethylene diamine from 6-aminocaproate) This example describes an exemplary production route for hexamethylene diamine.

A novel route for producing hexamethylene diamine (HMDA) and related products is described herein. In this pathway, HMDA is synthesized from 6-aminocaproate (6-ACA). These pathways involve activation of acid groups by phosphorylation and/or acylation. Acetylation of the terminal amino group protects pathway intermediates from spontaneous cyclization. Candidate enzymes and the risks associated with their implementation are discussed in Example XXI below.

The present invention relates, in part, to non-naturally occurring microorganisms that express genes encoding enzymes that catalyze the production of HMDA. Successful genetic engineering of these pathways involves identifying a suitable set of enzymes with sufficient activity and specificity, cloning their corresponding genes into the production host, and disseminating these enzymes in the production host. There is a need to optimize gene expression, optimize fermentation conditions, and assay for post-fermentation product formation.

Several routes for producing HMDA from 6-aminocaproate are detailed in Figure 13. All routes require that the carboxylic acid group is activated, then reduced and undergoes transamination. In three routes, the 6-aminocaproate is activated directly, while in the other route, the terminal amine group is protected by N-acetylation to prevent spontaneous cyclization.

In one route, 6-aminocaproate is phosphorylated to 6-AHOP by 6-aminocaproate kinase (Figure 13, Step A). 6-AHOP is then reduced to 6-aminocaproic acid semialdehyde (Figure 13, Step B), followed by transamination by an aminotransferase or aminating oxidoreductase (Figure 13, Step C).

Alternatively, 6-AHOP is converted to 6-aminocaproyl-CoA by an acyltransferase (Figure 13, Step L). 6-Aminocaproyl-CoA is then reduced to 6-aminocaproyl semialdehyde by a CoA-dependent aldehyde dehydrogenase (Figure 13, Step N). HMDA is then formed by transamination of 6-aminocaproic semialdehyde by an aminotransferase or an aminating oxidoreductase (FIG. 13, step C).

In yet another route, 6-aminocaproate is first activated to a CoA derivative by CoA transferase or CoA ligase (Figure 13, step M). The product, 6-aminocaproyl-CoA, can spontaneously cyclize or be converted to 6-aminocaproic acid semialdehyde by a CoA-dependent aldehyde dehydrogenase to form an aldehyde (Figure 13, Step N). 6-Aminocaproic semialdehyde is converted to HMDA by an aminotransferase or aminating oxidoreductase (Figure 13, Step C).

An additional route proceeds from 6-acetamidohexanoic acid, the acetylated product of 6-aminocaproate N-acetyltransferase. 6-acetamidohexanoic acid is converted to 6-acetamidohexanal by different routes (described below). In the last two steps of these routes, 6-acetamidohexanal is first converted to 6-acetamidohexanamine by an aminotransferase or aminating oxidoreductase (Figure 13, step G). 6-acetamidohekianamine is then converted to HMDA by amidohydrolase or N-acetyltransferase (Figure 13, Step H).

In one route, 6-acetamidohexanoic acid is phosphorylated by 6-acetamidohexanoate kinase (Figure 13, Step E). The product 6-AAHOP is reduced to form 6-acetamidohexanal (Figure 13, Step F), which is then converted to HMDA as described above.

In another route, 6-acetamidohexanoic acid is activated to 6-acetamidohexanoyl-CoA by CoA transferase or CoA ligase (Figure 13, Step I). The CoA derivative is then reduced to 6-acetamidohexanal by a CoA-dependent oxidoreductase that forms an aldehyde (Figure 13, Step J). 6-acetamidohexanal is then converted to HMDA as described above.

Alternatively, 6-acetamidohexanoic acid is phosphorylated to 6-AAHOP (Figure 13, Step E), which is then converted to 6-acetamidohexanoyl-CoA by an acyltransferase (Figure 13, Step K). 6-acetamidohexanoyl-CoA is then reduced to HMDA as described above.

(Example XXI) (Classification system of enzymes for producing 6-aminocaproic acid and hexamethylene diamine) In this example, Example XIX and the An enzyme classification scheme for the exemplary pathway described in Example XX is described.

All transformations shown in FIGS. 12 and 13 fall into the general categories of transformations shown in Table 9. Below we list a number of biochemically characterized genes within each category. Specifically listed are genes that, when properly cloned and expressed, can be applied to catalyze the appropriate transformations of Figures 12-13.

Table 9 shows the types of enzymes useful for converting common core metabolic intermediates to 6-aminocaproate and hexamethylene diamine. The first three numbers of each label correspond to the first three numbers of the enzyme commission number indicating a general type of conversion that is independent of substrate specificity.

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1.2.1.b Oxidoreductase (acyl-CoA to aldehyde). EC Catalyzed by enzymes that are class 1.2.1 CoA-dependent oxidoreductases. Adipyl-CoA is converted to adipate semialdehyde by adipyl-CoA oxidoreductase, an enzyme with similar functionality (Figure 12, Step O). Succinic semialdehyde dehydrogenase, which is an enzyme that forms succinic semialdehyde, the precursor in FIG. 12, from succinyl-CoA is also a CoA-dependent oxidoreductase. Oxidoreductases of EC class 1.2.1.- are capable of reducing acyl-CoA to its corresponding aldehyde. An exemplary gene encoding such an enzyme is Acinetobacter calcoaceticus acr1, which encodes fatty acyl-CoA reductase (Reiser and Somerville, Journal of Bacteriology 179:2969-2975 (1997)), fatty acyl-CoA reductase of Acinetobacter sp. Contains the Clostridium kluyveri sucD gene encoding acid semialdehyde dehydrogenase (Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996)). P. gingivalis sucD is another succinic semialdehyde dehydrogenase (Takahashi et al., J. Bacteriol. 182:4704-4710 (2000)). The acylating acetaldehyde dehydrogenase encoded by bphG in Pseudomonas species has been demonstrated to oxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde and formaldehyde (Powlowski et al., J. Bacteriol. 175:377-385 (1993)), is yet another candidate. In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxidize the branched-chain compound isobutyraldehyde to isobutyryl-CoA ( Kazahaya et al., J. Gen. Appl. Microbiol. 18:43-55 (1972); and Koo et al., Biotechnol Lett. 27:505-510 (2005)).

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An additional enzyme that converts acyl-CoA to its corresponding aldehyde is malonyl-CoA reductase, which converts malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionic acid cycle in thermoacidophilic archaea (Berg et al., Science 318:1782-1786 (2007); and Thauer , R.K., Science.318:1732-1733(2007)). This enzyme utilizes NADPH as a cofactor and has been characterized in Metallospaera and Sulfolobus species (Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Hugler et al., J. Bacteriol. 184: 2404-2410(2002)). This enzyme is encoded by Msed_0709 from Metallospaera cedula (Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Berg et al., Science. 318:1782-1786 (2007)). The gene encoding malonyl-CoA reductase from Sulfolobus tokodaiii was cloned into E. coli and heterologously expressed (Alber et al., J. Bacteriol. 188:8551-8559 (2006)). This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde (WIPO Patent Application WO/2007/141208 Type Code: A2). The functionality of these enzymes as aldehyde dehydrogenases is similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus, but there is little sequence similarity.

Both malonyl-CoA reductase enzyme candidates share high sequence similarity to aspartate semialdehyde dehydrogenase, an enzyme that catalyzes the reduction of aspartyl-4-phosphate to aspartate semialdehyde and concomitant dephosphorylation. have Additional gene candidates can be found by sequence homology to proteins of other organisms, including Sulfolobus solfataricus and Sulfolobus acidocaldarius, and are listed below. Yet another candidate for a CoA-acylating aldehyde dehydrogenase is the ald gene from Clostridium beijerinckii (Toth et al., Appl Environ Microbiol 65:4973-4980 (1999)). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to eutE, which encodes the acetaldehyde dehydrogenase of Salmonella Typhimurium and Escherichia coli (Toth et al., Appl Environ Microbiol 65:4973-4980 (1999)).

<tables><img file="JP7370366B2_D0077.tif" /></tables>

1.2.1.c Oxidoreductase (2-keto acid to acyl-CoA). Some of the transformations in Figure 12 require that 2-keto acids be converted to acyl-CoA by enzymes of EC class 1.2.1 (steps L, P and Q). Such reactions are catalyzed by a multienzyme complex that catalyzes a series of partial reactions leading to acylated oxidative decarboxylation of 2-keto acids. Exemplary enzymes include the multienzyme complex (PDHC) of 1) branched chain 2-keto acid dehydrogenase, 2) alpha-ketoglutarate dehydrogenase, and 3) pyruvate dehydrogenase. Each of the 2-keto acid dehydrogenase complexes occupies an important position in intermediary metabolism, and enzyme activity is generally tightly regulated (Fries et al., Biochemistry 42:6996-7002(2003)). Enzymes share a complex, but a common structure consists of multiple copies of three catalytic components: alpha-keto acid decarboxylase (E1), dihydrolipoamide acyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). be done. The E3 component is shared among all 2-keto acid dehydrogenase complexes in an organism, whereas the E1 and E2 components are encoded by different genes. The enzyme component is present in multiple copies in the complex and utilizes multiple cofactors to catalyze a directed series of reactions through substrate channeling. The overall size of these dehydrogenase complexes is very large, with a molecular weight of 4 to 10 million Da (ie, larger than a ribosome).

The activity of enzymes of the 2-keto acid dehydrogenase family is usually low or limited in E. coli under anaerobic conditions. Increased production of NADH (or NADPH) can lead to redox imbalance, with NADH itself acting as an inhibitor on enzyme function. Attempts at genetic engineering have increased the anaerobic activity of the pyruvate dehydrogenase complex in Escherichia coli (Kim et al., Appl. Environ. Microbiol. 73:1766-1771 (2007); Kim et al., J. Bacteriol. 190: 3851-3858 (2008); and Zhou et al., Biotechnol. Lett. 30:335-342 (2008)). For example, the inhibitory effect of NADH can be overcome by genetically engineering the H322Y mutation in the E3 component (Kim et al., J. Bacteriol. 190:3851-3858 (2008)). Structural studies of the individual components and how they operate together in complexes provide insight into the catalytic mechanism and organization of enzymes in this family (Aevarsson et al., Nat. Struct. Biol. 6:785-792 (1999); and Zhou et al., Proc. Natl. Acad. sci. USA 98:14802-14807 (2001)). The substrate specificity of dehydrogenase complexes varies in different organisms, but branched-chain keto acid dehydrogenases generally have the broadest substrate range.

Alpha-ketoglutarate dehydrogenase (AKGD) converts alpha-ketoglutarate to succinyl-CoA and is the major site that regulates metabolic flux through the TCA cycle (Hansford, Curr. Top. Bioenerg. 10:217-278). 1980)). Encoded by the sucA, sucB, and lpd genes in E. coli, the expression of the AKGD gene is downregulated during growth on glucose under anaerobic conditions (Park et al., Mol. Microbiol. 15:473-482 (1995 )). Although the substrate range of AKGD is narrow, structural studies of the catalytic core of the E2 component pinpoint specific residues responsible for substrate specificity (Knapp et al., J. Mol. Biol. 280:655-668 (1998)). The Bacillus subtilis AKGD, encoded by odhAB (E1 and E2) and pdhD (E3, shared domain), is regulated at the transcriptional level and depends on the carbon source and growth phase of the organism (Resnekov et al., Mol. Gen. Genet. 234:285-296(1992)). In yeast, the LPD1 gene encoding the E3 component is regulated at the transcriptional level by glucose (Roy and Dawe, J. Gen. Microbiol. 133:925-933 (1987)). The E1 component encoded by KGD1 is also regulated by glucose and activated by the products HAP2 and HAP3 (Repetto and Tzagoloff, Mol. Cell Biol. 9:2695-2705 (1989)). The AKGD enzyme complex, which is inhibited by the products NADH and succinyl-CoA, has been well studied in mammalian systems, as its dysfunction has been associated with several neurological diseases (Tretter and Dam-Vizi, Philos. Trans. R. Soc. Lond B Biol. Sci. 360:2335-2345 (2005)).

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The branched-chain 2-keto acid dehydrogenase complex (BCKAD), also known as 2-oxoisovalerate dehydrogenase, converts the 2-keto acid derivatives of valine, leucine, and isoleucine into their acyl-CoA derivatives and CO₂Involved in the branched chain amino acid degradation pathway that converts into This complex is associated with Bacillus subtilis (Wang et al., Eur. J. Biochem. 213:1091-1099 (1993)), rat (Namba et al., J. Biol. Chem. 244:4437-4447 (1969)) and Pseudomonas. It has been studied in many organisms, including P. putida (Sokatch et al., J. Bacteriol. 148:647-652 (1981)). In Bacillus subtilis, this enzyme is encoded by the pdhD gene (E3 component), bfmBB gene (E2 component), bfmBAA gene and bfmBAB gene (E1 component) (Wang et al., Eur. J. Biochem. 213:1091-1099( 1993)). In mammals, this complex is regulated by phosphorylation by specific phosphatases and protein kinases. This complex has been studied in rat hepatocytes (Chicco et al., J. Biol.Chem. 269:19427-19434 (1994)), Bckdha gene (E1 alpha), Bckdhb gene (E1 beta), Dbt gene (E2), and Dld gene (E3). The E1 and E3 components of the BCKAD complex of Pseudomonas putida have been crystallized (Aevarsson et al., Nat. Struct. Biol. 6:785-792 (1999); and Mattevi et al., Science. 255:1544-1550 ( (1992)), the enzyme complex has been studied (Sokatch et al., J. Bacteriol. 148:647-652 (1981)). Transcription of the P. putida BCKAD gene is activated by the bkdR gene product (Hesslinger et al., Mol. Microbiol 27:477-492 (1998)). Rat (Paxton et al., Biochem. J. 234:295-303 (1986)) and Saccharomyces cerevisiae (Sinclair et al., Biochem. Mol. Biol. Int. 31:911-922 (1993)), this complex contains linear oxoacids such as 2-oxobutanoate and alpha-ketoglutarate in addition to branched-chain amino acid precursors. It has been shown to have a broad substrate range including: The active site of bovine BCKAD was genetically engineered to prefer an alternative substrate, acetyl-CoA (Meng and Chuang, Biochemistry. 33:12879-12885 (1994)).

<tables><img file="JP7370366B2_D0079.tif" /></tables>

The pyruvate dehydrogenase complex, which catalyzes the conversion of pyruvate to acetyl-CoA, has also been extensively studied. In the E. coli enzyme, specific residues in the E1 component are responsible for substrate specificity (Bisswanger, J Biol Chem. 256:815-822 (1981); Bremer, Eur. J Biochem. 8:535-540 (1969) ); and Gong et al., J Biol Chem. 275:13645-13653 (2000)). As mentioned above, attempts to genetically engineer the enzyme have improved the enzymatic activity of E. coli PDH under anaerobic conditions (Kim et al., Appl. Environ. Microbiol. 73:1766-1771 (2007); Kim et al., J. Bacteriol. 190:3851-3858 (2008)); and Zhou et al., Biotechnol. Lett. 30:335-342 (2008)). In contrast to E. coli PDH, the Bacillus subtilis complex is active under anaerobic conditions and required for growth (Nakano et al., J. Bacteriol. 179:6749-6755(1997)). K. pneumoniae PDH, which was characterized during growth on glycerol, is also active under anaerobic conditions (Menzel et al., J. Biotechnol. 56:135-142 (1997)). Crystal structure of the enzyme complex from bovine kidney (Zhou et al., Proc. Natl. Acad. Sci. US A 98:14802-14807 (2001)) and the crystal structure of the E2 catalytic domain from Azotobacter vinelandii (Mattevi et al., Science. 255:1544-1550 (1992)) is available.

Although some mammalian PDH enzyme complexes may be responsive to alternative substrates such as 2-oxobutanoate, the comparative kinetics of rat PDH and BCKAD demonstrate that 2-oxobutanoate as a substrate BCKAD has been shown to have higher activity against (Paxton et al., Biochem. J. 234:295-303 (1986)).

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As an alternative to the large multienzyme 2-keto acid dehydrogenase complex described above, some anaerobic organisms utilize enzymes of the 2-keto acid oxidoreductase family (OFOR) to perform the acylated oxidative reduction of 2-keto acids. Catalyzes carbonic acid. Unlike the dehydrogenase complex, these enzymes contain iron-sulfur clusters, utilize different cofactors, and use ferredoxin or flavodoxin instead of NAD(P)H as electron acceptors. Although the majority of enzymes in this family are specific for pyruvate as a substrate (POR), some 2-keto acid:ferredoxin oxidoreductases have a broad range of enzymes, including alpha-ketoglutarate and 2-oxobutanoate. have been shown to tolerate 2-keto acids as substrates (Fukuda and Wakagi, Biochim. Biophys. Acta1597:74-80 (2002); and Zhang et al., J. Biochem. 120:587-599(1996)). One such enzyme is OFOR from the thermoacidophilic archaeon Sulfolobus tokodaiii 7, which contains alpha and beta subunits encoded by the ST2300 gene (Fukuda and Wakagi, Biochim. Biophys Acta 1597:74-80 (2002); and Zhang et al., J. Biochem. 120:587-599 (1996)). To efficiently express this protein in E. coli, a plasmid-based expression system has been developed (Fukuda et al., Eur. J. Biochem. 268:5639-5646 (2001)), and the residues involved in substrate specificity have been developed. (Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74-80 (2002)). Two OFORs from Aeropyrum pernix strain K1 were also recently cloned into E. coli, characterized, and found to react with a wide range of 2-oxoacids (Nishizawa et al., FEBS Lett. 579:2319-2322(2005)). The gene sequences of these OFOR candidates have no previously assigned GenBank identifiers, but are available. There is bioinformatic evidence for the presence of similar enzymes in all archaea, some anaerobic bacteria, and eukaryotes that do not have mitochondria (Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74-80 (2002)). This class of enzymes is also interesting from an energetic point of view since reduced ferredoxin can be used to generate NADH by ferredoxin-NAD reductase (Petitdemange et al., Biochim. Biophys. Acta 421:334-337 (1976)). . Similarly, enzymes can be genetically engineered for activity in anaerobic environments compared to enzymes of the 2-keto acid dehydrogenase complex family, as the majority of enzymes are designed to operate under anaerobic conditions. May require less manipulation.

<tables><img file="JP7370366B2_D0081.tif" /></tables>

1.2.1.d Oxidoreductase (phosphonic acid to aldehyde). The reduction of phosphonic acid to its corresponding aldehyde is catalyzed by EC class 1.2.1 oxidoreductases. Steps B and F of Figure 13 require enzymes to reduce 6-AHOP and 6-AAHOP to their corresponding aldehydes. Although these reactions are not catalyzed by known enzymes, a similar reaction is catalyzed by aspartate semialdehyde dehydrogenase (ASD, EC 1.2.1.11): 4-aspartyl phosphate to aspartate-4-semialdehyde. NADPH-dependent reduction. ASD is involved in amino acid biosynthesis and has recently been investigated as an antibacterial target (Hadfield et al., Biochemistry 40:14475-14483 (2001)). The structure of the E. coli ASD has been elucidated (Hadfield et al., J Mol. Biol. 289:991-1002 (1999)), and the enzyme tolerates an alternative substrate, beta-3-methylaspartyl phosphate. It has been shown that (Shames et al., J Biol. Chem. 259:15331-15339(1984)). The Haemophilus influenzae enzyme has been the subject of studies in which the enzyme is genetically engineered to alter the substrate binding affinity of the active site (Blanco et al., ActaCrystallogr. D. Biol. Crystallogr. 60:1388-1395). 2004); and Blanco et al., Acta Crystallogr. D. Biol. Crystallogr. 60:1808-1815 (2004)). Other ASD candidates are Mycobacterium tuberculosis (Shafiani et al., J Appl Microbiol 98:832-838 (2005)), Methanococcus jannaschii (Faehnle et al., J Mol. Biol. 353:1055-1068 (2005)) ), and the infectious microorganisms Vibrio cholerae and Helicobacter pylori (Moore et al., Protein Expr. Purif. 25:189-194 (2002)). A related enzyme candidate is acetylglutamyl phosphate reductase (EC 1.2.1.38), an enzyme that naturally reduces acetylglutamyl phosphate to acetylglutamic acid-5-semialdehyde, and has been reported in Saccharomyces cerevisiae (Pauwels et al., Eur. J Biochem. 270:1014-1024 (2003)), Bacillus subtilis (O'Reilly and Devine, Microbiology 140(Pt 5):1023-1025 (1994)) and other organisms.

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1.3.1.a Oxidoreductase (alkenes to alkanes). Some transformations fall into the category of oxidoreductases that reduce alkenes to alkanes (EC 1.3.1.-).

For example, steps C, G, K and N in Figure 12 are catalyzed by OHED reductase, 6-OHE reductase, 2-AHE reductase and 2,3-dehydroadipyl-CoA reductase, respectively, which fall into this category. . The enzymes enone reductase, alkenal reductase, and enoate reductase are suitable enzyme candidates for catalyzing the conversions of steps C, G, and K. The enzyme, enoyl-CoA reductase, catalyzes the conversion of 2,3-dehydroadipyl-CoA to adipyl-CoA (Step N).

Enzymes with enone reductase activity have been identified in prokaryotes, eukaryotes and plants (Shimoda et al., Bulletin of the chemical Society of Japan 77:2269-2 (2004); and Wanner and Tressl, Eur. J Biochem 255:271-278(1998)). Two enone reductases from the cytosolic fraction of Saccharomyces cerevisiae were purified and characterized and show that they tolerate various alkenals (similar to 6-OHE) and enoyl ketones (similar to OHED) as substrates. was found (Wanner and Tressl, Eur. J Biochem. 255:271-278(1998)). The genes encoding these enzymes have not been identified so far. Various enone substrates are reduced to their corresponding alkyl ketones by cell extracts of the cyanobacteria Synechococcus sp. PCC7942 (Shimoda et al., Bulletin of the chemical Society of Japan 77:2269-2 (2004)) . No genes have been linked to this activity in this organism. Enone reductases from other organisms may also catalyze this conversion.

A recombinant NADPH-dependent enone reductase from tobacco, encoded by NtRed1, was functionally expressed and characterized in E. coli (Matsushima et al., Bioorganic Chemistry 36:23-28 (2008)). This reductase is functional towards pulegone, an exocyclic enoyl ketone (Matsushima et al., Bioorganic Chemistry 36:23-28 (2008)). The enzyme candidate at the S. cerevisiae locus YML131W has 30% identity to NtRed1 (E value = 1 × 10^-26). The amino acid sequence of NtRed1 shares significant homology with 2-alkenal reductase from Arabidopsis, zeta-crystallin homolog from Arabidopsis, pulegone reductase from peppermint, and phenylpropenal alkene reductase from Loblolly pine. These enzymes are known to catalyze the reduction of alkenes to α,β-unsaturated ketones and α,β-unsaturated aldehydes.

<tables><img file="JP7370366B2_D0083.tif" /></tables>

2-Alkenal reductase catalyzes the reduction of α,β unsaturated double bonds of aldehydes and ketones. Barley alkenal hydrogenase ALH1 has been identified for activity against various α,β-unsaturated ketones and α,β-unsaturated aldehydes, including trans-2-nonenal, 2-hexenal, traumatin, and 1-octen-3-one. (Hambraeus and Nyberg, J Agric. Food Chem. 53:8714-8721 (2005)). The barley ALH1 cDNA was cloned and expressed in E. coli (Hambraeus and Nyberg, J Agric. Food Chem. 53:8714-8721 (2005)).

<tables><img file="JP7370366B2_D0084.tif" /></tables>

The enzyme 2-enoate reductase is known to catalyze the NAD(P)H-dependent reduction of a wide variety of α,β-unsaturated carboxylic acids and α,β-unsaturated aldehydes (Rohdich et al. J. Biol. Chem. 276:5779-5787(2001)). In the recently published genome sequence of C. kluyveri, nine coding sequences for enoate reductase have been reported, one of which has been characterized (Seedorf et al., Proc. Natl. Acad. Sci US A 105:2128- 2133(2008)). Both the enr genes from C. tyrobutylicum and M. thermoaceticum have been cloned and sequenced and they show 59% identity to each other. The former gene was also found to have approximately 75% similarity to genes characterized in C. kluyberg (Giesel and Simon, Arch. Microbiol 135:51-57(1983)). Based on these sequencing results, it has been reported that enr is very similar to E. coli dienoyl-CoA reductase (fadH) (Rohdich et al., J. Biol. Chem. 276:5779-5787 (2001) ). The enr gene of C. thermoaceticum has also been expressed in a catalytically active form in E. coli (Rohdich et al., J. Biol. Chem. 276:5779-5787 (2001)).

<tables><img file="JP7370366B2_D0085.tif" /></tables>

Another candidate enoate reductase is 3-oxoadipate oxidoreductase (maleyl acetate reductase), an enzyme that catalyzes the reduction of 2-maleyl acetate (4-oxohex-2-enedioate) to 3-oxoadipate. . This enzyme activity was identified and characterized in strain B13 of Pseudomonas sp. (Kaschabek and Reineke, J. Bacteriol 177:320-325 (1995); )), the encoding gene was cloned and sequenced (Kasberg et al., J. Bacteriol. 179:3801-3803 (1997)). Candidate genes for 3-oxoadipate oxidoreductase are the clcE gene from Pseudomonas sp. strain B13 (Kasberg et al., J. Bacteriol. 179:3801-3803 (1997)), the macA gene from Rhodococcus opacus (Seibert et al., J. Bacteriol. 180:3503-3508 (1998)), and the macA gene from Ralstonia eutropha (also known as Capriavidus necator) (Seibert et al., Microbiology 150:463-472 (2004)).

<tables><img file="JP7370366B2_D0086.tif" /></tables>

The enzyme enoyl-CoA reductase is a suitable enzyme for catalyzing the reduction of 2,3-dehydroadipyl-CoA to adipyl-CoA (Figure 12, Step N). One exemplary enoyl-CoA reductase is the gene product of bcd from C. acetobutylicum (Atsumi et al., MetabEng 10:305-311 (2008); and Boynton et al., J. Bacteriol. 178:3015-3024 ( (1996)), this enzyme naturally catalyzes the reduction of crotonyl-CoA to butyryl-CoA. The activity of this enzyme can be increased by expressing bcd in conjunction with expression of the C. acetobutylicum etfAB gene, which encodes an electron transfer flavoprotein. An additional candidate for the enoyl-CoA reductase step is the mitochondrial enoyl-CoA reductase from E. gracilis (Hoffmeister et al., J Biol. Chem. 280:4329-4338(2005)). A construct derived from this sequence was cloned into E. coli after removal of the E. gracilis mitochondrial targeting leader sequence, resulting in an active enzyme (Hoffmeister et al., J Biol. Chem. 280:4329-4338 (2005)). This approach is well known to those skilled in the art of expressing eukaryotic genes, especially genes with leader sequences capable of targeting the gene product to specific subcellular compartments of prokaryotes. A close homolog of this gene, TDE0597 from the prokaryote Treponema denticola, represents a third enoyl-CoA reductase that has been cloned and expressed in E. coli (Tucci and Martin, Febs Letters 581:1561-1566 (2007)).

<tables><img file="JP7370366B2_D0087.tif" /></tables>

Additional enoyl-CoA reductase enzyme candidates are found in organisms that degrade aromatic compounds. Rhodopseudomonas palustris, a model organism for the degradation of benzoic acid, has the enzymatic ability to degrade pimelic acid through beta-oxidation of pimeloyl-CoA. The adjacent genes pimC and pimD in the pim operon have sequence homology to bcd of C. acetobutylicum and are predicted to encode flavin-containing pimeloyl-CoA dehydrogenases (Harrison and Harwood, Microbiology 151:727-736 (2005 )). The genome of the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum also contains a pim operon consisting of genes with high sequence similarity to R. palustris pimC and pimD (Harrison and Harwood, Microbiology 151:727-736(2005)).

<tables><img file="JP7370366B2_D0088.tif" /></tables>

An additional candidate is 2-methyl-branched enoyl-CoA reductase (EC 1.3.1.52), an enzyme that catalyzes the reduction of sterically hindered trans-enoyl-CoA substrates. This enzyme is involved in the synthesis of branched chain fatty acids in the nematode Ascaris porcine, and produces a variety of linear fatty acids including 2-methylbutanoyl-CoA, 2-methylpentanoyl-CoA, octanoyl-CoA and pentanoyl-CoA. Substrates and branched-chain substrates can be reduced (Duran et al., J Biol. Chem. 268:22391-22396 (1993)). Two isoforms of this enzyme encoded by the acad1 gene and the acad gene have been characterized.

<tables><img file="JP7370366B2_D0089.tif" /></tables>

1.4.1.a Oxidoreductase (ketone or aldehyde to amino). Oxidoreductases of EC class 1.4.1, which convert aldehydes or ketones to their corresponding amine groups, catalyze several biosynthetic steps in the disclosed pathway. In Figure 12, the conversion of OHED to 2-AHE (Step J), the conversion of 2-OHD to 2-AHD (Step H), and the conversion of adipate semialdehyde to 6-aminocaproate (Step E) are , OHED aminating oxidoreductase, 2-OHD aminating oxidoreductase and adipate semialdehyde aminating oxidoreductase. In Figure 13, the conversion of 6-aminocaproic acid semialdehyde to HMDA (Step H) and the conversion of 6-acetamidohexanal to 6-acetamidohexanamine (Step G) are also catalyzed by an aminating oxidoreductase.

Most aminating oxidoreductases catalyze the reversible oxidative deamination of alpha-amino acids using NAD+ or NADP+ as acceptors, and the reaction is generally reversible. Exemplary enzymes include glutamate dehydrogenase (deamination) encoded by gdhA, leucine dehydrogenase (deamination) encoded by ldh, and aspartate dehydrogenase (deamination) encoded by nadX. gdhA gene product from Escherichia coli (Korber et al., J Mol. Biol. 234:1270-1273 (1993); and McPherson et al., Nucleic Acids Res. 11:5257-5266 (1983)), gdh from Thermotoga maritima (Kort et al., Extremophiles. 1:52-60 (1997); Lebbink et al., J Mol. Biol. 280:287-296 (1998); and Lebbink et al., J Mol. Biol. 289:357-369 (1999)) and gdhA1 from Halobacterium salinarum (Ingoldsby et al., Gene 349:237-244 (2005)), which each contain NADP(H), NAD(H), or both. Preferably, it catalyzes the reversible interconversion of glutamic acid with 2-oxoglutaric acid and ammonia. The B. cereus ldh gene encodes the LeuDH protein, which has a wide range of substrates including leucine, isoleucine, valine, and 2-aminobutanoate (Ansorge and Kula, Biotechnol Bioeng 68:557-562 (2000); and Stoyan et al., J Biotechnol. 54:77-80 (1997)). The nadX gene from Thermotoga maritima, which encodes aspartate dehydrogenase, is involved in the biosynthesis of NAD (Yang et al., J Biol. Chem. 278:8804-8808 (2003)).

<tables><img file="JP7370366B2_D0090.tif" /></tables>

Lysine 6-dehydrogenase (deamination), encoded by lysDH, catalyzes the oxidative deamination of the 6-amino group of L-lysine to form 2-aminoadipate-6-semialdehyde, which in turn by non-enzymatic cyclization of Δ¹Forms piperidine-6-carboxylate (Misono and Nagasaki, J. Bacteriol. 150:398-401 (1982)). Exemplary enzymes are Geobacillus stearothermophilus (Heydari et al., Appl Environ. Microbiol 70:937-942 (2004)), Agrobacterium tumefaciens (Hashimoto et al., J Biochem. 106:76-80 (1989)) ; and Misono and Nagasaki, J. Bacteriol. 150:398-401 (1982)), and Achromobacter denitrificans (Ruldeekulthamrong et al., BMB. Rep. 41:790-795 (2008)). can. Such enzymes are particularly good candidates for converting adipate semialdehyde to 6-aminocaproate, given the structural similarity between them and 2-aminoadipate-6-semialdehyde. .

<tables><img file="JP7370366B2_D0091.tif" /></tables>

2.3.1.a Acyltransferase (transfers CoA to phospho). Acyltransferases that exchange CoA moieties with phosphates are of EC class 2.3.1. Conversions in this category include the conversion of 6-AAHOP to 6-acetamidohexanoyl-CoA (Figure 13, Step K) and the conversion of 6-AHOP to 6-aminocaproyl-CoA (Figure 13, Step L). include. Exemplary phosphotransferases include phosphotransacetylase encoded by pta (EC 2.3.1.8) and phosphotransbutyrylase encoded by ptb (EC 2.3.1.19). The pta gene from E. coli encodes an enzyme that reversibly converts acetyl-CoA to acetyl phosphate (Suzuki, T., Biochim. Biophys. Acta 191:559-569 (1969)). This enzyme can also utilize propionyl-CoA as a substrate in this process to form propionic acid (Hesslinger et al., Mol. Microbiol 27:477-492(1998)). Similarly, the ptb gene from C. acetobutylicum encodes phosphate transbutyrylase, an enzyme that reversibly converts butyryl-CoA to butyryl phosphate (Walter et al., Gene 134:107-111 (1993); and Wiesenborn et al., Appl Environ. Microbiol 55:317-322 (1989)). Additional ptb genes have been found in the butyrate-producing bacterium L2-50 (Louis et al., J. Bacteriol. 186:2099-2106 (2004)) and in Bacterium macrobialis (Vazquez et al., Curr. Microbiol 42:345-349 (2001)). It will be done.

<tables><img file="JP7370366B2_D0092.tif" /></tables>

2.3.1.C Acyltransferase (N-acetyltransferase). N-acetyltransferase transfers an acetyl group to an amine, forming an N-acetyl group. N-acetylation serves diverse functions in biological systems including transcriptional regulation, nuclear import, chromosome set and nucleosome remodeling (Kouzarides, EMBO J 19:1176-1179 (2000)). N-acetylation of metabolic intermediates in the arginine biosynthetic pathway serves both to protect reactive intermediates from spontaneous cyclization and to sequester pathway intermediates from competing pathways (Caldovic et al. Tuchman, Biochem. J 372:279-290(2003)). Acetylation of 6-ACA (Figure 13, Step D) plays a similar role in the proposed HMDA biosynthetic route of Figure 13, protecting the reactive intermediate from spontaneous cyclization.

One candidate enzyme for acetylating 6-ACA is an enzyme that selectively transfers an acetyl moiety from acetyl phosphate to the terminal amino group of L-lysine, beta-L-lysine or L-ornithine, lysine. N-acetyltransferase (EC 2.3.1.32). Although this enzyme is not known to acetylate 6-ACA, this substrate is structurally similar to the natural substrate. Lysine N-acetyltransferase is commonly used in bovine (Paik. and Kim, Arch. Biochem. Biophys. 108:221-229, 1964) and Methanosarcina mazei (Pfluger et al., Appl Environ. Microbiol 69:6047-6055 (2003)). ) is characterized in The methanogenic archaea M. maripaldis, M. acetivorans, M. barkeri, and M. janaskii are also predicted to encode enzymes with this functionality (Pfluger et al., Appl Environ. Microbiol 69:6047- 6055(2003)).

<tables><img file="JP7370366B2_D0093.tif" /></tables>

Alternatively, acetylation of 6-ACA can be catalyzed by enzymes of the GNAT family of N-acetyltransferases. Such enzymes transfer an acetyl group from acetyl-CoA to a primary amine. The enzyme spermidine N-acetyltransferase (SSAT), also known as diamine N-acetyltransferase (EC 2.3.1.57), is capable of acetylating a variety of small molecule substrates. Enzymes purified from Ascaris porcine and Onchocerca volvulus exhibit a broad range of substrates, including HMDA (Davids et al., Mol. Biochem. Parasitol. 64:341-344 (1994); and Wittich and Walter , Mol. Biochem. Parasitol. 38:13-17 (1990)), and no related genes have been identified so far. Other enzymes with this functionality are known from Bacillus subtilis (Forouhar et al., J Biol. Chem. 280:40328-40336 (2005)) and humans (Casero and Pegg, FASEB J 7:653-661 (1993)). A closely related enzyme is C. elegans thiarisine N-acetyltransferase, an enzyme that tolerates a variety of substrates including lysine, ornithine, thiarisine, and others (bo-Dalo et al., Biochem. J 384: 129-137(2004)). Amino acid residues involved in substrate binding have been identified in thiarisin N-acetyltransferase from Leishmania sylvatica (Luersen, K., FEBS Lett. 579:5347-5352 (2005)). Additional candidates are Methylomicrobium alkalinephyllum (Reshetnikov et al., Arch. Microbiol 184:286-297 (2006)), C. salexigens (formerly Halomonas elongata) (Canovas et al., Syst. Diaminobutyrate acetyltransferase (EC 2.3.1.178), an enzyme involved in the biosynthesis of ectoine in Appl Microbiol 21:487-497 (1998)).

<tables><img file="JP7370366B2_D0094.tif" /></tables>

Additional enzyme candidates for acetylating 6-ACA (Figure 13, Step D) and deacetylating 6-acetamidohexanamine (Figure 13, Step H) are two steps in arginine biosynthesis. Ornithine acetyltransferase (OAT, EC 2.3.1.35 and EC 2.3.1.1) is a bifunctional enzyme that catalyzes (FIG. 14A). The first step in arginine biosynthesis (Figure 14A, step 1) is N-acetylation of glutamate, catalyzed by OAT, using acetyl-CoA as the acetyl donor (O'Reilly and Devine, Microbiology 140 (Pt 5):1023-1025(1994)). OAT is present in the fifth step of arginine biosynthesis (Figure 14A, step 2) also catalyzes. This conversion serves to reuse the acetyl group and regenerate N-acetylglutamate, conserving energy and thereby making the linear pathway a circular route. A similar strategy in the biosynthesis of HMDA from 6-aminocaproate using a single enzyme that acetylates 6-aminocaproate and deacetylates 6-acetamidohekianamine to form HMDA. can be used (Figure 14B). Exemplary OAT enzymes include Bacillus subtilis argJ (O'Reilly and Devine, Microbiology 140(Pt 5):1023-1025 (1994); and Sakanyan et al., Journal of General Microbiology 138:125-130 (1992)) and ECM40 of Saccharomyces cerevisiae (Abadjieva et al., J Biol. Chem. 275:11361-11367 (2000); and Liu et al., Eur. J Biochem. 228:291-296 (1995)). Crystal structure of enzyme from yeast (Maes et al., Acta Crystallogr. Sect. F. Struct. Biol. Cryst. Commun. 62:1294-1297 (2006)) and crystal structure of enzyme from Mycobacterium tuberculosis (Sankaranarayanan et al., Acta Crystallogr. Sect. F. Struct. Biol. Cryst. Commun. 65:173-176 (2009)) is available. The OAT enzyme is encoded in a single open reading frame but has distinct alpha and beta subunit peptides (Liu et al., Eur. J Biochem. 228:291-296 (1995)).

<tables><img file="JP7370366B2_D0095.tif" /></tables>

2.3.1.d Acyltransferase (formate C-acyltransferase). The acylation of the keto acids HODH, OHED and 2-OHD to their corresponding CoA derivatives (Figure 12, steps L, P and Q) and the concomitant liberation of formic acid is a form of formic acid C of EC class 2.3.1. -Catalyzed by enzymes that are acyltransferases. This class of enzymes includes pyruvate formate lyase and keto acid formate lyase. Pyruvate formate lyase (PFL, EC 2.3.1.54), encoded by pflB in E. coli, converts pyruvate to acetyl-CoA and formate. The active site of PFL contains a catalytically essential glycyl radical that is posttranslationally activated under anaerobic conditions by the PFL-activating enzyme (PFL-AE, EC 1.97.1.4) encoded by pflA. (Knappe et al., Proc. Natl. Acad. Sci US A 81:1332-1335 (1984); and Wong et al., Biochemistry 32:14102-14110 (1993)). Pyruvate formate lyase from Archaeoglobus fulgidus, encoded by pflD, has been cloned, expressed, and characterized in Escherichia coli (Lehtio, L. and A. Goldman, Protein Eng Des Sel 17:545-552 (2004 )). The crystal structures of A. fulgidus and E. coli enzymes have been solved (Lehtio et al., J Mol. Biol. 357:221-235 (2006)). Additional PFL and PFL-AE candidates include Clostridium pasteurianum (Weidner and Sawers, J. Bacteriol. 178:2AAO-2AAA (1996)) and the eukaryotic alga Conamiridium (Cary et al., Appl. Environ. Microbiol 56:1576-1583 (1990)). Keto acid formate lyase (EC 2.3.1.-), also known as 2-ketobutyrate formate lyase (KFL) and pyruvate formate lyase 4, is the gene product of tdcE of E. coli. This enzyme catalyzes the conversion of 2-ketobutyrate to propionyl-CoA and formate during anaerobic threonine degradation and can also replace pyruvate formate lyase in anaerobic catabolic reactions (Simanshu et al. J Biosci.32:1195-1206(2007)). This enzyme is oxygen sensitive and, like PflB, requires post-translational modification by PFL-AE to activate the glycyl radical in the active site (Hesslinger et al., Mol. Microbiol 27:477-492 (1998 )).

<tables><img file="JP7370366B2_D0096.tif" /></tables>

2.6.1.a Aminotransferases. Steps E, H, and J of Figure 12 and steps C and G of Figure 13 require conversion of an aldehyde or ketone to an amino group. This conversion can be achieved by aminotransferases (EC 2.6.1.-). The conversion of aldehydes to terminal amines (Figure 12, Step E; Figure 13, Steps C and G) can be catalyzed by gamma-aminobutyrate transaminase (GABA transaminase). One of the E. coli GABA transaminases, encoded by gabT, transfers an amino group from glutamic acid to the terminal aldehyde of succinic semialdehyde (Bartsch et al., J. Bacteriol. 172:7035-7042 (1990)). This enzyme exhibits a broad substrate range (Liu et al., Biochemistry 43:10896-10905 (2004)). The puuE gene product encodes another 4-aminobutyrate transaminase in E. coli (Kurihara et al., J. Biol. Chem. 280:4602-4608(2005)). Mouse, Pseudomonas fluorescens, and wild boar GABA transaminases have been shown to react with 6-aminocaproic acid (Cooper, Methods Enzymol. 113:80-82 (1985); and Scott and Jakoby, J Biol. Chem. 234:932-936(1959)).

<tables><img file="JP7370366B2_D0097.tif" /></tables>

Additional enzyme candidates include putrescine aminotransferase or other diamine aminotransferases. Such enzymes are particularly well suited for carrying out the conversion of 6-aminocaproate semialdehyde to HMDA. E. coli putrescine aminotransferase is encoded by the ygjG gene, and the purified enzyme was also able to transfer the amino groups of cadaverine and spermidine (Samsonova et al., BMC. Microbiol 3:2 (2003)). Furthermore, the activity of this enzyme toward 1,7-diaminoheptane using amino acceptors other than 2-oxoglutarate (e.g., pyruvate, 2-oxobutanoate) has been reported (Kim, J Biol.Chem 239:783-786 (1964); and Samsonova et al., BMC. Microbiol 3:2 (2003)). Putrescine aminotransferase, which is more active toward pyruvate than alpha-ketoglutarate as an amino receptor, is the spuC gene of Pseudomonas aeruginosa (Lu et al., J. Bacteriol. 184:3765-3773 (2002)).

<tables><img file="JP7370366B2_D0098.tif" /></tables>

Additional candidate enzymes include beta-alanine/alpha-ketoglutarate aminotransferase, which produces malonic acid semialdehyde from beta-alanine (WO08027742). The Saccharomyces kluyveri SkPYD4 gene product was shown to preferentially use beta-alanine as an amino group donor (Andersen and Hansen, Gene 124:105-109 (1993)).

SkUGA1 encodes a homolog of the Saccharomyces cerevisiae GABA aminotransferase, UGA1 (Ramos et al., Eur. J. Biochem. 149:401-404 (1985)), while SkPYD4 encodes the amino groups of both β-alanine and GABA. It encodes an enzyme involved in translocation (Andersen and Hansen, Gene 124:105-109 (1993)). 3-Amino-2-methylpropionic acid transaminase catalyzes the conversion of methylmalonic acid semialdehyde to 3-amino-2-methylpropionic acid. This enzyme has been characterized in rat and boar and is encoded by Abat 1968 (Kakimoto et al., Biochim. Biophys. Acta 156:374-380 (1968); and Tamaki et al., Methods Enzymol. 324:376-389 (2000)).

<tables><img file="JP7370366B2_D0099.tif" /></tables>

Steps J and H of Figure 12 are catalyzed by aminotransferases that convert amino acids to oxoacids. In step J, OHED is transaminated to form 2-AHE by an OHED aminotransferase. Transamination of 2-OHD to 2-AHD by 2-OHD aminotransferase (Step H) is a similar reaction. An exemplary enzyme candidate for catalyzing these reactions is aspartate aminotransferase, an enzyme that naturally transfers the oxo group from oxaloacetate to glutamate, forming alpha-ketoglutarate and aspartate. Aspartic acid is similar in structure to OHED and 2-AHD. The activity of aspartate aminotransferase can be determined by, for example, aspC from Escherichia coli (Yagi et al., FEBS Lett. 100:81-84, (1979); and Yagi et al., Methods Enzymol. 113:83-89 (1985)), Saccharomyces cerevisiae AAT2 (Yagi et al., J Biochem. 92:35-43 (1982)) and ASP5 from Arabidopsis (de la Torre et al., Plant J 46:414-425 (2006); Kwok and Hanson, J Exp. Bot. 55:595-604 (2004); and Wilkie and Warren, Protein Expr. Purif. 12:381-389 (1998)). Enzymes from rats have been shown to transfer amino groups of alternative substrates such as 2-aminohexanedioic acid and 2,4-diaminobutyrate (Recasens et al., Biochemistry 19:4583-4589 (1980) ). Aminotransferases acting on other amino acid substrates can catalyze this conversion. Valine aminotransferase catalyzes the conversion of valine and pyruvate to 2-ketoisovalerate and alanine. The avtA gene of E. coli encodes one such enzyme (Whalen and Berg, CJ Bacteriol. 150:739-746(1982)). This gene product also catalyzes the transamination of α-ketobutyrate to produce α-aminobutyrate, although the amine donor in this reaction has not been identified (Whalen and Berg, J. Bacteriol. 158:571- 574(1984)). The E. coli serC gene product catalyzes two reactions, phosphoserine aminotransferase and phosphohydroxythreonine aminotransferase (Lam and Winkler, J. Bacteriol. 172:6518-6528 (1990)), and has activity toward nonphosphorylated substrates. could not be detected (Drewke et al., FEBS. Lett. 390:179-182 (1996)).

<tables><img file="JP7370366B2_D0100.tif" /></tables>

2.7.2.a Phosphotransferases (carboxy receptors). Enzymes that are EC class 2.7.2 phosphotransferases convert carboxylic acids to phosphonic acids, with simultaneous hydrolysis of one ATP. Steps A and E of Figure 13 are for activating the carboxyl group of 6-ACA to its corresponding phosphonic acid (Step A) and for activating the carboxyl group of 6-acetamidohexanoic acid to its corresponding phosphonic acid. (Step E) requires phosphotransferase. Butyrate kinase performs the reversible conversion of butyryl phosphate to butyrate during acid fermentation in C. acetobutylicum (Cary et al., Appl. Environ. Microbiol 56:1576-1583 (1990)). This enzyme is encoded by either of the two buk gene products (Huang et al., J Mol. Microbiol Biotechnol 2:33-38 (2000)). A related enzyme, isobutyrate kinase from Thermotoga maritima, has also been expressed in E. coli and crystallized (Diao et al., Acta Crystallogr. D. Biol. Crystallogr. 59:1100-1102 (2003); and Diao and Hasson, J. Bacteriol. 191:2521-2529 (2009)). Aspartokinase catalyzes the ATP-dependent phosphorylation of aspartate and is involved in the synthesis of several amino acids. The E. coli aspartokinase III enzyme is encoded by lysC, has a broad substrate range, and the catalytic residues involved in substrate specificity have been elucidated (Keng and Viola, Arch. Biochem. Biophys. 335:73 -81(1996)). Two additional kinases from E. coli are also good candidates: acetate kinase and gamma-glutamyl kinase. The E. coli acetate kinase is encoded by ackA (Skarstedt and Silverstein, J. Biol. Chem. 251:6775-6783 (1976)) and phosphorylates propionate in addition to acetate (Hesslinger et al., Mol. Microbiol 27: 477-492(1998)). Escherichia coli gamma-glutamyl kinase is encoded by proB (Smith et al., J. Bacteriol. 157:545-551 (1984)), which phosphorylates the gamma carbonate group of glutamic acid.

<tables><img file="JP7370366B2_D0101.tif" /></tables>

Acetylglutamate kinase is an excellent candidate for phosphorylating acetylated glutamate and phosphorylating 6-acetamidohexanoate during arginine biosynthesis (Figure 13, Step E). This enzyme is not known to tolerate alternative substrates; however, by site-directed mutagenesis, several residues of the E. coli enzyme involved in substrate binding and phosphorylation have been elucidated ( Marco-Martin et al., J Mol. Biol. 334:459-476 (2003); and Ramon-Maiques et al., Structure. 10:329-342 (2002)). This enzyme is argB of Bacillus subtilis and E. coli (Parsot et al., Gene 68:275-283 (1988)), and ARG5,6 of Saccharomyces cerevisiae (Pauwels et al., Eur. JBiochem. 270:1014-1024(2003)). The Saccharomyces cerevisiae ARG5,6 gene is a polyprotein precursor that matures in the mitochondrial matrix to become acetylglutamate kinase and acetylglutamyl phosphate reductase, which are candidate enzymes for reducing 6-AAHOP (Figure 13, Step F). Code your body.

<tables><img file="JP7370366B2_D0102.tif" /></tables>

2.8.3.a Coenzyme A transferase. Coenzyme A (CoA) transferase catalyzes the reversible transfer of a CoA moiety from one molecule to another. In step M of Figure 13, 3-aminocaproyl-CoA is formed by transfer of a CoA group from acetyl-CoA, succinyl-CoA, or another CoA donor. A similar conversion is catalyzed by 6-acetamidohexanoate CoA-transferase and is shown in step I of FIG. 13. Exemplary CoA transferase candidates are the cat1 and cat2 gene products of Clostridium kluyveri, which have been shown to exhibit succinyl-CoA transferase activity, 4-hydroxybutyryl-CoA transferase activity, and butyryl-CoA transferase activity, respectively. , and catalyzed by the cat3 gene product (Seedorf et al., Proc. Natl. Acad. Sci US A 105:2128-2133 (2008); and Sohling and Gottschalk, J. Bacteriol. 178:871-880(1996)). Similar CoA transferase activity was observed in Trichomonas vaginalis (van Grinsven et al., J. Biol. Chem. 283:1411-1418 (2008)) and Trypanosoma brucei (Riviere et al., J. Biol. Chem. 279:45337-45346 (2004)). ) also exists.

<tables><img file="JP7370366B2_D0103.tif" /></tables>

A CoA transferase that can utilize acetyl-CoA as a CoA donor is acetoacetyl-CoA transferase encoded by the atoA (alpha subunit) and atoD (beta subunit) genes of Escherichia coli (Korolev et al., Acta Crystallogr. D. Biol. Crystallogr. 58:2116-2121 (2002); and Vanderwinkel et al., Biochem. Biophys. Res.Commun. 33:902-908 (1968)). This enzyme has a broad substrate range (Sramek and Frerman, Arch. Biochem. Biophys. 171:14-26 (1975)) and isobutyrate (Matthies and Schink, ApplEnviron. Microbiol 58:1435-1439 (1992)). , valeric acid (Vanderwinkel et al., Biochem. Biophys. Res. Commun. 33:902-908 (1968)) and butanoate (Vanderwinkel et al., Biochem. Biophys. Res. Commun. 33:902-908 (1968)) has been shown to transfer the CoA moiety to acetate from a variety of branched and linear acyl-CoA substrates. Since this enzyme is induced by acetoacetate at the transcriptional level, regulatory modifications may be required to genetically engineer this enzyme into the pathway (Pauli and Overath, Eur.J Biochem. 29 :553-562(1972)). Similar enzymes are found in Corynebacterium glutamicum ATCC13032 (Duncan et al., Appl. Environ. Microbiol 68:5186-5190 (2002)) and Clostridium acetobutylicum (Cary et al., Appl. Environ. Microbiol 56:1576-1583 (1990)). ; and Wiesenborn et al., Appl. Environ. Microbiol 55:323-329 (1989)), and Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem. 71:58-68 (2007)).

<tables><img file="JP7370366B2_D0104.tif" /></tables>

The glutaconyl-CoA-transferase (EC 2.8.3.12) enzyme from the anaerobic bacterium Acidaminococcus fermentans reacts with glutaconyl-CoA and 3-butenoyl-CoA (Mack et al., Eur. J. Biochem. 226 :41-51(1994)). The genes encoding this enzyme are gctA and gctB. This enzyme has reduced but detectable activity towards other CoA derivatives including glutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckel et al., Eur. J Biochem 118:315-321(1981)). This enzyme has been cloned and expressed in E. coli (Mack et al., Eur. J. Biochem. 226:41-51 (1994)).

<tables><img file="JP7370366B2_D0105.tif" /></tables>

Yet another CoA transferase is the two-unit succinyl-CoA:3:oxoacid CoA transferase encoded by pcaI and pcaJ in Pseudomonas putida (Kaschabek et al., J. Bacteriol. 184:207-215 (2002)). . A similar enzyme based on homology exists in Acinetobacter species ADP1 (Kowalchuk et al., Gene 146:23-30 (1994)). Additional exemplary succinyl-CoA:3:oxoacid CoA transferases are present in H. pylori (Corthesy-Theulaz et al., J Biol. Chem. 272:25659-25667 (1997)) and Bacillus subtilis (Stols et al., Protein Expr. Purif. 53:396-403(2007)).

<tables><img file="JP7370366B2_D0106.tif" /></tables>

3.5.1.a Hydrolases (acting on linear amides). Deacetylation of linear acetamides is catalyzed by amidohydrolases of the 3.5.1 family of enzymes. Such an enzyme is required to deacetylate 6-acetamidohekianamine to HMDA (Figure 13, Step H). An enzyme that catalyzes a similar conversion is 4-acetamidobutyrate deacetylase (EC 3.5.1.63), which naturally deacetylates 4-acetamidobutyrate. This enzyme was studied for its role in the degradation of putrescine in Candida boidinii (Gillyon et al., Journal of General Microbiology 133:2477-2485 (1987)) and deacetylated various substrates including 6-acetamidohexanoic acid. (Haywood and Large, Journal of General Microbiology 132:7-14 (1986)). Although 6-acetamidohexanoic acid is structurally similar to the desired substrate, deacetylation of this compound (Figure 13, Step D, reverse reaction) may prevent efficient production of HMDA. Genetic engineering or directed evolution of the protein may be necessary to improve specificity for 6-acetamidohexanamine. No genes associated with this activity have been identified so far.

Acetyl polyamine amide hydrolase (EC 3.5.1.62) is another candidate enzyme that forms the diamines putrescine and cadaverine from their acetylated precursors. Acetylpolyamine deacetylase (AphA) from Mycoplana ramosa has been cloned into Escherichia coli and characterized (Sakurada et al., J. Bacteriol. 178:5781-5786 (1996)), and a crystal structure is available. (Fujishiro et al., Biochem. Biophys. Res. Commun. 157:1169-1174 (1988)). This enzyme has also been studied in Micrococcus luteus, but no related genes have been identified so far (Suzuki et al., Biochim. Biophys. Acta 882:140-142 (1986)).

A histone deacetylase superfamily protein with high sequence similarity to AphA was identified in the M. luteus genome (E value = 1 × 10^-18, 37% identity). N-acetyl-L-ornithine deacetylase from E. coli is another candidate for amidohydrolase (EC 3.5.1.16). The Escherichia coli enzyme encoded by the argE gene (McGregor et al., J Am. Chem. Soc. 127:14100-14107 (2005); and Meinnel et al., J. Bacteriol. 174:2323-2331 (1992)) is capable of producing ornithine, It removes the N-acetyl group from a variety of substrates including lysine, glutamine, and other amino acids (Javid-Majd and Blanchard, Biochemistry 39:1285-1293 (2000)).

<tables><img file="JP7370366B2_D0107.tif" /></tables>

4.1.1.a Carboxylyase. Steps D and F of Figure 12 are catalyzed by an enzyme that is a 2-keto acid decarboxylase that generates 6-OHE (Step F) from OHED and adipate semialdehyde (Step D) from 2-OHD. Additionally, alpha-ketoglutarate is decarboxylated by the keto acid decarboxylase alpha-ketoglutarate decarboxylase to form the pathway precursor succinic semialdehyde. Decarboxylation of keto acids is performed using pyruvate decarboxylase (EC 4.1.1.1), benzoylformate decarboxylase (EC 4.1.1.7), is catalyzed by a variety of enzymes with varying substrate specificities, including alpha-ketoglutarate decarboxylase and branched-chain alpha-keto acid decarboxylase. Pyruvate decarboxylase (PDC), also called keto acid decarboxylase, is an important enzyme in alcohol fermentation, catalyzing the decarboxylation of pyruvate to acetaldehyde. The enzyme from Saccharomyces cerevisiae has a broad substrate range for aliphatic 2-keto acids, including 2-ketobutyrate, 2-ketovalerate, 3-hydroxypyruvate, and 2-phenylpyruvate (22). . This enzyme has been extensively studied in Escherichia coli, genetically engineered to alter its activity, and functionally expressed (Killenberg-Jabs et al., Eur. J. Biochem. 268:1698-1704 (2001 ); Li, H. and F. Jordan, Biochemistry. 38:10004-10012 (1999); and ter Schure et al., Appl. Environ. Microbiol. 64:1303-1307(1998)). PDC from Zymomonas mobilus, encoded by PDC, also has a broad substrate range and has been the subject of direct genetic engineering studies to alter its affinity for different substrates (Siegert et al., Protein Eng Des Sel 18:345-357(2005)). A crystal structure of this enzyme is available (Killenberg-Jabs et al., Eur. J. Biochem. 268:1698-1704 (2001)). Other well-characterized PDC candidates are Acetobacter pasteurians (Chandra et al., Arch. Microbiol. 176:443-451 (2001)) and Kluyveromyces lactis (Krieger et al., Eur. J. Biochem. 269 :3256-3263(2002)).

<tables><img file="JP7370366B2_D0108.tif" /></tables>

Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broad substrate range and has been the target of studies to genetically engineer the enzyme. The enzyme from Pseudomonas putida has been extensively studied and crystal structures of this enzyme are available (Hasson et al., Biochemistry 37:9918-9930 (1998); and Polovnikova et al., Biochemistry 42:1820-1830 ( 2003)). Site-directed mutagenesis of two residues in the active site of the Pseudomonas putida enzyme altered the affinity (Km) of naturally occurring and non-naturally occurring substrates (Siegert et al., Protein Eng Des Sel 18:345-357(2005)). The properties of this enzyme have been further modified by directed genetic engineering (Lingen et al., Protein Eng 15:585-593 (2002); and Lingen et al., ChembioChem. 4:721-726(2003)). The enzyme from Pseudomonas aeruginosa encoded by mdlC has also been characterized experimentally (Barrowman et al., FEMS Microbiology Letters 34:57-60 (1986)). Additional gene candidates from Pseudomonas stuzzeri, Pseudomonas fluorescens, and other organisms can be identified by sequence homology or using the growth selection system developed in Pseudomonas putida. (Henning et al., Appl. Environ. Microbiol. 72:7510-7517 (2006)).

<tables><img file="JP7370366B2_D0109.tif" /></tables>

A third enzyme that can decarboxylate 2-oxoacids is alpha-ketoglutarate decarboxylase (KGD). The substrate range of this class of enzymes has not been investigated to date. KDC from Mycobacterium tuberculosis (Tian et al., Proc Natl Acad Sci US A 102:10670-10675 (2005)) has been cloned and functionally expressed in another internal project at Genomatica. However, it is large (approximately 130 kD) and GC-rich, so it is not an ideal candidate for genetically engineering the strain. KDC enzymatic activity has been detected in several species of rhizobia, including Bradyrhizobium japonicum and Mesorhizobium loti (Green et al., J. Bacteriol. 182:2838-2844(2000)). Although the KDC-encoding gene(s) have not been isolated in these organisms, genome sequences are available and several genes within each genome are annotated as putative KDCs. KDC from Euglena gracilis has also been characterized, but the genes associated with this activity have not been identified so far (Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). The first 20 amino acids starting from the N-terminus were sequenced <img file="JP7370366B2_D0110.tif" /> (Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). Genes can be identified by testing candidate genes containing this N-terminal sequence for KDC activity.

<tables><img file="JP7370366B2_D0111.tif" /></tables>

A fourth candidate enzyme for catalyzing this step is branched-chain alpha-keto acid decarboxylase (BCKA). This class of enzymes has been shown to act on a variety of compounds varying in chain length from 3 to 6 carbons (Oku and Kaneda, J Biol Chem. 263:18386-18396 (1988); and Smit et al., Appl Environ Microbiol. 71:303-311 (2005)). Lactococcus lactis enzymes are 2-oxobutanoate, 2-oxohexanoic acid, 2-oxopentanoic acid, 3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate and isocaprone. It has been characterized for a variety of branched and linear substrates, including acids (Smit et al., Appl Environ Microbiol. 71:303-311(2005)). This enzyme has been structurally characterized (Berg et al., Science. 318:1782-1786 (2007)). Since sequence alignments between the Lactococcus lactis enzyme and the Zymomonas mobilus pyruvate decarboxylase have shown that the catalytic and substrate recognition residues are nearly identical (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)), making this enzyme a promising candidate for directed genetic engineering manipulation. Decarboxylation of alpha-ketoglutarate by BCKA was detected in B. subtilis; however, this activity was low (5%) compared to the activity toward other branched-chain substrates (Oku and Kaneda, J BiolChem. 263:18386-18396 (1988)), and the gene encoding this enzyme has not been identified to date. Additional BCKA gene candidates can be identified by homology to Lactococcus lactis protein sequences. Many of the high scoring BLASTp hits for this enzyme are annotated as indolepyruvate decarboxylase (EC 4.1.1.74). Indolepyruvate decarboxylase (IPDA) is an enzyme that catalyzes the decarboxylation of indolepyruvate to indoleacetaldehyde in plants and plant bacteria.

<tables><img file="JP7370366B2_D0112.tif" /></tables>

The enzyme, a recombinant branched-chain alpha-keto acid decarboxylase derived from the E1 subunit of the mitochondrial branched-chain keto acid dehydrogenase complex of human and bovine origin, has been cloned and functionally expressed in Escherichia coli (Davie et al., J. Biol. Chem. 267:16601-16606(1992); Wynn et al., J. Biol. Chem. 267:1881-1887(1992); and Wynn et al., J. Biol. Chem. 267:12400-12403 (1992)). In these studies, the authors found that co-expression of the chaperonins GroEL and GroES increased the specific activity of decarboxylase by up to 500-fold (Wynn et al., J. Biol. Chem. 267 :12400-12403(1992)). These enzymes are composed of two alpha subunits and two beta subunits.

<tables><img file="JP7370366B2_D0113.tif" /></tables>

Decarboxylation of 2-AHD to 6-aminocaproate (Figure 12, Step I) is catalyzed by an amino acid decarboxylase, such as aspartate decarboxylase. Aspartate decarboxylase is involved in the biosynthesis of pantothenic acid and is encoded by the gene panD in Escherichia coli (Dusch et al., Appl. Environ. Microbiol 65:1530-1539 (1999); Merke and Nichols, FEMS Microbiol Lett. 143 :247-252(1996); Ramjee et al., Biochem. J 323(Pt 3):661-669(1997); and Schmitzberger et al., EMBO J 22:6193-6204(2003)). Similar enzymes from Mycobacterium tuberculosis (Chopra et al., ProteinExpr. Purif. 25:533-540 (2002)) and Corynebacterium glutamicum (Dusch et al., Appl. Environ. Microbiol 65:1530-1539 (1999)) Expressed and characterized in E. coli.

<tables><img file="JP7370366B2_D0114.tif" /></tables>

4.1.2.a Aldehyde lyase. HOHD aldolase, also known as HHED aldolase, catalyzes the conversion of 4-hydroxy-2-oxo-heptane-1,7-dioate (HOHD) to pyruvate and succinic semialdehyde (Figure 12, Step A). This enzyme is a divalent metal ion-dependent class II aldolase that catalyzes the final step in the degradation of 4-hydroxyphenylacetate in E. coli C, E. coli W, and other organisms. In its native context, this enzyme functions in a degradative direction. The reverse (condensation) reaction is thermodynamically unfavorable; however, the equilibrium can be shifted by coupling the HOHD aldolase with downstream pathway enzymes that act efficiently on the reaction products. Such a strategy is effective to shift the equilibrium of other aldolases in the direction of condensation (Nagata et al., Appl MicrobiolBiotechnol 44:432-438 (1995); and Pollard et al., Appl Environ. Microbiol 64:4093-4094(1998)). The E. coli C enzyme encoded by hpcH has been extensively studied and recently crystallized (Rea et al., J Mol. Biol. 373:866-876 (2007); and Stringfellow et al., Gene 166:73 -76(1995)). The E. coli W enzyme is encoded by hpaI (Prieto et al., J. Bacteriol. 178:111-120 (1996)).

<tables><img file="JP7370366B2_D0115.tif" /></tables>

4.2.1.a Hydrolyase. The enzyme OHED hydratase is involved in the degradation of 4-hydroxyphenylacetate, converting 2-oxo-hept-4-ene-1,7-dioate (OHED) into 2-oxo-4-hydroxy- Convert to hepta-1,7-dioate (HODH) (Burks et al., J. Am. Chem. Soc. 120 (1998)) (Figure 12, Step B). Enzyme candidates that are OHED hydratases have been tested in E. coli C (Izumi et al., J Mol. Biol. 370:899-911 (2007); and Roper et al., Gene 156:47-51 (1995)) and E. coli W (Prieto et al., J Bacteriol. 178:111-120 (1996)). Sequence comparisons reveal a variety of bacterial, plant and animal homologs. Enzymes with highly similar sequences were found, among others, in Klebsiella pneumoniae (91% identity, E value = 2 × 10^-138) and Salmonella enterica (91% identity, E value = 4 × 10^-138).

<tables><img file="JP7370366B2_D0116.tif" /></tables>

Dehydration of 3-hydroxyadipyl-CoA to 2,3-dehydroadipyl-CoA (Figure 12, Step M) is catalyzed by an enzyme with enoyl-CoA hydratase activity. 3-Hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), also called crotonase, dehydrates 3-hydroxyisobutyryl-CoA to form crotonoyl-CoA (Figure 14, step 2). The enzyme crotonase is required for the formation of n-butanol in some organisms, particularly Clostridium species, and also in thermoacidophilic archaea of the genera Sulphorobus, acidianus, and Metallospera. /4-Hydroxybutyrate constitutes one step of the cycle. Exemplary genes encoding enzymes that are crotonases include C. acetobutylicum (Atsumi et al., Metab Eng 10:305-311 (2008); and Boynton et al., J. Bacteriol. 178:3015-3024 (1996)), C. .Kruyberg (Hillmer and Gottschalk, FEBS Lett. 21:351-354 (1972)) and Metallospaera cedula (Berg et al., Science. 318:1782-1786 (2007)), but the sequence of the latter gene is unknown.

<tables><img file="JP7370366B2_D0117.tif" /></tables>

Enoyl-CoA hydratase (EC 4.2.1.17) also catalyzes the dehydration of 3-hydroxyacyl-CoA substrates (Agnihotri and Liu., J. Bacteriol. 188:8551-8559 (2003); Conrad et al., J. Bacteriol. 118:103-111 (1974); and Roberts et al., Arch. Microbiol 117:99-108 (1978)). The Pseudomonas putida enoyl-CoA hydratase, encoded by ech, catalyzes the conversion of 3-hydroxybutyryl-CoA to crotonoyl-CoA (Roberts et al., Arch. Microbiol 117:99-108 (1978)). Additional enoyl-CoA hydratase candidates are phaA and phaB from P. putida and paaA and paaB from P. fluorescens (Olivera et al., Proc. Natl. Acad. Sci U. SA 95:6419-6424(1998)). The gene product of pimF in Rhodopseudomonas palustris is predicted to encode an enoyl-CoA hydratase involved in the degradation of pimeloyl-CoA (Harrison and Harwood, Microbiology 151:727-736 (2005)). Finally, maoC (Park and Lee, J. Bacteriol. 185:5391-5397 (2003)), paaF (Ismail et al., J Biochem. 270:3047-3054 (2003); Park and Lee, Appl. Biochem. Biotechnol 113 -116:335-346 (2004); and Park and Yup, Biotechnol Bioeng 86:681-686 (2004)) and paaG (Ismail et al., J Biochem. 270:3047-3054 (2003); Park and Lee, Appl. Biochem. A number of E. coli genes have been shown to exhibit enoyl-CoA hydratase functionality, including Biotechnol 113-116:335-346 (2004); and Park and Yup, Biotechnol Bioeng 86:681-686 (2004)). There is.

<tables><img file="JP7370366B2_D0118.tif" /></tables>

Alternatively, the E. coli fadA and fadB gene products encode a multienzyme complex that exhibits enoyl-CoA hydratase activity, which is involved in fatty acid oxidation (Nakahigashi and Inokuchi, Nucleic Acids Res. 18:4937 (1990) ); Yang, J. Bacteriol. 173:7405-7406 (1991); and Yang et al., Biochemistry 30:6788-6795 (1991)). Knockout of the negative regulator encoded by fadR can be used to activate the fadB gene product (Sato et al., J Biosci. Bioeng 103:38-44 (2007)). The fadI and fadJ genes encode similar functions and are naturally expressed under anaerobic conditions (Campbell et al., Mol. Microbiol 47:793-805 (2003)).

<tables><img file="JP7370366B2_D0119.tif" /></tables>

6.2.1.a Acid-thiol ligase (also called CoA synthetase). In steps I and M of Figure 13, acid-thiol ligase or CoA synthetase functionality is required to convert 6-ACA and 6-acetamidohexanoic acid into their corresponding CoA derivatives (ligase, synthetase, and synthase are used interchangeably herein and refer to the same enzyme class). The enzymes that catalyze these precise transformations have not been characterized to date; however, several enzymes with broad substrate specificity have been described in the literature. ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is an enzyme that couples the conversion of acyl-CoA esters to their corresponding acids, accompanied by the synthesis of ATP. ACD I from Archaeoglobus fulgidus, encoded by AF1211, was shown to act on a variety of linear and branched substrates, including isobutyrate, isopentanoic acid, and fumarate (Musfeldt and Schonheit, J. Bacteriol. 184:636-644(2002)). A second reversible ACD of Archaeoglobus fulgidus encoded by AF1983 was also shown to have a broad substrate range and high activity towards the cyclic compounds phenylacetate and indole acetate (Musfeldt et al. Schonheit, J. Bacteriol. 184:636-644(2002)). The enzyme from Haloarchula marismortii (annotated as succinyl-CoA synthetase) accepts propionic acid, butyrate, and branched-chain acids (isovaleric acid and isobutyrate) as substrates and can operate in the forward and reverse directions. (Brasen and Schonheit, Arch. Microbiol 182:277-287 (2004)). The ACD encoded by PAE3250 from the hyperthermophilic Crenoarchaeal phylum Pyrobaculum aerophyllum reacts with acetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA, and is one of the most characterized ACDs among all characterized ACDs. showed the broadest substrate range (Brasen and Schonheit, Arch. Microbiol 182:277-287(2004)). Directed evolution or directed genetic engineering can be used to modify this enzyme to operate at the physiological temperature of the host organism. Enzymes from A. fulgidus, H. marismortii, and P. aerophilum have all been cloned into E. coli, functionally expressed, and characterized (Brasen and Schonheit, Arch. Microbiol 182:277-287 (2004); and Musfeldt and Schonheit, J. Bacteriol. 184:636-644 (2002)). An additional candidate is the enzyme encoded by sucCD in E. coli, which naturally catalyzes the formation of succinyl-CoA from succinate with the consumption of one ATP, a reaction that is reversible in vivo (Buck et al., Biochemistry 24:6245-6252 (1985)).

<tables><img file="JP7370366B2_D0120.tif" /></tables>

Another candidate enzyme for this step is 6-carboxyhexanoic acid-CoA ligase, also known as pimeloyl-CoA ligase (EC 6.2.1.14), which inhibits pimelin during the biosynthesis of biotin in Gram-positive bacteria. Naturally activates acid to pimeloyl-CoA. An enzyme from Pseudomonas mendocina, cloned into E. coli, was shown to tolerate alternative substrates hexanedioate and nonanedioate (Binieda et al., Biochem. J 340(Pt 3):793-801 (1999) )). Other candidates are Bacillus subtilis (Bower et al., J. Bacteriol. 178:4122-4130 (1996)) and Ricinibacillus sphaericus (formerly Bacillus sphaericus) (Ploux et al., Biochem. J 287(Pt 3):685- 690 (1992)).

<tables><img file="JP7370366B2_D0121.tif" /></tables>

Additional CoA ligases include the rat dicarboxylic acid-CoA ligase (Vamecq et al., Biochem. J 230:683-693 (1985)), whose sequence has not yet been characterized, and two well-characterized species from P. chrysogenum. phenylacetate-CoA ligase (Lamas-Maceiras et al., Biochem. J395:147-155 (2006); and Wang et al., Biochem. Biophys. Res. Commun. 360:453-458 (2007)) and Pseudomonas. phenylacetate-CoA ligase from P. putida (Martinez-Blanco et al., J Biol. Chem. 265:7084-7090 (1990)). Additional candidate enzymes include mouse (Hasegawa et al., Biochim. Biophys. Acta 1779:414-419 (2008)) and human (Ohgami) enzymes that naturally catalyze the ATP-dependent conversion of acetoacetate to acetoacetyl-CoA. et al., Biochem. Pharmacol. 65:989-994 (2003)).

<tables><img file="JP7370366B2_D0122.tif" /></tables>

EXAMPLE XXII (Demonstration of Resistance to 6-Aminocaproate in E. coli) Resistance, metabolic activity and growth were observed during exposure of E. coli to various concentrations of 6-aminocaproate (6-ACA). was assayed for. Aerobically, cultures were able to grow in media with up to 10% 6-ACA, whereas anaerobic cultures could grow in media with approximately 6% 6-ACA. (Figure 15). All other further tests were performed under anaerobic conditions, as the pathway to produce 6-ACA may require anaerobic conditions. To assay for resistance, cultures were grown in mid-log phase (0.3 OD) and early stationary phase (0.6 OD). OD), cells were spun down and resuspended in medium containing various concentrations of 6-ACA. Cultures were grown in capped microtubes, grown overnight, and the OD of the cultures was assayed (Figure 16). Under these conditions, cultures were able to grow (doubling at least once) up to 10% in 6-ACA. Additional resistance may have been due to additional glucose by resuspending the culture in fresh M9-glucose medium or to the limited oxygen present in the capped microtube. . To determine whether the cells were metabolically active in the presence of 6-ACA, samples were obtained and assayed for ethanol production (Figure 17). Ethanol production (and thus metabolic activity) closely tracked using OD suggests that the cells, if present, may be metabolically active. This is helpful for understanding as it suggests that cells can still continue to produce products even though proliferation may be inhibited by product accumulation.

At high concentrations (&gt;65 g/L), the osmolarity of 6-ACA is ~0.5 M, which can cause osmotic stress. To determine osmotic stress as a criterion for growth inhibition of 6-ACA, cultures were grown in various concentrations of 6-ACA with and without the osmoprotectant glycine betaine. I let it happen. As seen in Figure 18, anaerobic growth in media with up to 10-12% 6-ACA can be achieved in the presence of glycine betaine, but without glycine betaine, only 4-6 %. Therefore, most of the 6-ACA toxicity can be attributed to osmotic stress. However, it should be noted that 6-ACA is similar to the amino acid lysine and can have significant toxic effects in the cytoplasm versus extracellularly.

EXAMPLE XXIII (Demonstration of Enzyme Activity for Condensing Succinyl-CoA and Acetyl-CoA to Form β-Ketoadipyl-CoA) Some β-ketothiolase enzymes convert β-ketoadipyl-CoA into acetylated It has been shown that it can be divided into -CoA and succinyl-CoA. For example, pcaF of Pseudomonas strain B13 (Kaschabek et al., J. Bacteriol, 184(1):207-15(2002)), phaD of Pseudomonas putida U (Olivera et al., Proc Natl Acad Sci USA, 95(11), 6419-24(1998)), Pseudomonas fluorescens ST encoded by paaE (Di Gennaro et al., Arch Microbiol, 188(2), 117-25(2007)) and paaJ from Escherichia coli (Nogales et al., Microbiology, 153(Pt 2), 357-65(2007)). The gene product catalyzes the conversion of 3-oxoadipyl-CoA to succinyl-CoA and acetyl-CoA during the degradation of aromatic compounds such as phenylacetate or styrene. To confirm that enzymes that are β-ketothiolases exhibit condensation activity, several thiolases (Table 10; SEQ ID NO:, respectively) were cloned into derivatives of pZE13 (Lutz et al., Nucleic Acids Res, 29(18)). , 3873-81 (2001)), 6×His tag at the carboxy terminus<u style="Single">(Sequence number 2)</u>A clone with .

<tables><img file="JP7370366B2_D0123.tif" /></tables><img file="JP7370366B2_D0124.tif" /><img file="JP7370366B2_D0125.tif" /><img file="JP7370366B2_D0126.tif" /><img file="JP7370366B2_D0127.tif" /><img file="JP7370366B2_D0128.tif" /><img file="JP7370366B2_D0129.tif" /><img file="JP7370366B2_D0130.tif" /><img file="JP7370366B2_D0131.tif" /><img file="JP7370366B2_D0132.tif" /><img file="JP7370366B2_D0133.tif" />

The gene was expressed in E. coli and the protein was purified and quantified using a Ni-NTA spin column. To assay enzymatic activity in vitro, a 5x CoA:DTNB (Ellman's reagent or 5,5'-dithiobis-(2-nitrobenzoic acid)) mixture was prepared. The mixture consisted of 10mM succinyl-CoA, 5mM acetyl-CoA, 30mM DTNB in 100mM Tris buffer, pH 7.4. Add 5 μL of CoA:DTNB mixture to 100 mM Tris buffer, pH The enzyme was added to 0.5 μM purified thiolase in 7.8 to bring the final volume to 50 μL. Reactions were incubated at 30 °C for 30 min, then quenched with 2.5 μL of 10% formic acid, and samples were frozen at 20 °C until ready for analysis by LC/MS. The production of acetoacetyl-CoA was examined because many thiolases can condense two acetyl-CoA molecules into acetoacetyl-CoA. Figure 19 shows that three thiolases exhibited thiolase activity in which acetoacetyl-CoA was formed. These were fadAx from Pseudomonas putida, thiA from Clostridium acetobutylicum, and thiB also from Clostridium acetobutylicum. When the enzymatic assay tested for the condensation of succinyl-CoA and acetyl-CoA to β-ketoadipyl-CoA, several candidates showed the desired activity; paaJ from E. coli (Nogales et al., Microbiol 153:357-365 (2007)), phaD from Pseudomonas putida (Olivera et al., Proc. Natl. Acad.Sci. USA 95:6419-6424 (1998)), bkt from Burkholderia ambiphalia AMMD, pcaF from Pseudomonas putida KT2440 (Harwood et al., J. Bacteriol. 176:6479-6488 (1994)), and P. aeruginosa pcaF from fungus PAO1. There was excellent specificity between the thiolases. Thiolases that produced significant amounts of β-ketoadipyl-CoA did not produce significant amounts of acetoacetyl-CoA, and thiolases that also produced acetoacetyl-CoA produced detectable amounts of β-ketoadipyl-CoA. There wasn't.

(Example XXIV) (Production route of hexamethylene diamine from glutamic acid, glutaryl-CoA or pyruvate and 4-aminobutanal) In this example, glutamic acid, glutaryl-CoA, pyruvate and 4-aminobutanal, or 2- An exemplary production route for hexamethylene diamine (HMDA) from amino-7-oxosubarate via homolysine, a seven carbon analog of lysine, is described. Homolysin is an attractive precursor to HMDA. Although homolysine is a potentially valuable precursor, it is not a known metabolic intermediate of any organism. Homolysine can be biocatalytically formed from the central metabolic precursors glutamate, glutaryl-CoA or pyruvate and 4-aminobutanal. Subsequent decarboxylation of homolysine by an enzyme similar to lysine decarboxylase yields HMDA.

This example describes an additional route proceeding from 2-amino-7-oxosabarate, or pyruvate and 4-aminobutanal, via the intermediate 6-aminohexanal. 6-aminohexanal can be easily converted to HMDA by aminotransferases or aminating oxidoreductases.

The maximum theoretical yield of HMDA is 0.71 moles per mole of glucose utilized (0.46 g/g). The routes disclosed in Figures 20-22 and Figure 26 achieve a maximum HMDA yield of 0.67 mol/mol (0.43 g/g).

C₆H₁₂O₆ +1.41NH₄0.71 C₆H₁₈N₂ + 1.76 CO₂ +2.47H₂O

A novel route for producing hexamethylene diamine (HMDA) and related products is described herein. Candidate enzymes and the risks associated with their implementation are discussed in Example XXVI below.

The present invention relates, in part, to non-naturally occurring microorganisms that express genes encoding enzymes that catalyze the production of HMDA. Successful genetic engineering of these pathways involves identifying a suitable set of enzymes with sufficient activity and specificity, cloning their corresponding genes into the production host, and disseminating these enzymes in the production host. There is a need to optimize gene expression, optimize fermentation conditions, and assay for post-fermentation product formation.

HMDA can be produced from glutamic acid via glutaryl-CoA in eight enzymatic steps shown in Figure 20. In this route, glutamic acid is acylated to glutamyl-CoA by CoA transferase or CoA ligase (Step A of Figure 20). Glutamyl-CoA and acetyl-CoA are linked by beta-ketothiolase to form the C7 compound 3-oxo-6-aminopimeloyl-CoA (Step B of Figure 20). The 3-oxo group of this product is then reduced and dehydrated, resulting in 6-amino-7-carboxy-hept-2-enoyl-CoA (Steps C and D of Figure 20). The double bond is reduced by enoyl-CoA reductase to form 6-aminopimeloyl-CoA (Step E in Figure 20). 6-aminopimeloyl-CoA is then converted to 2-amino-7-oxoheptanoic acid by CoA-dependent aldehyde dehydrogenase (Step F). Aldehydes undergo transamination to amines, resulting in homolysines (Step G in Figure 20). Finally, HMDA is formed as a decarboxylation product of homolysine (step H in Figure 20). The maximum theoretical HMDA yield for this route is 0.67 moles of HMDA per mole of glucose utilized. Yield calculations assume aerobic conditions and that CoA transferase is utilized in step A.

HMDA can also be produced from glutaryl-CoA by several routes. An exemplary route for producing HMDA is shown in FIG. 21. Glutaryl-CoA is a common metabolic intermediate in organisms that metabolize aromatic compounds. In the disclosed pathway for HMDA, glutaryl-CoA is first condensed with acetyl-CoA by beta-ketothiolase to form 3-oxopimeloyl-CoA (Step A of Figure 21). The CoA moiety of 3-oxopimeloyl-CoA is removed by CoA hydrolase, transferase and ligase (Step B of Figure 21). Several alternative routes for converting 3-oxopimelic acid to HMDA are outlined in Figure 21 and described herein. The final step of all routes to HMDA requires decarboxylation of homolysine (Step S in Figure 21).

One route requires the conversion of 3-oxopimelic acid to 3-oxo-1-carboxyheptanal. This conversion can be catalyzed by an ATP- and NAD(P)H-dependent enzyme with 3-oxopimelate reductase activity (step C in Figure 21), or the intermediate 5-oxopimeloyl-CoA can be It may proceed by activation (steps H, I of Figure 21) or by activation of 5-oxopimeloyl-phosphonic acid (steps F, G of Figure 21). Once 3-oxo-1-carboxyheptanal is formed, the 3-oxo-1-carboxyheptanal undergoes transamination at the 3-position (Step AB of Figure 21) or the 7-position (Step D of Figure 21). Subsequent transamination to 3-oxo-7-aminoheptanoic acid (Step E in Figure 21) or transamination to 3-amino-7-oxoheptanoic acid (Step Z in Figure 21) results in 3,7 -Diaminoheptanoic acid is provided. Homolysine is then formed by an enzyme with 3,7-diaminoheptanoate 2,3-aminomutase activity (Step R in Figure 21), which is decarboxylated to HMDA (Step S in Figure 21).

In an alternative route, 3-oxopimelic acid undergoes transamination to become 3-aminopimelic acid (Step J of Figure 21). 3-aminopimelic acid is then converted to 3-amino-7-oxoheptanoic acid directly (step O in Figure 21) or via a CoA intermediate (steps K, L in Figure 21) or a phosphonic acid intermediate. (Steps M and N in Figure 21). 3-Amino-7-oxoheptanoic acid is then converted to 2-amino-7-oxoheptanoic acid by 2,3-aminomutase (Step P in Figure 21). 2-amino-7-oxoheptanoic acid is converted to homolysine by aminotransferases or aminating oxidoreductases. Alternatively, 3-amino-7-oxoheptanoic acid first undergoes transamination (step Z in Figure 21) and is then converted to homolysine by aminomutase (step R in Figure 21).

3-aminopimelic acid can be converted to 2-aminopimelic acid by the enzyme 2,3-aminomutase (Step T of Figure 21). The HMDA pathway involving this intermediate requires the reduction of the 7-carboxylic acid to an aldehyde. This reduction is catalyzed by a bifunctional reductase (step W in Figure 21) or proceeds via a CoA intermediate (steps V, Y in Figure 21) or via a phosphonate intermediate. (Steps U, X in Figure 21) Catalyzed by two enzymes. The product, 2-amino-7-oxoheptanoic acid, is converted to HMDA as described above.

Two routes for producing HMDA from pyruvate and 4-aminobutanal are shown in Figure 22. This route provides the highest yield of 0.67 moles of HMDA (0.43 g/g) per mole of glucose utilized under anaerobic and aerobic conditions. 4-Aminobutanal occurs naturally by decarboxylation of ornithine to putrescine followed by transamination. 4-aminobutanal may originate from 4-aminobutanoate. In one route, 4-aminobutanal and pyruvate are linked via aldol condensation to form 2-oxo-4-hydroxy-7-aminoheptanoic acid (Step A of Figure 22). The condensation product is then dehydrated (Step B of Figure 22) and reduced (Step C of Figure 22). Transamination to 2-oxo-7-aminoheptanoic acid yields homolysine (Step D of Figure 22). HMDA is the decarboxylation product of homolysine decarboxylase (Step E of Figure 22). Alternatively, the pathway intermediate 2-oxo-7-aminoheptanoic acid is decarboxylated to form 6-aminohexanal (Step F of Figure 22). 6-aminohexanal is then converted to HMDA by an aminotransferase or an aminating oxidoreductase (Step G in Figure 22).

Several routes for producing HMDA from 2-amino-7-oxosabarate are shown in Figure 26. 2-Amino-7-oxosubarate is not known to be a naturally occurring metabolite. An exemplary route for synthesizing 2-amino-7-oxosubarate is shown in FIG. 27. This pathway begins with glutamic acid-5-semialdehyde, a metabolite that is naturally formed during ornithine biosynthesis. 2-Amino-7-oxosubarate is then synthesized in three enzymatic steps. In the first step, glutamate-5-semialdehyde is condensed with pyruvate by aldolase (Figure 27, Step A). The product 2-amino-5-hydroxy-7-oxosubarate is then dehydrated and the resulting alkene is reduced to form 2-amino-7-oxosabarate (Figure 27, Step B/ C). In one proposed route to HMDA from 2-amino-7-oxosubarate, the 2-oxo acid is first decarboxylated to form 2-amino-7-oxoheptanoic acid (step A in Figure 26). ). This product is again decarboxylated to form 6-aminohexanal (Step B of Figure 26). Finally, 6-aminohexanal is converted to HMDA by an aminotransferase or an aminating oxidoreductase (Step C of Figure 26).

Alternatively, the intermediate 2-amino-7-oxoheptanoic acid is first converted to homolysine by an aminotransferase or aminating oxidoreductase (Step M in Figure 26). Homolysine is decarboxylated to HMDA as described above (Step H of Figure 26).

In yet another route, the 2-amino acid group of 2-amino-7-oxosubarate is decarboxylated to yield 2-oxo-7-aminoheptanoic acid (Step I in Figure 26). This product can then be further decarboxylated to 6-aminohexanal (Step G of Figure 26) or transaminated to homolysine (Step J of Figure 26). Homolysine or 6-aminohexanal is then converted to HMDA as described above.

In yet another route, the 2-oxo group of 2-amino-7-oxosubarate is converted to an amino group to form 2,7-diaminosubarate (Step K in Figure 26). HMDA is then obtained by two decarboxylation reactions (steps L, H in Figure 26).

As shown in FIG. 22 (Steps AE), described herein is the production of a microbial organism genetically engineered to produce HMDA from pyruvate and 4-aminobutanal.

This example also teaches methods for genetically engineering strains that overproduce HMDA.

E. coli is used as a target organism to genetically engineer the HMDA production pathway shown in FIG. 22. E. coli provides an excellent host for producing non-naturally occurring microorganisms capable of producing HMDA. E. coli is amenable to genetic manipulation and can effectively produce a variety of products such as ethanol, acetic acid, formic acid, lactic acid and succinic acid under anaerobic, microaerobic or aerobic conditions. is known to be possible.

An E. coli strain is genetically engineered to produce HMDA from 4-aminobutanal via the route outlined in Figure 22. For the first step of constructing the pathway, assemble on the vector the genes encoding the enzymes for converting 4-aminobutanal and pyruvate to homolysine (Figure 3, steps A-D). In particular, 2-oxo-4-hydroxy-7-aminoheptanoate aldolase, 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase, 2-oxo-7-aminohept-3-enoate reductase and 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase, respectively. -The hpcH gene (CAA87759), hpcG gene (CAA57202), enr gene (YP_430895) and lysN() gene encoding oxo-7-aminoheptanoate aminotransferase were cloned into the pZE13 vector under the control of the PA1/lacO promoter. (Expressys, Ruelzheim, Germany). The plasmid is introduced into E. coli strain MG1655 and transformed to express the proteins and enzymes required for the synthesis of HMDA from 4-aminobutanal. E. coli naturally encodes two enzymes, lysine decarboxylases, that convert homolysine to HMDA.

The resulting genetically engineered organisms are cultured in glucose-containing media according to procedures well known in the art (see, eg, Sambrook et al., supra, 2001). HMDA pathway genes are confirmed using methods well known in the art for determining polypeptide expression or enzymatic activity, including, for example, Northern blots, PCR amplification of mRNA, and immunoblots. . The enzymatic activity of the expressed enzyme is confirmed using assays specific for the individual activity. Confirm the ability of genetically engineered E. coli strains to produce HMDA via this pathway using HPLC, gas chromatography-mass spectrometry (GCMS) or liquid chromatography-mass spectrometry (LCMS) .

Microbial strains genetically engineered to have a functional HMDA synthesis pathway from 4-aminobutanal are further enhanced by optimizing efficient utilization of the pathway. Briefly, genetically engineered strains are evaluated to determine whether any of the exogenous genes are expressed at rate-limiting levels. For any enzymes expressed at low levels where flux through the pathway may be limited, expression is increased, for example, by introducing additional gene copy numbers.

After successfully demonstrating that the activity of exogenous enzymes increases the production of HMDA, the genes encoding these enzymes were inserted into the chromosome of a wild-type E. coli host using methods known in the art. insert. Such methods include, for example, the sequential single crossover (Gay et al., J. Bacteriol. 3:153 (1983)) and the Red/ET method from GeneBridges (Zhang et al., European Patent Application No. 01117 (2001)). ))including. Chromosomal insertion offers several advantages over plasmid-based systems, including significant stability and the ability to co-express pathway genes.

Optimize growth conditions using metabolic modeling to generate good producers. Modeling is also used to design gene knockouts that further optimize pathway utilization (e.g., U.S. Patent Application Publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003). /0059792, US 2002/0168654 and US 2004/0009466, and US Pat. No. 7,127,379). Modeling analysis allows reliable prediction of the effect on cell proliferation of shifting metabolism towards efficient production of HMDA. One modeling method is a two-layer optimization technique, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)) and is applied to select gene knockouts that collectively result in good production of HMDA. Adaptive evolution can also be used, for example, to generate good producers of 2-oxo-4-hydroxy-7-aminoheptanoic acid intermediates or HMDA products. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic manipulation, and adaptive evolution can be applied to the HMDA producers to further increase production.

To produce HMDA on a large scale, organisms containing the HMDA pathway described above are grown in fermenters using media known to those skilled in the art to support the growth of the organisms under anaerobic conditions. Cultivate with Fermentation is carried out either in batch mode, fed-batch mode, or continuous mode. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel (eg, the flask can be sealed with a septum and crimp cap). Microaerobic conditions can also be exploited by providing small holes to restrict ventilation. The pH of the medium is maintained at pH 7 by adding an acid such as H2SO4. Growth rate is measured by optical density using a spectrophotometer (600 (nm) and the rate of glucose uptake is determined by monitoring the depletion of the carbon source over time. By-products such as undesirable alcohols, organic acids and residual glucose were detected by HPLC (Shimadzu) with an HPX-087 column (BioRad) using a refractive index detector for glucose and alcohols and for organic acids. can be quantified using a UV detector. Lin et al., Biotechnol. Bioeng., 775-779 (2005).

(Example XXV) (Production route of 6-aminocaproate from glutamic acid, glutaryl-CoA, homolysine, or 2-amino-7-oxosabarate) Herein, 6-aminocaproate (6- A novel route for producing ACA) and related products has been described. Candidate enzymes and the risks associated with their implementation are discussed in Example XXVI below.

The present invention relates, in part, to non-naturally occurring microorganisms that express genes encoding enzymes that catalyze the production of 6-ACA. Successful genetic engineering of these pathways involves identifying a suitable set of enzymes with sufficient activity and specificity, cloning their corresponding genes into the production host, and disseminating these enzymes in the production host. There is a need to optimize gene expression, optimize fermentation conditions, and assay for post-fermentation product formation.

6-ACA can then be produced from glutamate as the starting molecule. Glutamic acid is converted to 6-aminopimeloyl-CoA as described above (Figure 20, steps A-E). Removal of the CoA moiety of 6-aminopimeloyl-CoA by CoA hydrolase, transferase or ligase yields 2-aminopimelic acid (Step I in Figure 20). Decarboxylation of this product yields 6-ACA (Step J in Figure 20).

6-ACA can also be produced from glutaryl-CoA as a starting molecule. In the pathway disclosed for 6-ACA, similar to the HMDA pathway described above, glutaryl-CoA is first condensed with acetyl-CoA by beta-ketothiolase to form 3-oxopimeloyl-CoA (step A in Figure 21). ). The CoA moiety of 3-oxopimeloyl-CoA is removed by CoA hydrolase, transferase and ligase (Step B of Figure 21). 3-oxopimelic acid then undergoes transamination to become 3-aminopimelic acid (Step J of Figure 21). 3-aminopimelic acid can be converted to 2-aminopimelic acid by the enzyme 2,3-aminomutase (Step T of Figure 21). Aminopimelic acid can then be decarboxylated to form 6-aminocaproic acid (Step AA of Figure 21).

Homolysine is also an attractive precursor for producing 6-aminocaproate (6-ACA). Although homolysine is a potentially valuable precursor, it is not a known metabolic intermediate of any organism. Oxidation of homolysine by lysine 2-monooxygenase under aerobic conditions yields 6-aminohexanamide, which is immediately hydrolyzed to 6-ACA in dilute acid or basic solutions (Fig. twenty three).

6-ACA can also be produced from 2-amino-7-oxosabarate as the starting molecule (Figure 26). 2-Amino-7-oxosubarate is not known to be a naturally occurring metabolite. An exemplary route for synthesizing 2-amino-7-oxosubarate is shown in FIG. 27. This pathway begins with glutamic acid-5-semialdehyde, a metabolite that is naturally formed during ornithine biosynthesis. 2-Amino-7-oxosubarate is then synthesized in three enzymatic steps. In the first step, glutamate-5-semialdehyde is condensed with pyruvate by aldolase (Figure 27, Step A). The product 2-amino-5-hydroxy-7-oxosubarate is then dehydrated and the resulting alkene is reduced to form 2-amino-7-oxosabarate (Figure 27, Step B/ C). In one proposed route, 2-amino-7-oxosabarate is decarboxylated to form 2-amino-7-oxoheptanoic acid (Step A of Figure 26). The aldehyde of 2-amino-7-oxoheptanoic acid is oxidized by oxidoreductase to form 2-aminopimelic acid (Step D of Figure 26). 6-ACA is the decarboxylation product of 2-aminopimelic acid (Step E of Figure 26). Alternatively, the 2-amino-7-oxoheptanoic acid intermediate is decarboxylated to form 6-aminohexanal (step B in Figure 26), which undergoes transamination to 6-ACA (step B in Figure 26). Step F). In the third proposed route, the 2-amino acid group of 2-amino-7-oxosubarate is decarboxylated to yield 2-oxo-7-aminoheptanoic acid (Step I in Figure 26). This product can then be further decarboxylated to 6-aminohexanal (Step G of Figure 26). Finally, 6-aminohexanal undergoes transamination to become 6-ACA (Step F of Figure 26).

(Example XXVI) (Classification system of enzymes for producing hexamethylene diamine and 6-aminocaproic acid) In this example, hexamethylene diamine or 6-aminocaproate described in Example XXIV and Example XXV was used. A classification system of enzymes for exemplary routes for production is described.

All transformations shown in FIGS. 20-23 and 26 fall into the general category of transformations shown in Table 11. Below we list a number of biochemically characterized genes in each category. Specifically listed are genes that, when properly cloned and expressed, can be applied to catalyze the appropriate transformations of Figures 20-23 and 26.

Table 11 shows the types of enzymes useful for converting common core metabolic intermediates to 6-aminocaproate and hexamethylene diamine. The first three numbers of each label correspond to the first three numbers of the enzyme commission number indicating a general type of conversion that is independent of substrate specificity.

<tables><img file="JP7370366B2_D0134.tif" /></tables>

1.1.1.a Oxidoreductase (oxo to alcohol). The reduction of 3-oxo-6-aminopimeloyl-CoA to 3-hydroxy-6-aminopimeloyl-CoA is catalyzed by 3-oxoacyl-CoA dehydrogenase (Figure 20, Step C). Such enzymes convert 3-oxoacyl-CoA molecules into 3-hydroxyacyl-CoA molecules and are often involved in beta-oxidation of fatty acids or catabolism of phenylacetate. For example, in E. coli, two fatty acid oxidation complex subunits encoded by fadB and fadJ function as 3-hydroxyacyl-CoA dehydrogenases (Binstock et al., Methods Enzymol. 71 Pt C:403-411 (1981)). . Additionally, the gene product encoded by phaC in Pseudomonas putida U (Olivera et al., Proc. Natl. Acad. Sci US A 95:6419-6424 (1998)) and the gene product encoded by paaC in Pseudomonas fluorescens ST (Di Arch et al., Microbiol 188:117-125 (2007)), the reverse reaction of step B in Figure 10, That is, it catalyzes the oxidation of 3-hydroxyadipyl-CoA during phenylacetate or styrene catabolism reactions to form 3-oxoadipyl-CoA. Note that the reactions catalyzed by such enzymes are reversible. Furthermore, in E. coli, paaH and other genes are in close proximity within the phenylacetate-degrading operon (Nogales et al., Microbiology 153:357-365 (2007)) and paaH mutants grow in contact with phenylacetate. (Ismail et al., Eur. J Biochem. 270:3047-3054 (2003)), it is expected that the E. coli paaH gene encodes 3-hydroxyacyl-CoA dehydrogenase.

<tables><img file="JP7370366B2_D0135.tif" /></tables>

Additional exemplary oxidoreductases that can convert 3-oxoacyl-CoA molecules to their corresponding 3-hydroxyacyl-CoA molecules include 3-hydroxybutyryl-CoA dehydrogenase. The enzyme from Clostridium acetobutylicum encoded by hbd has been cloned and functionally expressed in E. coli (Youngleson et al., J Bacteriol. 171:6800-6807 (1989)). Additional gene candidates include Hbd1 (C-terminal domain) and Hbd2 (N-terminal domain) of Clostridium kluyveri (Hillmer et al., FEBS Lett. 21:351-354 (1972)) and HSD17B10 of bovine (Wakil et al., J Biol. Chem. 207:631-638 (1954)). Yet another gene candidate demonstrated to reduce acetoacetyl-CoA to 3-hydroxybutyryl-CoA is phbB from Zooglea lamigera (Ploux et al., Eur. J Biochem. 174:177-182 (1988)) and phaB from Rhodobacter sphaeroides (Alber et al., Mol. Microbiol. 61:297-309 (2006))). The former gene candidate is NADPH-dependent, its nucleotide sequence has been determined (Peoples et al., Mol. Microbiol 3:349-357 (1989)), and the gene has been expressed in E. coli. Substrate specificity studies for this gene led to the conclusion that 3-oxopropionyl-CoA could be tolerated as an alternative substrate (Peoples et al., Mol. Microbiol 3:349-357 (1989)).

<tables><img file="JP7370366B2_D0136.tif" /></tables>

A number of similar enzymes have been found in other species of Clostridium and Metallospaera cedula (Berg et al., Science. 318:1782-1786 (2007)).

<tables><img file="JP7370366B2_D0137.tif" /></tables>

1.13.12.a Monooxygenase (O₂built-in). To convert homolysine to 6-aminohexanamide, O₂(Step A of Figure 23). Lysine 2-monooxygenase (EC 1.13.12.2) from Pseudomonas fluorescens reacts with homolysine as a substrate (Nakazawa et al., J Biol. Chem. 247:3439-3444 (1972)). The enzyme from P. putida has been biochemically characterized and its gene identified (Karyakin et al., Prikladnaya Biokhimiya i Mikrobiologiya 27:825-832 (1991)). In P. fluorescens (eval=0.0, 90% identity), Streptomyces coelicolor (eval=0.0, 58% identity), Rhodococcus jostii (eval=0.0, 56% identity), etc. A gene encoding an enzyme, lysine 2-monooxygenase, was identified by sequence homology of the protein to the P. putida enzyme.

<tables><img file="JP7370366B2_D0138.tif" /></tables>

1.2.1.a Oxidoreductase (aldehyde to acid). Two transformations in Figure 26 require the conversion of aldehydes to acids: the conversion of 2-amino-7-oxoheptanoic acid to 2-aminopimelic acid (Step D) and the conversion of 6-aminohexal to 6- Conversion to aminocaproate (Step F). Such reactions are catalyzed by NAD(P)+-dependent oxidoreductases of EC class 1.2.1, which convert aldehydes to acids. The candidate enzyme is NAD+ dependent aldehyde dehydrogenase (EC 1.2.1.3). Two aldehyde dehydrogenases found in the human liver, ALDH-1 and ALDH-2, have a broad substrate range for various aliphatic, aromatic and polycyclic aldehydes (Klyosov et al., Biochemistry 35 :4457-4467(1996)). Active ALDH-2 has been efficiently expressed in E. coli using the GroEL protein as a chaperonin (Lee et al., Biochem. Biophys. Res. Commun. 298:216-224(2002)). Rat mitochondrial aldehyde dehydrogenase also has a broad substrate range, including enoyl-aldehyde crotonaldehyde (Siew et al., Arch. Biochem. Biophys. 176:638-649 (1976)). The E. coli gene astD also encodes an NAD+-dependent aldehyde dehydrogenase that converts succinic semialdehyde to succinate (Kuznetsova et al., FEMS Microbiol Rev 29:263-279 (2005)).

<tables><img file="JP7370366B2_D0139.tif" /></tables>

1.2.1.b Oxidoreductase (acyl-CoA to aldehyde). Reductive desorption of 3-oxopimeloyl-CoA (Figure 21, Step I), 5-aminopimeloyl-CoA (Figure 21, Step L) and 6-aminopimeloyl-CoA (Figure 21, Step Y) to their corresponding aldehydes. Acylation is catalyzed by enzymes of EC class 1.2.1. Exemplary acyl-CoA dehydrogenases that reduce acyl-CoA to its corresponding aldehyde include Acinetobacter calcoaceticus (Reiser et al., Journal of Bacteriology 179:2969-2975 (1997)) and Acinetobacter sp. M-1 (Ishige et al., Appl. Environ. Microbiol. 68:1192-1195 (2002)), a fatty acyl-CoA reductase enzyme, and a CoA- and NADP-dependent succinic semialdehyde encoded by the sucD gene in Clostridium kluyveri. Dehydrogenase (Sohling et al., J Bacteriol. 178:871-880 (1996); and Sohling et al., J Bacteriol 178:871-80 (1996)). P. gingivalis sucD is another succinic semialdehyde dehydrogenase (Takahashi et al., J. Bacteriol. 182:4704-4710 (2000)). In Pseudomonas species, the bphG-encoded enzyme acylating acetaldehyde dehydrogenase is yet another enzyme, as it has been demonstrated to oxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde, and formaldehyde ( Powlowski et al., J Bacteriol. 175:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxidize the branched-chain compound isobutyraldehyde to isobutyryl-CoA (Koo et al. , Biotechnol Lett. 27:505-510(2005)).

<tables><img file="JP7370366B2_D0140.tif" /></tables>

An additional type of enzyme that converts acyl-CoA to its corresponding aldehyde is malonyl-CoA reductase, which converts malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is an important enzyme in autotrophic carbon fixation via the 3-hydroxypropionic acid cycle in thermoacidophilic archaea (Berg et al., Science. 318:1782-1786 (2007); and Thauer et al., Science.318:1732-1733(2007)). This enzyme utilizes NADPH as a cofactor and has been characterized in Metallospaera and Sulfolobus species (Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Hugler et al., J. Bacteriol. 184: 2404-2410(2002)). This enzyme is encoded by Msed_0709 in Metallospaera cedula (Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Berg et al., Science. 318:1782-1786 (2007)). The gene encoding malonyl-CoA reductase from Sulfolobus tokodaiii was cloned into Escherichia coli and heterologously expressed (Alber et al., J. Bacteriol. 188:8551-8559(2006)). This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde (WO/2007/141208). Although the aldehyde dehydrogenase functionality of these enzymes is similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little sequence similarity. Both malonyl-CoA reductase enzyme candidates share high sequence similarity to aspartate semialdehyde dehydrogenase, an enzyme that catalyzes the reduction of aspartyl-4-phosphate to aspartate semialdehyde and concomitant dephosphorylation. have Additional gene candidates can be found by sequence homology to proteins of other organisms, including Sulfolobus solfataricus and Sulfolobus acidocaldarius. Yet another candidate as a CoA-acylating aldehyde dehydrogenase is the ald gene from Clostridium beijerinckii (Toth et al., Appl Environ. Microbiol 65:4973-4980(1999)). This enzyme is reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to eutE, which encodes the acetaldehyde dehydrogenase of Salmonella Typhimurium and Escherichia coli (Toth et al., Appl Environ. Microbiol 65:4973-4980 (1999)).

<tables><img file="JP7370366B2_D0141.tif" /></tables>

1.2.1.d Oxidoreductase (phosphonate reductase). The reduction of phosphonic acid to its corresponding aldehyde is catalyzed by EC class 1.2.1 oxidoreductases. Steps G, N and be done. These reactions are not catalyzed by known enzymes. A similar reaction is catalyzed by aspartate semialdehyde dehydrogenase (ASD, EC 1.2.1.11): NADPH-dependent reduction of 4-aspartyl phosphate to aspartate-4-semialdehyde. ASD is involved in amino acid biosynthesis and has recently been studied as an antibacterial target (Hadfield et al., Biochemistry 40:14475-14483 (2001)). The structure of E. coli ASD has been elucidated (Hadfield et al., J Mol. Biol. 289:991-1002 (1999)), this enzyme has been shown to tolerate the alternative substrate beta-3-methylaspartyl phosphate (Shames, et al., J Biol. Chem. 259:15331- 15339(1984)). The Haemophilus influenzae enzyme has been the subject of research in genetically engineering the enzyme to alter the binding affinity of substrates in the active site (Blanco et al., Crystallogr. 60:1388-1395 (2004)). Other ASD candidates are Mycobacterium tuberculosis (Shafiani et al., J Appl Microbiol 98:832-838 (2005)), Methanococcus janaskii (Faehnle et al., J Mol. 353:1055-1068 (2005)) and infectious microorganisms. Vibrio cholerae and Helicobacter pylori (Moore et al., Protein Expr. Purif. 25:189-194 (2002)). Acetylglutamyl phosphate reductase (EC 1.2.1.38) is a related enzyme that naturally reduces acetylglutamyl phosphate to acetylglutamic acid-5-semialdehyde. The gene encoding this enzyme is found in Saccharomyces cerevisiae (Pauwels et al., Eur. JBiochem. 270:1014-1024(2003)) and Bacillus subtilis (O'Reilly et al., Microbiology 140(Pt 5):1023-1025(1994)). and found in other organisms.

<tables><img file="JP7370366B2_D0142.tif" /></tables>

Enzymes that are other exemplary phosphonate reductases include glyceraldehyde-3-phosphate dehydrogenase (e.g., E. coli gapA (Branlant et al., Eur. J. Biochem. 150:61-66(1985)).23)), converting N-acetyl-L-glutamic acid-5-semialdehyde to N-acetyl-L-glutamyl-5-phosphate. N-acetyl-gamma-glutamyl-phosphate reductase (e.g., Escherichia coli argC (Parsot et al., Gene. 68:275-283 (1988)) and L-glutamate-5-semialdehyde to L-glutamyl-5-phosphate. Contains glutamic acid-5-semialdehyde dehydrogenase (e.g. proA of Escherichia coli (Smith et al., J. Bacteriol. 157:545-551 (1984))) that converts to acid; Salmonella typhimurium (Mahan et al., J. Bacteriol. 156:1249) -1262 (1983)) and Campylobacter jejuni (Louie et al., Mol. Gen. Genet. 240:29-35 (1993)) was cloned and expressed in Escherichia coli.

<tables><img file="JP7370366B2_D0143.tif" /></tables>

1.2.1.e Acid reductase. Some transformations in Figure 21 require conversion of acids to aldehydes (Figure 21, Steps C, O, W). Such transformations are thermodynamically unfavorable and generally require energy-rich cofactors and multiple enzymatic steps. For example, in butanol biosynthesis, the conversion of butyrate to butyraldehyde involves activation of butyrate to its corresponding acyl-CoA by CoA transferase or CoA ligase, and subsequent reduction to butyraldehyde by a CoA-dependent aldehyde dehydrogenase. catalyzed by Alternatively, the acid can be activated to an acyl phosphate and then reduced by phosphate reductase. Direct conversion of acids to aldehydes by a single enzyme is catalyzed by the 1.2.1 family of enzymes. Exemplary enzymes that catalyze these conversions include carboxylic acid reductase, alpha-aminoadipate reductase, and retinoic acid reductase.

Carboxylic acid reductase, found in Nocardia siowensis, converts carboxylic acids to their corresponding aldehydes.Carboxylic acid reductases found in Nocardia siowensis catalyze the magnesium-, ATP- and NADPH-dependent reduction of carboxylic acids to their corresponding (Venkitasubramanian et al., J Biol. Chem. 282:478-485) 2007)). This enzyme, encoded by car, was cloned and functionally expressed in E. coli (Venkitasubramanian et al., J Biol. Chem. 282:478-485(2007)). Expression of the npt gene product improved the activity of the enzyme through post-transcriptional modification. The npt gene encodes a specific phosphopantetheine transferase (PPTase) that converts an inactive apoenzyme into an active holoenzyme. The natural substrate of this enzyme is vanillic acid, and the enzyme shows wide tolerance to aromatic and aliphatic substrates (Venkitasubramanian et al., Biocatalytic Reduction of Carboxylic Acids: Mechanisms and Applications). RN Patel, CRC Press LLC, Boca Raton, FL. (2006)).

<tables><img file="JP7370366B2_D0144.tif" /></tables>

An enzyme with similar properties, alpha-aminoadipate reductase (AAR, EC 1.2.1.31), is involved in the lysine biosynthetic pathway in some fungal species. This enzyme naturally reduces alpha-aminoadipate to alpha-aminoadipate semialdehyde. First, the carboxyl group is activated by the ATP-dependent formation of adenylic acid, which is then reduced by NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizes magnesium and requires activation by PPTase. AAR and its corresponding enzyme candidates as PPTases have been identified in Saccharomyces cerevisiae (Morris et al., Gene 98:141-145 (1991)), Candida albicans (Guo et al., Mol. Genet. Genomics 269:271-279 (2003)) ), and fission yeast (Ford et al., Curr. Genet. 28:131-137 (1995)). AAR from fission yeast showed significant activity when expressed in E. coli (Guo et al., Yeast 21:1279-1288 (2004)). AAR from Penicillium chrysogenum tolerates S-carboxymethyl-L-cysteine as an alternative substrate but did not react with adipate, L-glutamic acid or diaminopimelic acid (Hijarrubia et al., J Biol. Chem 278:8250 -8256(2003)). The gene encoding PPTase in P. chrysogenum has not been identified to date, and sequence comparison homology searches did not identify any reliable hits. Directed evolution or other methods of genetically engineering the enzyme may be required to increase activity towards the substrates of Figure 21.

<tables><img file="JP7370366B2_D0145.tif" /></tables>

1.3.1.a Oxidoreductase (alkenes to alkanes). The three transformations that reduce alkenes to alkanes fall into the category of oxidoreductases (EC 1.3.1.-). Conversion of 6-amino-7-carboxy-hept-2-enoyl-CoA to 6-aminopimeloyl-CoA (Figure 20, Step E), 2-oxo-7-aminohept-3-onoate (onoate) Conversion to -7-aminoheptanoic acid (Figure 22, Step C) and conversion of 2-amino-5-ene-7-oxosubarate to 2-amino-7-oxosabarate (Figure 27, Step C) is catalyzed by 2-enoate reductase. The enzyme 2-enoate reductase is known to catalyze the NAD(P)H-dependent reduction of a wide variety of α,β-unsaturated carboxylic acids and α,β-unsaturated aldehydes (Rohdich, et al. , J Biol. Chem. 276:5779- 5787 (2001)). In the recently published genome sequence of C. kluyveri, nine coding sequences for enoate reductase have been reported, one of which has been characterized (Seedorf et al., Proc. Natl. Acad. Sci US A 105:2128-2133(2008)). Both the enr genes from C. tyrobutylicum and M. thermoaceticum have been cloned and sequenced and they show 59% identity to each other. The former gene was also found to have approximately 75% similarity to genes characterized in C. kluyveri (Giesel et al., Arch. Microbiol 135:51-57 (1983)). Based on these sequencing results, it has been reported that enr is very similar to E. coli dienoyl-CoA reductase (fadH) (Rohdich, et al., J Biol. Chem. 276:5779-5787 (2001 )). The enr gene of Moorella thermoaceticum (formerly C. thermoaceticum) has also been expressed in a catalytically active form in E. coli (Ohdich, et al., J Biol. Chem. 276:5779-5787 (2001)).

<tables><img file="JP7370366B2_D0146.tif" /></tables>

Another candidate 2-enoate reductase is maleyl acetate reductase (MAR), an enzyme that catalyzes the reduction of 2-maleyl acetate (4-oxohex-2-enedioate) to 3-oxoadipate. MAR enzymes are naturally involved in aromatic degradation pathways (Camara et al., J Bacteriol. (2009); Huang et al., Appl Environ. Microbiol 72:7238-7245 (2006)); Kaschabek et al., J Bacteriol. 177:320 -325 (1995) and Kaschabek et al., J Bacteriol. 175:6075-6081 (1993)). This enzyme activity was identified and characterized in Pseudomonas sp. strain B13 (Kaschabek et al., J Bacteriol 177:320-325 (1995); and Kaschabek et al., J Bacteriol 175:6075-6081 (1993)), and the encoding gene Cloned and sequenced (Kasberg et al., J Bacteriol. 179:3801-3803(1997)). Additional MAR gene candidates are the clcE gene from Pseudomonas sp. 3508 (1998)), the macA gene from Ralstonia eutropha (also known as Capriavidus necator) (Seibert et al., Microbiology 150:463-472 (2004)), and the tfdFII from Ralstonia eutropha (Seibert et al., JBacteriol). 175:6745-6754 (1993)) and NCgl1112 of Corynebacterium glutamicum (Huang et al., Appl Environ. Microbiol 72:7238-7245(2006)). A MAR encoded by ccaD was recently identified in Pseudomonas reinekei MT1, and its nucleotide sequence is available under DBJ/EMBL Genbank accession number EF159980 (Camara et al., J Bacteriol. (2009)).

<tables><img file="JP7370366B2_D0147.tif" /></tables>

The enzyme that is enoyl-CoA reductase is a suitable enzyme for catalyzing the reduction of 6-amino-7-carboxy-hepta-2-enoyl-CoA to 6-aminopimeloyl-CoA (Figure 20, Step E). . One exemplary enoyl-CoA reductase is the gene product of bcd from C. acetobutylicum (Atsumi et al., Metab Eng. 10:305-311 (2008)); and Boynton et al., J Bacteriol. 178:3015- 3024 (1996)), which naturally catalyzes the reduction of crotonyl-CoA to butyryl-CoA. The activity of this enzyme can be increased by expressing bcd in conjunction with expression of the C. acetobutylicum etfAB gene, which encodes an electron transfer flavoprotein. An additional candidate for the enoyl-CoA reductase step is the mitochondrial enoyl-CoA reductase from E. gracilis (Hoffmeister et al., J Biol. Chem. 280:4329-4338 (2005)).

A construct derived from this sequence was cloned into E. coli after removing the mitochondrial targeting leader sequence of this sequence, resulting in an active enzyme (Hoffmeister et al., J Biol. Chem. 280:4329-4338 (2005) ). This approach is well known to those skilled in the art of expressing eukaryotic genes, especially genes with leader sequences capable of targeting the gene product to specific subcellular compartments of prokaryotes. A close homolog of this gene, TDE0597 from the prokaryote Treponema denticola, represents a third enoyl-CoA reductase that has been cloned and expressed in E. coli (Tucci et al., Febs Letters 581:1561-1566) 2007)).

<tables><img file="JP7370366B2_D0148.tif" /></tables>

Additional enoyl-CoA reductase enzyme candidates are found in organisms that degrade aromatic compounds. Rhodopseudomonas palustris, a model organism for the degradation of benzoic acid, has the enzymatic ability to degrade pimelic acid through beta-oxidation of pimeloyl-CoA. The adjacent genes pimC and pimD in the pim operon have sequence homology to bcd of C. acetobutylicum and are predicted to encode a flavin-containing pimeloyl-CoA dehydrogenase (Harrison et al., Microbiology 151:727-736 (2005) ). The genome of the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum also contains a pim operon consisting of genes with high sequence similarity to R. palustris pimC and pimD (Harrison et al., Microbiology 151 :727-736(2005)).

<tables><img file="JP7370366B2_D0149.tif" /></tables>

An additional candidate is 2-methyl-branched enoyl-CoA reductase (EC 1.3.1.52), an enzyme that catalyzes the reduction of sterically hindered trans-enoyl-CoA substrates. This enzyme is involved in the synthesis of branched chain fatty acids in the nematode Ascaris porcine, and produces a variety of linear fatty acids including 2-methylbutanoyl-CoA, 2-methylpentanoyl-CoA, octanoyl-CoA and pentanoyl-CoA. Substrates and branched-chain substrates can be reduced (Duran et al., J Biol. Chem. 268:22391-22396 (1993)). Two isoforms of this enzyme encoded by the acad1 gene and the acad gene have been characterized.

<tables><img file="JP7370366B2_D0150.tif" /></tables>

1.4.1.a Oxidoreductase (amination). Some of the reactions in Figures 20-23 require the conversion of a ketone or aldehyde to an amine group. Such conversion can be achieved by aminating oxidoreductases of EC class 1.4.1. This EC class of enzymes catalyzes the oxidative deamination of amino groups using NAD+ or NADP+ as acceptors, and the reaction is generally reversible.

In Step D of Figure 22, the 2-oxo acid 2-oxo-7-aminoheptanoic acid is converted to homolysine, a molecule similar to amino acids (Figure 22, Step D; Figure 26, Step J). The conversion of 2-amino-7-oxosabarate to 2,7-diaminosabarate (Step K in Figure 26) is a similar conversion. Exemplary enzymes for catalyzing these reactions include glutamate dehydrogenase (EC 1.4.1.2), leucine dehydrogenase (EC 1.4.1.9), and aspartate dehydrogenase (EC 1.4.1.21). gdhA gene product from Escherichia coli (Korber et al., J Mol. Biol. 234:1270-1273. (1993)); gdh from Thermotoga maritima (Kort et al., Extremophiles 1:52-60. 1997); Lebbink et al. J Mol. Biol. 280:287-296 (1998) and Lebbink et al., J Mol. Biol. 289:357-369(1999))) and gdhA1 from Halobacterium salinarum (Ingoldsby et al., Gene 349:237-244(2005)) are NADP(H), NAD(H), or both, respectively. catalyzes the reversible conversion of glutamic acid to 2-oxoglutaric acid and ammonia. The B. cereus ldh gene encodes the LeuDH protein, which has a broad substrate range including leucine, isoleucine, valine, and 2-aminobutanoate (Ansorge et al., Biotechnol Bioeng. 68:557-562 (2000)) ; and Stoyan et al., J Biotechnol 54:77-80 (1997)). The nadX gene from Thermotoga maritima, which encodes aspartate dehydrogenase, is involved in the biosynthesis of NAD (Yang et al., J Biol. Chem. 278:8804-8808 (2003)).

<tables><img file="JP7370366B2_D0151.tif" /></tables>

Two reactions require the conversion of 3-oxo acids to 3-amino acids: 3-oxo-7-aminoheptanoic acid to 3,7-diaminoheptanoic acid (Figure 21, Step E), 3- Oxopimelic acid to 3-aminopimelic acid (Figure 21, Step J) and 3-oxo-1-carboxyheptanal to 3-amino-7-oxoheptanoic acid (Figure 21, Step AB). The enzyme that reacts with 3-oxoacids is 3,5-diaminohexanoate dehydrogenase (EC 1.4.1.11), an enzyme found in organisms that ferment lysine. The gene encoding this enzyme, kdd, was recently identified in Fusobacterium nucleatum (Kreimeyer et al., J Biol. Chem. 282:7191-7197 (2007)). Although this enzyme has been purified and characterized in other organisms (Baker et al., Chem. 247:7724-7734 (1972)); and Baker et al., Biochemistr. 13:292-299 (1974)), the genes associated with these enzymes are unknown. Candidates in Myxococcus xanthus, Porphyromonas gingivalis W83 and other sequenced organisms can be inferred by sequence homology.

<tables><img file="JP7370366B2_D0152.tif" /></tables>

Conversion of 2-amino-7-oxoheptanoic acid to homolysine (Figure 20, Step G; Figure 21, Step Q; Figure 26, Step M), 3-oxo-7- of 3-oxo-1-carboxyheptanal Conversion to aminoheptanoic acid (Figure 21, Step D) Conversion of 3-amino-7-oxoheptanoic acid to 3,7-diaminoheptanoic acid (Figure 21, Step Z) and Conversion of 6-aminohexal to HMDA (Figure 26, Step C; Figure 22, Step G) is catalyzed by aminating oxidoreductases that convert aldehydes to their corresponding primary amines. An enzyme that catalyzes a similar reaction is lysine 6-dehydrogenase (EC 1.4.1.18), encoded by the lysDH gene. This enzyme catalyzes the reversible oxidative deamination of the 6-amino group of L-lysine in which 2-aminoadipate-6-semialdehyde is formed (Misono et al., J Bacteriol. 150:398-401). 1982)). An exemplary enzyme candidate is Geobacillus stearothermophilus (Heydari et al., Appl Environ. Microbiol 70:937-942(2004)), Agrobacterium tumefaciens (Hashimoto et al., J Biochem 106:76-80(1989); and Misono et al., J Bacteriol. 150:398-401(1982)), Found in Mobacter denitrificans (Ruldeekulthamrong et al., BMP Rep. 41:790-795 (2008)).

<tables><img file="JP7370366B2_D0153.tif" /></tables>

2.3.1.b Acyltransferases (beta-ketothiolases). In step A of Figure 21, glutaryl-CoA and acetyl-CoA are condensed to form 3-oxopimeloyl-CoA by oxopimeloyl-CoA:glutaryl-CoA acyltransferase, a beta-ketothiolase (EC 2.3.1.16). The enzyme that catalyzes this conversion is found in Ralstonia eutropha (previously known as Alcaligenes eutrophus) and is encoded by the bktB and bktC genes (Haywood et al., FEMS Microbiology Letters 52: 91-96 (1988); and Slater et al., J. Bacteriol. 180:1979-1987 (1998)).

The sequence of the BktB protein is known; however, the sequence of the BktC protein has not been reported. The pim operon of Rhodopseudomonas palustris also encodes a beta-ketothiolase encoded by pimB and is predicted to catalyze this conversion in a degradative direction during the degradation of benzoyl-CoA (Harrison et al., Microbiology 151:727-736(2005)). An enzyme candidate, a beta-ketothiolase in S. aciditrophicus, was identified by sequence homology to bktB (43% identity, E value = 1 × 10^-93)。

<tables><img file="JP7370366B2_D0154.tif" /></tables>

Enzymes that are beta-ketothiolases that catalyze the formation of beta-ketovalerate from acetyl-CoA and propionyl-CoA may also be capable of catalyzing the formation of 3-oxopimeloyl-CoA. Zooglea ramigera possesses two ketothiolases that can form β-ketovaleryl-CoA from propionyl-CoA and acetyl-CoA, and R. eurotofa possesses a β-oxidation-ketothiolase that can catalyze this conversion as well. (Gruys et al., US Pat. No. 5,958,745 (1999)). Although the sequences of these genes or their translated proteins have not been reported, sequence homology to bktB from R. eurotofa makes some candidates in R. eurotofa, Z. lamigera, or other organisms. can be identified based on These include:<tables><img file="JP7370366B2_D0155.tif" /></tables>

Additional candidates include beta-ketothiolase (EC 2.1.3.9), which is known to convert two molecules of acetyl-CoA to acetoacetyl-CoA. Enzymes that are exemplary acetoacetyl-CoA thiolases include the atoB gene product from E. coli (Martin et al., Nat. Biotechnol 21:796-802 (2003)), the thlA gene product from C. acetobutylicum, and the thlB gene product. (Hanai et al., Appl Environ Microbiol 73:7814-7818(2007)); and Winzer et al., J. Mol. Microbiol Biotechnol 2:531-541(2000)), and the gene product of ERG10 from Saccharomyces cerevisiae (Hiser et al. , J. Biol. Chem. 269:31383-31389 (1994)).

<tables><img file="JP7370366B2_D0156.tif" /></tables>

Beta-ketoadipyl-CoA thiolase (EC 2.3.1.174), also called 3-oxoadipyl-CoA thiolase, converts beta-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA, and converts beta-ketoadipyl-CoA into succinyl-CoA and acetyl-CoA, producing beta-ketoadipyl-CoA for degrading aromatic compounds. -Is an important enzyme in the ketoadipate pathway. This enzyme is commonly used in soil bacteria, including Pseudomonas putida (Harwood et al., J Bacteriol. 176:6479-6488 (1994)) and Acinetobacter calcoaceticus (Doten et al., J Bacteriol. 169:3168-3174 (1987)). and widespread in soil fungi. pcaF of Pseudomonas strain B13 (Kaschabek et al., J Bacteriol. 184:207-215 (2002)), phaD of Pseudomonas putida U (Olivera et al., Proc. Natl. Acad. Sci US A 95:6419-6424(1998)), paaE from Pseudomonas fluorescens ST (Di Arch et al., Microbiol 188:117-125(2007)), and paaJ from E. coli (Nogales et al., Microbiology 153:357-365( The gene product encoded by (2007)) also catalyzes this conversion. Several beta-ketothiolases, including bkt from Pseudomonas putida, pcaF and bkt from Pseudomonas aeruginosa PA01, bkt from Burkholderia ambiphalia AMMD, paaJ from E. coli, and phaD from P. putida, It exhibits significant and selective activity in the direction in which oxoadipyl-CoA is formed. These enzymes can also be used to synthesize 3-oxopimeloyl-CoA, a compound structurally similar to 3-oxoadipyl-CoA.

<tables><img file="JP7370366B2_D0157.tif" /></tables>

Beta-ketothiolase is also required to condense glutamyl-CoA and acetyl-CoA (Figure 20, Step B). This conversion is not known to occur naturally. The beta-ketothiolase candidates described above are also exemplary candidates for catalyzing this conversion.

2.6.1.a Aminotransferases. Some of the reactions in Figures 20-26 are catalyzed by EC class 2.6.1 aminotransferases. Such enzymes reversibly transfer amino groups from aminated donors to acceptors such as pyruvate and alpha-ketoglutarate.

Aminotransferases selective for aldehydes transaminate to 2-amino-7-oxoheptanoic acid (Figure 20, Step G; Figure 21, Step Q; Figure 26, Step M), 3-oxo-1 -Transamination of 3-amino-7-oxoheptanoic acid to carboxyheptanal (Figure 21, Step D) and of 3-amino-7-oxoheptanoic acid (Figure 21, Step Z) and to 6-aminohexanal (Figure 26) , step C; required in Figure 22, step G). An exemplary enzyme for converting aldehydes to primary amines is lysine-6-aminotransferase (EC 2.6.1.36). This enzyme function to convert lysine to alpha-aminoadipate semialdehyde has been demonstrated in yeast and bacteria. Torula yeast (Candida utilis) (Hammer et al., J Basic Microbiol 32:21-27 (1992)), Flavobacterium lutescens (Fujii et al., J Biochem. 128:391-397 (2000)) and Streptomyces clavuligenus (Romero et al., Microbiol Biotechnol 18:241-246 (1997)). A recombinant lysine-6-aminotransferase from S. clavuligenus has been functionally expressed in E. coli (Tobin et al., J Bacteriol. 173:6223-6229 (1991)). The enzyme of F. lutescens is specific for alpha-ketoglutarate as an amino receptor (Soda et al., Biochemistry 7:4110-4119 (1968)). Other enzymes that convert aldehydes to terminal amines include the dat gene product encoding 2,4-diaminobutanoate:2-ketoglutarate 4-transaminase in Acinetobacter baumannii (Ikai et al., J Bacteriol. 179:5118-5125(1997)). In addition to its natural substrate 2,4-diaminobutyrate, DAT transaminates the terminal amines of lysine, 4-aminobutyrate, and ornithine.

<tables><img file="JP7370366B2_D0158.tif" /></tables>

Additional enzyme candidates include putrescine aminotransferase or other diamine aminotransferases. E. coli putrescine aminotransferase is encoded by the ygjG gene, and the purified enzyme can also transfer the amino groups of cadaverine and spermidine (Samsonova et al., Microbiol 3:2 (2003)). Additionally, activity of this enzyme toward 1,7-diaminoheptane with amino acceptors other than 2-oxoglutarate (e.g., pyruvate, 2-oxobutanoate) has been reported (Kim et al., J Biol. Chem. 239:783-786 (1964); and Samsonova et al., Microbiol 3:2 (2003)). The spuC gene of Pseudomonas aeruginosa encodes putrescine aminotransferase, which is more active toward pyruvate than alpha-ketoglutarate as an amino receptor (Lu et al., J Bacteriol. 184:3765-3773 (2002)).

<tables><img file="JP7370366B2_D0159.tif" /></tables>

Conversion of aldehydes to terminal amines can also be catalyzed by gamma-aminobutyrate transaminase (GABA transaminase). This enzyme naturally interconverts succinic semialdehyde and glutamate with 4-aminobutyrate and alpha-ketoglutarate and is known to have a broad substrate range (Liu et al., Biochemistry 43:10896- 10905 (2004); and Schulz et al., Appl Environ Microbiol 56:1-6 (1990)). The two GABA transaminases of E. coli are encoded by gabT (Bartsch et al., J Bacteriol. 172:7035-7042 (1990)) and puuE (Kurihara et al., J. Biol. Chem. 280:4602-4608 (2005)). . Mouse, Pseudomonas fluorescens, and wild boar GABA transaminases have been shown to react with a variety of alternative substrates, including 6-aminocaproic acid (Cooper, Methods Enzymol. 113:80-82 (1985)); and Scott et al., J Biol. Chem. 234:932-936(1959)).

<tables><img file="JP7370366B2_D0160.tif" /></tables>

Enzymes that transfer amino groups to 3-oxo acids convert 3-oxo-7-aminoheptanoic acid to 3,7-diaminoheptanoic acid (Figure 21, Step E), 3-oxopimelic acid to 3-aminopimelic acid (Figure 21, Step J) and the conversion of 3-oxo-1-carboxyheptanal to 3-amino-7-oxoheptanoic acid (Figure 21, Step AB). The enzymes that catalyze these precise conversions have not been identified so far. Beta-alanine/alpha-ketoglutarate aminotransferase (WO08027742) reacts with beta-alanine to form malonic semialdehyde, a 3-oxoacid. The gene product of Saccharomyces kluybergi SkPYD4 was shown to preferentially use beta-alanine as an amino group donor (Andersen et al., Gene. 124:105-109 (1993)). SkUGA1 encodes a homolog of the Saccharomyces cerevisiae GABA aminotransferase, UGA1 (Ramos et al., Eur. J. Biochem. 149:401-404 (1985)), while SkPYD4 encodes an enzyme involved in the transamination of both beta-alanine and GABA (Andersen et al., Gene. 124:105-109 (1993)). 3-Amino-2-methylpropionic acid transaminase catalyzes the conversion of methylmalonic acid semialdehyde to 3-amino-2-methylpropionic acid. This enzyme has been characterized in rat and boar and is encoded by Abat (Kakimoto et al., Biochim. Biophys. Acta 156:374-380 (1968); and Tamaki et al., Methods Enzymol. 324:376-389 ( 2000)).

<tables><img file="JP7370366B2_D0161.tif" /></tables>

Some aminotransferases transaminate the amino group of 2-oxoacids to form amino acids. Such enzymes are useful because 2-oxo-7-aminoheptanoic acid undergoes transamination to homolysine (Figure 22, Step D; Figure 26, Step M) and 2-amino-7-oxosubarate Required to undergo transamination to 2,7-diaminosabarate (Figure 26, Step K). A promising enzyme candidate is alpha-aminoadipate aminotransferase (EC 2.6.1.39), an enzyme involved in the biosynthesis and degradation of lysine in some organisms. This enzyme interconverts 2-aminoadipate and 2-oxoadipate using alpha-ketoglutarate as the amino acceptor. Gene candidates include humans (Okuno et al., Enzyme Protein 47:136-148 (1993)) and hyperthermophiles (Miyazaki et al., Microbiology 150:2327-2334 (2004)). The hyperthermophilic enzyme encoded by lysN is sensitive to several alternative substrates, including oxaloacetate, 2-oxoisocaproic acid, 2-oxoisovalerate, and 2-oxo-3-methylvalerate. It is active.

<tables><img file="JP7370366B2_D0162.tif" /></tables>

Another candidate is aspartate aminotransferase, an enzyme that naturally transfers the oxo group from oxaloacetate to glutamate, forming alpha-ketoglutarate and aspartate. The activity of aspartate aminotransferase can be determined by, for example, the gene product of aspC from Escherichia coli (Yagi et al., FEBS Lett. 100:81-84 (1979); and Yagi et al., Methods Enzymol. 113:83-89 (1985)), The gene product of AAT2 from Saccharomyces cerevisiae (Yagi et al., J Biochem. 92:35-43 (1982)) and the gene product of ASP5 from Arabidopsis (de la et al., Plant J 46:414-425 (2006); Kwok et al. , J Exp. Bot. 55:595-604 (2004) and Wilkie et al., Protein Expr. Purif. 12:381-389 (1998)). Enzymes from rats have been shown to transfer amino groups of alternative substrates such as 2-aminohexanedioic acid and 2,4-diaminobutyrate (Recasens et al., Biochemistry 19:4583-4589 (1980) ). Aminotransferases acting on other amino acid substrates may also be able to catalyze this conversion. Valine aminotransferase catalyzes the conversion of valine and pyruvate to 2-ketoisovalerate and alanine.

The avtA gene of E. coli encodes one such enzyme (Whalen et al., J. Bacteriol. 150:739-746 (1982)). This gene product also catalyzes the transamination of α-aminobutyrate to α-ketobutyrate, although the amine donor in this reaction has not been identified (Whalen et al., J. Bacteriol. 158:571- 574(1984)). The E. coli serC gene product catalyzes two reactions, phosphoserine aminotransferase and phosphohydroxythreonine aminotransferase (Lam, J. et al., Bacteriol. 172:6518-6528 (1990)), and has activity against non-phosphorylated substrates. could not be detected (Drewke et al., FEBS. Lett. 390:179-182 (1996)).

<tables><img file="JP7370366B2_D0163.tif" /></tables>

2.7.2.a Phosphotransferases (carboxy receptors). Enzymes that are EC class 2.7.2 phosphotransferases convert carboxylic acids to phosphonic acids, with simultaneous hydrolysis of one ATP. In Steps F, M and U of Figure 21, the activity of the carboxyl groups of 3-oxopimelic acid (Step F), 3-aminopimelic acid (Step M) and 2-aminopimelic acid (Step U) towards their corresponding phosphonic acids For oxidation, phosphotransferases are required. Butyrate kinase (EC 2.7.2.7) performs the reversible conversion of butyryl phosphate to butyrate during acid fermentation in C. acetobutylicum (Cary et al., Appl. Environ. Microbiol 56:1576-1583 (1990) ). This enzyme is encoded by either of the two buk gene products (Huang et al., J Mol. Microbiol Biotechnol 2:33-38 (2000)). Other butyrate kinase enzymes include C. butyricum and Clostridium tetani (C. tetanomorphum) (Twarog et al., J Bacteriol. 86:112-117 (1963)). A related enzyme, isobutyrate kinase from Thermotoga maritima, has also been expressed in E. coli and crystallized (Diao et al., E. Biol. Crystallogr. 59:1100-1102 (2003); and Diao et al., J Bacteriol. 191:2521-2529(2009)). Aspartokinase catalyzes the ATP-dependent phosphorylation of aspartate and is involved in the synthesis of several amino acids. The aspartokinase III enzyme encoded by lysC in E. coli has a broad substrate range, and the catalytic residues involved in substrate specificity have been elucidated (Keng et al., Arch. Biochem. Biophys. 335:73- 81(1996)). Two additional kinases from E. coli are also good candidates: acetate kinase and gamma-glutamyl kinase. The E. coli acetate kinase encoded by ackA (Skarstedt et al., J. Biol. Chem. 251:6775-6783 (1976)), phosphorylates propionic acid in addition to acetate (Hesslinger et al., Mol. Microbiol 27:477-492 (1998)). Escherichia coli gamma-glutamyl kinase, encoded by proB (Smith et al., J. Bacteriol. 157:545-551 (1984)), phosphorylates the gamma carbonate group of glutamic acid.

<tables><img file="JP7370366B2_D0164.tif" /></tables>

2.8.3.a Coenzyme A transferase. CoA transferase catalyzes the reversible transfer of a CoA moiety from one molecule to another. In some of the transformations in Figures 20 and 21, CoA transferase is required for activation of carboxylic acids to their corresponding acyl-CoA derivatives (Figure 20, Steps A and I; Figure 21, Step H, J, V). A candidate enzyme to catalyze these conversions is cat1 of Clostridium kluyveri, which has been shown to exhibit succinyl-CoA transferase activity, 4-hydroxybutyryl-CoA transferase activity, and butyryl-CoA transferase activity, respectively. gene products, cat2 gene products, and cat3 gene products (Seedorf et al., Proc. Natl. Acad. Sci US A 105:2128-2133 (2008); and Sohling et al., J Bacteriol. 178:871-880 ( 1996)). Similar CoA transferase activity was observed in Trichomonas vaginalis (van Grinsven et al., J. Biol. Chem. 283:1411-1418 (2008)) and Trypanosoma brucei (Riviere et al., J. Biol. Chem. 279:45337-45346 (2004)).

<tables><img file="JP7370366B2_D0165.tif" /></tables>

The glutaconyl-CoA-transferase (EC 2.8.3.12) enzyme from the anaerobic bacterium Acidaminococcus fermentans uses substrates glutaconyl-CoA and 2,3-dehydroadipyl-CoA, which are structurally similar. Reacts with 3-butenoyl-CoA (Mack et al., Eur. J. Biochem. 226:41-51 (1994)). The genes encoding this enzyme are gctA and gctB. This enzyme has reduced but detectable activity toward other CoA derivatives, including glutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA, crotonyl-CoA, and acrylyl-CoA (Buckel et al. , Eur. J Biochem. 118:315-321 (1981)). This enzyme has been cloned and expressed in E. coli (Mack et al., Eur. J. Biochem. 226:41-51 (1994)).

<tables><img file="JP7370366B2_D0166.tif" /></tables>

A CoA transferase that can utilize acetyl-CoA as a CoA donor is the acetoacetyl-CoA transferase encoded by the atoA (alpha subunit) and atoD (beta subunit) genes of Escherichia coli (Korolev et al., Biol. Crystallogr. 58:2116-2121 (2002); and Vanderwinkel et al., Biophys. Res. Commun. 33:902-908 (1968)). This enzyme has a broad substrate range (Sramek et al., Arch. Biochem. Biophys. 171:14-26 (1975)), isobutyrate (Matthies et al., Appl Environ. Microbiol 58:1435-1439 (1992)), Valeric acid (Vanderwinkel et al., Biophys. Res. Commun. 33:902-908 (1968)) and butanoate (Vanderwinkel et al., Biophys. Res. Commun. 33:902-908 (1968)) has been shown to transfer the CoA moiety to acetate from a variety of branched and linear acyl-CoA substrates. Since this enzyme is induced by acetoacetate at the transcriptional level, regulatory modifications may be required to genetically engineer this enzyme into the pathway (Pauli et al., Eur. J Biochem. 29: 553-562(1972)). Similar enzymes are found in Corynebacterium glutamicum ATCC 13032 (Duncan et al., Appl. Environ. Microbiol 68:5186-5190 (2002)) and Clostridium acetobutylicum (Cary et al., Appl. Environ. Microbiol 56:1576-1583 (1990)). and Wiesenborn et al., Appl. Environ. Microbiol 55:323-329 (1989)), and Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem 71:58-68 (2007)).

<tables><img file="JP7370366B2_D0167.tif" /></tables>

Deacylation of 3-oxopimeloyl-CoA to 3-oxopimelic acid (Figure 21, Step B) is catalyzed by 3-oxo acid-CoA transferase (EC 2.8.3.6). Succinyl-CoA:3-oxoacid-CoA transferase, also known as beta-ketoadipyl-CoA transferase, is encoded by pcaI and pcaJ in Pseudomonas putida (Kaschabek et al., J Bacteriol. 184:207-215). 2002)). An enzyme that is similar based on protein sequence homology exists in Acinetobacter sp. ADP1 (Kowalchuk et al., Gene 146:23-30 (1994)). Additional exemplary succinyl-CoA:3:oxoacid-CoA transferases are H. pylori (Corthesy-Theulaz et al., J Biol. Chem. 272:25659-25667 (1997)) and Bacillus subtilis (Stols et al., Protein Expr. Purif. 53:396-403 (2007)).

<tables><img file="JP7370366B2_D0168.tif" /></tables>

3.1.2.a CoA hydrolase. Hydrolysis of 6-aminopimeloyl-CoA to 6-aminopimelic acid (Figure 20, Step I) is carried out by enzymes that are acyl-CoA hydrolases of the 3.1.2 family. No enzyme has been demonstrated to catalyze this conversion. Several eukaryotic acetyl-CoA hydrolases (EC 3.1.2.1) have broad substrate specificity, thus making them suitable candidate enzymes for hydrolyzing 6-aminopimelic acid. For example, an enzyme from rat brain (Robinson et al., Res. Commun. 71:959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA and malonyl-CoA. Although its sequence has not been reported, the enzyme from pea leaf mitochondria also has broad substrate specificity, including acetyl-CoA, propionyl-CoA, butyryl-CoA, palmitoyl-CoA, oleoyl-CoA, and succinyl. -CoA, and crotonyl-CoA have been demonstrated (Zeiher et al., Plant. Physiol. 94:20-27(1990)). ACH1, an acetyl-CoA hydrolase from Saccharomyces cerevisiae, is another candidate hydrolase (Buu et al., J. Biol. Chem. 278:17203-17209 (2003)).

<tables><img file="JP7370366B2_D0169.tif" /></tables>

Another candidate hydrolase is the human dicarboxylic acid thioesterase, acot8 (Westin et al., J Biol. Chem. 280 :38125-38132 (2005)), and the closest E. coli homolog, tesB, which can also hydrolyze a wide range of CoA thioesters (Naggert et al., J Biol. Chem. 266:11044-11050 (1991) ). A similar enzyme has been characterized in rat liver (Deana et al., Biochem. Int. 26:767-773 (1992)). Other potential E. coli thioester hydrolases include the gene product of tesA (Bonner et al., Chem. 247:3123-3133 (1972)), the gene product of ybgC (Kuznetsova et al., FEMS Microbiol Rev 29:263-279 (2005)) ; and (Zhuang et al., FEBS Lett. 516:161-163 (2002)), and the gene product of paaI (Song et al., J Biol. Chem. 281:11028-11038 (2006)), and the ybdB gene product (Leduc et al., J Bacteriol. 189:7112-7126 (2007)).

<tables><img file="JP7370366B2_D0170.tif" /></tables>

Yet another candidate hydrolase is glutaconic acid CoA-transferase from Acidaminococcus fermentans. This enzyme has been transformed by site-directed mutagenesis into an acyl-CoA hydrolase with activity towards glutaryl-CoA, acetyl-CoA, and 3-butenoyl-CoA (Mack et al., FEBS. Lett 405:209-212(1997)). This means that the enzymes encoding succinyl-CoA:3-ketoacid-CoA transferase and acetoacetyl-CoA:acetyl-CoA transferase could also function as candidates for this reaction step, but to alter their function. This suggests that specific mutations may be required.

<tables><img file="JP7370366B2_D0171.tif" /></tables>

4.1.1.a Carboxylyase. Decarboxylation of homolysine to HMDA (Figure 20, Step H; Figure 21, Step S; Figure 22, Step E; Figure 26, Step H), decarboxylation of 2-aminopimelic acid to 6-ACA (Figure 20) , Step J, Figure 21, Step AA and Figure 26, Step E), Decarboxylation of 2,7-diaminosabarate to homolysine (Figure 26, Step L), 6 of 2-amino-7-oxoheptanoic acid -Decarboxylation to aminohexanal (Figure 26, Step B; Figure 22, Step F) and decarboxylation of 2-amino-7-oxosabarate to 2-oxo-7-aminoheptanoic acid (Figure 26, Step I) is catalyzed by an enzyme that is an amino acid decarboxylase. Lysine decarboxylase (EC 4.1.1.18) catalyzes a similar transformation: decarboxylate lysine to form cadaverine.

Two isozymes of this enzyme are encoded by the cadA and ldcC genes in the E. coli genome. cadA is involved in acid tolerance and is positively regulated by the cadC gene product (Lemonnier et al., Microbiology 144(Pt 3):751-760 (1998)). cadC tolerates hydroxylysine and S-aminoethylcysteine as alternative substrates, whereas 2-aminopimelic acid and 6-ACA act as competitive inhibitors on this enzyme (Sabo et al., Biochemistry 13:662- 670(1974)). Decarboxylating 2-aminopimelic acid with this enzyme may require directed evolution or methods of genetically engineering other enzymes. The constitutively expressed ldc gene product is less active than CadA (Lemonnier et al., Microbiology 144(Pt 3):751-760 (1998)). A lysine decarboxylase similar to CadA was recently identified in Vibrio parahaemolyticus (Tanaka et al., J Appl Microbiol 104:1283-1293(2008)). Lysine decarboxylase from Selenomonas ruminantium, encoded by ldc, has sequence similarity to eukaryotic ornithine decarboxylase and tolerates both L-lysine and L-ornithine as substrates (Takatsuka et al., Biosci Biotechnol Biochem. 63:1843-1846 (1999)). To alter the substrate specificity of the enzyme, active site residues have been identified and genetically engineered (Takatsuka et al., J Bacteriol. 182:6732-6741 (2000)).

<tables><img file="JP7370366B2_D0172.tif" /></tables>

Several enzymes, ornithine decarboxylase (EC 4.1.1.17), exhibit activity towards lysine and other similar compounds. Such enzymes include Nicotiana glutinosa (Lee et al., Biochem. J 360:657-665 (2001)), Lactobacillus sp. 30a (Guirard et al., J Biol. Chem. 255:5960-5964 (1980)) and Vibrio - Found in P. vulnificus (Lee et al., J Biol. Chem. 282:27115-27125 (2007)). Enzymes from Lactobacillus sp. 30a (Momany et al., J Mol. Biol. 252:643-655 (1995)) and V. vulnificus have been crystallized. The V. vulnificus enzyme efficiently catalyzes the decarboxylation of lysine, and the residues involved in substrate specificity have been elucidated (Lee et al., J Biol. Chem. 282:27115-27125 (2007)). A similar enzyme has been characterized in Trichomonas vaginalis, but the gene encoding this enzyme is unknown (Yarlett et al., Biochem. J 293(Pt 2):487-493(1993)).

<tables><img file="JP7370366B2_D0173.tif" /></tables>

The enzyme, a keto acid decarboxylase, converts 2-oxo-7-aminoheptanoic acid to 6-aminohexanal (Step F in Figure 22; Step G in Figure 26) and converts 2-amino-7-oxosabarate into Required for conversion to 2-amino-7-oxoheptanoic acid (Step A of Figure 26). Keto acid decarboxylation is performed by a variety of enzymes, including pyruvate decarboxylase (EC 4.1.1.1), benzoylformate decarboxylase (EC 4.1.1.7), alpha-ketoglutarate decarboxylase, and branched-chain alpha-keto acid decarboxylase. Catalyzed by various enzymes with substrate specificity. Pyruvate decarboxylase (PDC), also called keto acid decarboxylase, is an important enzyme in alcohol fermentation, catalyzing the decarboxylation of pyruvate to acetaldehyde. The enzyme from Saccharomyces cerevisiae has a broad substrate range for aliphatic 2-keto acids, including 2-ketobutyrate, 2-ketovaleric acid, 3-hydroxypyruvate, and 2-phenylpyruvate (Henning et al. Appl. Environ. Microbiol. 72:7510-7517(2006)). This enzyme has been extensively studied, genetically engineered to alter its activity, and functionally expressed in E. coli (Killenberg-Jabs et al., Eur. J. Biochem. 268:1698-1704 (2001 ); Li, H. and F. Jordan, Biochemistry. 38:10004-10012 (1999); and ter Schure et al., Appl. Environ. Microbiol. 64:1303-1307 (1998)). PDC from Zymomonas mobilus, encoded by pdc, also has a broad substrate range and has been the subject of directed genetic engineering studies to alter its affinity for different substrates (Siegert et al. Protein Eng Des Sel 18:345-357(2005)). A crystal structure of this enzyme is available (Killenberg-Jabs et al., Eur. J. Biochem. 268:1698-1704 (2001)). Another well-characterized PDC candidate is Acetobacter pasteurians (Chandra et al., Arch. Microbiol. 176:443-451 (2001)) and Kluyveromyces lactis (Krieger et al., Eur. J. Biochem. 269:3256-3263 (2002)).

<tables><img file="JP7370366B2_D0174.tif" /></tables>

Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broad substrate range and has been the target of studies to genetically engineer the enzyme. The enzyme from Pseudomonas putida has been extensively studied and crystal structures of this enzyme are available (Hasson et al., Biochemistry 37:9918-9930 (1998); and Polovnikova et al., Biochemistry 42:1820-1830 ( 2003)). Site-directed mutagenesis of two residues in the active site of the Pseudomonas putida enzyme altered the affinity (Km) of naturally occurring and non-naturally occurring substrates (Siegert et al., Protein Eng Des Sel 18:345-357(2005)). The properties of this enzyme have been further modified by directed genetic engineering (Lingen et al., Protein Eng 15:585-593 (2002); and Lingen et al., ChembioChem. 4:721-726(2003)). The enzyme from Pseudomonas aeruginosa encoded by mdlC has also been characterized experimentally (Barrowman et al., FEMS Microbiology Letters 34:57-60 (1986)). Additional gene candidates from Pseudomonas stuzzeri, Pseudomonas fluorescens and other organisms can be inferred by sequence homology or identified using the growth selection system developed in Pseudomonas putida. (Henning et al., Appl. Environ. Microbiol. 72:7510-7517 (2006)).

<tables><img file="JP7370366B2_D0175.tif" /></tables>

A third enzyme that can decarboxylate 2-oxoacids is alpha-ketoglutarate decarboxylase (KGD). The substrate range of this class of enzymes has not been investigated so far. KDC from Mycobacterium tuberculosis (Tian et al., Proc Natl Acad Sci US. A 102:10670-10675 (2005)) has been cloned and functionally expressed in another internal project at Genomatica. However, it is large (approximately 130 kD) and GC-rich, so it is not an ideal candidate for genetically engineering the strain. KDC enzymatic activity has been detected in several species of rhizobia, including Bradyrhizobium japonicum and Mesorhizobium loti (Green et al., J. Bacteriol. 182:2838-2844(2000)). Although the KDC-encoding gene(s) have not been isolated in these organisms, genome sequences are available and several genes within each genome are annotated as putative KDCs. KDC from Euglena gracilis has also been characterized, but the genes associated with this activity have not been identified so far (Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). The first 20 amino acids starting from the N-terminus were sequenced <img file="JP7370366B2_D0176.tif" /> (Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). This gene could be identified by testing candidate genes containing this N-terminal sequence for KDC activity.

<tables><img file="JP7370366B2_D0177.tif" /></tables>

A fourth candidate enzyme for catalyzing this reaction is branched-chain alpha-keto acid decarboxylase (BCKA). This class of enzymes has been shown to act on a variety of compounds varying in chain length from 3 to 6 carbons (Oku and Kaneda, J Biol Chem. 263:18386-18396 (1988); and Smit et al., Appl Environ Microbiol. 71:303-311 (2005)). Lactococcus lactis enzymes are 2-oxobutanoate, 2-oxohexanoic acid, 2-oxopentanoic acid, 3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate and isocaprone. It has been characterized for a variety of branched and linear substrates, including acids (Smit et al., Appl Environ Microbiol. 71:303-311(2005)). This enzyme has been structurally characterized (Berg et al., Science. 318:1782-1786 (2007)). Since sequence alignments between the Lactococcus lactis enzyme and the Zymomonas mobilus pyruvate decarboxylase have shown that the catalytic and substrate recognition residues are nearly identical (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)), making this enzyme a promising candidate for directed genetic engineering manipulation. Decarboxylation of alpha-ketoglutarate by BCKA was detected in Bacillus subtilis; however, this activity was low (5%) compared to the activity towards other branched-chain substrates (Oku and Kaneda, J Biol Chem. 263: 18386-18396 (1988)), and the gene encoding this enzyme has not been identified so far.

Additional BCKA gene candidates can be identified by homology to Lactococcus lactis protein sequences. Many of the high scoring BLASTp hits for this enzyme are annotated as indolepyruvate decarboxylase (EC 4.1.1.74). Indolepyruvate decarboxylase (IPDA) is an enzyme that catalyzes the decarboxylation of indolepyruvate to indoleacetaldehyde in plants and plant bacteria.

<tables><img file="JP7370366B2_D0178.tif" /></tables>

The enzyme, a recombinant branched-chain alpha-keto acid decarboxylase derived from the E1 subunit of the mitochondrial branched-chain keto acid dehydrogenase complex of human and bovine origin, has been cloned and functionally expressed in Escherichia coli (Davie et al., J. Biol. Chem. 267:16601-16606(1992); Wynn et al., J. Biol. Chem. 267:1881-1887(1992); and Wynn et al., J. Biol. Chem. 267:12400-12403 (1992)). In these studies, the authors found that co-expression of the chaperonins GroEL and GroES increased the specific activity of decarboxylase by up to 500-fold (Wynn et al., J. Biol. Chem. 267 :12400-12403(1992)). These enzymes are composed of two alpha subunits and two beta subunits.

<tables><img file="JP7370366B2_D0179.tif" /></tables>

4.1.2.a The condensation of pyruvate with 4-aminobutanal (Figure 22, Step A) or with glutamate-5-semialdehyde (Figure 27, Step A) is an aldehyde lyase of EC class 4.1.2. catalyzed by Various aldehyde lyase enzymes utilize pyruvate as an acceptor; however, no enzymes have been demonstrated to utilize 4-aminobutanal or glutamate-5-semialdehyde as donors. The enzyme 4-hydroxy-2-oxopimelic acid (HODH) aldolase (EC 4.1.2.-) catalyzes the condensation of succinic semialdehyde and pyruvate to form 4-hydroxy-2-oxopimelic acid. This enzyme is a divalent metal ion-dependent class II aldolase that catalyzes the final step in the degradation of 4-hydroxyphenylacetate in E. coli C, E. coli W, and other organisms. In its native context, this enzyme functions in a degradative direction. The reverse (condensation) reaction is thermodynamically unfavorable; however, the equilibrium can be shifted by coupling the HOHD aldolase with downstream pathway enzymes that act efficiently on the reaction products. Such a strategy is effective to shift the equilibrium of other aldolases in the direction of condensation (Nagata et al., Appl Microbiol Biotechnol 44:432-438 (1995); and Pollard et al., Appl Environ. Microbiol 64:4093 -4094(1998)). The E. coli C enzyme encoded by hpcH can condense various aldehyde receptors with pyruvate and was recently crystallized (Rea et al., J Mol. Biol. 373:866-876 (2007); and Stringfellow et al., Gene 166:73-76 (1995)). The E. coli W enzyme is encoded by hpaI (Prieto et al., J Bacteriol. 178:111-120 (1996)).

<tables><img file="JP7370366B2_D0180.tif" /></tables>

Another pyruvate-utilizing aldehyde lyase is 2-dehydro-3-deoxyglucarate aldolase (DDGA, EC 4.1. 2.20). The natural donor for this enzyme is tartronic acid semialdehyde, but this enzyme has broad substrate specificity and has been shown to reversibly condense a wide range of aldehydes with pyruvate (Fish et al., Methods Enzymol 9:529-534 (1966)). The crystal structure of this enzyme has been determined and the catalytic mechanism has been proposed (Izard et al., EMBO J 19:3849-3856 (2000)). Additional candidate DDGA enzymes are found in Leptospira interrogans (118) and Sulphorobus solfataricus (Buchanan et al., Biochem. J 343 Pt 3:563-570 (1999)). The S. solfataricus enzyme is highly thermostable and was cloned and expressed in E. coli (Buchanan et al., Biochem. J 343 Pt 3:563-570(1999)).

<tables><img file="JP7370366B2_D0181.tif" /></tables>

4.2.1.a Hydrolyase. In the two reactions of Figures 20 and 22, enzymes of the dehydratase class (EC4.1.2) are used. Dehydration of 3-hydroxy-6-aminopimeloyl-CoA (Figure 20, Step D) is catalyzed by enoyl-CoA hydratase. This reaction is not known to occur naturally; however, the ability to dehydrate 3-hydroxyacyl-CoA derivatives is widespread. Enoyl-CoA hydratase (EC 4.2.1.17) catalyzes the dehydration of various 3-hydroxyacyl-CoA substrates (Agnihotri et al., Bioorg. Med. Chem. 11:9-20 (2003; Conrad et al., J Bacteriol. 118:103-111 (1974) and Roberts et al., Arch. Microbiol 117:99-108 (1978)). The Pseudomonas putida enoyl-CoA hydratase encoded by ech is a crotonyl-CoA hydratase of 3-hydroxybutyryl-CoA. catalyzes the conversion to CoA (Roberts et al., Arch. Microbiol 117:99-108(1978)). Additional enoyl-CoA hydratase candidates are phaA and phaB from P. putida and paaA and paaB from P. fluorescens (Olivera et al., Proc. Natl. Acad. Sci US A 95:6419-6424 (1998 )). The Rhodopseudomonas palustris pimF gene product is predicted to encode an enoyl-CoA hydratase involved in the degradation of pimeloyl-CoA (Harrison et al., Microbiology 151:727-736 (2005)). Finally, maoC (Park et al., J Bacteriol. 185:5391-5397. 2003), paaF (Ismail et al., Eur. J Biochem. 270:3047-3054 (2003); Park et al., Appl. Biochem. Biotechnol 113-116 :335-346(2004) and (Park et al., Biotechnol Bioeng86:681-686(2004)) and paaG (Park et al., J Bacteriol. 185:5391-5397. 2003), paaF (Ismail et al., Eur. J Biochem. 270:3047-3054 (2003); Park et al., Appl. Biochem. Biotechnol 113-116:335-346 (2004) and (Park et al. , Biotechnol Bioeng 86:681-686 (2004)) have been shown to exhibit enoyl-CoA hydratase functionality.

<tables><img file="JP7370366B2_D0182.tif" /></tables>

3-Hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), also called crotonase, is an enoyl-CoA hydratase that dehydrates 3-hydroxyisobutyryl-CoA to form crotonyl-CoA. The enzyme crotonase is required for the formation of n-butanol in some organisms, particularly Clostridium species, and also in thermoacidophilic archaea of the genera Sulphorobus, acidianus, and Metallospera. /4-Hydroxybutyrate constitutes one step of the cycle. An exemplary gene encoding an enzyme that is crotonase is C. acetobutylicum (Atsumi et al., Metab Eng. 10:305-311 (2008); and Boynton et al., J Bacteriol. 178:3015-3024 (1996)), C. Kluyberg (Hillmer et al., FEBS Lett. 21:351-354. 1972)) and Metallospaera cedula (Berg et al., Science. 318:1782-1786 (2007)), although the sequence of the latter gene I don't know.

<tables><img file="JP7370366B2_D0183.tif" /></tables>

Alternatively, the E. coli fadA and fadB gene products encode a multienzyme complex that exhibits enoyl-CoA hydratase activity, which is involved in fatty acid oxidation (Nakahigashi et al., Nucleic Acids Res. 18:4937 (1990)). ; Yang et al., J Bacteriol. 173:7405-7406 (1991) and Yang et al., Biochemistry 30:6788-6795 (1991)). Knockout of the negative regulator encoded by fadR can be used to activate the fadB gene product (Sato et al., J Biosci. Bioeng 103:38-44 (2007)). The fadI and fadJ genes encode similar functions and are naturally expressed under anaerobic conditions (Campbell et al., Mol. Microbiol 47:793-805 (2003)).

<tables><img file="JP7370366B2_D0184.tif" /></tables>

2-Oxo-7-aminohepta-3-enoate is formed upon dehydration of 2-oxo-4-hydroxy-7-aminoheptanoic acid (Figure 22, Step B). Dehydration of 2-amino-5-hydroxy-7-oxosubarate to 2-amino-5-ene-7-oxosabarate (Figure 27, Step B) is a similar conversion. The enzymes that catalyze these precise reactions are not known to occur naturally. A candidate enzyme that catalyzes a similar reaction naturally dehydrates 2-oxo-4-hydroxy-hepta-1,7-dioate (HODH) to 2-oxohept-4-ene-1,7-dioate (OHED). It is an OHED hydratase.

HODH is structurally similar to the desired substrate. This enzyme requires magnesium as a cofactor (Burks et al., J. Am. Chem. Soc. 120 (1998)). Enzyme candidates that are OHED hydratases have been proposed in E. coli C (Izumi et al., J Mol. Biol. 370:899-911 (2007; and Roper et al., Gene 156:47-51 (1995)) and E. coli W (Prieto et al., J Bacteriol. 178:111-120 (1996)). Sequence comparisons reveal homologues in various bacteria, plants, and animals. Enzymes with highly similar sequences are In particular, Klebsiella pneumoniae (91% identity, E value = 2 × 10^-138) and Salmonella enterica (91% identity, E value = 4 × 10^-138).

<tables><img file="JP7370366B2_D0185.tif" /></tables>

An alternative enzyme candidate for catalyzing this reaction is fumarase, also known as fumarate hydratase (EC 4.2.1.2). E. coli has three fumarases that are controlled by growth conditions: FumA, FumB and FumC. FumB is oxygen sensitive and active only under anaerobic conditions. FumA is active under microanaerobic conditions and FumC is an enzyme active only in aerobic growth (Guest et al., J Gen Microbiol. 131:2971-2984 (1985); Tseng et al., J Bacteriol 183:461 -467 (2001) and Woods et al., Biochim Biophys Acta 954:14-26 (1988)). FumC has been shown to dehydrate alternative substrates including tartrate and threo-hydroxyaspartate (Teipel et al., J Biol. Chem. 243:5684-5694 (1968)). A wealth of structural information is available about FumC, and researchers have successfully genetically engineered this enzyme to alter activity, inhibition, and localization (Weaver et al., D Biol Crystallogr. 61:1395-1401(2005)). Additional fumarate hydratase enzymes are found in Escherichia coli (Estevez et al., Protein Sci 11:1552-1557 (2002); Hong et al., Biotechnol. BioprocessEng. 9:252-255 (2005)) and Rose et al., Proc Natl. Acad Sci U S. A 101:3393-3397(2004)), Corynebacterium glutamicum (Genda et al., Biotechnol Biochem. 70:1102-1109(2006)), Campylobacter jejuni (Smith et al., Cell Biol 31:961) -975 (1999)), highly thermophilic bacteria (Mizobata et al., Arch. Biochem. Biophys. 355:49-55 (1998)), and rats (Kobayashi et al., J Biochem. 89:1923-1931 (1981)). MmcBC fumarase from Perotomaculum thermopropionicum is another class of fumarase with two subunits (Shimoyama et al., FEMS Microbiol Lett 270:207-213 (2007)).

<tables><img file="JP7370366B2_D0186.tif" /></tables>

Another enzyme candidate is citramalate hydrolyase (EC 4.2.1.34), an enzyme that naturally dehydrates 2-methylmalate to mesaconic acid. This enzyme has been studied in Methanochaldococcus janaskii in the context of the pyruvate pathway to 2-oxobutanoate and has been shown to have broad substrate specificity (Drevland et al., J Bacteriol . 189:4391-4400(2007)). This enzyme activity was also detected in Clostridium tetanomorphum, Morganella morganii, and Citrobacter amalonaticus, which are thought to be involved in the degradation of glutamate (Kato et al., Arch. Microbiol. 168:457-463 1997)). The protein sequences of M. janaskii have no significant homology to the genes of these organisms.

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5.4.3.a Aminomutase. Some reactions in Figure 21 involve the transfer of a secondary amine from the 3-position to the 2-position (Figure 21, steps P, R, T). Promising enzyme candidates for catalyzing these transformations are enzymes that reversibly move the amine group of lysine from the 2- to 3-position, naturally converting it to (3S)-3,6-diaminohexanoic acid. , lysine 2,3-aminomutase (EC 5.4.3.2). This enzyme is derived from Fusobacterium nucleatum (kamA) (Barker et al., J. Bacteriol. 152:201-207 (1982)) and Clostridium subterminale (kamA) (Chirpich et al., J. Biol. Chem. 245:1778-1789 ( It is found in bacteria that ferment lysine to acetate and butyrate, including (1970)). The enzyme from Clostridium subterminale has been crystallized (117). The enzyme encoding this function is also encoded by yodO of Bacillus subtilis (Chen et al., Biochem. J. 348 Pt 3:539-549(2000)). This enzyme utilizes pyridoxal 5'-phosphate as a cofactor, requires activation by S-adenosylmethionine, and is stereoselective for L-lysine. This enzyme has not been shown to react with alternative substrates, so we tested it with the non-natural substrates 3-amino-7-oxohexanoic acid, 3,7-diaminoheptanoic acid and 3-aminopimelic acid. Directed evolution or other genetic engineering methods may be required to effect the reaction. For example, Cargill developed an enzyme that is a novel 2,3-aminomutase derived from lysine-2,3-aminomutase that converts L-alanine to β-alanine (Liao et al., United States Patent 20050221466 (2005) ).

<tables><img file="JP7370366B2_D0188.tif" /></tables>

Other enzymes with 2,3-aminomutase activity include tyrosine 2,3-aminomutase (EC 5.4.3.6) and leucine 2,3-aminomutase (EC 5.4.3.7). Tyrosine 2,3-aminomutase is involved in tyrosine biosynthesis and reversibly converts tyrosine to 3-amino-3-(4-hydroxyphenyl)-propionic acid by transferring the amine from the 2- to 3-position. do. In Streptomyces globisporus, this enzyme has also been shown to react with tyrosine derivatives (Christenson et al., Biochemistry 42:12708-12718 (2003)); however, the sequence of this enzyme is not yet available. Can not. Leucine 2,3-aminomutase converts L-leucine to beta-leucine during leucine biosynthesis and degradation. Although assays specific for leucine 2,3-aminomutase have detected enzyme activity in many organisms (Poston et al., Methods Enzymol. 166:130-135 (1988)), the gene encoding this enzyme is has not been identified until now.

6.2.1.a Acid-thiol ligase. Activation of carboxylic acids to acyl-CoA derivatives is catalyzed by EC class 6.2.1 CoA acid-thiol ligases or CoA synthetases (the terms ligase, synthetase, and synthase are used interchangeably herein). and refer to the same enzyme class). Such enzymes couple the energetic cost of the thioester bond formation with the hydrolysis of ATP to ADP or AMP. Some ADP-forming CoA ligases have been demonstrated to react in the opposite direction, removing the CoA moiety from acyl-CoA molecules and forming ATP at the same time. Reversible CoA ligase is required to deacylate 6-aminopimeloyl-CoA (Figure 20, Step I) and 3-oxopimeloyl-CoA (Figure 21, Step B), while acylate 3-oxopimelic acid (Figure 21, Step H), acylate 3-aminopimelic acid (Figure 21, Step K) and 2-aminopimelic acid with a ligase to form AMP or ADP. acylation (Figure 21, Step V). The enzymes that catalyze these precise transformations have not been characterized to date; however, several enzymes with broad substrate specificity have been described in the literature.

Acetyl-CoA synthetase (ACD, EC 6.2.1.13), which forms ADP, is an enzyme that couples the conversion of acyl-CoA esters to their corresponding acids, accompanied by the synthesis of ATP. ACD I from Archaeoglobus fulgidus is encoded by AF1211 and was shown to act on a variety of linear and branched substrates including isobutyrate, isopentanoic acid, and fumarate (Musfeldt et al., J Bacteriol 184:636-644(2002)). A second reversible ACD encoded by AF1983 in Archaeoglobus fulgidus was also shown to have a broad substrate range and high activity towards the cyclic compounds phenylacetate and indole acetate (Musfeldt et al. , J Bacteriol. 184:636-644(2002)). The enzyme from Haloacula marismortii (annotated as succinyl-CoA synthetase) accepts propionic acid, butyrate, and branched-chain acids (isovaleric acid and isobutyrate) as substrates and can operate in the forward and reverse directions. (Brasen et al., Arch. Microbiol 182:277-287(2004)). The ACD encoded by PAE3250 from the hyperthermophilic Crenoarchaeal phylum Pyrobaculum aerophyllum reacts with acetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA, and is one of the most characterized ACDs among all characterized ACDs. showed the broadest substrate range (Brasen et al., Arch. Microbiol 182:277-287 (2004)). Directed evolution or directed genetic engineering can be used to modify this enzyme to operate at the physiological temperature of the host organism. Enzymes from A. fulgidus, H. marismortii, and P. aerophilum have all been cloned into E. coli, functionally expressed, and characterized (Brasen et al., Arch. Microbiol 182:277-287 (2004); and Musfeldt et al., J Bacteriol. 184:636-644(2002)). An additional candidate is the enzyme encoded by sucCD in E. coli, which naturally catalyzes the formation of succinyl-CoA from succinate with the consumption of one ATP, a reaction that is reversible in vivo (Buck et al., Biochemistry 24:6245-6252 (1985)).

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Another candidate enzyme is the AMP-forming pimeloyl-CoA ligase (EC 6.2.1.14), which naturally activates pimelic acid to pimeloyl-CoA during the biosynthesis of biotin in Gram-positive bacteria. An enzyme from Pseudomonas mendocina, cloned into E. coli, was shown to tolerate alternative substrates hexanedioate and nonanedioate (Binieda et al., Biochem. J 340(Pt 3):793-801 (1999) )). Other pimeloyl-CoA ligase candidates include Bacillus subtilis (Bower et al., J Bacteriol. 178:4122-4130 (1996)) and Ricinibacillus sphaericus (formerly Bacillus sphaericus) (Ploux et al., Biochem. J 287 (Pt 3) :685-690 (1992)).

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Additional CoA ligases include the rat dicarboxylic acid-CoA ligase (Vamecq et al., Biochem J 230:683-693 (1985)), whose sequence has not yet been characterized, and two characterized species from P. chrysogenum. either phenylacetic acid-CoA ligase (Lamas-et al., Maceiras, J 395:147-155 (2006); and Wang et al., Biophys. Res. Commun. 360:453-458 (2007)) and Pseudomonas putida (Martinez-Blanco et al., J Biol. Chem. 265:7084-7090 (1990)). Acetoacetyl-CoA synthetase from mice (Hasegawa et al., Biochim. Biophys. Acta 1779:414-419 (2008)) and humans (Ohgami et al., Biochem. Pharmacol. 65:989-994 (2003)) Naturally catalyzes the ATP-dependent conversion to acetoacetyl-CoA.

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EXAMPLE XXVII (Additional Production Route for Hexamethylene Diamine from 6-Aminocaproate) Figure 24 provides an additional route for producing HMDA and is similar to Figure 13 and Example XX above. It is something to add. The arrows in steps O and P indicate the direct conversion of 6-aminocaproate to 6-aminocaproic acid semialdehyde and the direct conversion of 6-acetamidohexanoic acid to 6-acetamidohexanal, respectively. These reactions are catalyzed by reductases of EC class 1.2.1.e. For details on enzyme candidates, see Example XXVI (EC 1.2.1.e).

Example XXVIII (Production Route of 6-Aminocaproate from Adipate) Figure 25 provides an additional route for producing 6-ACA and is in addition to Figure 10 and Example XVI above. It is. The conversion of adipate to adipate semialdehyde (Figure 25, Step X) is catalyzed by an enzyme with adipate reductase functionality. Adipate kinase catalyzes the formation of adipyl phosphate from adipate (Figure 25, Step Y). Adipate semialdehyde is formed from adipyl phosphate by adipyl phosphate reductase (Figure 25, Step Z). Enzyme candidates for catalyzing these transformations are described in Example XXVI.

(Example XXIX) (Production pathway of levulinic acid) Levulinic acid (LA), also known as 4-oxopentanoic acid and 4-ketovaleric acid, is a precursor of nylon-like polymers, synthetic rubbers and plastics. be. Levulinic acid is also a precursor for other commodity chemicals such as methyltetrahydrofuran, valerolactone and ethyl levulinate. Other potential applications include use as a fuel extender and biodegradable herbicide/insecticide. Levulinic acid is traditionally produced by treating cellulosic biomass with strong acids such as hydrochloric acid and sulfuric acid. This process has the disadvantages of low LA yield and numerous by-products. More recently, a biofine process has been developed that converts cellulosic biomass to LA, formic acid, and furfural with a theoretical maximum yield of 70% (Hayes et al., "Biofine Process - Lignocellulosic Raw Materials"). The biofine process-production of levulinic acid, furfural and formic acid from lignocellulosic feedstock), p. 139-164. In Biorefineries: Industrial Processes and Products. Wiley, Weinheim, Germany (2006)). Described herein is a process for selectively producing LA from sugar or syngas feedstocks using microbial organisms.

The maximum theoretical yield of LA from glucose is 1.45 moles of LA per mole of glucose utilized (0.938 g/g) according to the following equation: Glucose (C₆H₁₂O₂)+1.27 CO₂1.45 LA(C₅H₈O₃)+0.18 H₂OLA is produced in three enzymatic steps from the central metabolites succinyl-CoA and acetyl-CoA. In the first step, acetyl-CoA and succinyl-CoA are condensed by beta-ketothiolase to form 3-oxoadipyl-CoA (Step A of Figure 25). The CoA moiety is then removed by CoA hydrolase, transferase or ligase (steps E/F/G in Figure 25). In the final step of this pathway, 3-oxoadipate is decarboxylated to LA (step AA in Figure 25).

Decarboxylation of 3-oxoadipate to LA can occur enzymatically or spontaneously. In Escherichia coli, several 3-oxoacids produced during amino acid biosynthesis have been shown to undergo spontaneous decarboxylation (Boylan et al., Biochem. Biophys. Res Commun. 85:190-197( 1978)).

An enzyme that catalyzes the decarboxylation of 3-oxoadipate to LA has not been demonstrated to our knowledge. An exemplary candidate enzyme that catalyzes a similar reaction is acetoacetate decarboxylase (EC 4.1.1.4). Acetoacetate decarboxylase from Clostridium acetobutylicum, encoded by adc, has broad substrate specificity, including 3-oxopentanoic acid, 2-oxo-3-phenylpropionic acid, and 2-methyl-3-oxobutylic acid. Benner et al., J. Am. Chem. Soc. 103:993-994 (1981) and Rozzel et al., J. Am. Chem. Soc. 106:4937-4941 (1984). )). Acetoacetate decarboxylase is produced by Clostridium beijerinckii (Ravagnani et al., Mol. Microbiol 37:1172-1185 (2000)). Acetoacetate decarboxylase from Bacillus polymyxa, which was characterized in cell-free extracts, similarly has broad substrate specificity for 3-keto acids and decarboxylates 3-oxopentanoic acid. (Matiasek et al., Curr. Microbiol 42:276-281 (2001)). The gene encoding this enzyme has not been identified to date, and the genome sequence of M. polymyxa is not yet available. Another adc is found in Clostridium saccharoperbutylacetonicum (Kosaka, et al., Biosci.Biotechnol Biochem. 71:58-68 (2007)).

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(Example XXX) (In silico knockout strategy for producing adipate, 6-ACA and HMDA) In this example, for producing adipate 6-aminocaproate (6-ACA) and hexamethylene diamine (HMDA), We describe the gene disruption strategy for.

Below, we have genetically engineered adipate, 6-aminocaproate (6-ACA) and hexamethylene diamine (HMDA) production pathways, including those that use succinyl-CoA and acetyl-CoA as precursors. A set of enzyme activities that can be reduced in engineered production hosts by disruption or deletion of appropriate genes is detailed.

OptKnock is a two-layer computational framework designed with the overall goal of developing genetically stable overproducing microorganisms. In particular, this framework examines the complete network of microorganisms in order to propose genetic manipulations that would result in desired biochemicals becoming essential by-products of cell growth. Coupling biochemical production and cell growth through the disruption or deletion of strategically placed genes reduces the growth burden imposed on genetically engineered strains after extended periods in bioreactors. Selection pressure improves performance as a result of forced, growth-coupled production of biochemicals. Finally, in the case of gene deletions, the genes selected by OptKnock are completely removed from the genome, so the possibility of engineered strains reverting to their wild-type state is negligible.

Biochemical production coupled to proliferation can be visualized in the context of the biochemical production limits of an exemplary metabolic network calculated using an in silico model. These limits establish the rate(s) of uptake of the limiting substrate(s) relative to their experimental measurement(s), and determine the rate of growth at each achievable level of proliferation. It is obtained by calculating the maximum and minimum rates of production of a chemical. Although exceptions exist, in general the production of desired biochemicals directly competes with biomass formation for intracellular resources. Therefore, increasing the rate of biochemical production generally results in submaximal rates of proliferation. Design the knockouts proposed by OptKnock to limit the range of acceptable solutions that alter metabolic behavior from the wild-type strain. The range of actual solutions for a given strain will expand or contract as the substrate uptake rate(s) increases or decreases, but each experimental point should be within the range of solutions calculated for it. be. Such a plot makes it possible to visualize how close stocks are to their performance limits, or in other words, how much room there is for improvement. The OptKnock framework has been used to identify promising gene deletion strategies for biochemical overproduction and establish a systematic framework that will yield future improvements in naturally occurring metabolic and regulatory modeling frameworks. .

Addition of biosynthetic pathways proceeding via succinyl-CoA and acetyl-CoA should be absent in order to create a host organism in which growth-coupled production of adipate, 6-ACA or HMDA is achieved. The set of enzyme activities that are present, should be diluted, or should be eliminated are described below. Perform an optimization technique called integer cut, which involves solving the OptKnock problem iteratively by incorporating an additional constraint called integer cut at each iteration to enumerate all potential strategies .

A design was identified based on a stoichiometric model of E. coli metabolism using the OptKnock algorithm. The assumptions are that (i) the glucose uptake rate is 10 mmol/gdw/h; (ii) anaerobic or microaerobic conditions; and (iii) the minimum maintenance requirements not related to growth are Contains 4mmol/gdw/hour. Table 12 provides a list of all reaction stoichiometries and relevant genes known to be involved in the reactions identified for deletion in this strategy. In Table 13, the abbreviations, corresponding names and positions of all metabolites involved in the reactions listed in Table 12 are provided. Growth coupled production designs for adipic acid, 6ACA and HMDA are provided in Tables 14-16. The product formation rates shown in Tables 14-16 are mmol/g DCW time. The basic glucose uptake rate is 10 mmol/g DCW time, and the biomass formation rate is expressed in units of 1/hr. These tables list the reactions, expected product yields, and biomass yields that will be knocked out with a particular strategy. Although the design was identified using a metabolic model, E. coli metabolism, and the names of the genes listed are specific to E. coli, the method of selecting the strategy to genetically engineer the metabolism and the design itself are based on HMDA. , 6-ACA or adipate-producing organisms. Therefore, the design is essentially a list of enzymatic transformations whose activity should be eliminated, diluted, or absent in the microorganism to result in the production of adipate, 6ACA, and HMDA coupled to growth.

An important criterion for prioritizing the final selection of designs was the yield coupled with the respective growth of the product. To test this, for each strategy, a production cone was constructed by first maximizing and then minimizing the product yield at different biomass formation rates, as described above. If the right-most boundary of all possible phenotypes of a mutant network is a single point, it means that the only optimal yield of product is the maximum possible biomass formation in the network. Means at speed. In other cases, the rightmost boundary of the likely phenotype is a vertical line, which is calculated in the network at the time of greatest biomass, including the lowest abundance at the bottom point of the vertical line. This shows that any amount of product within the given range can be produced. Such designs were given low priority.

The metabolic engineering strategy described below assumes that an organism can produce adipate, 6-ACA or HMDA via a pathway that uses succinyl-CoA and acetyl-CoA. The construction of recombinant host organisms capable of producing these products via that pathway is described herein.

Strain Construction: Strains were constructed, evolved, and tested to test the computational predictions proposed in this report. E. coli K-12 MG1655, which harbors the succinyl-CoA-acetyl-CoA pathway, serves as the strain into which the deletion is introduced. This strain was constructed by incorporating in-frame deletions using homologous recombination with the Datsenko and Wanner λRed recombinase system (Proc. Natl. Acad. Sci. USA 97(12):6640-6645 2000). do. This approach involves replacing the chromosomal sequence, ie, the target gene for removal, with a selectable antibiotic resistance gene that is itself subsequently removed. Incorporate the knockouts one by one into the recipient strain. No antibiotic resistance markers remain after each deletion, allowing multiple mutations to accumulate in each target strain. Deletion techniques completely remove the gene targeted for deletion, such that the likelihood that the constructed mutant will revert to the wild type is substantially reduced.

Shake flask characterization: Once the intermediate strain is constructed, strain performance is quantified by performing shake flask fermentations. Anaerobic conditions are obtained by sealing the flask with a rubber septum and then sparging the medium with nitrogen. For strains where growth is not observed under strictly anaerobic conditions, microaerobic conditions can be applied by covering the flask with foil and poking small holes to limit ventilation. All experiments were performed using M9 minimal medium supplemented with glucose unless otherwise specified. The pre-culture is grown overnight and used as an inoculum for fresh batch cultures where readings are taken during exponential growth. Growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and glucose uptake rate is determined by monitoring carbon source depletion over time. The products, ethanol and organic acids, are analyzed by GC-MS or HPLC using routine procedures. Triplicate cultures were grown for each strain.

Batch Fermenter Test: The performance of selected strains was tested in an anaerobic, pH-controlled batch fermentation. This not only allows reliable quantification of growth rates, glucose uptake and formation rates of all products, but also ensures that cell growth is not limited by the accumulation of acidic fermentation products. Furthermore, it becomes possible to accurately determine volumetric productivity and yield of product formation, two of the most important parameters in benchmarking strain performance. Fermentation is carried out in a 1 L bioreactor with a working volume of 600 mL, equipped with temperature and pH regulation. N to the reactor₂sparge continuously at approximately 0.5 L/min to ensure that dissolved oxygen (DO) levels remain below detection levels. The medium is the same as above except that the glucose concentration is increased depending on the high cell density achievable in the fermentation vessel.

Chemostat studies: Chemostat experiments were performed to directly measure how switching the mode of fermentation from batch to continuous affects product yield and volumetric productivity. The bioreactor described above using batch mode was operated in chemostatic mode by continuously feeding medium and removing spent culture. Set the inlet flow rate to maintain a constant dilution of 80% of the maximum growth rate observed for each strain in the batch, and adjust the outlet flow to maintain the level. Glucose is the limiting nutrient in the medium and is set to achieve the desired optical density within the container.

Adaptive evolution: Knockout strains are expected to initially exhibit suboptimal growth rates until their metabolic networks are adjusted for the function they are deficient in. Strains are adaptively evolved to enable this adjustment. By subjecting the strain to adaptive evolution, cell proliferation rate becomes the primary selection pressure and mutant cells are forced to rearrange their metabolic flux to increase their proliferation rate. . This metabolic reprogramming was recently demonstrated for several E. coli mutants that evolved adaptively on different substrates to reach growth rates predicted a priori by in silico models (Fong et al. Palsson, Nat. Genet. 36(10):1056-1058 (2004). Strains generated by OptKnock have previously been tested in E. coli such that one strain potentially has superior production quality over the other. Having demonstrated the different evolutionary patterns that can result, we adaptively evolve (run in parallel) in triplicates (Fong and Palsson, Nat Genet. 36(10):1056-1058 (2004); Fong et al. J. Bacteriol. 185(21):6400-6408(2003); Ibarra et al., Nature 420(6912):186-189(2002)). The evolution is performed for 2-6 weeks depending on the growth rate improvement achieved. Generally, evolution is stopped once a stable phenotype is achieved. The concept of biochemical production coupled to proliferation behind the OptKnock approach results in the generation of genetically stable overproducers.

Although described as a deletion set, as disclosed herein, a gene set can be deleted or disrupted such that the activity of the gene product it encodes is reduced or eliminated. be understood. Therefore, the gene deletion sets in Tables 14-16 can be used to delete or disrupt gene sets in a host organism in which it is desirable to increase the production of 6-ACA, adipate and/or HMDA. . Any of the disclosed gene deletion sets can be used to generate knockout strains with disrupted or deleted genes that confer increased production of 6-ACA, adipate and/or HMDA. is understood.

Table 12: List of all reaction stoichiometries and associated genes known to be associated with reactions in which deletions were identified in the strategies listed in Tables 1 and 2.<tables><img file="JP7370366B2_D0193.tif" /></tables><img file="JP7370366B2_D0194.tif" /><img file="JP7370366B2_D0195.tif" /><img file="JP7370366B2_D0196.tif" />

Table 13: List of abbreviations, corresponding names and positions of all metabolites involved in the reactions listed in Table 12<tables><img file="JP7370366B2_D0197.tif" /></tables><img file="JP7370366B2_D0198.tif" /><img file="JP7370366B2_D0199.tif" />

Table 14. Design of knockout strains to increase 6-ACA production, with 6-ACA yield and biomass yield shown.

<tables><img file="JP7370366B2_D0200.tif" /></tables><img file="JP7370366B2_D0201.tif" /><img file="JP7370366B2_D0202.tif" /><img file="JP7370366B2_D0203.tif" /><img file="JP7370366B2_D0204.tif" /><img file="JP7370366B2_D0205.tif" /><img file="JP7370366B2_D0206.tif" /><img file="JP7370366B2_D0207.tif" /><img file="JP7370366B2_D0208.tif" /><img file="JP7370366B2_D0209.tif" /><img file="JP7370366B2_D0210.tif" /><img file="JP7370366B2_D0211.tif" /><img file="JP7370366B2_D0212.tif" /><img file="JP7370366B2_D0213.tif" /><img file="JP7370366B2_D0214.tif" /><img file="JP7370366B2_D0215.tif" /><img file="JP7370366B2_D0216.tif" /><img file="JP7370366B2_D0217.tif" /><img file="JP7370366B2_D0218.tif" /><img file="JP7370366B2_D0219.tif" /><img file="JP7370366B2_D0220.tif" /><img file="JP7370366B2_D0221.tif" /><img file="JP7370366B2_D0222.tif" /><img file="JP7370366B2_D0223.tif" /><img file="JP7370366B2_D0224.tif" /><img file="JP7370366B2_D0225.tif" /><img file="JP7370366B2_D0226.tif" /><img file="JP7370366B2_D0227.tif" /><img file="JP7370366B2_D0228.tif" /><img file="JP7370366B2_D0229.tif" /><img file="JP7370366B2_D0230.tif" />

Table 15. Design of knockout strains to increase adipate production, with adipate yield and biomass yield shown.

<tables><img file="JP7370366B2_D0231.tif" /></tables><img file="JP7370366B2_D0232.tif" /><img file="JP7370366B2_D0233.tif" /><img file="JP7370366B2_D0234.tif" /><img file="JP7370366B2_D0235.tif" /><img file="JP7370366B2_D0236.tif" /><img file="JP7370366B2_D0237.tif" /><img file="JP7370366B2_D0238.tif" /><img file="JP7370366B2_D0239.tif" /><img file="JP7370366B2_D0240.tif" /><img file="JP7370366B2_D0241.tif" /><img file="JP7370366B2_D0242.tif" /><img file="JP7370366B2_D0243.tif" /><img file="JP7370366B2_D0244.tif" /><img file="JP7370366B2_D0245.tif" /><img file="JP7370366B2_D0246.tif" /><img file="JP7370366B2_D0247.tif" /><img file="JP7370366B2_D0248.tif" />

Table 16. Design of knockout strains to increase HMDA production, HMDA yield and biomass yield are shown.

<tables><img file="JP7370366B2_D0249.tif" /></tables><img file="JP7370366B2_D0250.tif" /><img file="JP7370366B2_D0251.tif" /><img file="JP7370366B2_D0252.tif" /><img file="JP7370366B2_D0253.tif" /><img file="JP7370366B2_D0254.tif" /><img file="JP7370366B2_D0255.tif" /><img file="JP7370366B2_D0256.tif" /><img file="JP7370366B2_D0257.tif" /><img file="JP7370366B2_D0258.tif" /><img file="JP7370366B2_D0259.tif" /><img file="JP7370366B2_D0260.tif" /><img file="JP7370366B2_D0261.tif" /><img file="JP7370366B2_D0262.tif" /><img file="JP7370366B2_D0263.tif" /><img file="JP7370366B2_D0264.tif" /><img file="JP7370366B2_D0265.tif" /><img file="JP7370366B2_D0266.tif" /><img file="JP7370366B2_D0267.tif" /><img file="JP7370366B2_D0268.tif" /><img file="JP7370366B2_D0269.tif" /><img file="JP7370366B2_D0270.tif" /><img file="JP7370366B2_D0271.tif" /><img file="JP7370366B2_D0272.tif" /><img file="JP7370366B2_D0273.tif" />

A minimal set of gene deletions to increase production of 6-ACA. A strain design strategy for improving 6-aminocaproate (6-ACA) production in microorganisms with a 6-ACA pathway via acetyl-CoA and succinyl-CoA was described above. Based on extensive analysis of the design of strains to produce 6-ACA listed in Table 14, a minimal set of deletions required for growth-coupled production of 6-ACA has been identified. Note that phosphoenolpyruvate carboxykinase (PPCK) was assumed to be reversible.

Briefly, deletions in acetaldehyde dehydrogenase (ADHEr) and lactate dehydrogenase (LDH_D) are required to prevent the formation of competitive byproducts, ethanol and lactate. Therefore, the minimal deletion set includes deletions of acetaldehyde dehydrogenase (ADHEr) and lactate dehydrogenase (LDH_D). Additional deletion strains include, in addition to ADHHer and LDH_D, strains lacking at least one of the following activities: malate dehydrogenase (MDH), aspartase (ASPT), NAD(P) transhydrogenase (THD2). and glutamate dehydrogenase (GLUDy). Such additional deletions result in a tight coupling of production and cell proliferation. Figures 28-31 show the calculated 6-ACA yield versus growth yield for deletion mutants lacking at least ADHHer and LDH_D (Figure 28). Calculated yields for strains with additional deletions are shown in Figures 29-31.

A minimal set of additional deletions includes phosphoglucoisomerase (PGI). This design focuses on generating reducing equivalents via the pentose phosphate pathway. Additional advantageous deletions include: acetaldehyde dehydrogenase (ADHEr), hexokinase (HEX1), 2-dehydro-3-deoxy-phosphogluconate aldolase (EDA) and phosphogluconate dehydratase (PGDHy). Calculated 6-ACA yield versus growth yield for deletion mutants lacking at least PGI are shown in Figures 32-34, and additional exemplary mutants are shown in Figures 32-34. Shown in 34.

If it is determined that the expected strain design does not sufficiently couple product formation with biomass formation, or in order to increase the efficiency of the coupling of product formation with biomass formation, these strains may be Each of these can be filled in with additional deletions. Alternatively, some other enzymes not known to have significant activity under growth conditions may become active through adaptive evolution or random mutagenesis. Such enzymatic activity can also be knocked out. For example, succinate dehydrogenase, which oxidizes succinate to fumarate and is known to be active only under aerobic conditions, can assume significant activity even under anaerobic conditions and therefore its Such activity can be knocked out. However, the list of gene deletion sets provided herein serves as an excellent starting point for constructing high-yield, growth-coupled 6-ACA producing strains.

A minimal set of gene deletions to increase adipate production. A strain design strategy for improving adipate production in microorganisms with an adipate pathway via acetyl-CoA and succinyl-CoA has been described above. Based on an extensive analysis of the design of strains to produce adipate listed in Table 15, a minimal set of deletions required for adipate production coupled with growth in the network has been identified. Note that phosphoenolpyruvate carboxykinase (PPCK) was assumed to be reversible in the network.

Briefly, deletions in acetaldehyde dehydrogenase (ADHEr) and lactate dehydrogenase (LDH_D) are required to prevent the formation of competitive byproducts, ethanol and lactate. Therefore, the minimal deletion set includes deletions of acetaldehyde dehydrogenase (ADHEr) and lactate dehydrogenase (LDH_D). Additional deletion strains include strains lacking at least one of the following activities in addition to acetaldehyde dehydrogenase (ADHEr) and lactate dehydrogenase (LDH_D): fumarase (FUM), phosphoglucose isomerase (PGI), PEP carboxy kinase (PPCK), hexokinase (HEX1), malate dehydrogenase (MDH) and NADH dehydrogenase (NADH6).

Additional deletions have been identified by the OptKnock framework to improve adipate formation coupled to proliferation. These include one or more of the following: malic enzyme (ME2), aspartate aminotransferase (ASPT), acetate kinase (ACKr), phosphotransacetylase (PTAr), pyruvate formate lyase (PFL), transhydrogenase ( THD2), and glutamate dehydrogenase (GLUDy), and the PTS system of glucose uptake (GLCpts). Further improvements in yield can be achieved by additional deletion of any of the following enzymes: ATP synthase (ATPS4r), phosphogluconate dehydratase (PGDHY), 2-dehydro-3-deoxy-phosphoglucone. acid aldolase (EDA), 6-phosphogluconolactonase (PGL), glucose 6-phosphate dehydrogenase (G6PDHY), and phosphogluconate dehydrogenase (PGDH).

If it is determined that the expected strain design does not sufficiently couple product formation with biomass formation, or in order to increase the efficiency of the coupling of product formation with biomass formation, these strains may be Each of these can be filled in with additional deletions. Alternatively, some other enzymes not known to have significant activity under growth conditions may become active through adaptive evolution or random mutagenesis. Such enzymatic activity can also be knocked out. However, the list of gene deletion sets provided herein serves as an excellent starting point for constructing high-yielding, growth-coupled adipate producing strains.

A minimal set of gene deletions to increase HMDA production. A strain design strategy for improving HMDA production in microorganisms with hexamethylenediamine (HMDA) pathway via acetyl-CoA and succinyl-CoA was described above. Based on extensive analysis of the design of strains to produce HMDA listed in Table 16, a minimal set of deletions required for production of HMDA coupled with growth in the network has been identified. Note that phosphoenolpyruvate carboxykinase (PPCK) was assumed to be reversible in the network.

Briefly, deletions in acetaldehyde dehydrogenase (ADHEr) and lactate dehydrogenase (LDH_D) are required to prevent the formation of competitive byproducts, ethanol and lactate. Therefore, the minimal deletion set includes deletions of acetaldehyde dehydrogenase (ADHEr) and lactate dehydrogenase (LDH_D). Additional deletion strains include strains lacking at least one of the following activities in addition to acetaldehyde dehydrogenase (ADHEr) and lactate dehydrogenase (LDH_D): fumarate reductase (FRD2), fumarase (FUM), phosphoglucose isomerase (PGI), or PEP carboxykinase (PPCK).

Additional deletions have been identified by the OptKnock framework to improve HMDA formation in conjunction with proliferation. These include one or more of the following: hexokinase (HEX1), malate enzyme (ME2), malate dehydrogenase (MDH), aspartate transaminase (ASPT), acetate kinase (ACKr), phosphotransacetylase (PTAr), pyruvate formate lyase (PFL), and pyruvate kinase (PYK). The yield of HMDA can be further improved by further deletion of one or more of the following enzymes: transhydrogenase (THD2), glutamate dehydrogenase (GLUDy), ATP synthase (ATPS4r), GLCpts (for glucose uptake). PTS system), PGDHY (phosphogluconate dehydratase) and EDA (2-dehydro-3-deoxy-phosphogluconate aldolase).

If it is determined that the expected strain design does not sufficiently couple product formation with biomass formation, or in order to increase the efficiency of the coupling of product formation with biomass formation, these strains may be Each of these can be filled in with additional deletions. Alternatively, some other enzymes not known to have significant activity under growth conditions may become active through adaptive evolution or random mutagenesis. Such enzymatic activity can also be knocked out. For example, succinate dehydrogenase, which oxidizes succinate to fumarate and is known to be active only under aerobic conditions, can assume significant activity even under anaerobic conditions and therefore its Such activity can be knocked out. However, the list of gene deletion sets provided herein serves as an excellent starting point for constructing high-yield, growth-coupled HMDA producing strains.

Design of Optknock strains for growth-coupled adipate production. Further illustrations of deletion strategies for genetically engineering strains to synthesize adipate using the succinyl-CoA pathway are described below. All high-priority growth-coupled designs to synthesize adipate are directed to strains lacking acetylaldehyde-CoA dehydrogenase (ADHEr) and lactate dehydrogenase (LDH_D) activities to prevent the formation of fermentation byproducts, as described above. Based on. Further deletion of malate dehydrogenase (MDH) also reduces byproduct production. Figure 35 shows the characteristics of adipate production coupled to growth of the high priority strain design (gray) compared to the characteristics of adipate production coupled to growth of wild-type E. coli (black). Assume that the glucose uptake rate is 10 mmol/gDW/hour. A strain deficient in ADHHer, LDH_D, and MDH activities (Design 1 in Figure 35) achieved an adipate yield of 0.51 grams of adipate per gram of glucose utilized (g/g) at maximum biomass yield. It is expected that

Designs 2 to 4 are based on Design 1 as the basic design. Design 2 requires removal of phosphoenolpyruvate carboxykinase (PPCK). This design improves the adipate yield to 3.6 g/g at maximum biomass yield. The additional deletion of pyruvate formate lyase (PFLi) activity in design 3 further improves the yield by preventing the secretion of formate as a byproduct. The predicted adipate yield for this design is 5.8 g/g. Design 4 features the deletion of NAD(P) transhydrogenase (THD2) in addition to ADHHer, LDH_D, MDH, PPCK and PFLi. This results in an adipate yield of 6.8 g/g at a growth rate of 0.117 1/hr. Design 4 works to tightly couple adipate production with cell proliferation, achieving a theoretical maximum yield of 91%.

(Example XXXI) (Biosynthesis of adipate semialdehyde from adipate and biosynthesis of 6-aminocaproic acid semialdehyde from 6-aminocaproate) In this example, the biosynthesis of adipate semialdehyde from adipate will be described. The production and biosynthetic production of 6-aminocaproic acid semialdehyde from 6-aminocaproate is described.

The conversion of adipate to adipate semialdehyde (Figure 25, Step X) can be catalyzed by carboxylic acid reductase (CAR). This was demonstrated by the following results. Chemically competent cells of E. coli strain ECKh-422 harboring F'pKLJ33s (ΔadhE, ΔldhA, ΔpflB, ΔlpdA, lpdA incorporated from Klebsiella pneumoniae::Ε354K, Δmdh, ΔarcA, gltA-R163L) were incubated with various Transformed with pZs*13s plasmid carrying CAR gene (Table 17) or control plasmid without any CAR gene. A single colony of transformants was selected and grown overnight in LB at 37°C with 100 μg/ml carbenecillin and 10 μg/ml chloramphenicol. Cells were subcultured at a ratio of 1:50 and incubated with 200 μM IPTG at an OD600 of 0.6. Cells were incubated at 37°C for 5 hours before harvesting.

The cell culture was aliquoted into 15 ml samples and pelleted. Cell pellets were stored at 80°C until used in assays.

Table 17. CAR genes used in this example<tables><img file="JP7370366B2_D0274.tif" /></tables>

The cell pellet was lysed by adding 500 μl of B-PER along with 0.5 μl of lysozyme and benzonase. 2 μl of the crude lysate was added to a 96-well format microplate in a total volume of 250 μl with 50 mM Tris (pH 7.2), 1 mM EDTA, 10 mM MgCl2, 1 mM DTT, 10% (v/v) glycerol, 1 mM ATP, 0.5 mM NADPH and CAR activity was measured by adding 20mM adipate or 50mM 6-aminocaproate to the assay solution. NADP of NADPH⁺The oxidation to was monitored by absorbance at 340 nm for 30 minutes at room temperature. The activity of various CAR proteins was calculated using the NADPH depletion rate. Total protein concentration of each lysate was determined by Bradford and activity was normalized to total protein concentration (units/mg).

Conversion of adipate to adipate semialdehyde (Figure 24, step). As shown in Figure 36, significant CAR activity using adipate as a substrate was observed for both CAR genes 889 and 891, whereas control lysates showed no CAR activity.

Additionally, 500μl of 50mM Tris (pH 7.2), 1mM EDTA, 10mM MgCl₂, 1mM DTT, 10% (v/v) glycerol, 5mM ATP, 3mM NADPH and 20mM adipate. The reaction was incubated for 30 minutes at room temperature and stopped by adding 1% formic acid. The samples were then centrifuged and the supernatants analyzed by LC-MS. Low mM levels of adipate semialdehyde were detected, thereby confirming the conversion of adipate to adipate semialdehyde.

Conversion of 6-aminocaproate to 6-aminocaproic acid semialdehyde. As shown in Figure 37, significant CAR activity using 6-aminocaproate as substrate was observed for several CAR genes, 720, 889, 890, 891 and 892, whereas lysis of control showed no CAR activity. These results suggest the conversion of 6-aminocaproate to 6-aminocaproic acid semialdehyde.

Throughout this application, various publications have been referenced. The disclosures of these publications are hereby incorporated by reference in their entirety to fully describe the current state of the art to which this invention pertains. Although the invention has been described with reference to the embodiments provided above, it should be understood that various modifications may be made without departing from the spirit of the invention.

<u style="Single"> The present application provides an invention having the following configuration.</u><u style="Single">(Configuration 1)</u><u style="Single"> 6-aminocaproic acid pathway enzymes expressed in sufficient amounts to produce 6-aminocaproic acid</u><u style="Single">a microorganism having a 6-aminocaproic acid pathway comprising at least one exogenous nucleic acid encoding</u><u style="Single">A non-naturally occurring microbial organism, including a biological organism, wherein the 6-aminocaproic acid pathway is</u><u style="Single">, 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA</u><u style="Single">Reductase; 3-hydroxy-6-aminohexanoyl-CoA dehydratase; 6-aminohexanoyl-</u><u style="Single">2-enoyl-CoA reductase; and 6-aminocaproyl-CoA/acyl-CoA transferer</u><u style="Single">enzyme, 6-aminocaproyl-CoA synthase, or 6-aminocaproyl-CoA hydrolase.</u><u style="Single">The non-naturally occurring microbial organism.</u><u style="Single">(Configuration 2)</u><u style="Single"> Construct 1 comprising two exogenous nucleic acids each encoding a 6-aminocaprone pathway enzyme</u><u style="Single">non-naturally occurring microbial organisms.</u><u style="Single">(Configuration 3)</u><u style="Single"> Construct 1 described, comprising three exogenous nucleic acids each encoding a 6-aminocaprone pathway enzyme</u><u style="Single">non-naturally occurring microbial organisms.</u><u style="Single">(Configuration 4)</u><u style="Single"> Construct 1 described, comprising four exogenous nucleic acids each encoding a 6-aminocaprone pathway enzyme</u><u style="Single">non-naturally occurring microbial organisms.</u><u style="Single">(Configuration 5)</u><u style="Single"> Construct 1 described, comprising five exogenous nucleic acids each encoding a 6-aminocaprone pathway enzyme</u><u style="Single">non-naturally occurring microbial organisms.</u><u style="Single">(Configuration 6)</u><u style="Single"> The five exogenous nucleic acids are 3-oxo-6-aminohexanoyl-CoA thiolase;</u><u style="Single">6-aminohexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoA dehyde</u><u style="Single">latase; 6-aminohex-2-enoyl-CoA reductase; and 6-aminocaproyl-CoA/a</u><u style="Single">syl-CoA transferase, 6-aminocaproyl-CoA synthase, or 6-aminocaproyl</u><u style="Single">5. The non-naturally occurring microbial organism of configuration 5, which encodes an yl-CoA hydrolase.</u><u style="Single">(Configuration 7)</u><u style="Single"> The non-naturally occurring nucleic acid according to configuration 1, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.</u><u style="Single">microbial organisms.</u><u style="Single">(Configuration 8)</u><u style="Single"> The non-naturally occurring microbial organism of configuration 1 in a substantially anaerobic medium.</u><u style="Single">(Configuration 9)</u><u style="Single"> A method for producing 6-aminocaproic acid, the method comprising:</u><u style="Single">culturing the non-naturally occurring microbial organism according to configuration 1 under conditions for a sufficient period of time;</u><u style="Single">The method described above, comprising:</u><u style="Single">(Configuration 10)</u><u style="Single"> 9, wherein said non-naturally occurring microbial organism is in a substantially anaerobic medium.</u><u style="Single">Method.</u><u style="Single">(Configuration 11)</u><u style="Single"> The microbial organism has two exogenous enzymes, each encoding a 6-aminocaproic acid pathway enzyme.</u><u style="Single">The method according to constitution 9, comprising the nucleic acid of.</u><u style="Single">(Configuration 12)</u><u style="Single"> The microbial organism contains three exogenous enzymes, each encoding a 6-aminocaproic acid pathway enzyme.</u><u style="Single">The method according to constitution 9, comprising the nucleic acid of.</u><u style="Single">(Configuration 13)</u><u style="Single"> The microbial organism has four exogenous enzymes, each encoding a 6-aminocaproic acid pathway enzyme.</u><u style="Single">The method according to constitution 9, comprising the nucleic acid of.</u><u style="Single">(Configuration 14)</u><u style="Single"> The microbial organism has four exogenous enzymes, each encoding a 6-aminocaproic acid pathway enzyme.</u><u style="Single">The method according to constitution 9, comprising the nucleic acid of.</u><u style="Single">(Configuration 15)</u><u style="Single"> The four exogenous nucleic acids are 3-oxo-6-aminohexanoyl-CoA thiolase;</u><u style="Single">6-aminohexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoA dehyde</u><u style="Single">latase; 6-aminohex-2-enoyl-CoA reductase; and 6-aminocaproyl-CoA/a</u><u style="Single">syl-CoA transferase, 6-aminocaproyl-CoA synthase, or 6-aminocaproyl</u><u style="Single">15. The method of construction 14, wherein the method encodes an yl-CoA hydrolase.</u><u style="Single">(Configuration 16)</u><u style="Single"> 10. The method of configuration 9, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.</u><u style="Single">(Configuration 17)</u><u style="Single"> 6-aminocaproic acid pathway enzymes expressed in sufficient amounts to produce 6-aminocaproic acid</u><u style="Single">a microorganism having a 6-aminocaproic acid pathway comprising at least one exogenous nucleic acid encoding</u><u style="Single">A non-naturally occurring microbial organism, including a biological organism, wherein the 6-aminocaproic acid pathway is</u><u style="Single">,3-oxo-6-aminohexanoyl-CoA thiolase;3-oxo-6-aminohexanoyl-CoA/</u><u style="Single">Acyl-CoA transferase, 3-oxo-6-aminohexanoyl-CoA synthase, or 3</u><u style="Single">-oxo-6-aminohexanoyl-CoA hydrolase; 3-oxo-6-aminohexanoate reda</u><u style="Single">3-hydroxy-6-aminohexanoate dehydratase; and 6-aminohexa-2-</u><u style="Single">Said non-naturally occurring microbial organism comprising enoate reductase.</u><u style="Single">(Configuration 18)</u><u style="Single"> Construct 17 comprising two exogenous nucleic acids each encoding a 6-aminocaproic acid pathway enzyme</u><u style="Single">Non-naturally occurring microbial organisms as described.</u><u style="Single">(Configuration 19)</u><u style="Single"> Construct 17, comprising three exogenous nucleic acids each encoding a 6-aminocaproic acid pathway enzyme.</u><u style="Single">Non-naturally occurring microbial organisms as described.</u><u style="Single">(Configuration 20)</u><u style="Single"> Construct 17 containing four exogenous nucleic acids, each encoding a 6-aminocaproic acid pathway enzyme.</u><u style="Single">Non-naturally occurring microbial organisms as described.</u><u style="Single">(Configuration 21)</u><u style="Single"> Construct 17 containing five exogenous nucleic acids, each encoding a 6-aminocaproic acid pathway enzyme.</u><u style="Single">Non-naturally occurring microbial organisms as described.</u><u style="Single">(Configuration 22)</u><u style="Single"> The five exogenous nucleic acids are 3-oxo-6-aminohexanoyl-CoA thiolase;</u><u style="Single">6-aminohexanoyl-CoA/acyl-CoA transferase, 3-oxo-6-aminohexano</u><u style="Single">yl-CoA synthase, or 3-oxo-6-aminohexanoyl-CoA hydrolase; 3-oxo-6</u><u style="Single">-aminohexanoate reductase; 3-hydroxy-6-aminohexanoate dehydrata</u><u style="Single">and 6-aminohex-2-enoate reductase;</u><u style="Single">Non-existent microbial organisms.</u><u style="Single">(Configuration 23)</u><u style="Single"> The naturally occurring nucleic acid according to structure 17, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.</u><u style="Single">No microbial organisms.</u><u style="Single">(Configuration 24)</u><u style="Single"> 18. The non-naturally occurring microbial organism of composition 17 in a substantially anaerobic medium.</u><u style="Single">(Configuration 25)</u><u style="Single"> A method for producing 6-aminocaproic acid, the method comprising:</u><u style="Single">cultivating the non-naturally occurring microbial organism described in Structure 17 under conditions for a sufficient period of time;</u><u style="Single">The method described above, comprising:</u><u style="Single">(Configuration 26)</u><u style="Single"> 26, wherein said non-naturally occurring microbial organism is in a substantially anaerobic medium.</u><u style="Single">Method.</u><u style="Single">(Configuration 27)</u><u style="Single"> The microbial organism has two exogenous enzymes, each encoding a 6-aminocaproic acid pathway enzyme.</u><u style="Single">26. The method according to constitution 25, comprising the nucleic acid of.</u><u style="Single">(Configuration 28)</u><u style="Single"> The microbial organism contains three exogenous enzymes, each encoding a 6-aminocaproic acid pathway enzyme.</u><u style="Single">26. The method according to constitution 25, comprising the nucleic acid of.</u><u style="Single">(Configuration 29)</u><u style="Single"> The microbial organism has four exogenous enzymes, each encoding a 6-aminocaproic acid pathway enzyme.</u><u style="Single">26. The method according to constitution 25, comprising the nucleic acid of.</u><u style="Single">(Configuration 30)</u><u style="Single"> The microbial organism has five exogenous enzymes, each encoding a 6-aminocaproic acid pathway enzyme.</u><u style="Single">26. The method according to constitution 25, comprising the nucleic acid of.</u><u style="Single">(Configuration 31)</u><u style="Single"> The five exogenous nucleic acids are 3-oxo-6-aminohexanoyl-CoA thiolase;</u><u style="Single">6-aminohexanoyl-CoA/acyl-CoA transferase, 3-oxo-6-aminohexano</u><u style="Single">yl-CoA synthase, or 3-oxo-6-aminohexanoyl-CoA hydrolase; 3-oxo-6</u><u style="Single">-aminohexanoate reductase; 3-hydroxy-6-aminohexanoate dehydrata</u><u style="Single">and 6-aminohex-2-enoate reductase; and 6-aminohex-2-enoate reductase.</u><u style="Single">(Configuration 32)</u><u style="Single"> 26. The method of configuration 25, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.</u><u style="Single">(Configuration 33)</u><u style="Single"> Encodes a caprolactam pathway enzyme expressed in sufficient amounts to produce caprolactam</u><u style="Single">a microbial organism having a caprolactam pathway containing at least one exogenous nucleic acid that</u><u style="Single">a non-naturally occurring microbial organism, the caprolactam pathway is</u><u style="Single">yl-CoA/acyl-CoA transferase or 6-aminocaproyl-CoA synthase</u><u style="Single">, said non-naturally occurring microbial organism.</u><u style="Single">(Configuration 34)</u><u style="Single"> the exogenous nucleic acid encodes 6-aminocaproyl-CoA/acyl-CoA transferase;</u><u style="Single">The non-naturally occurring microbial organism according to composition 33, which comprises:</u><u style="Single">(Configuration 35)</u><u style="Single"> According to construction 33, the exogenous nucleic acid encodes 6-aminocaproyl-CoA synthase.</u><u style="Single">Microbial organisms that do not occur in nature.</u><u style="Single">(Configuration 36)</u><u style="Single"> The non-naturally occurring microbial organism of composition 33, further comprising a 6-aminocaproic acid pathway.</u><u style="Single">(Configuration 37)</u><u style="Single"> The 6-aminocaproic acid pathway is a CoA-dependent aldehyde dehydrogenase;</u><u style="Single">The naturally occurring substance according to composition 36, comprising saminase or 6-aminocaproate dehydrogenase.</u><u style="Single">Non-existent microbial organisms.</u><u style="Single">(Configuration 38)</u><u style="Single"> The 6-aminocaproic acid pathway is linked to 3-oxo-6-aminohexanoyl-CoA thiolase;</u><u style="Single">xo-6-aminohexanoyl-CoA/acyl-CoA transferase, 3-oxo-6-aminohexanoyl-CoA/acyl-CoA transferase</u><u style="Single">xanoyl-CoA synthase, or 3-oxo-6-aminohexanoyl-CoA hydrolase;</u><u style="Single">Xo-6-aminohexanoate reductase; 3-hydroxy-6-aminohexanoate dehyde</u><u style="Single">dolatase; and 6-aminohex-2-enoate reductase.</u><u style="Single">Non-existent microbial organisms.</u><u style="Single">(Configuration 39)</u><u style="Single"> The naturally occurring nucleic acid according to construction 33, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.</u><u style="Single">No microbial organisms.</u><u style="Single">(Configuration 40)</u><u style="Single"> The non-naturally occurring microbial organism of composition 33 in a substantially anaerobic medium.</u><u style="Single">(Configuration 41)</u><u style="Single"> A method for producing caprolactam, the conditions for producing caprolactam</u><u style="Single">culturing the non-naturally occurring microbial organism described in Constitution 33 for a sufficient period of time under</u><u style="Single">The method, comprising:</u><u style="Single">(Configuration 42)</u><u style="Single"> Caprolactam is produced by spontaneous cyclization of 6-aminocaproyl-CoA to caprolactam.</u><u style="Single">42. The method according to composition 41, wherein the method is produced by:</u><u style="Single">(Configuration 43)</u><u style="Single"> 42, wherein the non-naturally occurring microbial organism is in a substantially anaerobic medium.</u><u style="Single">Method.</u><u style="Single">(Configuration 44)</u><u style="Single"> the exogenous nucleic acid encodes 6-aminocaproyl-CoA/acyl-CoA transferase;</u><u style="Single">The method described in Configuration 41.</u><u style="Single">(Configuration 45)</u><u style="Single"> 42, wherein the exogenous nucleic acid encodes 6-aminocaproyl-CoA synthase.</u><u style="Single">Method.</u><u style="Single">(Configuration 46)</u><u style="Single"> 42. The method of configuration 41, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.</u><u style="Single">(Configuration 47)</u><u style="Single"> hexamethylene diamine expressed in sufficient amounts to produce hexamethylene diamine</u><u style="Single">Hexamethylenediamine pathway comprising at least one exogenous nucleic acid encoding a pathway enzyme</u><u style="Single">a non-naturally occurring microbial organism comprising a microbial organism having a hexamethylene</u><u style="Single">6-aminocaproyl-CoA/acyl-CoA transferase or 6-aminodiamine pathway</u><u style="Single">Nocaproyl-CoA synthase; 6-aminocaproyl-CoA reductase (aldehyde formation); and</u><u style="Single">and hexamethylenediamine transaminase or hexamethylenediamine dehydrogenase</u><u style="Single">said non-naturally occurring microbial organism.</u><u style="Single">(Configuration 48)</u><u style="Single"> 48. The non-naturally occurring microbial organism of composition 47, further comprising a 6-aminocaproic acid pathway.</u><u style="Single">(Configuration 49)</u><u style="Single"> The 6-aminocaproic acid pathway is a CoA-dependent aldehyde dehydrogenase;</u><u style="Single">Naturally occurring enzymes according to composition 48, comprising suaminase or 6-aminocaproate dehydrogenase.</u><u style="Single">Non-existent microbial organisms.</u><u style="Single">(Configuration 50)</u><u style="Single"> The 6-aminocaproic acid pathway is linked to 3-oxo-6-aminohexanoyl-CoA thiolase;</u><u style="Single">xo-6-aminohexanoyl-CoA/acyl-CoA transferase, 3-oxo-6-aminohexanoyl-CoA/acyl-CoA transferase</u><u style="Single">xanoyl-CoA synthase, or 3-oxo-6-aminohexanoyl-CoA hydrolase;</u><u style="Single">Xo-6-aminohexanoate reductase; 3-hydroxy-6-aminohexanoate dehyde</u><u style="Single">and 6-aminohex-2-enoate reductase.</u><u style="Single">Non-existent microbial organisms.</u><u style="Single">(Configuration 51)</u><u style="Single"> A construct containing two exogenous nucleic acids, each encoding a hexamethylenediamine pathway enzyme.</u><u style="Single">The non-naturally occurring microbial organism described in 47.</u><u style="Single">(Configuration 52)</u><u style="Single"> A construct containing three exogenous nucleic acids, each encoding a hexamethylenediamine pathway enzyme.</u><u style="Single">The non-naturally occurring microbial organism described in 47.</u><u style="Single">(Configuration 53)</u><u style="Single"> The three exogenous nucleic acids are linked to 6-aminocaproyl-CoA/acyl-CoA transferase or</u><u style="Single">is 6-aminocaproyl-CoA synthase; 6-aminocaproyl-CoA reductase (aldehyde</u><u style="Single">formation); and hexamethylenediamine transaminase or hexamethylenediamine dehyde</u><u style="Single">53. The non-naturally occurring microbial organism of composition 52, which encodes a drogenase.</u><u style="Single">(Configuration 54)</u><u style="Single"> The naturally occurring nucleic acid according to construction 47, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.</u><u style="Single">No microbial organisms.</u><u style="Single">(Configuration 55)</u><u style="Single"> 48. The non-naturally occurring microbial organism of composition 47 in a substantially anaerobic medium.</u><u style="Single">(Configuration 56)</u><u style="Single"> A method for producing hexamethylene diamine, the method comprising: producing hexamethylene diamine;</u><u style="Single">a non-naturally occurring microbial organism according to constitution 47 under conditions and for a sufficient period of time to enable it to survive;</u><u style="Single">The method described above, comprising culturing.</u><u style="Single">(Configuration 57)</u><u style="Single"> 57, wherein the non-naturally occurring microbial organism is in a substantially anaerobic medium.</u><u style="Single">Method.</u><u style="Single">(Configuration 58)</u><u style="Single"> The microbial organism has two foreign molecules each encoding a hexamethylenediamine pathway enzyme.</u><u style="Single">57.</u><u style="Single">(Configuration 59)</u><u style="Single"> The microbial organism has three external enzymes, each encoding a hexamethylenediamine pathway enzyme.</u><u style="Single">57.</u><u style="Single">(Configuration 60)</u><u style="Single"> The three exogenous nucleic acids are linked to 6-aminocaproyl-CoA/acyl-CoA transferase or</u><u style="Single">is 6-aminocaproyl-CoA synthase; 6-aminocaproyl-CoA reductase (aldehyde</u><u style="Single">formation); and hexamethylenediamine transaminase or hexamethylenediamine dehyde</u><u style="Single">59. The method of composition 59, encoding a drogenase.</u><u style="Single">(Configuration 61)</u><u style="Single"> 57. The method of construction 56, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.</u><u style="Single">(Configuration 62)</u><u style="Single"> Encodes a caprolactam pathway enzyme expressed in sufficient amounts to produce caprolactam</u><u style="Single">a microbial organism having a caprolactam pathway containing at least one exogenous nucleic acid that</u><u style="Single">is a non-naturally occurring microbial organism, in which the caprolactam pathway is</u><u style="Single">Minohexanoyl-CoA thiolase;3-oxo-6-aminohexanoyl-CoA reductase;3-</u><u style="Single">Hydroxy-6-aminohexanoyl-CoA dehydratase; and 6-aminohex-2-enoyl-</u><u style="Single">Said non-naturally occurring microbial organism comprising CoA reductase.</u><u style="Single">(Configuration 63)</u><u style="Single"> Construct 62 comprising two exogenous nucleic acids each encoding a caprolactam pathway enzyme</u><u style="Single">non-naturally occurring microbial organisms.</u><u style="Single">(Configuration 64)</u><u style="Single"> Construct 62 comprising three exogenous nucleic acids each encoding a caprolactam pathway enzyme</u><u style="Single">non-naturally occurring microbial organisms.</u><u style="Single">(Configuration 65)</u><u style="Single"> Construct 62 comprising four exogenous nucleic acids each encoding a caprolactam pathway enzyme</u><u style="Single">non-naturally occurring microbial organisms.</u><u style="Single">(Configuration 66)</u><u style="Single"> The four exogenous nucleic acids are 3-oxo-6-aminohexanoyl-CoA thiolase;</u><u style="Single">6-aminohexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoA dehyde</u><u style="Single">latase; and 6-aminohex-2-enoyl-CoA reductase, according to construction 65.</u><u style="Single">Microbial organisms that do not occur in nature.</u><u style="Single">(Configuration 67)</u><u style="Single"> The naturally occurring nucleic acid according to construction 62, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.</u><u style="Single">No microbial organisms.</u><u style="Single">(Configuration 68)</u><u style="Single"> 63. The non-naturally occurring microbial organism of composition 62 in a substantially anaerobic medium.</u><u style="Single">(Configuration 69)</u><u style="Single"> A method for producing caprolactam, the conditions for producing caprolactam</u><u style="Single">culturing the non-naturally occurring microbial organism described in Constitution 62 for a sufficient period of time under</u><u style="Single">The method, comprising:</u><u style="Single">(Configuration 70)</u><u style="Single"> Caprolactam is produced by spontaneous cyclization of 6-aminocaproyl-CoA to caprolactam.</u><u style="Single">69. The method according to constitution 69, wherein the method is produced by:</u><u style="Single">(Configuration 71)</u><u style="Single"> 69, wherein said non-naturally occurring microbial organism is in a substantially anaerobic medium.</u><u style="Single">Method.</u><u style="Single">(Configuration 72)</u><u style="Single"> The microbial organism has two exogenous nuclei each encoding a caprolactam pathway enzyme.</u><u style="Single">69. The method of constitution 69, comprising an acid.</u><u style="Single">(Configuration 73)</u><u style="Single"> The microbial organism has three exogenous nuclei each encoding a caprolactam pathway enzyme.</u><u style="Single">69. The method of constitution 69, comprising an acid.</u><u style="Single">(Configuration 74)</u><u style="Single"> The microbial organism has four exogenous nuclei, each encoding a caprolactam pathway enzyme.</u><u style="Single">69. The method of constitution 69, comprising an acid.</u><u style="Single">(Configuration 75)</u><u style="Single"> The four exogenous nucleic acids are 3-oxo-6-aminohexanoyl-CoA thiolase;</u><u style="Single">6-aminohexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoA dehyde</u><u style="Single">latase; and 6-aminohex-2-enoyl-CoA reductase, according to construction 74.</u><u style="Single">Method.</u><u style="Single">(Configuration 76)</u><u style="Single"> 70. The method of construction 69, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.</u><u style="Single">(Configuration 77)</u><u style="Single"> hexamethylene diamine expressed in sufficient amounts to produce hexamethylene diamine</u><u style="Single">Hexamethylenediamine pathway comprising at least one exogenous nucleic acid encoding a pathway enzyme</u><u style="Single">a non-naturally occurring microbial organism comprising a microbial organism having a hexamethylene</u><u style="Single">3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-amino</u><u style="Single">Hexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoA dehydratase</u><u style="Single">;6-aminohex-2-enoyl-CoA reductase;6-aminohex-2-enoyl-CoA reductase</u><u style="Single">hexamethylene diamine transaminase or hexamethylene diamine transaminase</u><u style="Single">Said non-naturally occurring microbial organism comprising mine dehydrogenase.</u><u style="Single">(Configuration 78)</u><u style="Single"> A construct containing two exogenous nucleic acids, each encoding a hexamethylenediamine pathway enzyme.</u><u style="Single">The non-naturally occurring microbial organism described in 77.</u><u style="Single">(Configuration 79)</u><u style="Single"> A construct containing three exogenous nucleic acids, each encoding a hexamethylenediamine pathway enzyme.</u><u style="Single">The non-naturally occurring microbial organism described in 77.</u><u style="Single">(Configuration 80)</u><u style="Single"> A construct containing four exogenous nucleic acids, each encoding a hexamethylenediamine pathway enzyme.</u><u style="Single">The non-naturally occurring microbial organism described in 77.</u><u style="Single">(Configuration 81)</u><u style="Single"> A construct containing five exogenous nucleic acids, each encoding a hexamethylenediamine pathway enzyme.</u><u style="Single">The non-naturally occurring microbial organism described in 77.</u><u style="Single">(Configuration 82)</u><u style="Single"> A construct containing six exogenous nucleic acids, each encoding a hexamethylenediamine pathway enzyme.</u><u style="Single">The non-naturally occurring microbial organism described in 77.</u><u style="Single">(Configuration 83)</u><u style="Single"> The six exogenous nucleic acids are 3-oxo-6-aminohexanoyl-CoA thiolase;</u><u style="Single">6-aminohexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoA dehyde</u><u style="Single">Latase; 6-aminohex-2-enoyl-CoA reductase; 6-aminocaproyl-CoA reductase</u><u style="Single">transaminase (aldehyde formation); and hexamethylene diamine transaminase or hexame</u><u style="Single">The non-naturally occurring microbial organism of composition 82, which encodes tylene diamine dehydrogenase.</u><u style="Single">object.</u><u style="Single">(Configuration 84)</u><u style="Single"> The naturally occurring nucleic acid according to construction 77, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.</u><u style="Single">No microbial organisms.</u><u style="Single">(Configuration 85)</u><u style="Single"> 78. The non-naturally occurring microbial organism of composition 77 in a substantially anaerobic medium.</u><u style="Single">(Configuration 86)</u><u style="Single"> A method for producing hexamethylene diamine, the method comprising: producing hexamethylene diamine;</u><u style="Single">a non-naturally occurring microbial organism according to constitution 77 under conditions and for a sufficient period of time to allow it to survive;</u><u style="Single">The method described above, comprising culturing.</u><u style="Single">(Configuration 87)</u><u style="Single"> 87, wherein the non-naturally occurring microbial organism is in a substantially anaerobic medium.</u><u style="Single">Method.</u><u style="Single">(Configuration 88)</u><u style="Single"> The microbial organism has two foreign molecules each encoding a hexamethylenediamine pathway enzyme.</u><u style="Single">87. The method according to constitution 86, comprising a pathogenic nucleic acid.</u><u style="Single">(Configuration 89)</u><u style="Single"> The microbial organism has three external enzymes, each encoding a hexamethylenediamine pathway enzyme.</u><u style="Single">87. The method according to constitution 86, comprising a pathogenic nucleic acid.</u><u style="Single">(Configuration 90)</u><u style="Single"> The microbial organism has four external enzymes, each encoding a hexamethylenediamine pathway enzyme.</u><u style="Single">87. The method according to constitution 86, comprising a pathogenic nucleic acid.</u><u style="Single">(Configuration 91)</u><u style="Single"> The microbial organism has five external enzymes, each encoding a hexamethylenediamine pathway enzyme.</u><u style="Single">87. The method according to constitution 86, comprising a pathogenic nucleic acid.</u><u style="Single">(Configuration 92)</u><u style="Single"> The microbial organism contains six external enzymes, each encoding a hexamethylenediamine pathway enzyme.</u><u style="Single">87. The method according to constitution 86, comprising a pathogenic nucleic acid.</u><u style="Single">(Configuration 93)</u><u style="Single"> The four exogenous nucleic acids are 3-oxo-6-aminohexanoyl-CoA thiolase;</u><u style="Single">6-aminohexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoA dehyde</u><u style="Single">Latase; 6-aminohex-2-enoyl-CoA reductase; 6-aminocaproyl-CoA reductase</u><u style="Single">transaminase (aldehyde formation); and hexamethylene diamine transaminase or hexame</u><u style="Single">93. The method of constitution 92, wherein the method encodes tylene diamine dehydrogenase.</u><u style="Single">(Configuration 94)</u><u style="Single"> 87. The method of construction 86, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.</u><u style="Single">(Configuration 95)</u><u style="Single"> It encodes a 6-ACA pathway enzyme expressed in sufficient quantities to produce 6-aminocaproic acid (6-ACA).</u><u style="Single">a microbial organism with a 6-ACA pathway that contains at least one exogenous nucleic acid that</u><u style="Single">microbial organisms that are not present in the 6-ACA pathway, the 6-ACA pathway is</u><u style="Single">,7-dioate (HODH) aldolase, 2-oxohept-4-ene-1,7-dioate (OHED) hydride</u><u style="Single">latase, 2-oxohept-4-ene-1,7-dioate (OHED) reductase, 2-oxohepta</u><u style="Single">-1,7-dioate (2-OHD) decarboxylase, adipate semialdehyde aminotolane</u><u style="Single">Spherase, adipate semialdehyde oxidoreductase (amination), 2-oxohe</u><u style="Single">pt-4-ene-1,7-dioate (OHED) decarboxylase, 6-oxohex-4-enoate (</u><u style="Single">6-OHE) reductase, 2-oxoheptane-1,7-dioate (2-OHD) aminotransferer</u><u style="Single">2-oxoheptane-1,7-dioate (2-OHD) oxidoreductase (amination), 2-amino</u><u style="Single">Noheptane-1,7-dioate (2-AHD) decarboxylase, 2-oxohept-4-ene-1,7-di</u><u style="Single">Oate (OHED) aminotransferase, 2-oxohept-4-ene-1,7-dioate (OHE</u><u style="Single">D) Oxidoreductase (amination), 2-aminohept-4-ene-1,7-dioate (2-AHE)</u><u style="Single">ductase, 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) formate lyase, 4-hydroxy-2-oxoheptane-1,7-dioate (HODH)</u><u style="Single">Droxy-2-oxoheptane-1,7-dioate (HODH) dehydrogenase, 3-hydroxyazide</u><u style="Single">Pill-CoA dehydratase, 2,3-dehydroadipyl-CoA reductase, adipyl-CoA dehyde</u><u style="Single">Rogenase, 2-oxohept-4-ene-1,7-dioate (OHED) formate lyase, 2-oxohep</u><u style="Single">ter-4-ene-1,7-dioate (OHED) dehydrogenase, 2-oxoheptane-1,7-dioate (</u><u style="Single">2-OHD) formate lyase, 2-oxoheptane-1,7-dioate (2-OHD) dehydrogenase, or</u><u style="Single">Said non-naturally occurring microbial organism comprising a pyruvate formate lyase activating enzyme.</u><u style="Single">(Configuration 96)</u><u style="Single"> at least one exogenous nucleic acid each encoding a 6-ACA pathway enzyme;</u><u style="Single">HODH aldolase; OHED hydratase; OHED reductase; 2-OHD decarboxylase</u><u style="Single">; or adipate semialdehyde aminotransferase or adipate semial</u><u style="Single">a non-naturally occurring microbial organism according to composition 95, comprising dehyde oxidoreductase (amination);</u><u style="Single">object.</u><u style="Single">(Configuration 97)</u><u style="Single"> at least one exogenous nucleic acid each encoding a 6-ACA pathway enzyme;</u><u style="Single">HODH aldolase; OHED hydratase; OHED decarboxylase; 6-OHE reductase</u><u style="Single">; or adipate semialdehyde aminotransferase or adipate semial</u><u style="Single">a non-naturally occurring microbial organism according to composition 95, comprising dehyde oxidoreductase (amination);</u><u style="Single">object.</u><u style="Single">(Configuration 98)</u><u style="Single"> at least one exogenous nucleic acid each encoding a 6-ACA pathway enzyme;</u><u style="Single">HODH aldolase; OHED hydratase; OHED aminotransferase or OHED</u><u style="Single">Contains oxidoreductase (amination); 2-AHE reductase; or 2-AHD decarboxylase.</u><u style="Single">The non-naturally occurring microbial organism according to composition 95.</u><u style="Single">(Configuration 99)</u><u style="Single"> at least one exogenous nucleic acid each encoding a 6-ACA pathway enzyme;</u><u style="Single">HODH aldolase; OHED hydratase; OHED reductase; 2-OHD aminotransferase</u><u style="Single">or 2-OHD oxidoreductase (amination); or 2-AHD decarboxylase</u><u style="Single">95. The non-naturally occurring microbial organism of composition 95, comprising:</u><u style="Single">(Configuration 100)</u><u style="Single"> at least one exogenous nucleic acid each encoding a 6-ACA pathway enzyme;</u><u style="Single">HODH aldolase; HODH formate lyase and pyruvate formate lyase activating enzyme or</u><u style="Single">HODH dehydrogenase; 3-hydroxyadipyl-CoA dehydratase; 2,3-dehydroazide</u><u style="Single">Pyr-CoA reductase; adipyl-CoA dehydrogenase; or adipyl semialdehyde</u><u style="Single">Minotransferase or adipate semialdehyde oxidoreductase (aminotransferase)</u><u style="Single">95. The non-naturally occurring microbial organism of composition 95, comprising:</u><u style="Single">(Configuration 101)</u><u style="Single"> at least one exogenous nucleic acid each encoding a 6-ACA pathway enzyme;</u><u style="Single">HODH aldolase; OHED hydratase; OHED formate lyase and pyruvate formate lyase</u><u style="Single">enzyme-activating enzyme or OHED dehydrogenase; 2,3-dehydroadipyl-CoA reductase;</u><u style="Single">dipyr-CoA dehydrogenase; or adipate semialdehyde aminotransferase</u><u style="Single">or comprising adipate semialdehyde oxidoreductase (amination), described in structure 95</u><u style="Single">non-naturally occurring microbial organisms.</u><u style="Single">(Configuration 102)</u><u style="Single"> at least one exogenous nucleic acid each encoding a 6-ACA pathway enzyme;</u><u style="Single">HODH aldolase; OHED hydratase; OHED reductase; 2-OHD formate lyase and pi</u><u style="Single">Rubate formate lyase activating enzyme or 2-OHD dehydrogenase; adipyl-CoA dehydro</u><u style="Single">genase; or adipate semialdehyde aminotransferase or adipate</u><u style="Single">Containing semialdehyde oxidoreductase (amination), non-naturally occurring according to composition 95</u><u style="Single">Microbial organisms.</u><u style="Single">(Configuration 103)</u><u style="Single"> a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, wherein the set of exogenous nucleic acids</u><u style="Single">, HODH aldolase; OHED hydratase; OHED reductase; 2-OHD decarboxylase;</u><u style="Single">and adipate semialdehyde aminotransferase or adipate semialdehyde aminotransferase</u><u style="Single">Non-naturally occurring microbial organism according to composition 95 encoding an oxidoreductase (amination)</u><u style="Single">body.</u><u style="Single">(Configuration 104)</u><u style="Single"> a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, wherein the set of exogenous nucleic acids</u><u style="Single">, HODH aldolase; OHED hydratase; OHED decarboxylase; 6-OHE reductase;</u><u style="Single">and adipate semialdehyde aminotransferase or adipate semialdehyde aminotransferase</u><u style="Single">Non-naturally occurring microbial organism according to composition 95 encoding an oxidoreductase (amination)</u><u style="Single">body.</u><u style="Single">(Configuration 105)</u><u style="Single"> a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, wherein the set of exogenous nucleic acids</u><u style="Single">, HODH aldolase; OHED hydratase; OHED aminotransferase or OHED oxide</u><u style="Single">Encodes reductase (amination); 2-AHE reductase; and 2-AHD decarboxylase</u><u style="Single">, a non-naturally occurring microbial organism according to composition 95.</u><u style="Single">(Configuration 106)</u><u style="Single"> a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, wherein the set of exogenous nucleic acids</u><u style="Single">, HODH aldolase; OHED hydratase; OHED reductase; 2-OHD aminotransferer</u><u style="Single">or 2-OHD reductase (amination); and 2-AHD decarboxylase, construct 9</u><u style="Single">5. The non-naturally occurring microbial organism described in 5.</u><u style="Single">(Configuration 107)</u><u style="Single"> a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, wherein the set of exogenous nucleic acids</u><u style="Single">, HODH aldolase; HODH formate lyase and pyruvate formate lyase activating enzyme or HODH</u><u style="Single">Dehydrogenase; 3-hydroxyadipyl-CoA dehydratase; 2,3-dehydroadipyl-CoA</u><u style="Single">reductase; adipyl-CoA dehydrogenase; and adipate semialdehyde aminotra</u><u style="Single">encodes sferase or adipate semialdehyde oxidoreductase (amination)</u><u style="Single">95. The non-naturally occurring microbial organism according to composition 95.</u><u style="Single">(Configuration 108)</u><u style="Single"> 6 - a set of exogenous nucleic acids encoding ACA pathway enzymes, wherein the set of exogenous nucleic acids</u><u style="Single">, HODH aldolase; OHED hydratase; OHED formate lyase and pyruvate formate lyase activities</u><u style="Single">sexase or OHED dehydrogenase; 2,3-dehydroadipyl-CoA reductase; adipyl-C</u><u style="Single">oA dehydrogenase; and adipate semialdehyde aminotransferase or adipate semialdehyde aminotransferase</u><u style="Single">The natural compound according to composition 95, encoding pate semialdehyde oxidoreductase (amination)</u><u style="Single">Microbial organisms not present in</u><u style="Single">(Configuration 109)</u><u style="Single"> a set of exogenous nucleic acids encoding 6-ACA pathway enzymes, wherein the set of exogenous nucleic acids</u><u style="Single">, HODH aldolase; OHED hydratase; OHED reductase; 2-OHD formate lyase and pyrubi</u><u style="Single">formate lyase activating enzyme or 2-OHD dehydrogenase; adipyl-CoA dehydrogenase</u><u style="Single">; and adipate semialdehyde aminotransferase or adipate semialdehyde</u><u style="Single">Non-naturally occurring microorganism according to composition 95, encoding dooxidoreductase (amination)</u><u style="Single">biological body.</u><u style="Single">(Configuration 110)</u><u style="Single"> The naturally occurring nucleic acid of construction 95, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.</u><u style="Single">No microbial organisms.</u><u style="Single">(Configuration 111)</u><u style="Single"> 96. The non-naturally occurring microbial organism of composition 95 in a substantially anaerobic medium.</u><u style="Single">(Configuration 112)</u><u style="Single"> A method for producing 6-aminocaproic acid (6-ACA), comprising conditions for producing 6-ACA.</u><u style="Single">cultivating a non-naturally occurring microbial organism according to configuration 1 under conditions and for a sufficient period of time;</u><u style="Single">The method described above.</u><u style="Single">(Configuration 113)</u><u style="Single"> 113. The method of construction 112, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 114)</u><u style="Single"> 113. The method of construction 112, wherein the conditions further include an osmoprotectant.</u><u style="Single">(Configuration 115)</u><u style="Single"> 115. The method of construction 114, wherein the osmoprotectant is glycine betaine.</u><u style="Single">(Configuration 116)</u><u style="Single"> HMDA pathway enzymes expressed in sufficient amounts to produce hexamethylene diamine (HMDA)</u><u style="Single">a microbial organism having an HMDA pathway that contains at least one exogenous nucleic acid that encodes</u><u style="Single">6-aminocaproate kinase, 6-aminocaproate kinase,</u><u style="Single">[(6-aminohexanoyl)oxy]phosphonate (6-AHOP) oxidoreductase, 6-amino</u><u style="Single">Caproic acid semialdehyde aminotransferase, 6-aminocaproic acid semialdehyde</u><u style="Single">Doxidoreductase (amination), 6-aminocaproate N-acetyltransferase</u><u style="Single">, 6-acetamidohexanoate kinase, [(6-acetamidohexanoyl)oxy]phos</u><u style="Single">Honate (6-AAHOP) oxidoreductase, 6-acetamidohexanal aminotrans</u><u style="Single">Ferrase, 6-acetamidohexanal oxidoreductase (amination), 6-aceto</u><u style="Single">Midohexanamine N-acetyltransferase, 6-acetamidohexanamine hydride</u><u style="Single">Lorase (amide), 6-acetamidohexanoate CoA transferase, 6-aceto</u><u style="Single">Midohexanoate CoA ligase, 6-acetamidohexanoyl-CoA oxidoreductor</u><u style="Single">[(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) acyl transfer</u><u style="Single">[(6-aminohexanoyl)oxy]phosphonate (6-AHOP) acyltransferase</u><u style="Single">6-aminocaproate CoA transferase, and 6-aminocaproate CoA ligator</u><u style="Single">said non-naturally occurring microbial organism.</u><u style="Single">(Configuration 117)</u><u style="Single"> at least one exogenous nucleic acid each encoding an HMDA pathway enzyme;</u><u style="Single">but 6-aminocaproate kinase; 6-AHOP oxidoreductase; or 6-aminocaproate</u><u style="Single">Acid semialdehydoxide reductase (amination) or 6-aminocaproic acid semialde</u><u style="Single">117. The non-naturally occurring microbial organism of composition 116, comprising a hydraminotransferase.</u><u style="Single">(Configuration 118)</u><u style="Single"> at least one exogenous nucleic acid each encoding an HMDA pathway enzyme;</u><u style="Single">6-aminocaproate kinase; 6-AHOP acyltransferase; 6-aminocaproate kinase;</u><u style="Single">-CoA oxidoreductase; or 6-aminocaproic acid semialdehydoxide reductase</u><u style="Single">(amination) or 6-aminocaproic acid semialdehyde aminotransferase.</u><u style="Single">The non-naturally occurring microbial organism according to composition 116.</u><u style="Single">(Configuration 119)</u><u style="Single"> at least one exogenous nucleic acid each encoding an HMDA pathway enzyme;</u><u style="Single">However, 6-aminocaproate CoA transferase or 6-aminocaproate CoA ligase</u><u style="Single">6-aminocaproyl-CoA oxidoreductase; or 6-aminocaproic acid semialde</u><u style="Single">Hydroxide reductase (amination) or 6-aminocaproic acid semialdehyde aminot</u><u style="Single">117. The non-naturally occurring microbial organism of composition 116, comprising a transferase.</u><u style="Single">(Configuration 120)</u><u style="Single"> at least one exogenous nucleic acid each encoding an HMDA pathway enzyme;</u><u style="Single">6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate</u><u style="Single">tokinase; 6-AAHOP oxidoreductase; 6-acetamidohexanal aminotrans</u><u style="Single">ferase or 6-acetamidohexanal oxidoreductase (amination); or 6</u><u style="Single">-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine</u><u style="Single">Non-naturally occurring microbial organism according to composition 116, comprising a xanamine hydrolase (amide)</u><u style="Single">body.</u><u style="Single">(Configuration 121)</u><u style="Single"> at least one exogenous nucleic acid each encoding an HMDA pathway enzyme;</u><u style="Single">6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate</u><u style="Single">CoA transferase or 6-acetamidohexanoate CoA ligase; 6-aceto</u><u style="Single">Amidohexanoyl-CoA oxidoreductase; 6-acetamidohexanal aminotra</u><u style="Single">spherase or 6-acetamidohexanal oxidoreductase (amination);</u><u style="Single">or 6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine</u><u style="Single">Non-naturally occurring microorganism according to composition 116, comprising dohexanamine hydrolase (amide)</u><u style="Single">biological body.</u><u style="Single">(Configuration 122)</u><u style="Single"> at least one exogenous nucleic acid each encoding an HMDA pathway enzyme;</u><u style="Single">6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate</u><u style="Single">tokinase; 6-AAHOP oxidoreductase; 6-acetamidohexanal aminotrans</u><u style="Single">ferase or 6-acetamidohexanal oxidoreductase (amination); or 6</u><u style="Single">-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine</u><u style="Single">Non-naturally occurring microbial organism according to composition 116, comprising a xanamine hydrolase (amide)</u><u style="Single">body.</u><u style="Single">(Configuration 123)</u><u style="Single"> a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set of exogenous nucleic acids comprising:</u><u style="Single">6-aminocaproate kinase; 6-AHOP oxidoreductase;</u><u style="Single">Mialdehydoxide reductase (amination) or 6-aminocaproic acid semialdehyde amine</u><u style="Single">117. The non-naturally occurring microbial organism of construction 116, which encodes a notransferase.</u><u style="Single">(Configuration 124)</u><u style="Single"> a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set of exogenous nucleic acids comprising:</u><u style="Single">6-aminocaproate kinase; 6-AHOP acyltransferase; 6-aminocaproyl-C</u><u style="Single">oA oxidoreductase; and 6-aminocaproic acid semialdehydoxide reductase (A</u><u style="Single">6-aminocaproic acid semialdehyde aminotransferase</u><u style="Single">, composition 116.</u><u style="Single">(Configuration 125)</u><u style="Single"> a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set of exogenous nucleic acids comprising:</u><u style="Single">6-aminocaproate CoA transferase or 6-aminocaproate CoA ligase; 6-</u><u style="Single">Aminocaproyl-CoA oxidoreductase; and 6-aminocaproic acid semialdehyde</u><u style="Single">Side reductase (amination) or 6-aminocaproic acid semialdehyde aminotransfer</u><u style="Single">116. The non-naturally occurring microbial organism of construction 116, which encodes a microbial enzyme.</u><u style="Single">(Configuration 126)</u><u style="Single"> a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set of exogenous nucleic acids comprising:</u><u style="Single">6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate</u><u style="Single">6-AAHOP oxidoreductase; 6-acetamidohexanal aminotransferase</u><u style="Single">or 6-acetamidohexanal oxidoreductase (amination); and 6-acetamidohexanal oxidoreductase;</u><u style="Single">Amidohexanamine N-acetyltransferase or 6-acetamidohexanamine</u><u style="Single">117. The non-naturally occurring microbial organism of composition 116, which encodes a hydrolase (amide).</u><u style="Single">(Configuration 127)</u><u style="Single"> a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set of exogenous nucleic acids comprising:</u><u style="Single">6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate CoA</u><u style="Single">Transferase or 6-acetamidohexanoate CoA ligase; 6-acetamidohexanoate</u><u style="Single">Sanoyl-CoA oxidoreductase; 6-acetamidohexanal aminotransfera</u><u style="Single">-ase or 6-acetamidohexanal oxidoreductase (amination); and 6-acetoacetate</u><u style="Single">Midohexanamine N-acetyltransferase or 6-acetamidohexanamine</u><u style="Single">117. The non-naturally occurring microbial organism of composition 116, which encodes dololase (amide).</u><u style="Single">(Configuration 128)</u><u style="Single"> a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set of exogenous nucleic acids comprising:</u><u style="Single">6-aminocaproate N-acetyltransferase; 6-acetamidohexanoate</u><u style="Single">6-AAHOP oxidoreductase; 6-acetamidohexanal aminotransferase</u><u style="Single">or 6-acetamidohexanal oxidoreductase (amination); and 6-acetamidohexanal oxidoreductase;</u><u style="Single">Amidohexanamine N-acetyltransferase or 6-acetamidohexanamine</u><u style="Single">117. The non-naturally occurring microbial organism of composition 116, which encodes a hydrolase (amide).</u><u style="Single">(Configuration 129)</u><u style="Single"> The naturally occurring nucleic acid of construction 116, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.</u><u style="Single">No microbial organisms.</u><u style="Single">(Configuration 130)</u><u style="Single"> 117. The non-naturally occurring microbial organism of composition 116 in a substantially anaerobic medium.</u><u style="Single">(Configuration 131)</u><u style="Single"> A method for producing hexamethylene diamine (HMDA), the method comprising:</u><u style="Single">cultivating a non-naturally occurring microbial organism according to composition 116 under conditions and for a period sufficient thereto;</u><u style="Single">The method described above.</u><u style="Single">(Configuration 132)</u><u style="Single"> 132. The method of construction 131, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 133)</u><u style="Single"> 132. The method of construction 131, wherein the conditions further include an osmoprotectant.</u><u style="Single">(Configuration 134)</u><u style="Single"> 134. The method of structure 133, wherein the osmoprotectant is glycine betaine.</u><u style="Single">(Configuration 135)</u><u style="Single"> HMDA pathway enzymes expressed in sufficient amounts to produce hexamethylene diamine (HMDA)</u><u style="Single">a microbial organism having an HMDA pathway that contains at least one exogenous nucleic acid that encodes</u><u style="Single">In microbial organisms that do not exist naturally, the HMDA pathway is linked to the glutamyl-CoA transferer.</u><u style="Single">glutamyl-CoA ligase, beta-ketothiolase, 3-oxo-6-aminopimeloyl-C</u><u style="Single">oA oxidoreductase, 3-hydroxy-6-aminopimeloyl-CoA dehydratase, 6-a</u><u style="Single">Mino-7-carboxyhept-2-enoyl-CoA reductase, 6-aminopimeloyl-CoA reductase</u><u style="Single">tase (aldehyde formation), 2-amino-7-oxoheptanoate aminotransferase</u><u style="Single">, 2-amino-7-oxoheptanoate aminating oxidoreductase, or homolysine oxidoreductase</u><u style="Single">Said non-naturally occurring microbial organism comprising a carboxylase.</u><u style="Single">(Configuration 136)</u><u style="Single"> 135, wherein said HMDA pathway comprises at least two exogenous nucleic acids.</u><u style="Single">microbial organisms.</u><u style="Single">(Configuration 137)</u><u style="Single"> 135, wherein said HMDA pathway comprises at least three exogenous nucleic acids.</u><u style="Single">microbial organisms.</u><u style="Single">(Configuration 138)</u><u style="Single"> 135, wherein said HMDA pathway comprises at least four exogenous nucleic acids.</u><u style="Single">microbial organisms.</u><u style="Single">(Configuration 139)</u><u style="Single"> The non-naturally occurring microbial organism according to composition 135, wherein the exogenous nucleic acid is a heterologous nucleic acid.</u><u style="Single">body.</u><u style="Single">(Configuration 140)</u><u style="Single"> 136. The non-naturally occurring microbial organism of composition 135 in a substantially anaerobic medium.</u><u style="Single">(Configuration 141)</u><u style="Single"> A method for producing hexamethylene diamine (HMDA), the method comprising:</u><u style="Single">cultivating a non-naturally occurring microbial organism according to composition 135 under conditions and for a period sufficient thereto;</u><u style="Single">The method described above.</u><u style="Single">(Configuration 142)</u><u style="Single"> 142. The method of construction 141, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 143)</u><u style="Single"> HMDA pathway enzymes expressed in sufficient amounts to produce hexamethylene diamine (HMDA)</u><u style="Single">naturally occurring microbial organisms that have a HMDA pathway that contains a set of exogenous nucleic acids that encode</u><u style="Single">a microbial organism that does not undergo glutamyl-CoA transfection, said set of exogenous nucleic acids</u><u style="Single">glutamyl-CoA ligase; beta-ketothiolase; 3-oxo-6-aminopimeroy</u><u style="Single">-CoA oxidoreductase;3-hydroxy-6-aminopimeloyl-CoA dehydratase;6-</u><u style="Single">Amino-7-carboxyhept-2-enoyl-CoA reductase; 6-aminopimeloyl-CoA reductase</u><u style="Single">ctase (aldehyde formation); 2-amino-7-oxoheptanoate aminotransferase</u><u style="Single">or aminating oxidoreductase; and encoding a homolysine decarboxylase,</u><u style="Single">Said non-naturally occurring microbial organism.</u><u style="Single">(Configuration 144)</u><u style="Single"> A method for producing hexamethylene diamine (HMDA), the method comprising:</u><u style="Single">cultivating a non-naturally occurring microbial organism according to composition 143 under conditions and for a period sufficient thereto;</u><u style="Single">The method described above.</u><u style="Single">(Configuration 145)</u><u style="Single"> 144. The method of construction 143, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 146)</u><u style="Single"> HMDA pathway enzymes expressed in sufficient amounts to produce hexamethylene diamine (HMDA)</u><u style="Single">a microbial organism having an HMDA pathway that contains at least one exogenous nucleic acid that encodes</u><u style="Single">In a microbial organism that does not exist naturally, the HMDA pathway</u><u style="Single">3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferase</u><u style="Single">3-oxopimeloyl-CoA ligase, 3-oxopimelate reductase, 3-oxo-1</u><u style="Single">-Carboxyheptanal aminotransferase, 3-oxo-1-carboxyheptaner</u><u style="Single">oxidoreductase, 3-oxo-7-aminoheptanoate 3-aminotrans</u><u style="Single">ferase, 3-oxo-7-aminoheptanoate 3-aminated oxidoreductase, 3-o</u><u style="Single">xopimelate kinase, 5-oxopimeloylphosphonate reductase, 3-oxopimelate</u><u style="Single">rate CoA transferase, 3-oxopimelate ligase, 5-oxopimeloyl-CoA</u><u style="Single">reductase (aldehyde formation), 3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase,</u><u style="Single">Sopimelate aminating oxidoreductase, 3-aminopimelate CoA transferer</u><u style="Single">enzyme, 3-aminopimelate ligase, 5-aminopimeloyl-CoA reductase (aldehyde form</u><u style="Single">synthesis), 3-aminopimelate kinase, 5-aminopimeloylphosphonate reductase, 3-</u><u style="Single">Aminopimelate reductase, 3-amino-7-oxoheptanoate 2,3-aminomutase</u><u style="Single">, 2-amino-7-oxoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoate</u><u style="Single">Ptanoate amination oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomer</u><u style="Single">homolysine decarboxylase, 3-aminopimelate 2,3-aminomutase, 2-aminopimelate 2,3-aminomutase,</u><u style="Single">minopimelate kinase, 2-aminopimelate CoA transferase, 2-aminopimelate</u><u style="Single">CoA ligase, 2-aminopimelate reductase, 6-aminopimeloylphosphonate</u><u style="Single">reductase, 6-aminopimeloyl-CoA reductase (aldehyde formation), 3-amino-7-o</u><u style="Single">xoheptanoate 7-aminotransferase, or 3-amino-7-oxoheptanoate</u><u style="Single">said non-naturally occurring microbial organism comprising a toaminating oxidoreductase.</u><u style="Single">(Configuration 147)</u><u style="Single"> The HMDA pathway is linked to glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA</u><u style="Single">Dolorase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA riga</u><u style="Single">3-oxopimelate reductase, 3-oxo-1-carboxyheptanal 7-amino</u><u style="Single">Transferase, 3-oxo-1-carboxyheptanal 7-aminated oxidoreductor</u><u style="Single">3-oxo-7-aminoheptanoate 3-aminotransferase, 3-oxo-7-aminotransferase, 3-oxo-7-aminoheptanoate</u><u style="Single">noheptanoate 3-aminated oxidoreductase, 3,7-diaminoheptanoate 2,3-a</u><u style="Single">The naturally occurring compound according to composition 146, comprising minomutase, or homolysine decarboxylase.</u><u style="Single">No microbial organisms.</u><u style="Single">(Configuration 148)</u><u style="Single"> The HMDA pathway is linked to glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA</u><u style="Single">Dolorase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA riga</u><u style="Single">enzyme, 3-oxopimelate kinase, 5-oxopimeloylphosphonate reductase, 3-</u><u style="Single">Oxo-1-carboxyheptanal 7-aminotransferase, 3-oxo-1-carboxy</u><u style="Single">Heptanal 7-aminating oxidoreductase, 3-oxo-7-aminoheptanoate 3-a</u><u style="Single">Minotransferase, 3-oxo-7-aminoheptanoate 3-aminated oxidoreduct</u><u style="Single">3,7-diaminoheptanoate 2,3-aminomutase, or homolysine decarboxylate</u><u style="Single">147.</u><u style="Single">(Configuration 149)</u><u style="Single"> The HMDA pathway is linked to glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA</u><u style="Single">Dolorase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA riga</u><u style="Single">3-oxopimelate-CoA transferase, 3-oxopimelate-CoA ligase, 5-</u><u style="Single">Oxopimeloyl-CoA reductase (aldehyde formation), 3-oxo-1-carboxyheptana</u><u style="Single">7-aminotransferase, 3-oxo-1-carboxyheptanal 7-aminotransferase</u><u style="Single">side reductase, 3-oxo-7-aminoheptanoate 3-aminotransferase, 3-</u><u style="Single">Oxo-7-aminoheptanoate 3-aminated oxidoreductase, 3,7-diaminohepta</u><u style="Single">Composition 146, comprising noate 2,3-aminomutase or homolysine decarboxylase</u><u style="Single">non-naturally occurring microbial organisms.</u><u style="Single">(Configuration 150)</u><u style="Single"> The HMDA pathway is linked to glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA</u><u style="Single">Dolorase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA riga</u><u style="Single">3-oxopimelate reductase, 3-oxo-1-carboxyheptanal 3-amino</u><u style="Single">Transferase, 3-oxo-1-carboxyheptanal 3-aminated oxidoreductor</u><u style="Single">3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate</u><u style="Single">soheptanoate 7-aminated oxidoreductase, 3,7-diaminoheptanoate 2,3-a</u><u style="Single">The naturally occurring compound according to composition 146, comprising minomutase, or homolysine decarboxylase.</u><u style="Single">No microbial organisms.</u><u style="Single">(Configuration 151)</u><u style="Single"> The HMDA pathway is linked to glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA</u><u style="Single">Dolorase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA riga</u><u style="Single">enzyme, 3-oxopimelate kinase, 5-oxopimeloylphosphonate reductase, 3-</u><u style="Single">Oxo-1-carboxyheptanal 3-aminotransferase, 3-oxo-1-carboxy</u><u style="Single">Heptanal 3-aminating oxidoreductase, 3-amino-7-oxoheptanoate 7-a</u><u style="Single">Minotransferase, 3-amino-7-oxoheptanoate 7-aminated oxidoreduct</u><u style="Single">3,7-diaminoheptanoate 2,3-aminomutase, or homolysine decarboxylate</u><u style="Single">147.</u><u style="Single">(Configuration 152)</u><u style="Single"> The HMDA pathway is linked to glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA</u><u style="Single">Dolorase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA riga</u><u style="Single">3-oxopimelate-CoA transferase or 3-oxopimelate-CoA ligator</u><u style="Single">5-oxopimeloyl-CoA reductase (aldehyde formation), 3-oxo-1-carboxyl</u><u style="Single">putanal 3-aminotransferase, 3-oxo-1-carboxyheptanal 3-amino</u><u style="Single">oxidoreductase, 3-amino-7-oxoheptanoate 7-aminotransferer</u><u style="Single">3-amino-7-oxoheptanoate 7-aminated oxidoreductase, 3,7-diamino</u><u style="Single">Composition 14 comprising heptanoate 2,3-aminomutase or homolysine decarboxylase</u><u style="Single">6. The non-naturally occurring microbial organism described in 6.</u><u style="Single">(Configuration 153)</u><u style="Single"> The HMDA pathway is linked to glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA</u><u style="Single">Dolorase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA riga</u><u style="Single">3-oxopimelate aminotransferase or 3-oxopimelate amino</u><u style="Single">Oxidoreductase, 3-aminopimelate reductase, 3-amino-7-oxohepta</u><u style="Single">Noate 2,3-aminomutase, 2-amino-7-oxoheptanoate 7-aminotransfer</u><u style="Single">oxidoreductase, 2-amino-7-oxoheptanoate aminating oxidoreductase, or homolytic</u><u style="Single">147. The non-naturally occurring microbial organism of composition 146, comprising a zine decarboxylase.</u><u style="Single">(Configuration 154)</u><u style="Single"> The HMDA pathway is linked to glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA</u><u style="Single">Dolorase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA riga</u><u style="Single">3-oxopimelate aminotransferase or 3-oxopimelate amino</u><u style="Single">oxidoreductase, 3-aminopimelate kinase, 5-aminopimeloylphosphonate</u><u style="Single">Toreductase, 3-amino-7-oxoheptanoate 2,3-aminomutase, 2-amino-7-o</u><u style="Single">xoheptanoate 7-aminotransferase, 2-amino-7-oxoheptanoate</u><u style="Single">Composition 146 described, comprising a minating oxidoreductase or a homolysine decarboxylase</u><u style="Single">non-naturally occurring microbial organisms.</u><u style="Single">(Configuration 155)</u><u style="Single"> The HMDA pathway is linked to glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA</u><u style="Single">Dolorase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA riga</u><u style="Single">3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase,</u><u style="Single">Side reductase, 3-aminopimelate CoA transferase, 3-aminopimelate CoA</u><u style="Single">Ligase, 5-aminopimeloyl-CoA reductase (aldehyde formation), 3-amino-7-oxo</u><u style="Single">heptanoate 2,3-aminomutase, 2-amino-7-oxoheptanoate 7-aminotolane</u><u style="Single">spherase, 2-amino-7-oxoheptanoate aminating oxidoreductase, or</u><u style="Single">147. The non-naturally occurring microbial organism of composition 146, comprising a homolysine decarboxylase.</u><u style="Single">(Configuration 156)</u><u style="Single"> The HMDA pathway is linked to glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA</u><u style="Single">Dolorase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA riga</u><u style="Single">3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase,</u><u style="Single">Side reductase, 3-aminopimelate reductase, 3-amino-7-oxoheptanoe</u><u style="Single">7-aminotransferase, 3-amino-7-oxoheptanoate 7-aminated oxy</u><u style="Single">doreductase, 3,7-diaminoheptanoate 2,3-aminomutase, or homolysine deca</u><u style="Single">147. The non-naturally occurring microbial organism of composition 146, comprising a ruboxylase.</u><u style="Single">(Configuration 157)</u><u style="Single"> The HMDA pathway is linked to glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA</u><u style="Single">Dolorase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA riga</u><u style="Single">3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase,</u><u style="Single">Side reductase, 3-aminopimelate CoA transferase, 3-aminopimelate CoA</u><u style="Single">Ligase, 5-aminopimeloyl-CoA reductase (aldehyde formation), 3-amino-7-oxo</u><u style="Single">heptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate amino</u><u style="Single">oxidoreductase, 3,7-diaminoheptanoate 2,3-aminomutase, or homolytic</u><u style="Single">147. The non-naturally occurring microbial organism of composition 146, comprising a zine decarboxylase.</u><u style="Single">(Configuration 158)</u><u style="Single"> The HMDA pathway is linked to glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA</u><u style="Single">Dolorase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA riga</u><u style="Single">3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase,</u><u style="Single">Side reductase, 3-aminopimelate kinase, 5-aminopimeloylphosphonate reductase</u><u style="Single">ctase, 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-</u><u style="Single">Oxoheptanoate aminating oxidoreductase, 3,7-diaminoheptanoate 2,3-</u><u style="Single">The naturally occurring compound according to composition 146, comprising aminomutase or homolysine decarboxylase.</u><u style="Single">Microbial organisms that do not.</u><u style="Single">(Configuration 159)</u><u style="Single"> The HMDA pathway is linked to glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA</u><u style="Single">Dolorase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA riga</u><u style="Single">3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase,</u><u style="Single">Side reductase, 3-aminopimelate 2,3-aminomutase, 2-aminopimelate reductase</u><u style="Single">2-amino-7-oxoheptanoate 7-aminotransferase, 2-amino-7-o</u><u style="Single">xoheptanoate aminating oxidoreductase or homolysine decarboxylase</u><u style="Single">147. The non-naturally occurring microbial organism of composition 146, comprising:</u><u style="Single">(Configuration 160)</u><u style="Single"> The HMDA pathway is linked to glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA</u><u style="Single">Dolorase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA riga</u><u style="Single">3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase,</u><u style="Single">Side reductase, 3-aminopimelate 2,3-aminomutase, 2-aminopimelate kinase</u><u style="Single">enzyme, 6-aminopimeloylphosphonate reductase, 2-amino-7-oxoheptanoate 7</u><u style="Single">-Aminotransferase, 2-amino-7-oxoheptanoate aminated oxidoreduct</u><u style="Single">the non-naturally occurring microorganism of composition 146, including</u><u style="Single">Biological organism.</u><u style="Single">(Configuration 161)</u><u style="Single"> The HMDA pathway is linked to glutaryl-CoA beta-ketothiolase, 3-oxopimeloyl-CoA</u><u style="Single">Dolorase, 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA riga</u><u style="Single">3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase, 3-oxopimelate aminotransferase,</u><u style="Single">Side reductase, 3-aminopimelate 2,3-aminomutase, 2-aminopimelate CoA</u><u style="Single">Transferase, 2-aminopimelate-CoA ligase, 6-aminopimeloyl-CoA reductor</u><u style="Single">(aldehyde formation), 2-amino-7-oxoheptanoate 7-aminotransferase</u><u style="Single">, 2-amino-7-oxoheptanoate aminating oxidoreductase, or homolysine oxidoreductase</u><u style="Single">147. The non-naturally occurring microbial organism of composition 146, comprising a carboxylase.</u><u style="Single">(Configuration 162)</u><u style="Single"> a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set of exogenous nucleic acids comprising:</u><u style="Single">Glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxo</u><u style="Single">pimeloyl-CoA transferase, or 3-oxopimeloyl-CoA ligase; 3-oxopimeloyl-CoA ligase;</u><u style="Single">Melate reductase; 3-oxo-1-carboxyheptanal 7-aminotransferase</u><u style="Single">or 3-oxo-1-carboxyheptanal 7-aminated oxidoreductase; 3-oxo-7-</u><u style="Single">Aminoheptanoate 3-aminotransferase or 3-oxo-7-aminoheptanoate</u><u style="Single">3-aminated oxidoreductase; 3,7-diaminoheptanoate 2,3-aminomutase; and</u><u style="Single">the non-naturally occurring microorganism according to composition 146, which encodes homolysine decarboxylase and homolysine decarboxylase;</u><u style="Single">biological body.</u><u style="Single">(Configuration 163)</u><u style="Single"> a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set of exogenous nucleic acids comprising:</u><u style="Single">Glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxo</u><u style="Single">Pimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimeloyl-CoA transferase</u><u style="Single">rate kinase; 5-oxopimeloylphosphonate reductase; 3-oxo-1-carboxy</u><u style="Single">heptanal 7-aminotransferase or 3-oxo-1-carboxyheptanal 7-aminotransferase</u><u style="Single">minated oxidoreductase; 3-oxo-7-aminoheptanoate 3-aminotransfera</u><u style="Single">oxidoreductase or 3-oxo-7-aminoheptanoate 3-amination oxidoreductase; 3,7-diami</u><u style="Single">encodes noheptanoate 2,3-aminomutase; and homolysine decarboxylase</u><u style="Single">, composition 146.</u><u style="Single">(Configuration 164)</u><u style="Single"> a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set of exogenous nucleic acids comprising:</u><u style="Single">Glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxo</u><u style="Single">Pimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimeloyl-CoA transferase</u><u style="Single">rate CoA transferase or 3-oxopimelate CoA ligase; 5-oxopimeloyl-</u><u style="Single">CoA reductase (aldehyde formation); 3-oxo-1-carboxyheptanal 7-aminotran</u><u style="Single">Spherase or 3-oxo-1-carboxyheptanal 7-aminated oxidoreductase;</u><u style="Single">3-oxo-7-aminoheptanoate 3-aminotransferase or 3-oxo-7-aminoheptanoate</u><u style="Single">Ptanoate 3-amination oxidoreductase; 3,7-diaminoheptanoate 2,3-amino</u><u style="Single">mutase; and homolysine decarboxylase, according to construction</u><u style="Single">No microbial organisms.</u><u style="Single">(Configuration 165)</u><u style="Single"> a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set of exogenous nucleic acids comprising:</u><u style="Single">Glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxo</u><u style="Single">Pimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimeloyl-CoA transferase</u><u style="Single">rate reductase; 3-oxo-1-carboxyheptanal 3-aminotransferase or</u><u style="Single">is 3-oxo-1-carboxyheptanal 3-aminated oxidoreductase; 3-amino-7-o</u><u style="Single">xoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate 7</u><u style="Single">-aminated oxidoreductase; 3,7-diaminoheptanoate 2,3-aminomutase; and</u><u style="Single">A non-naturally occurring microbial organism according to composition 146, which encodes a homolysine decarboxylase.</u><u style="Single">object.</u><u style="Single">(Configuration 166)</u><u style="Single"> a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set of exogenous nucleic acids comprising:</u><u style="Single">Glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxo</u><u style="Single">Pimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimeloyl-CoA transferase</u><u style="Single">rate kinase; 5-oxopimeloylphosphonate reductase; 3-oxo-1-carboxy</u><u style="Single">Heptanal 3-aminotransferase or 3-oxo-1-carboxyheptanal 3-aminotransferase</u><u style="Single">Mininated oxidoreductase; 3-amino-7-oxoheptanoate 7-aminotransfera</u><u style="Single">oxidoreductase or 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; 3,7-diami</u><u style="Single">encodes noheptanoate 2,3-aminomutase; and homolysine decarboxylase</u><u style="Single">, composition 146.</u><u style="Single">(Configuration 167)</u><u style="Single"> a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set of exogenous nucleic acids comprising:</u><u style="Single">Glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxo</u><u style="Single">Pimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimeloyl-CoA transferase</u><u style="Single">rate CoA transferase or 3-oxopimelate CoA ligase; 5-oxopimeloyl-</u><u style="Single">CoA reductase (aldehyde formation); 3-oxo-1-carboxyheptanal 3-aminotran</u><u style="Single">Spherase or 3-oxo-1-carboxyheptanal 3-aminated oxidoreductase;</u><u style="Single">3-Amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate</u><u style="Single">Ptanoate 7-amination oxidoreductase; 3,7-diaminoheptanoate 2,3-amino</u><u style="Single">mutase; and homolysine decarboxylase, according to construction</u><u style="Single">No microbial organisms.</u><u style="Single">(Configuration 168)</u><u style="Single"> a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set of exogenous nucleic acids comprising:</u><u style="Single">Glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxo</u><u style="Single">Pimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimeloyl-CoA transferase</u><u style="Single">rate aminotransferase or 3-oxopimelate aminating oxidoreductase</u><u style="Single">;3-aminopimelate reductase;3-amino-7-oxoheptanoate 2,3-aminomuter</u><u style="Single">2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate</u><u style="Single">soheptanoate aminating oxidoreductase; and homolysine decarboxylase</u><u style="Single">146. A non-naturally occurring microbial organism according to composition 146, encoding the non-naturally occurring microbial organism of composition 146.</u><u style="Single">(Configuration 169)</u><u style="Single"> a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set of exogenous nucleic acids comprising:</u><u style="Single">Glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxo</u><u style="Single">Pimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimeloyl-CoA transferase</u><u style="Single">rate aminotransferase or 3-oxopimelate aminating oxidoreductase</u><u style="Single">;3-aminopimelate kinase;5-aminopimeloylphosphonate reductase;3-amino-</u><u style="Single">7-oxoheptanoate 2,3-aminomutase;2-amino-7-oxoheptanoate 7-amino</u><u style="Single">Transferase or 2-amino-7-oxoheptanoate aminating oxidoreductor</u><u style="Single">and homolysine decarboxylase;</u><u style="Single">Biological organism.</u><u style="Single">(Configuration 170)</u><u style="Single"> a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set of exogenous nucleic acids comprising:</u><u style="Single">Glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxo</u><u style="Single">Pimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimeloyl-CoA transferase</u><u style="Single">rate aminotransferase or 3-oxopimelate aminating oxidoreductase</u><u style="Single">;3-aminopimelate-CoA transferase or 3-aminopimelate-CoA ligase;5-aminopimelate-CoA ligase;</u><u style="Single">Minopimeloyl-CoA reductase (aldehyde formation); 3-amino-7-oxoheptanoate 2,</u><u style="Single">3-aminomutase; 2-amino-7-oxoheptanoate 7-aminotransferase or 2-</u><u style="Single">Amino-7-oxoheptanoate aminating oxidoreductase; and homolysine decarbo</u><u style="Single">147. The non-naturally occurring microbial organism of composition 146, which encodes a xylase.</u><u style="Single">(Configuration 171)</u><u style="Single"> a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set of exogenous nucleic acids comprising:</u><u style="Single">Glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxo</u><u style="Single">Pimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimeloyl-CoA transferase</u><u style="Single">rate aminotransferase or 3-oxopimelate aminating oxidoreductase</u><u style="Single">;3-aminopimelate reductase;3-amino-7-oxoheptanoate 7-aminotrans</u><u style="Single">ferase or 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; 3,7-</u><u style="Single">Diaminoheptanoate 2,3-aminomutase; and homolysine decarboxylase.</u><u style="Single">146. The non-naturally occurring microbial organism according to composition 146.</u><u style="Single">(Configuration 172)</u><u style="Single"> a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set of exogenous nucleic acids comprising:</u><u style="Single">Glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxo</u><u style="Single">Pimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimeloyl-CoA transferase</u><u style="Single">rate aminotransferase or 3-oxopimelate aminating oxidoreductase</u><u style="Single">;3-aminopimelate-CoA transferase or 3-aminopimelate-CoA ligase;5-aminopimelate-CoA ligase;</u><u style="Single">Nopimeloyl-CoA reductase (aldehyde formation); 3-amino-7-oxoheptanoate 7-a</u><u style="Single">Minotransferase or 3-amino-7-oxoheptanoate aminated oxidreduc</u><u style="Single">3,7-diaminoheptanoate 2,3-aminomutase; and homolysine decarboxylase</u><u style="Single">147.</u><u style="Single">(Configuration 173)</u><u style="Single"> a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set of exogenous nucleic acids comprising:</u><u style="Single">Glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxo</u><u style="Single">Pimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimeloyl-CoA transferase</u><u style="Single">rate aminotransferase or 3-oxopimelate aminating oxidoreductase</u><u style="Single">;3-aminopimelate kinase;5-aminopimeloylphosphonate reductase;3-amino-</u><u style="Single">7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoe</u><u style="Single">3,7-diaminoheptanoate 2,3-aminomutase;</u><u style="Single">the non-naturally occurring microorganism according to composition 146, which encodes homolysine decarboxylase and homolysine decarboxylase;</u><u style="Single">biological body.</u><u style="Single">(Configuration 174)</u><u style="Single"> a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set of exogenous nucleic acids comprising:</u><u style="Single">Glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxo</u><u style="Single">Pimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimeloyl-CoA transferase</u><u style="Single">rate aminotransferase or 3-oxopimelate aminating oxidoreductase</u><u style="Single">;3-aminopimelate 2,3-aminomutase;2-aminopimelate reductase;2-amino-7-</u><u style="Single">Oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate</u><u style="Single">A construct encoding a toaminating oxidoreductase; and a homolysine decarboxylase.</u><u style="Single">A non-naturally occurring microbial organism described in 146.</u><u style="Single">(Configuration 175)</u><u style="Single"> a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set of exogenous nucleic acids comprising:</u><u style="Single">Glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxo</u><u style="Single">Pimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimeloyl-CoA transferase</u><u style="Single">rate aminotransferase or 3-oxopimelate aminating oxidoreductase</u><u style="Single">;3-aminopimelate 2,3-aminomutase;2-aminopimelate kinase;6-aminopimero</u><u style="Single">phosphonate reductase; 2-amino-7-oxoheptanoate 7-aminotransferase</u><u style="Single">or 2-amino-7-oxoheptanoate aminating oxidoreductase;</u><u style="Single">147. The non-naturally occurring microbial organism of construction 146, which encodes a zine decarboxylase.</u><u style="Single">(Configuration 176)</u><u style="Single"> a set of exogenous nucleic acids encoding HMDA pathway enzymes, the set of exogenous nucleic acids comprising:</u><u style="Single">Glutaryl-CoA beta-ketothiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxo</u><u style="Single">Pimeloyl-CoA transferase or 3-oxopimeloyl-CoA ligase; 3-oxopimeloyl-CoA transferase</u><u style="Single">rate aminotransferase or 3-oxopimelate aminating oxidoreductase</u><u style="Single">;3-aminopimelate 2,3-aminomutase;2-aminopimelate CoA transferase or</u><u style="Single">is 2-aminopimelate-CoA ligase; 6-aminopimeloyl-CoA reductase (aldehyde form</u><u style="Single">2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate</u><u style="Single">soheptanoate aminating oxidoreductase; and homolysine decarboxylase</u><u style="Single">146. A non-naturally occurring microbial organism according to composition 146, encoding the non-naturally occurring microbial organism of composition 146.</u><u style="Single">(Configuration 177)</u><u style="Single"> A method for producing hexamethylene diamine (HMDA), the method comprising:</u><u style="Single">cultivating a non-naturally occurring microbial organism according to composition 146 under conditions and for a period sufficient thereto;</u><u style="Single">The method described above.</u><u style="Single">(Configuration 178)</u><u style="Single"> 178. The method of construction 177, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 179)</u><u style="Single"> HMDA pathway enzymes expressed in sufficient amounts to produce hexamethylene diamine (HMDA)</u><u style="Single">a microbial organism having an HMDA pathway that contains at least one exogenous nucleic acid that encodes</u><u style="Single">A microbial organism that does not exist in nature, the HMDA pathway</u><u style="Single">Heptanoate aldolase, 2-oxo-4-hydroxy-7-aminoheptanoate dehydra</u><u style="Single">tase, 2-oxo-7-aminohepta-3-enoate reductase, 2-oxo-7-aminohep</u><u style="Single">tanoate aminotransferase, 2-oxo-7-aminoheptanoate aminotran</u><u style="Single">Spherase amination oxidoreductase, homolysine decarboxylase, 2-oxo</u><u style="Single">-7-aminoheptanoate decarboxylase, 6-aminohexanal aminotransfer</u><u style="Single">6-aminohexanalaminating oxidoreductase.</u><u style="Single">Non-existent microbial organisms.</u><u style="Single">(Configuration 180)</u><u style="Single"> 179, wherein said HMDA pathway comprises at least two exogenous nucleic acids.</u><u style="Single">microbial organisms.</u><u style="Single">(Configuration 181)</u><u style="Single"> 179, wherein said HMDA pathway comprises at least three exogenous nucleic acids.</u><u style="Single">microbial organisms.</u><u style="Single">(Configuration 182)</u><u style="Single"> 179, wherein said HMDA pathway comprises at least four exogenous nucleic acids.</u><u style="Single">microbial organisms.</u><u style="Single">(Configuration 183)</u><u style="Single"> A method for producing hexamethylene diamine (HMDA), the method comprising:</u><u style="Single">cultivating the non-naturally occurring microbial organism described in Constitution 179 under conditions and for a period sufficient thereto;</u><u style="Single">The method described above.</u><u style="Single">(Configuration 184)</u><u style="Single"> 184. The method of construction 183, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 185)</u><u style="Single"> HMDA pathway enzymes expressed in sufficient amounts to produce hexamethylene diamine (HMDA)</u><u style="Single">naturally occurring microbial organisms that have a HMDA pathway that contains a set of exogenous nucleic acids that encode</u><u style="Single">a microbial organism that does not contain 2-oxo-4-hydroxy-7-alpha</u><u style="Single">Minoheptanoate aldolase; 2-oxo-4-hydroxy-7-aminoheptanoate dehyde</u><u style="Single">Dolatase; 2-oxo-7-aminohepta-3-enoate reductase; 2-oxo-7-aminoheptase</u><u style="Single">Ptanoate aminotransferase or 2-oxo-7-aminoheptanoate amination</u><u style="Single">oxidoreductase; and homolysine decarboxylase.</u><u style="Single">Non-existent microbial organisms.</u><u style="Single">(Configuration 186)</u><u style="Single"> A method for producing hexamethylene diamine (HMDA), the method comprising:</u><u style="Single">cultivating a non-naturally occurring microbial organism according to composition 185 under conditions and for a period sufficient thereto;</u><u style="Single">The method described above.</u><u style="Single">(Configuration 187)</u><u style="Single"> 187. The method of construction 186, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 188)</u><u style="Single"> HMDA pathway enzymes expressed in sufficient amounts to produce hexamethylene diamine (HMDA)</u><u style="Single">naturally occurring microbial organisms that have a HMDA pathway that contains a set of exogenous nucleic acids that encode</u><u style="Single">a microbial organism that does not contain 2-oxo-4-hydroxy-7-alpha</u><u style="Single">Minoheptanoate aldolase; 2-oxo-4-hydroxy-7-aminoheptanoate dehyde</u><u style="Single">Dolatase; 2-oxo-7-aminohepta-3-enoate reductase; 2-oxo-7-aminoheptase</u><u style="Single">Ptanoate decarboxylase; and 6-aminohexanal aminotransferase</u><u style="Single">or said naturally occurring enzyme encoding a 6-aminohexanalaminating oxidoreductase.</u><u style="Single">Microbial organisms that do not.</u><u style="Single">(Configuration 189)</u><u style="Single"> A method for producing hexamethylene diamine (HMDA), the method comprising:</u><u style="Single">cultivating a non-naturally occurring microbial organism according to composition 188 under conditions and for a period sufficient thereto;</u><u style="Single">The method described above.</u><u style="Single">(Configuration 190)</u><u style="Single"> 189. The method of construction 189, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 191)</u><u style="Single"> HMDA pathway enzymes expressed in sufficient amounts to produce hexamethylene diamine (HMDA)</u><u style="Single">a microbial organism having an HMDA pathway that contains at least one exogenous nucleic acid that encodes</u><u style="Single">A microbial organism that does not exist naturally, the HMDA pathway</u><u style="Single">6-aminocaproic acid semialdehyde aminotransferase, 6-aminocaproic acid</u><u style="Single">Semialdehydoxide reductase (amination), 6-aminocaproate N-acetyltran</u><u style="Single">Spherase, 6-acetamidohexanoate reductase, 6-acetamidohexanor</u><u style="Single">aminotransferase, 6-acetamidohexanal oxidoreductase (aminotransferase)</u><u style="Single">6-acetamidohexanamine N-acetyltransferase, or acetamidohexanamine</u><u style="Single">Said non-naturally occurring microbial organism comprising dohexanamine hydrolase (amide).</u><u style="Single">(Configuration 192)</u><u style="Single"> 192, wherein said HMDA pathway comprises at least two exogenous nucleic acids.</u><u style="Single">microbial organisms.</u><u style="Single">(Configuration 193)</u><u style="Single"> 192, wherein said HMDA pathway comprises at least three exogenous nucleic acids.</u><u style="Single">microbial organisms.</u><u style="Single">(Configuration 194)</u><u style="Single"> A method for producing hexamethylene diamine (HMDA), the method comprising:</u><u style="Single">culturing the non-naturally occurring microbial organism described in Constitution 191 under conditions and for a period sufficient thereto;</u><u style="Single">The method described above.</u><u style="Single">(Configuration 195)</u><u style="Single"> 195. The method of construction 194, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 196)</u><u style="Single"> HMDA pathway enzymes expressed in sufficient amounts to produce hexamethylene diamine (HMDA)</u><u style="Single">naturally occurring microbial organisms that have a HMDA pathway that contains a set of exogenous nucleic acids that encode</u><u style="Single">a microbial organism that does not contain 6-aminocaproate reductase, and the set of exogenous nucleic acids</u><u style="Single">and 6-aminocaproic acid semialdehyde aminotransferase or 6-amino</u><u style="Single">Containing caproic acid semialdehydoxide reductase (aminated), said non-naturally occurring</u><u style="Single">Microbial organisms.</u><u style="Single">(Configuration 197)</u><u style="Single"> A method for producing hexamethylene diamine (HMDA), the method comprising:</u><u style="Single">culturing the non-naturally occurring microbial organism described in Constitution 196 under conditions and for a period sufficient thereto;</u><u style="Single">The method described above.</u><u style="Single">(Configuration 198)</u><u style="Single"> 198. The method of construction 197, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 199)</u><u style="Single"> HMDA pathway enzymes expressed in sufficient amounts to produce hexamethylene diamine (HMDA)</u><u style="Single">naturally occurring microbial organisms that have a HMDA pathway that contains a set of exogenous nucleic acids that encode</u><u style="Single">a microbial organism that does not contain 6-aminocaproate N-acetate, said set of exogenous nucleic acids</u><u style="Single">chilltransferase; 6-acetamidohexanoate reductase; 6-acetamidohexanoate reductase;</u><u style="Single">Xanal aminotransferase or 6-acetamidohexanal oxidoreductor</u><u style="Single">6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine N-acetyltransferase (amination);</u><u style="Single">- said non-naturally occurring enzyme encoding acetamidohexanamine hydrolase (amide)</u><u style="Single">microbial organisms.</u><u style="Single">(Configuration 200)</u><u style="Single"> A method for producing hexamethylene diamine (HMDA), the method comprising:</u><u style="Single">culturing the non-naturally occurring microbial organism described in Constitution 199 under conditions and for a period sufficient thereto;</u><u style="Single">The method described above.</u><u style="Single">(Configuration 201)</u><u style="Single"> 201. The method of composition 200, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 202)</u><u style="Single"> HMDA pathway enzymes expressed in sufficient amounts to produce hexamethylene diamine (HMDA)</u><u style="Single">a microbial organism having an HMDA pathway that contains at least one exogenous nucleic acid that encodes</u><u style="Single">A microbial organism that does not exist in nature, the HMDA pathway is</u><u style="Single">to-acid decarboxylase, 2-amino-7-oxoheptanoate decarboxylase, 6-a</u><u style="Single">Minohexanalaminooxidoreductase, 6-aminohexanalaminotrans</u><u style="Single">ferase, 2-amino-7-oxoheptanoate aminating oxidoreductase, 2-amino</u><u style="Single">Nor-7-oxoheptanoate aminotransferase, homolysine decarboxylase</u><u style="Single">, 2-amino-7-oxosabarate amino acid decarboxylase, 2-oxo-7-aminohepta</u><u style="Single">Noate aminating oxidoreductase, 2-oxo-7-aminoheptanoate aminotra</u><u style="Single">spherase, 2-oxo-7-aminoheptanoate decarboxylase, 2-amino-7-o</u><u style="Single">Xosabarate aminating oxidoreductase, 2-amino-7-oxosabarate aminot</u><u style="Single">transferase, or 2,7-diaminosabarate decarboxylase.</u><u style="Single">Non-existent microbial organisms.</u><u style="Single">(Configuration 203)</u><u style="Single"> The naturally occurring non-naturally occurring compound of composition 202, wherein said HMDA pathway comprises at least two exogenous nucleic acids.</u><u style="Single">microbial organisms.</u><u style="Single">(Configuration 204)</u><u style="Single"> 203, wherein said HMDA pathway comprises at least three exogenous nucleic acids.</u><u style="Single">microbial organisms.</u><u style="Single">(Configuration 205)</u><u style="Single"> 203, wherein said HMDA pathway comprises at least four exogenous nucleic acids.</u><u style="Single">microbial organisms.</u><u style="Single">(Configuration 206)</u><u style="Single"> 2-amino-7-oxo expressed in sufficient amounts to produce 2-amino-7-oxosabarate</u><u style="Single">2-amino-7-oxo comprising at least one exogenous nucleic acid encoding a sabalate pathway enzyme</u><u style="Single">further comprising a sabalate pathway, wherein the 2-amino-7-oxosabalate pathway is 2-amino-5-hydro-</u><u style="Single">Roxy-7-oxosabarate aldolase, 2-amino-5-hydroxy-7-oxosabarate</u><u style="Single">Composition 20 comprising dehydratase or 2-amino-5-ene-7-oxosubare reductase</u><u style="Single">2. The non-naturally occurring microbial organism described in 2.</u><u style="Single">(Configuration 207)</u><u style="Single"> A method for producing hexamethylene diamine (HMDA), the method comprising:</u><u style="Single">cultivating a non-naturally occurring microbial organism according to composition 202 under conditions and for a period sufficient thereto;</u><u style="Single">The method described above.</u><u style="Single">(Configuration 208)</u><u style="Single"> 208. The method of configuration 207, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 209)</u><u style="Single"> HMDA pathway enzymes expressed in sufficient amounts to produce hexamethylene diamine (HMDA)</u><u style="Single">naturally occurring microbial organisms that have a HMDA pathway that contains a set of exogenous nucleic acids that encode</u><u style="Single">a microbial organism that does not contain 2-amino-7-oxosabare</u><u style="Single">toaminated oxidoreductase or 2-amino-7-oxosabarate aminotransfer</u><u style="Single">2,7-diaminosabarate decarboxylase; and homolysine decarboxylase</u><u style="Single">said non-naturally occurring microbial organism encoding.</u><u style="Single">(Configuration 210)</u><u style="Single"> 2-amino-7-oxo expressed in sufficient amounts to produce 2-amino-7-oxosabarate</u><u style="Single">a 2-amino-7-oxosa containing a second set of exogenous nucleic acids encoding Sabalate pathway enzymes;</u><u style="Single">further comprising the Barreto pathway, wherein the second set of exogenous nucleic acids is 2-amino-5-hydroxy-7</u><u style="Single">-Oxosabarate aldolase; 2-amino-5-hydroxy-7-oxosabarate dehydrata</u><u style="Single">and 2-amino-5-ene-7-oxosubare reductase, according to structure 209.</u><u style="Single">non-naturally occurring microbial organisms.</u><u style="Single">(Configuration 211)</u><u style="Single"> A method for producing hexamethylene diamine (HMDA), the method comprising:</u><u style="Single">cultivating a non-naturally occurring microbial organism according to composition 209 under conditions and for a period sufficient thereto;</u><u style="Single">The method described above.</u><u style="Single">(Configuration 212)</u><u style="Single"> 212. The method of configuration 211, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 213)</u><u style="Single"> HMDA pathway enzymes expressed in sufficient amounts to produce hexamethylene diamine (HMDA)</u><u style="Single">naturally occurring microbial organisms that have a HMDA pathway that contains a set of exogenous nucleic acids that encode</u><u style="Single">a microbial organism that does not contain 2-amino-7-oxosabare</u><u style="Single">amino acid decarboxylase; 2-oxo-7-aminoheptanoate aminated oxidoreda</u><u style="Single">ctase or 2-oxo-7-aminoheptanoate aminotransferase; and homolyzate</u><u style="Single">said non-naturally occurring microbial organism encoding a decarboxylase.</u><u style="Single">(Configuration 214)</u><u style="Single"> 2-amino-7-oxo expressed in sufficient amounts to produce 2-amino-7-oxosabarate</u><u style="Single">a 2-amino-7-oxosa containing a second set of exogenous nucleic acids encoding Sabalate pathway enzymes;</u><u style="Single">further comprising the Barreto pathway, wherein the second set of exogenous nucleic acids is 2-amino-5-hydroxy-7</u><u style="Single">-Oxosabarate aldolase; 2-amino-5-hydroxy-7-oxosabarate dehydrata</u><u style="Single">and 2-amino-5-ene-7-oxosubare reductase, according to structure 213.</u><u style="Single">non-naturally occurring microbial organisms.</u><u style="Single">(Configuration 215)</u><u style="Single"> A method for producing hexamethylene diamine (HMDA), the method comprising:</u><u style="Single">cultivating a non-naturally occurring microbial organism according to composition 213 under conditions and for a period sufficient thereto;</u><u style="Single">The method described above.</u><u style="Single">(Configuration 216)</u><u style="Single"> 216. The method of construction 215, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 217)</u><u style="Single"> HMDA pathway enzymes expressed in sufficient amounts to produce hexamethylene diamine (HMDA)</u><u style="Single">naturally occurring microbial organisms that have a HMDA pathway that contains a set of exogenous nucleic acids that encode</u><u style="Single">a microbial organism that does not contain 2-amino-7-oxosabare</u><u style="Single">amino acid decarboxylase; 2-oxo-7-aminoheptanoate decarboxylase;</u><u style="Single">and 6-aminohexanalaminating oxidoreductase or 6-aminohexanalamide</u><u style="Single">Said non-naturally occurring microbial organism encoding a notransferase.</u><u style="Single">(Configuration 218)</u><u style="Single"> 2-amino-7-oxo expressed in sufficient amounts to produce 2-amino-7-oxosabarate</u><u style="Single">a second set of exogenous nucleic acids encoding subalternate pathway enzymes;</u><u style="Single">The second set is 2-amino-5-hydroxy-7-oxosabarate aldolase;</u><u style="Single">Hydroxy-7-oxosabarate dehydratase; and 2-amino-5-en-7-oxosabarate</u><u style="Single">217 compositions further comprising the 2-amino-7-oxosabalate pathway encoding toreductase</u><u style="Single">Non-naturally occurring microbial organisms.</u><u style="Single">(Configuration 219)</u><u style="Single"> A method for producing hexamethylene diamine (HMDA), the method comprising:</u><u style="Single">cultivating a non-naturally occurring microbial organism according to composition 217 under conditions and for a period sufficient thereto;</u><u style="Single">The method described above.</u><u style="Single">(Configuration 220)</u><u style="Single"> 220. The method of construction 219, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 221)</u><u style="Single"> HMDA pathway enzymes expressed in sufficient amounts to produce hexamethylene diamine (HMDA)</u><u style="Single">naturally occurring microbial organisms that have a HMDA pathway that contains a set of exogenous nucleic acids that encode</u><u style="Single">a microbial organism that does not contain 2-amino-7-oxosabare</u><u style="Single">Toketo acid decarboxylase; 2-amino-7-oxoheptanoate decarboxylase; and</u><u style="Single">and 6-aminohexanalamino oxidoreductase or 6-aminohexanalamino</u><u style="Single">Said non-naturally occurring microbial organism encoding a transferase.</u><u style="Single">(Configuration 222)</u><u style="Single"> 2-amino-7-oxo expressed in sufficient amounts to produce 2-amino-7-oxosabarate</u><u style="Single">a 2-amino-7-oxosa containing a second set of exogenous nucleic acids encoding Sabalate pathway enzymes;</u><u style="Single">further comprising the Barreto pathway, wherein the second set of exogenous nucleic acids is 2-amino-5-hydroxy-7</u><u style="Single">-Oxosabarate aldolase; 2-amino-5-hydroxy-7-oxosabarate dehydrata</u><u style="Single">and 2-amino-5-ene-7-oxosubare reductase, according to structure 221.</u><u style="Single">non-naturally occurring microbial organisms.</u><u style="Single">(Configuration 223)</u><u style="Single"> A method for producing hexamethylene diamine (HMDA), the method comprising:</u><u style="Single">cultivating a non-naturally occurring microbial organism according to composition 221 under conditions and for a period sufficient thereto;</u><u style="Single">The method described above.</u><u style="Single">(Configuration 224)</u><u style="Single"> 224. The method of construction 223, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 225)</u><u style="Single"> HMDA pathway enzymes expressed in sufficient amounts to produce hexamethylene diamine (HMDA)</u><u style="Single">naturally occurring microbial organisms that have a HMDA pathway that contains a set of exogenous nucleic acids that encode</u><u style="Single">a microbial organism that does not contain 2-amino-7-oxosabare</u><u style="Single">Toketo acid decarboxylase; 2-amino-7-oxoheptanoate aminated oxidoreduct</u><u style="Single">tase or 2-amino-7-oxoheptanoate aminotransferase; and homolysine</u><u style="Single">Said non-naturally occurring microbial organism encoding a decarboxylase.</u><u style="Single">(Configuration 226)</u><u style="Single"> 2-amino-7-oxo expressed in sufficient amounts to produce 2-amino-7-oxosabarate</u><u style="Single">a 2-amino-7-oxosa containing a second set of exogenous nucleic acids encoding Sabalate pathway enzymes;</u><u style="Single">further comprising the Barreto pathway, wherein the second set of exogenous nucleic acids is 2-amino-5-hydroxy-7</u><u style="Single">-Oxosabarate aldolase; 2-amino-5-hydroxy-7-oxosabarate dehydrata</u><u style="Single">and 2-amino-5-ene-7-oxosubare reductase, according to structure 225.</u><u style="Single">non-naturally occurring microbial organisms.</u><u style="Single">(Configuration 227)</u><u style="Single"> A method for producing hexamethylene diamine (HMDA), the method comprising:</u><u style="Single">culturing a non-naturally occurring microbial organism according to composition 225 under conditions and for a period sufficient thereto;</u><u style="Single">The method described above.</u><u style="Single">(Configuration 228)</u><u style="Single"> 228. The method of construction 227, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 229)</u><u style="Single"> It encodes a 6-ACA pathway enzyme expressed in sufficient quantities to produce 6-aminocaproic acid (6-ACA).</u><u style="Single">a microbial organism with a 6-ACA pathway that contains at least one exogenous nucleic acid that</u><u style="Single">microbial organisms that are not present in the 6-ACA pathway, the 6-ACA pathway</u><u style="Single">glutamyl-CoA ligase, beta-ketothiolase, 3-oxo-6-aminopimeloyl-C</u><u style="Single">oA oxidoreductase, 3-hydroxy-6-aminopimeloyl-CoA dehydratase, 6-a</u><u style="Single">Mino-7-carboxyhept-2-enoyl-CoA reductase, 6-aminopimeloyl-CoA reductase</u><u style="Single">(aldehyde formation), or 2-aminopimelate decarboxylase.</u><u style="Single">Microbial organisms not present in</u><u style="Single">(Configuration 230)</u><u style="Single"> 229, wherein said 6-ACA pathway comprises at least two exogenous nucleic acids.</u><u style="Single">No microbial organisms.</u><u style="Single">(Configuration 231)</u><u style="Single"> 229, wherein said 6-ACA pathway comprises at least three exogenous nucleic acids.</u><u style="Single">No microbial organisms.</u><u style="Single">(Configuration 232)</u><u style="Single"> 229, wherein said 6-ACA pathway comprises at least four exogenous nucleic acids.</u><u style="Single">No microbial organisms.</u><u style="Single">(Configuration 233)</u><u style="Single"> A method for producing 6-aminocaproic acid (6-ACA), comprising conditions for producing 6-ACA.</u><u style="Single">culturing a non-naturally occurring microbial organism described in Constitution 229 under conditions and for a period sufficient thereto;</u><u style="Single">and the method.</u><u style="Single">(Configuration 234)</u><u style="Single"> 234. The method of construction 233, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 235)</u><u style="Single"> It encodes a 6-ACA pathway enzyme expressed in sufficient quantities to produce 6-aminocaproic acid (6-ACA).</u><u style="Single">naturally occurring microbial organisms that have a 6-ACA pathway that contains a set of exogenous nucleic acids that</u><u style="Single">a microbial organism that does not undergo glutamyl-CoA transfection, said set of exogenous nucleic acids</u><u style="Single">glutamyl-CoA ligase; beta-ketothiolase; 3-oxo-6-aminopimeroy</u><u style="Single">-CoA oxidoreductase;3-hydroxy-6-aminopimeloyl-CoA dehydratase;6-</u><u style="Single">Amino-7-carboxyhept-2-enoyl-CoA reductase; 6-aminopimeloyl-CoA reductase</u><u style="Single">ctase (aldehyde formation); and 2-aminopimelate decarboxylase,</u><u style="Single">Said non-naturally occurring microbial organism.</u><u style="Single">(Configuration 236)</u><u style="Single"> A method for producing 6-aminocaproic acid (6-ACA), comprising conditions for producing 6-ACA.</u><u style="Single">Cultivating a non-naturally occurring microbial organism described in Constitution 235 under conditions and for a period sufficient thereto;</u><u style="Single">and the method.</u><u style="Single">(Configuration 237)</u><u style="Single"> 237. The method of construction 236, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 238)</u><u style="Single"> It encodes a 6-ACA pathway enzyme expressed in sufficient quantities to produce 6-aminocaproic acid (6-ACA).</u><u style="Single">a microbial organism with a 6-ACA pathway that contains at least one exogenous nucleic acid that</u><u style="Single">microbial organisms that are not present in the 6-ACA pathway, the 6-ACA pathway</u><u style="Single">3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transferer</u><u style="Single">enzyme, 3-oxopimeloyl-CoA ligase, 3-oxopimelate aminotransferase,</u><u style="Single">3-oxopimelate aminating oxidoreductase, 3-aminopimelate 2,3-aminomuta</u><u style="Single">2-aminopimelate decarboxylase, or 2-aminopimelate decarboxylase.</u><u style="Single">biological body.</u><u style="Single">(Configuration 239)</u><u style="Single"> 238, wherein said 6-ACA pathway comprises at least two exogenous nucleic acids.</u><u style="Single">No microbial organisms.</u><u style="Single">(Configuration 240)</u><u style="Single"> 238, wherein said 6-ACA pathway comprises at least three exogenous nucleic acids.</u><u style="Single">No microbial organisms.</u><u style="Single">(Configuration 241)</u><u style="Single"> 238, wherein said 6-ACA pathway comprises at least four exogenous nucleic acids.</u><u style="Single">No microbial organisms.</u><u style="Single">(Configuration 242)</u><u style="Single"> A method for producing 6-aminocaproic acid (6-ACA), comprising conditions for producing 6-ACA.</u><u style="Single">Cultivating a non-naturally occurring microbial organism described in Constitution 238 under conditions and for a period sufficient thereto;</u><u style="Single">and the method.</u><u style="Single">(Configuration 243)</u><u style="Single"> 243. The method of construction 242, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 244)</u><u style="Single"> It encodes a 6-ACA pathway enzyme expressed in sufficient quantities to produce 6-aminocaproic acid (6-ACA).</u><u style="Single">naturally occurring microbial organisms that have a 6-ACA pathway that contains a set of exogenous nucleic acids that</u><u style="Single">a microbial organism that does not contain glutaryl-CoA beta-keto</u><u style="Single">Thiolase; 3-oxopimeloyl-CoA hydrolase, 3-oxopimeloyl-CoA transfer</u><u style="Single">3-oxopimelate aminotransferase, or 3-oxopimeloyl-CoA ligase;</u><u style="Single">erase or 3-oxopimelate aminating oxidoreductase; 3-aminopimelate 2,3</u><u style="Single">- aminomutase; and 2-aminopimelate decarboxylase;</u><u style="Single">Non-existent microbial organisms.</u><u style="Single">(Configuration 245)</u><u style="Single"> A method for producing 6-aminocaproic acid (6-ACA), comprising conditions for producing 6-ACA.</u><u style="Single">Cultivating a non-naturally occurring microbial organism described in Structure 244 under conditions and for a period sufficient thereto;</u><u style="Single">and the method.</u><u style="Single">(Configuration 246)</u><u style="Single"> 246. The method of construction 245, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 247)</u><u style="Single"> It encodes a 6-ACA pathway enzyme expressed in sufficient quantities to produce 6-aminocaproic acid (6-ACA).</u><u style="Single">a microbial organism with a 6-ACA pathway that contains at least one exogenous nucleic acid that</u><u style="Single">microbial organisms that are not present in the 6-ACA pathway, the 6-ACA pathway</u><u style="Single">said non-naturally occurring microbial organism.</u><u style="Single">(Configuration 248)</u><u style="Single"> A method for producing 6-aminocaproic acid (6-ACA), comprising conditions for producing 6-ACA.</u><u style="Single">culturing the non-naturally occurring microbial organisms described in Constitution 247 under conditions and for a period sufficient thereto;</u><u style="Single">and the method.</u><u style="Single">(Configuration 249)</u><u style="Single"> 249. The method of construction 248, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 250)</u><u style="Single"> at least one encoding a 6-ACA pathway enzyme expressed in sufficient amounts to produce 6-ACA</u><u style="Single">Naturally occurring microbial organisms with a 6-aminocaproic acid (6-ACA) pathway that contain exogenous nucleic acids of</u><u style="Single">In a microbial organism that does not exist naturally, the 6-ACA pathway is activated by adipate reductase,</u><u style="Single">pate kinase, or adipyl phosphate reductase, said non-naturally occurring</u><u style="Single">Microbial organisms.</u><u style="Single">(Configuration 251)</u><u style="Single"> The non-naturally occurring microorganism of composition 250, wherein said 6-ACA pathway comprises an adipate reductase.</u><u style="Single">Biological organism.</u><u style="Single">(Configuration 252)</u><u style="Single"> The 6-ACA pathway includes adipate kinase and adipylphosphate reductase.</u><u style="Single">, composition 250.</u><u style="Single">(Configuration 253)</u><u style="Single"> A method for producing 6-aminocaproic acid (6-ACA), comprising conditions for producing 6-ACA.</u><u style="Single">Cultivating a non-naturally occurring microbial organism described in Constitution 250 under conditions and for a period sufficient thereto;</u><u style="Single">and the method.</u><u style="Single">(Configuration 254)</u><u style="Single"> 254. The method of construction 253, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 255)</u><u style="Single"> It encodes a 6-ACA pathway enzyme expressed in sufficient quantities to produce 6-aminocaproic acid (6-ACA).</u><u style="Single">a microbial organism with a 6-ACA pathway that contains at least one exogenous nucleic acid that</u><u style="Single">microbial organisms that are not present in the 6-ACA pathway, the 6-ACA pathway</u><u style="Single">Acid decarboxylase, 2-amino-7-oxoheptanoate decarboxylase, 2-amino</u><u style="Single">Nor-7-oxoheptanoate oxidoreductase, 2-aminopimelate decarboxyla</u><u style="Single">6-aminohexanal oxidoreductase, 2-amino-7-oxoheptanoate</u><u style="Single">decarboxylase or 2-amino-7-oxosabarate amino acid decarboxylase</u><u style="Single">said non-naturally occurring microbial organism.</u><u style="Single">(Configuration 256)</u><u style="Single"> 255, wherein said 6-ACA pathway comprises at least two exogenous nucleic acids.</u><u style="Single">No microbial organisms.</u><u style="Single">(Configuration 257)</u><u style="Single"> 255, wherein said 6-ACA pathway comprises at least three exogenous nucleic acids.</u><u style="Single">No microbial organisms.</u><u style="Single">(Configuration 258)</u><u style="Single"> 2-amino-7-oxo expressed in sufficient amounts to produce 2-amino-7-oxosabarate</u><u style="Single">2-amino-7-oxo comprising at least one exogenous nucleic acid encoding a sabalate pathway enzyme</u><u style="Single">further comprising a sabalate pathway, wherein the 2-amino-7-oxosabalate pathway is 2-amino-5-hydro-</u><u style="Single">Roxy-7-oxosabarate aldolase, 2-amino-5-hydroxy-7-oxosabarate</u><u style="Single">Composition 25, comprising dehydratase or 2-amino-5-ene-7-oxosubare reductase</u><u style="Single">5. The non-naturally occurring microbial organism described in 5.</u><u style="Single">(Configuration 259)</u><u style="Single"> A method for producing 6-aminocaproic acid (6-ACA), comprising conditions for producing 6-ACA.</u><u style="Single">Cultivating a non-naturally occurring microbial organism described in Constitution 255 under conditions and for a period sufficient thereto;</u><u style="Single">and the method.</u><u style="Single">(Configuration 260)</u><u style="Single"> 259. The method of construction 259, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 261)</u><u style="Single"> It encodes a 6-ACA pathway enzyme expressed in sufficient quantities to produce 6-aminocaproic acid (6-ACA).</u><u style="Single">naturally occurring microbial organisms that have a 6-ACA pathway that contains a set of exogenous nucleic acids that</u><u style="Single">a microbial organism that does not contain 2-amino-7-oxosabare</u><u style="Single">Toketo acid decarboxylase; 2-amino-7-oxoheptanoate oxidoreductase;</u><u style="Single">and the non-naturally occurring microorganism encoding 2-aminopimelate decarboxylase.</u><u style="Single">biological body.</u><u style="Single">(Configuration 262)</u><u style="Single"> A method for producing 6-aminocaproic acid (6-ACA), comprising conditions for producing 6-ACA.</u><u style="Single">Cultivating a non-naturally occurring microbial organism described in Structure 261 under conditions and for a period sufficient thereto;</u><u style="Single">and the method.</u><u style="Single">(Configuration 263)</u><u style="Single"> 263. The method of construction 262, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 264)</u><u style="Single"> It encodes a 6-ACA pathway enzyme expressed in sufficient quantities to produce 6-aminocaproic acid (6-ACA).</u><u style="Single">naturally occurring microbial organisms that have a 6-ACA pathway that contains a set of exogenous nucleic acids that</u><u style="Single">a microbial organism that does not contain 2-amino-7-oxosabare</u><u style="Single">Toketo acid decarboxylase; 2-amino-7-oxoheptanoate decarboxylase; and</u><u style="Single">and 6-aminohexanal oxidoreductase.</u><u style="Single">physical organism.</u><u style="Single">(Configuration 265)</u><u style="Single"> A method for producing 6-aminocaproic acid (6-ACA), comprising conditions for producing 6-ACA.</u><u style="Single">Cultivating a non-naturally occurring microbial organism described in Constitution 264 under conditions and for a period sufficient thereto;</u><u style="Single">and the method.</u><u style="Single">(Configuration 266)</u><u style="Single"> 266. The method of construction 265, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 267)</u><u style="Single"> It encodes a 6-ACA pathway enzyme expressed in sufficient quantities to produce 6-aminocaproic acid (6-ACA).</u><u style="Single">naturally occurring microbial organisms that have a 6-ACA pathway that contains a set of exogenous nucleic acids that</u><u style="Single">a microbial organism that does not contain 2-amino-7-oxosabare</u><u style="Single">amino acid decarboxylase; 2-amino-7-oxoheptanoate decarboxylase;</u><u style="Single">and the non-naturally occurring microorganism encoding 6-aminohexanal oxidoreductase.</u><u style="Single">Biological organism.</u><u style="Single">(Configuration 268)</u><u style="Single"> 2-amino-7-oxo expressed in sufficient amounts to produce 2-amino-7-oxosabarate</u><u style="Single">a 2-amino-7-oxosa containing a second set of exogenous nucleic acids encoding Sabalate pathway enzymes;</u><u style="Single">further comprising the Barreto pathway, wherein the second set of exogenous nucleic acids is 2-amino-5-hydroxy-7</u><u style="Single">-Oxosabarate aldolase; 2-amino-5-hydroxy-7-oxosabarate dehydrata</u><u style="Single">and 2-amino-5-ene-7-oxosubare reductase, described in structure 267</u><u style="Single">non-naturally occurring microbial organisms.</u><u style="Single">(Configuration 269)</u><u style="Single"> The non-naturally occurring microbial organism according to composition 267, wherein the exogenous nucleic acid is a heterologous nucleic acid.</u><u style="Single">body.</u><u style="Single">(Configuration 270)</u><u style="Single"> 267. The non-naturally occurring microbial organism of composition 267 in a substantially anaerobic medium.</u><u style="Single">(Configuration 271)</u><u style="Single"> A method for producing 6-aminocaproic acid (6-ACA), comprising conditions for producing 6-ACA.</u><u style="Single">cultivating a non-naturally occurring microbial organism described in Constitution 267 under conditions and for a period sufficient thereto;</u><u style="Single">and the method.</u><u style="Single">(Configuration 272)</u><u style="Single"> 272. The method of construction 271, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 273)</u><u style="Single"> at least one enzyme encoding an LA pathway enzyme expressed in sufficient quantities to produce levulinic acid (LA).</u><u style="Single">Non-naturally occurring microorganisms, including microbial organisms with LA pathways that also contain one exogenous nucleic acid.</u><u style="Single">3-oxoadipyl-CoA thiolase, 3-oxoadipyl-C</u><u style="Single">oA/acyl-CoA transferase, 3-oxoadipyl-CoA synthase, 3-oxoadipyl-CoA synthase</u><u style="Single">3-CoA hydrolase, or 3-oxoadipate decarboxylase.</u><u style="Single">Non-existent microbial organisms.</u><u style="Single">(Configuration 274)</u><u style="Single"> The non-naturally occurring method of construction 273, wherein said LA pathway comprises at least two exogenous nucleic acids.</u><u style="Single">Microbial organisms.</u><u style="Single">(Configuration 275)</u><u style="Single"> The non-naturally occurring method of construction 273, wherein said LA pathway comprises at least three exogenous nucleic acids.</u><u style="Single">Microbial organisms.</u><u style="Single">(Configuration 276)</u><u style="Single"> A method for producing levulinic acid (LA), comprising:</u><u style="Single">culturing the non-naturally occurring microbial organism of composition 273 for a sufficient period of time.</u><u style="Single">Method.</u><u style="Single">(Configuration 277)</u><u style="Single"> 277. The method of construction 276, wherein the conditions include substantially anaerobic culture conditions.</u><u style="Single">(Configuration 278)</u><u style="Single"> Exogenous enzymes encoding LA pathway enzymes expressed in sufficient quantities to produce levulinic acid (LA)</u><u style="Single">Non-naturally occurring microbial organisms, including microbial organisms that have an LA pathway that includes a set of nucleic acids</u><u style="Single">and the set of exogenous nucleic acids is 3-oxoadipyl-CoA thiolase;</u><u style="Single">Pyr-CoA/acyl-CoA transferase, 3-oxoadipyl-CoA synthase, or 3-oxoadipyl-CoA</u><u style="Single">encodes xoadipyl-CoA hydrolase; and 3-oxoadipate decarboxylase.</u><u style="Single">said non-naturally occurring microbial organism.</u><u style="Single">(Configuration 279)</u><u style="Single"> The non-naturally occurring microbial organism according to composition 278, wherein the exogenous nucleic acid is a heterologous nucleic acid.</u><u style="Single">object.</u><u style="Single">(Configuration 280)</u><u style="Single"> 279. The non-naturally occurring microbial organism of composition 278 in a substantially anaerobic medium.</u><u style="Single">(Configuration 281)</u><u style="Single"> A method for producing levulinic acid (LA), comprising:</u><u style="Single">culturing the non-naturally occurring microbial organism of composition 278 for a sufficient period of time.</u><u style="Single">Method.</u><u style="Single">(Configuration 282)</u><u style="Single"> 282. The method of construction 281, wherein the conditions include substantially anaerobic culture conditions.</u>