Microbial engineering for the production of chemical and pharmaceutical products from the isoprenoid pathway

MICROBIAL ENGINEERING FOR THE PRODUCTION OF CHEMICAL AND PHARMACEUTICAL PRODUCTS FROM THE ISOPRENOID PATHWAY

<b><u>Related applications</u></b>

This application is incorporated herein by reference in its entirety in U.S. Provisional Application Serial No. 61/280,877, entitled "Microbial Engineering for the Production of Chemical and Pharmaceutical Products from Isoprenoid Pathway," filed on November 10, 2009, and the United States Provisional Application Serial No. 61/388,543 entitled "Microbial Engineering for the Production of Chemical and Pharmaceutical Products from Isoprenoid Pathway," filed September 30, 2010, claims the benefit under 35 USC § 119(e).

<b><u>government interest</u></b>

This invention was funded in part by the National Institutes of Health under Grant Number 1-R01-GM085323-01A1. The United States Government reserves certain rights in this invention.

<b><u>field of invention</u></b>

The present invention relates to the production of one or more terpenoids through microbial engineering.

Taxol and its structural analogues are recognized as the most potent and commercially successful anticancer agents introduced in the past decade.¹. Taxol was initially isolated from the bark of the Pacific yew tree.², the initial production method required the sacrifice of two to four fully grown trees to supply a sufficient dose for one patient.³. Due to the structural complexity of Taxol, a complex chemical synthesis route requiring 35-51 steps was required, with the highest yield of 0.4%.⁴^{, 5, 6}. However, a semi-synthetic route has been devised to first isolate the biosynthetic intermediate baccatin III from plant sources and subsequently convert it to taxol.⁷. Although this approach and subsequent plant cell culture-based production efforts have reduced the need for harvesting yew trees, production has limited productivity and scalability, and the number of taxol derivatives that can be synthesized in the search for more efficacious drugs.⁹^{, 10}A plant-based process with the limitations of⁸continue to depend on

<b><u>Summary of the invention</u></b>

Recent advances in metabolic engineering and synthetic biology offer new possibilities for the overproduction of complex natural products through technologically more processable microbial hosts.¹¹^{, 12}. Taxus (<i>Taxus</i>Although there have been remarkable advances in the elucidation of the biosynthetic mechanism of taxol in^13-16, in commercially relevant Taxol-producing strains, attempts aimed at delivering this complex biosynthetic machinery into a microbial host have failed.¹⁷^{, 18}. However, like other natural products, production by microorganisms through metabolically engineered strains offers attractive economics and great potential for synthesizing a wide variety of new compounds with anticancer and other pharmaceutical activities.¹⁹^{, 20}.

The metabolic pathways of Taxol and its analogs are described in E. coli (<i>E. </i><i>coli</i>) is composed of an upstream isoprenoid pathway, which is native to ), and a heterologous downstream terpenoid pathway ( FIG. 6 ). The upstream mevalonic acid (MVA) or methylerythritol phosphate (MEP) pathway can produce two common building blocks, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), from which taxol and other iso Prenoid compounds are formed¹². Recent studies have focused on the manipulation of this upstream pathway to support the biosynthesis of heterologous isoprenoids such as lycopene and artemisinic acid.^21-²³. The downstream taxadiene pathway is this. Reconstituted in E. coli, but to date titers have not exceeded 1.3 mg/L²⁴.

This rational metabolic engineering approach focused on either the upstream (MVA or MEP) or downstream terpenoid pathways, with the absolute assumption that the modifications exhibit additive, ie, linear behavior.²⁵^-27. While this method can result in moderate increases in flux, it can lead to non-specific effects such as toxicity of intermediate metabolites, cellular effects of the vector used for expression, and the direction of flux that competes with the major pathway. Unknown pathways that may divert from the target are generally ignored. A combinatorial approach can avoid this problem because it provides an opportunity to properly sample the parameter space and to elucidate the complex non-linear interactions.²¹^{, 28, 29, 30}. However, they require high-efficiency screening that is often not available for many desirable natural products.³¹. Another class of pathway optimization methods investigated the combinatorial space of different sources of heterologous genes containing pathways of interest.³². As this method also relies on high-efficiency assays, the method generally ignores the need to determine optimal expression levels of individual pathway genes, and thus has proven to be less effective in constructing optimal pathways.

In the present invention, as an example of an aspect of the present invention, we have focused on the optimal balance between the upstream IPP-forming pathway and the downstream terpenoid pathway of taxadiene synthesis. This is achieved by classifying the 9-enzyme pathway into two modules - a native (MEP) pathway module upstream of the 4-gene and a heterologous pathway downstream of the 2-gene to taxadiene ( FIG. 1 ). Using this basic conformation, parameters such as the effect of plasmid copy number on cell physiology, gene order and promoter strength in the expression cassette, and chromosomal integration are evaluated for their effect on taxadiene production. Using the above modular and multivariable combinatorial approach, we can efficiently sample the key parameters affecting the pathway flux without the need to perform high-throughput screening. A multivariate search across multiple promoters and copy numbers for each pathway module showed a 15,000-fold increase in taxadiene production compared to controls at an overall maximum, producing 300 mg/L of taxadiene in small-scale fermentations, It shows a highly non-linear taxadiene flux landscape. In addition, the present inventors Engineered P450-based oxidation chemistry in taxol biosynthesis of E. coli, our engineered strain improves taxadien-5α-ol production by 2400 fold compared to prior art counterparts. These improvements show the potential for large-scale production of thousands of valuable terpenoids by well-established microbial systems.

Aspects of the invention provide for recombinantly expressing a taxadiene synthase enzyme and a geranylgeranyl diphosphate synthase (GGPPS) enzyme in a cell overexpressing one or more components of the non-mevalonate (MEP) pathway. It's about how to entail making things happen. In some embodiments, the cell is a bacterial cell, such as Escherichia coli (<i>Escherichia</i><i> coli</i>) are cells. In some embodiments, the bacterial cell is a Gram-positive cell, such as Bacillus (<i>Bacillus</i>) are cells. In some embodiments, the cell is a yeast cell, such as Saccharomyces (<i>saccharomyces</i>) cells or Yarrowia (<i>Yarrowia</i>) are cells. In some embodiments, the cell is an algal cell or a plant cell.

In some embodiments, the taxadiene synthase enzyme is a Taxus enzyme, such as Taxus brevifolia (<i>Taxus</i><i></i><i>brevifolia</i>) are enzymes. In some embodiments, the GGPPS enzyme is a Taxus enzyme, such as Taxus canadenis (<i>Taxus</i><i></i><i>canadenis</i>) are enzymes. In some embodiments, a gene encoding a taxadiene synthase enzyme and/or a gene encoding a GGPPS enzyme and/or a gene encoding one or more components of the MEP pathway are expressed from one or more plasmids. In some embodiments, the gene encoding the taxadiene synthase enzyme and/or the gene encoding the GGPPS enzyme and/or the gene encoding one or more components of the MEP are integrated into the genome of the cell.

In some embodiments, one or more components of the non-mevalonate (MEP) pathway are <i>dxs</i>, <i>ispC</i>, <i>ispD</i>, <i>ispE</i>, <i>ispF</i>, <i>ispG</i>, <i>ispH</i>, <i>idi</i>, <i>ispA</i> and <i>ispB</i>is selected from the group consisting of In certain embodiments,<i>dxs</i>,<i> idi</i>,<i> ispD </i>and<i> ispF</i>is overexpressed. E.g,<i>dxs</i>,<i> idi</i>,<i> ispD </i>and<i> ispF</i>can be overexpressed in the operon dxs-idi-idpDF. In some embodiments, a gene encoding a taxadiene synthase enzyme and a gene encoding a GGPPS enzyme are expressed together in an operon.

In some embodiments, the cell further expresses taxadiene 5α-hydroxylase (T5αOH) or a catalytically active portion thereof. In certain embodiments, the T5αOH enzyme or catalytically active portion thereof is fused to a cytochrome P450 reductase enzyme or a catalytically active portion thereof. For example, the T5αOH enzyme may be At24T5αOH-tTCPR.

Expression of the taxadiene synthase enzyme, the GGPPS enzyme, and one or more components of the MEP pathway can be balanced to maximize the production of taxadiene. The methods associated with the present invention may further comprise culturing the cells to produce taxadiene or taxadien-5α-ol. In some embodiments, at least 10 mg L^-1of taxadiene is produced. In certain embodiments, at least 250 mg L^-1of taxadiene is produced. In some embodiments, at least 10 mg L^-1of taxadien-5α-ol is produced. In certain embodiments, at least 50 mg L^-1of taxadien-5α-ol is produced. In some embodiments, the percentage conversion of taxadiene to taxadien-5α-ol and the byproduct 5(12)-oxa-3(11)-cyclotaxane is at least 50%, at least 75%, or at least 95%.

The method associated with the present invention may further comprise recovering taxadiene or taxadien-5α-ol from the cell culture medium. In some embodiments, the taxadiene or taxadien-5α-ol is recovered from the gas phase, in other embodiments the organic layer is added to the cell culture, and the taxadiene or taxadien-5α-ol is recovered from the organic layer .

Aspects of the invention relate to cells that overexpress one or more components of the non-mevalonate (MEP) pathway and recombinantly express a taxadiene synthase enzyme and a geranylgeranyl diphosphate synthase (GGPPS) enzyme. . In some embodiments, the cell is a bacterial cell, such as an Escherichia coli cell. In some embodiments, the bacterial cell is a Gram-positive cell, such as a Bacillus cell. In some embodiments, the cell is a yeast cell, such as a Saccharomyces cell or a Yarrowia cell. In some embodiments, the cell is an algal cell or a plant cell.

In some embodiments, the taxadiene synthase enzyme is a Taxus enzyme, such as a Taxus brevifolia enzyme. In some embodiments, the GGPPS enzyme is a Taxus enzyme, such as a Taxus canadenis enzyme. In some embodiments, a gene encoding a taxadiene synthase enzyme and/or a gene encoding a GGPPS enzyme and/or a gene encoding one or more components of the MEP pathway are expressed from one or more plasmids. In some embodiments, the gene encoding the taxadiene synthase enzyme and/or the gene encoding the GGPPS enzyme and/or the gene encoding one or more components of the MEP are integrated into the genome of the cell.

In some embodiments, one or more components of the non-mevalonate (MEP) pathway are <i>dxs</i>, <i>ispC</i>, <i>ispD</i>, <i>ispE</i>, <i>ispF</i>, <i>ispG</i>, <i>ispH</i>, <i>idi</i>, <i>ispA</i> and <i>ispB</i>is selected from the group consisting of In certain embodiments,<i>dxs</i>,<i> idi</i>,<i> ispD </i>and<i> ispF</i>is overexpressed. E.g,<i>dxs</i>,<i> idi</i>,<i> ispD </i>and<i> ispF</i>is overexpressed in the operon dxs-idi-idpDF. In some embodiments, the gene encoding the taxadiene synthase enzyme and the gene encoding the GGPPS enzyme are co-expressed in an operon. In some embodiments, expression of the taxadiene synthase enzyme, the GGPPS enzyme, and one or more components of the MEP pathway is balanced to maximize the production of taxadiene.

In some embodiments, the cell further expresses taxadiene 5α-hydroxylase (T5αOH) or a catalytically active portion thereof. In certain embodiments, the T5αOH enzyme or a catalytically active portion thereof is fused to a cytochrome P450 reductase enzyme or a catalytically active portion thereof. For example, the T5αOH enzyme may be At24T5αOH-tTCPR. In some embodiments, the cell produces taxadiene and/or taxadien-5α-ol.

Aspects of the invention generate or obtain cells that overexpress one or more components of the non-mevalonate (MEP) pathway, produce terpenoids from the cells, and determine the amount of terpenoids produced from the cells in control cells. In a method for selecting a cell exhibiting enhanced production of terpenoid comprising selecting a first improved cell that produces a greater amount of terpenoid than a control cell compared to the amount of terpenoid produced wherein the first improved cell that produces a greater amount of terpenoid than a control cell is a cell that exhibits enhanced production of terpenoid.

In some embodiments, the cell recombinantly expresses a terpenoid synthase enzyme and/or a geranylgeranyl diphosphate synthase (GGPPS) enzyme. The method comprises one or more components of the non-mevalonate (MEP) pathway, a terpenoid synthase enzyme and/or a geranylgeranyl diphosphate synthase in the first improved cell to produce a second improved cell. (GGPPS) altering the expression level of the enzyme and comparing the amount of terpenoid produced in the second improved cell with the amount of terpenoid produced in the first improved cell, wherein the second improved cell that produces a greater amount of terpenoid than the first improved cell is a cell that exhibits enhanced production of terpenoid. In some embodiments, the terpenoid synthase enzyme is a taxadiene synthase enzyme. The cell may further express any polypeptide associated with the present invention in a recombinant manner.

Aspects of the invention relate to isolated polypeptides comprising a taxadiene 5α-hydroxylase (T5αOH) enzyme or a catalytically active portion thereof fused to a cytochrome P450 reductase enzyme or a catalytically active portion thereof. In some embodiments, the cytochrome P450 reductase enzyme is Taxus cytochrome P450 reductase (TCPR). In certain embodiments, the taxadiene 5α-hydroxylase and TCPR are linked by a linker, such as GSTGS (SEQ ID NO: 50). In some embodiments, the taxadiene 5α-hydroxylase and/or TCPR is truncated to remove all or part of the transmembrane region. In certain embodiments, 8, 24, or 42 N-terminal amino acids of the taxadiene 5α-hydroxylase are truncated. In certain embodiments, 74 amino acids of the TCPR are truncated. In some embodiments, additional peptides are fused to taxadiene 5α-hydroxylase. In certain embodiments, the additional peptide is from bovine 17α hydroxylase. In certain embodiments, the peptide is MALLLAVF (SEQ ID NO:51). In certain embodiments, the isolated polypeptide is At24T5αOH-tTCPR. Aspects of the invention also encompass nucleic acid molecules encoding any polypeptide associated with the invention and cells recombinantly expressing any polypeptide associated with the invention.

Aspects of the invention relate to methods for increasing terpenoid production in a cell that produces one or more terpenoids. The method involves controlling the accumulation of indole in a cell or in a culture of the cell, thereby increasing terpenoid production in the cell. bacterial cells such as Escherichia coli cells; Gram-positive cells such as Bacillus cells; yeast cells such as Saccharomyces cells or Yarrowia cells; algal cells; plant cells; and any engineered cell described herein, any cell described herein can be used in this method.

In some embodiments, controlling the accumulation of an indole in the cell or in a culture of the cell comprises balancing an upstream non-mevalonate isoprenoid pathway with a downstream product synthesis pathway and/or modifying or modulating the indole pathway. do. In other embodiments, controlling the accumulation of indole in the cell or in the culture of the cell comprises or further comprises removing the accumulated indole from the fermentation through a chemical method, such as by using an absorbent or scavenger.

The one or more terpenoids produced by the cell(s) or in culture may be monoterpenoids, sesquiterpenoids, diterpenoids, triterpenoids or tetraterpenoids. In certain embodiments, the terpenoid is taxadiene or any taxol precursor.

Aspects of the present invention relate to a method comprising determining the amount or concentration of an indole in a cell producing one or more terpenoids or in a culture of a cell producing one or more terpenoids. The method may comprise measuring the amount or concentration of the indole two or more times. In some embodiments, the measured amount or concentration of indole is used to guide a method for producing one or more terpenoids. In some embodiments, the measured amount or concentration of indole is used to guide strain construction.

These and other aspects of the invention, and various embodiments thereof, will become more apparent upon reference to the drawings and detailed description of the invention.

The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component shown in the various figures is represented by the same numerical value. In the interest of clarity, not all components may be represented in all figures. From the drawing:<b>Fig. 1. </b><b>multivariate</b><b>-module </b><b>isoprenoids</b><b> path manipulation </b><b>terpenoid</b><b> It shows a strong non-linear response in accumulation.</b> To increase the flux through the upstream MEP pathway, we present a reported bottleneck enzymatic step for overexpression by the operon (dxs-idi-ispDF) (<i>dxs</i>,<i> idi</i>,<i> ispD </i>and<i> ispF</i>) was targeted²⁸. To direct excess flux from the universal isoprenoid precursors IPP and DMAPP towards taxol biosynthesis, the downstream genes GGPP synthase (G) and taxadiene synthase (T)¹⁶of the synthetic operon was constructed. The upstream isoprenoid and downstream synthetic taxadiene pathways were placed under the control of an inducible promoter to control their relative gene expression. (A) Schematic of the two modules, the native upstream MEP isoprenoid pathway (left) and the synthetic taxadiene pathway (right). this. In the E. coli biosynthetic network, the MEP isoprenoid pathway is initiated by the condensation of the precursors glyceraldehyde-3 phosphate (G3P) and pyruvate (PYR) from glycolysis. The Taxol pathway branch starts from the universal isoprenoid precursors IPP and DMAPP to form first the "linear" precursor geranylgeranyl diphosphate, followed by the "cyclic" taxadiene, an absolutely key intermediate to Taxol. The cyclic olefin taxadiene ultimately undergoes benzoylation with multiple rounds of stereospecific oxidation, acylation, and side chain assembly to form taxol. (b) Schematic diagram of a multivariate-module isoprenoid pathway engineering approach to investigate non-linear responses in terpenoid accumulation from upstream and downstream pathway engineered cells. Expression of the upstream and downstream pathways is modulated by altering promoter strength (Trc, T5 and T7) or increasing copy number using different plasmids. Altering upstream and downstream pathway expression alters the maxima of taxadiene accumulation.<b>Figure 2. By regulating the expression of upstream and downstream modular pathways </b><b>taxadiene</b><b> Optimization of production.</b> (a) Response of taxadiene accumulation to an increase in upstream pathway intensity at constant values of the downstream pathway. (b) Dependence of the downstream path on a constant increase in the strength of the upstream path. The multiple local maxima observed in the taxadiene response depend on an increase in the expression intensity of the upstream or downstream pathway. (c) taxadiene responses from strains engineered with high upstream pathway overexpression (20-100), two different downstream expression (-30 and -60) to confirm taxadiene responses with balanced expression (17) -24). Expression of the downstream pathways from low-copy plasmids (p5 and p10) under the strong promoter T7TG operon was used to modulate their expression. Note that both upstream and downstream pathways expressed from different plasmids with different promoters can impose a metabolic burden on the plasmid. (d) Adjusting the upstream pathway (strain 25-32) while increasing promoter strength from the chromosome with two different downstream expressions (-30 and -60) to identify a lost search space with reduced toxic effects. (e) Details of the gene of the taxadiene producing strain. Numbers corresponding to different strains and their corresponding genotypes, E - E. E. coli K12mG1655 ΔrecAΔendA, EDE3E. with the T7 RNA polymerase DE3 construct in its chromosome. E. coli K12mG1655 ΔrecAΔendA, MEP - dxs-idi-ispDF operon, GT - GPPS-TS operon, TG - TS-GPPS operon, Ch1 - 1 copy in chromosome, Trc - Trc promoter, T5 - T5 promoter, T7 - T7 promoter, p5 , p10, p20 - ˜5 (SC101), ˜10 (p15), and ˜20 (pBR322) copy plasmids.<b>Fig. 3. </b><b>metabolites are</b><b></b><b>taxadiene</b><b> production department </b><b>inverse proportion</b><b> show the relationship</b> (a) The mass spectrum of the detected metabolite, showing an inverse relationship with taxadiene production in the strain construct of FIG. 2 . The characteristic peaks observed for metabolites are 233, 207, 178, 117, 89 and 62. (b) Correlation of taxadiene with isoprenoid by-products of FIG. 3a. Strains 26-29 and 30-32, both with chromosomally integrated upstream pathway expression, were selected for consistent comparison. In strains 26-29 and 30-32, upstream expression was increased by changing the promoter from Trc to T5 and T7, respectively. The two sets of strains differ only in the expression of downstream pathways, where the second set (30-32) has twice the expression level compared to the first set. In a first set, optimal balancing is achieved with strain 26 which utilizes the Trc promoter for upstream pathway expression and also exhibits the lowest metabolite accumulation. In strains 30-32, strain 31 shows the lowest accumulation of metabolites and the highest production of taxadiene. The data show the observed inverse association between unknown metabolites and taxadiene production. <b>Figure 4. Changes in upstream and downstream pathway transcriptional gene expression levels and cell physiology of engineered strains.</b> The relative expression of the first gene in the operons of the upstream (DXS) and downstream (TS) pathways is quantified by qPCR. A similar expression profile was observed for genes downstream of the operon. Corresponding strain numbers are shown in the graph. (A) Relative transcript level DXS gene expression quantified from different upstream expression coordinated using promoters and plasmids under two different downstream expressions. (b) Relative transcript level TS gene expression quantified from two different downstream expression modulated using p5T7 and p10T7 plasmids under different upstream expression. Our gene expression analysis directly supported the hypothesis that expression of the upstream and downstream pathways could be modulated with increasing plasmid copy number (5, 10 and 20) and promoter strength (Trc, T5 and T7). (c) Cell growth of engineered strains 25-29. Growth phenotype was affected by activation of isoprenoid metabolism (strain 26), recombinant protein expression (strain 25) and metabolic load by the plasmid (control versus engineered strain). and, (d) the growth phenotype of strains 17, 22, 25-32. The black line is the engineered strain producing taxadiene and the gray line is the control strain without downstream expression, carrying an empty plasmid with a promoter and multiple cloning sites. Growth correlates with activation of terpenoid metabolism, metabolic load by plasmids, and expression of recombinant proteins.<b>Fig. 5. E. </b><b>in coli</b><b></b><b>Taxol</b><b></b><b>p450</b><b> Manipulation of oxidation chemistry.</b> (a) Schematic diagram of conversion of taxadiene to taxadiene 5α-ol, taxol. (b) Transmembrane engineering and construction of one-component chimeric proteins from taxadien 5α-ol hydroxylase (T5αOH) and taxus cytochrome p450 reductase (TCPR). 1 and 2 represent the full-length proteins of T5αOH and TCPR, which were identified to have a TM region of 42 and 74 amino acids, respectively, and 3, a 74 AA truncated TCPR (tTCPR) using a 5-residue GSTGS linker peptide and generated from three different TM engineered T5αOH constructs (At8T5αOH, At24T5αOH and At42T5αOH constructed by fusing an 8 residue synthetic peptide (A) to 8, 24 and 42 AA truncated T5αOH) via translational fusion of is a chimeric enzyme. (c) Functional activity of At8T5αOH-tTCPR, At24T5αOH-tTCPR and At42T5αOH-tTCPR constructs transformed into taxadiene producing strain 18. (d) Time pathway profile of taxadien-5α-ol accumulation and growth profile of strain 18-At24T5αOH-tTCPR fermented in a 1 L bioreactor. <b>Fig. 6. E. </b><b>in coli</b><b></b><b>Taxol</b><b> Biosynthetic methods for production.</b> Schematic of the two modules, the native upstream isoprenoid pathway (left) and the synthetic Taxol pathway (right). this. In the E. coli biosynthetic network, branching of the MEP isoprenoid pathway originates from the precursors of glycolysis (IV) glyceraldehyde-3 phosphate (G3P) and pyruvate (PYR). The taxol route is branching out from this. Starting with the "linear" precursor geranylgeranyl diphosphate (VIII), "cyclic" taxadiene (IX), "oxidized" taxadiene 5α-ol (X) from E. coli isoprenoid precursors IPP and DMAPP, initial This leads to multiple rounds of stereospecific oxidation to the precursor baccatin III, acylation, benzoylation and epoxidation (XII) and finally side chain assembly to Taxol (XIII). DXP - 1-deoxy-D-xylulose-5-phosphate, MEP - 2C-methyl-D-erythritol-4-phosphate, CDP - ME-4-diphosphocytidyl-2C-methyl-D-eryth Ritol, CDP-MEP - 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate, ME-cPP - 2C-methyl-D-erythritol-2,4-cyclodiphosphate, IPP - iso Pentenyl diphosphate, DMAPP - dimethylallyl diphosphate. The genes included biosynthetic pathways from G3P and PYR to Taxol. DXS - 1-deoxy-D-xylulose-5-phosphate synthase, ispC - 1-deoxy-D-xylulose-5-phosphate reductoisomerase, IspD - 4-diphosphocytidyl- 2C-Methyl-D-erythritol synthase, IspE - 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, IspF - 2C-methyl-D-erythritol-2,4-cyclodiphosphate Synthase, IspG-1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase, IspH-4-hydroxy-3-methyl-2-(E)-butenyl- 4-diphosphate reductase, IDI - isopentenyl-diphosphate isomerase, GGPPS - geranylgeranyl diphosphate synthase, taxadiene synthase, taxoid 5α-hydroxylase, taxoid-5α-O -acetyltransferase, taxoid 13α-hydroxylase, taxoid 10β-hydroxylase, taxoid 2α-hydroxylase, taxoid 2-O-benzoyl transferase, taxoid 7β-hydroxylase , Taxoid 10-O-acetyltransferase, Taxoid 1β-hydroxylase^*, Taxoid 9α-hydroxylase, Taxoid 9-keto-oxidase^*, Taxoid C4,C20-β-epoxidase^*, phenylalanine aminomutase, side chain CoA-ligase^*, Taxoid 13 O-phenylpropanoyltransferase, Taxoid 2'-hydroxylase^*, Taxoid 3'-N-benzoyltransferase²¹⁶^{, 219}. * Marked genes must be identified or characterized in the future.<b>Figure 7. From a modular pathway expression search </b><b>taxadiene</b><b> Improved multiples of production.</b> (A) Fold improvement in taxadiene response from all maxima observed from Figures 2a, b, and c compared to strain 1. A 2.5-fold difference between the two highest maxima (strains 17 and 26) and a 23-fold difference from the trough (strain 26 and 10) indicate that loss of optimal response significantly lowered titers.<b>Fig. 8. </b><b>metabolites</b>. (a) Correlation of taxadiene on metabolite accumulation. Metabolite accumulation from engineered strains is inversely related to taxadiene production in an exponential manner. The association coefficient for this association was determined to be 0.92. (b) Representative GC-profiles from strains 26-28 showing changes in taxadiene and metabolite accumulation. Numbers in chromatograms 1 and 2 correspond to metabolite and taxadiene peaks, respectively. (c) GC-MS profiles of metabolites (1) and taxadiene (2), respectively. The characteristic peaks observed for metabolites are 233, 207, 178, 117, 89 and 62. Taxa-4(20),11,12-diene characteristic ion m/z 272 (P⁺), 257 (P⁺-CH₃), 229 (P⁺-C₃H₇); 121, 122, 123 (C-ring fragment cluster)⁶⁰. The peak marked with an asterisk is the internal standard caryophyllene.<b>Figure 9. From an artificial chimeric enzyme engineered in strain 26 </b><b>GC</b><b>-MS Profile and </b><b>Taksa</b><b>diene/</b><b>taxadiene</b><b>-5α-ol production.</b> (A) GC profile of hexane:ether (8:2) extracts from three constructs (A-At8T5αOH-tTCPR, t24T5αOH-tTCPR and At42T5αOH-tTCPR) transferred to strain 26 and fermented for 5 days. Labels 1, 2 and 3 in the peak correspond to taxadiene, taxadien-5α-ol and 5(12)-oxa-3(11)-cyclotaxane (OCT), respectively. (b) Quantified production of taxa-4(20),11,12-dien-5α-ol and OCT from three strains. (c) and (d) GC-MS profiles of taxa-4(20),11,12-dien-5α-ol and OCT and peaks corresponding to fragmentation were compared with reliable standards and previous reports.⁴²^{, 47}. GC-MS analysis revealed the mass spectrum to the characteristic ion m/z 288 (P⁺), 273 (P⁺-H₂O), 255 (P⁺-H₂O-CH₃) was confirmed as a reliable taxa-4(20),11,12-dien-5α-ol with <b>Fig. 10</b>shows a schematic diagram depicting the terpenoid biosynthetic pathway and the natural products produced by the pathway. <b>Fig. 11</b>shows a schematic diagram depicting the modulation of the upstream pathway to amplify taxadiene production. <b>Fig. 12</b>shows a schematic diagram depicting the modulation of downstream pathways to amplify taxadiene production. <b>do </b><b>13</b>shows a schematic diagram indicating that the newly identified pathway switch is not characteristic of a downstream synthetic pathway. <b>Figure 14. Pathway strength correlates with transcriptional gene expression levels.</b> (a) relative expression of the idi, ispD and ispF genes with increasing upstream and downstream intensities in 31 arbitrary units, and (b) increasing upstream and downstream intensities in 61 arbitrary units, idi , Relative expression of ispD and ispF genes. As expected, gene expression increased with increasing upstream pathway intensity. Corresponding strain numbers are shown in bar graphs. Relative manifestations are housekeeping<i>rrsA</i> It was quantified using the expression of the gene. Data are mean +/- SD for 4 identical experiments.<b>15. </b><b>taxadiene</b><b> for production and growth </b><b>metabolites</b><b> Effect of by-product indole accumulation.</b> (a) Inverse association between taxadiene and indole. Strains 26-28 and 30-32, both with chromosomally integrated upstream pathway expression, were selected for consistent comparison. The two sets of strains differ only in the expression of downstream pathways, where the second set (30-32) has twice the expression level compared to the first set. In strains 26-28 and 30-32, upstream expression was increased by changing the promoter from Trc to T5 and T7, respectively. In the first set, optimal balancing is achieved with strain 26 which utilizes the Trc promoter for upstream pathway expression and also exhibits the lowest indole accumulation. In strains 30-32, strain 31 shows the lowest accumulation of indole and the highest production of taxadiene. The improvement fold is for strains 25 and 29, respectively, in both sets. (b) Effect of externally introduced indole on taxadiene production in high-producing strain 26. Different concentrations of indole were introduced into cell cultures cultured in minimal medium with 0.5% yeast extract. Taxadiene production decreased significantly as the indole concentration increased from 50 mg/L to 100 mg/L. (c) manipulated teeth. Effect of exogenously introduced indoles on cell growth in E. coli strains. Data are mean +/- SD for three identical experiments. Strains (1, 2, 6, 21, 40 and 100) lacking a downstream pathway and having an upstream pathway of different intensities were selected. Strain 26, a high taxadiene producer, showed the strongest inhibition.<b>Fig. 16. </b><b>indolo</b><b> confirmed unknown </b><b>metabolites</b>. (a) and (c) gas chromatograms and mass spectra of unknown metabolites extracted from cell culture using hexane. (b) and (d) correspond to gas chromatograms and mass spectra of pure indole dissolved in hexane. In addition to identifying the type of chemical, metabolites were extracted from the fermentation broth using hexane extraction, and purified by silica column chromatography using hexane:ethyl acetate (8:2) as an eluent. The purity of the compound was confirmed by TLC and GC-MS.¹HNMR and ¹³The CNMR spectrum confirmed that the chemical substance of the metabolite was indole. (e) of the indole extracted from the cell culture medium¹HNMR spectrum (CDC13, 400 MHz) δ: 6.56 (d, 1H, Ar CH), 7.16 (m, 3H, Ar CH), 7.38 (d, 1H, Ar CH), 7.66 (d, 1H, Ar CH), 8.05 (b, 1H, indole NH). (f)¹³CNMR δ: 135.7, 127.8, 124.2, 122, 120.7, 119.8, 111, 102.6. (g) of pure indole¹HNMR spectrum. <b>Figure 17. of strain engineered in 1L-bioreactor </b><b>fed-batch</b><b> culture</b>. Taxadiene accumulation (a), cell growth during 5 days of fed-batch bioreactor culture in a 1 L-bioreactor vessel with minimal medium and 0.5% yeast extract under controlled pH and oxygen conditions in strains 22, 17 and 26 (b), time course of acetic acid accumulation (c) and total substrate (glycerol) addition (d). After glycerol was depleted in the fermentor to about 0.5-1 g/L, 3 g/L of glycerol was introduced into the bioreactor during fermentation. Data are averages of two replicate test bioreactors.

<b><u>DETAILED DESCRIPTION OF THE INVENTION</u></b>

Taxol is a potent anticancer agent first isolated as a natural product from the Taxus brevifolia Pacific yew tree. However, reliable and cost-effective production of Taxol or Taxol analogs by traditional production routes from plant extracts is limited. Here, we report a multivariate-modular method for metabolic pathway engineering to amplify the production of taxadiene by about 15000-fold in engineered Escherichia coli. The first important taxol intermediate, taxadiene, is an E. coli, which contains two modules: a natural upstream pathway to form isopentenyl pyrophosphate (IPP) and a heterologous downstream terpenoid-forming pathway. It is a biosynthetic product of the non-mevalonate pathway in E. coli. A systematic multivariate search identified conditions that optimally balance the two pathway modules to minimize accumulation of inhibitory intermediates and flux conversion to by-products. We also engineered the next step after taxadiene in taxol biosynthesis, namely the P450-based oxidation step, leading to &gt;98% substrate conversion and E. A first example of the in vivo production of any functionalized Taxol intermediate in E. coli is presented. The modular pathway engineering approach not only highlights the complexity of the multi-step pathway, but also allows the accumulation of high taxadiene and taxadien-5α-ol titers (approximately 300 mg/L and 60 mg/L, respectively) in small-scale fermentations, The potential for microbial production of derivatives thereof is illustrated.

The present invention, in its application, is not limited to the manufacture and arrangement of the components set forth in the following description or illustrated in the drawings. The invention is capable of being practiced in other embodiments or of being practiced or carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be construed as limiting the present invention. As used herein, "comprising", "including" or "having", "comprising", "containing" and variations thereof are meant to include the hereinafter recited subject matter and equivalents thereof and additional subject matter.

The production by microorganisms of terpenoids such as taxadiene is exemplified herein. When expressed at satisfactory levels, the microbial pathway greatly reduces the cost of producing the compound. Additionally, this route uses inexpensive, abundant, renewable feedstocks (eg, sugars and other carbohydrates) and can be a source for the synthesis of many derivatives that may exhibit properties far superior to the original compound. A key element of the cost-competitive production of compounds of the isoprenoid pathway using the microbial pathway is the amplification of this pathway to allow for overproduction of these molecules. Described herein are methods of enhancing or amplifying the flux towards terpenoid production in Escherichia coli (E. coli). Specifically, isopentenyl pyrophosphate (IPP) (key intermediate for the production of isoprenoid compounds), dimethylallyl pyrophosphate (DMAPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranyl geranyl diphosphate (GGPP), and farnesyl geranyl diphosphate (FGPP), paclitaxel (taxol), gingcolide, geraniol, farnesol, geranylgeraniol, linalool, isoprene, monoterpenoids, Methods for amplifying metabolic flux, such as by synthesis of menthol, carotenoids such as lycopene, polyisoprenoids such as polyisoprene or natural gum, diterpenoids such as eluterobin, and sesquiterpenoids such as artemisinin this is provided

Aspects of the present invention relate to the production of terpenoids. As used herein, terpenoids, also referred to as isoprenoids, are organic chemicals derived from 5 carbon isoprene units. Some non-limiting examples of terpenoids classified based on the number of isoprene units they contain include: hemiterpenoids (1 isoprene unit), monoterpenoids (2 isoprene units), sesqui terpenoids (3 isoprene units), diterpenoids (4 isoprene units), sesterpenoids (5 isoprene units), triterpenoids (6 isoprene units), tetraterpenoids (8 isoprene units), and polyterpenoids having a greater number of isoprene units. In some embodiments, the terpenoid produced is taxadiene. In some embodiments, the terpenoids produced are citronellol, cubebol, nootkatone, cineol, limonene, eluterobin, sarcodictin, pseudoterosine, gingcolide, stevioside, rebaudioside A, sclareol, lapdendiol, levopimaradiene, sandracopimaradien, or isofemaradien.

Methods and compositions are described herein for optimizing the production of terpenoids in cells by controlling the expression of genes or proteins that participate in the upstream and downstream pathways. The upstream pathway consists of two different metabolic pathways, the mevalonic acid (MVA) pathway and the MEP (2-C-methyl-D-erythritol 4-phosphate) pathway, namely the MEP/DOXP (2-C-methyl-D-eryth Isopentyl pyrophosphate (IPP), which can be achieved by the non-mevalonate route or the mevalonic acid-independent route, also called the lithol 4-phosphate/1-deoxy-D-xylulose 5-phosphate) route. ) and the production of dimethylallyl pyrophosphate (DMAPP).

The downstream pathway is a synthetic pathway that induces the production of terpenoids and involves recombinant gene expression of the enzyme terpenoid synthase (also called terpene cyclase), and the enzyme geranylgeranyl diphosphate synthase (GGPPS). In some embodiments, the terpenoid synthase enzyme is a diterpenoid synthase enzyme. Some non-limiting examples of diterpenoid synthase enzymes are casben synthase, taxadiene synthase, levopimaradiene synthase, abietadiene synthase, isopimaradiene synthase, ent-copalyl di Phosphate synthase, syn-Stemar-13-ene synthase, syn-Stemod-13(17)-ene synthase, syn-pimara-7,15-diene synthase, ent-Sandaracopimaradiene synthase , ent-casa-12,15-diene synthase, ent-fimara-8(14),15-diene synthase, ent-kaur-15-ene synthase, ent-kaur-16-ene synthase , apidicolan-16β-ol synthase, pilocladan-16α-ol synthase, fusicoca-2,10(14)-diene synthase, and terpentetriene cyclase.

Surprisingly, as demonstrated in the Examples section, the optimization of terpenoid synthesis by manipulation of the upstream and downstream pathways described herein is not a simple linear or additive process. Instead, through complex combinatorial analysis, optimization was achieved by balancing the components of the upstream and downstream pathways. Unexpectedly, as shown in Figures 1 and 2, taxadiene accumulation showed a strong non-linear dependence on the relative intensities of the upstream MEP and downstream synthetic taxadiene pathways.

Aspects of the present invention relate to the control of expression of genes and proteins of the MEP pathway for the optimized production of terpenoids such as taxadiene. Optimized production of terpenoids means producing higher amounts of terpenoids following an optimization strategy than would be achieved in the absence of the optimized strategy. It should be understood that any genes and/or proteins within the MEP pathway are included in the methods and compositions described herein. In some embodiments, the gene in the MEP pathway is one of:<i>dxs</i>,<i></i><i>ispC</i>,<i></i><i>ispD</i>,<i></i><i>ispE</i>,<i> ispF</i>,<i></i><i>ispG</i>,<i></i><i>ispH</i>,<i></i><i>idi</i>,<i></i><i>ispA</i><i></i>or<i></i><i>ispB</i>. Expression of one or more genes and/or proteins in the MEP pathway may be up-regulated and/or down-regulated. In certain embodiments, upregulation of one or more genes and/or proteins in the MEP pathway may be combined with downregulation of one or more genes and/or proteins in the MEP pathway.

It should be understood that genes and/or proteins may be regulated alone or in combination. E.g,<i>dxs</i>expression alone or <i>ispC</i>,<i></i><i>ispD</i>,<i></i><i>ispE</i>,<i></i><i>ispF</i>,<i></i><i>ispG</i>,<i></i><i>ispH</i>,<i></i><i>idi</i>,<i></i><i>ispA</i><i></i>and<i></i><i>ispB</i> may be upregulated or downregulated in combination with upregulation or downregulation of the expression of one or more of the <i>ispC</i>expression alone or <i>dxs</i>,<i></i><i>ispD</i>,<i> ispE</i>,<i></i><i>ispF</i>,<i></i><i>ispG</i>,<i></i><i>ispH</i>,<i></i><i>idi</i>,<i></i><i>ispA</i><i></i>and<i></i><i>ispB</i> may be upregulated or downregulated in combination with upregulation or downregulation of the expression of one or more of the <i>ispD</i>the expression of <i>dxs</i>,<i></i><i>ispC</i>,<i> ispE</i>,<i></i><i>ispF</i>,<i></i><i>ispG</i>,<i></i><i>ispH</i>,<i></i><i>idi</i>,<i></i><i>ispA</i><i></i>and<i></i><i>ispB</i> may be upregulated or downregulated in combination with upregulation or downregulation of the expression of one or more of the <i>ispE</i>the expression of <i>dxs</i>,<i></i><i>ispC</i>,<i></i><i>ispD</i>,<i></i><i>ispF</i>,<i></i><i>ispG</i>,<i></i><i>ispH</i>,<i></i><i>idi</i>,<i></i><i>ispA</i><i></i>and<i></i><i>ispB</i> may be upregulated or downregulated in combination with upregulation or downregulation of the expression of one or more of the <i>ispF</i>the expression of <i>dxs</i>,<i></i><i>ispC</i>,<i></i><i>ispD</i>,<i></i><i>ispE</i>,<i></i><i>ispG</i>,<i></i><i>ispH</i>,<i></i><i>idi</i>,<i></i><i>ispA</i><i></i>and<i></i><i>ispB</i> may be upregulated or downregulated in combination with upregulation or downregulation of the expression of one or more of the <i>ispG</i>the expression of <i>dxs</i>,<i></i><i>ispC</i>,<i> ispD</i>,<i></i><i>ispE</i>,<i></i><i>ispF</i>,<i></i><i>ispH</i>,<i></i><i>idi</i>,<i></i><i>ispA</i><i></i>and<i></i><i>ispB</i> may be upregulated or downregulated in combination with upregulation or downregulation of the expression of one or more of the <i>ispH</i>the expression of <i>dxs</i>,<i></i><i>ispC</i>,<i> ispD</i>,<i></i><i>ispE</i>,<i></i><i>ispF</i>,<i></i><i>ispG</i>,<i></i><i>idi</i>,<i></i><i>ispA</i><i></i>and<i></i><i>ispB</i> may be upregulated or downregulated in combination with upregulation or downregulation of the expression of one or more of the <i>idi</i>the expression of <i>dxs</i>,<i></i><i>ispC</i>,<i> ispD</i>,<i></i><i>ispE</i>,<i></i><i>ispF</i>,<i></i><i>ispG</i>,<i></i><i>ispH</i>,<i></i><i>ispA</i><i></i>and<i></i><i>ispB</i> may be upregulated or downregulated in combination with upregulation or downregulation of the expression of one or more of the <i>ispA</i>the expression of <i>dxs</i>,<i> ispC</i>,<i></i><i>ispD</i>,<i></i><i>ispE</i>,<i></i><i>ispF</i>,<i></i><i>ispG</i>,<i></i><i>ispH</i>,<i></i><i>idi</i><i></i>and<i></i><i>ispB</i> may be upregulated or downregulated in combination with upregulation or downregulation of the expression of one or more of the <i>ispB</i>the expression of <i>dxs</i>,<i></i><i>ispC</i>,<i></i><i>ispD</i>,<i></i><i>ispE</i>,<i></i><i>ispF</i>,<i></i><i>ispG</i>,<i></i><i>ispH</i>,<i></i><i>idi</i><i></i>and<i></i><i>ispA</i> may be upregulated or downregulated in combination with upregulation or downregulation of the expression of one or more of the In some embodiments,<i>dxs</i>,<i></i><i>ispC</i>,<i></i><i>ispD</i>,<i></i><i>ispE</i>,<i></i><i>ispF</i>,<i></i><i>ispG</i>,<i></i><i>ispH</i>,<i></i>and<i></i><i>idi</i> the expression of one or more genes and/or proteins in <i>ispA</i> and/or <i>ispB</i>The expression of genes and/or proteins of

Expression of genes within the MEP pathway can be regulated in a modular manner. As used herein, regulation by a modular method refers to the regulation of multiple genes together. For example, in some embodiments, multiple genes in the MEP pathway are recombinantly expressed in contiguous regions of DNA, such as operons. It should also be understood that cells expressing the module may recombinantly or endogenously express one or more other genes within the MEP pathway.

A non-limiting example of a module of a gene within the MEP pathway is presented in the Examples section and referred to herein as dxs-idi-ispDF. <i>dxs</i>,<i> idi</i>,<i> ispD </i>and<i> ispF</i>A module containing It should be understood that a module of genes within the MEP pathway consistent with aspects of the present invention may contain any gene within the MEP pathway in any order.

In addition, the expression of genes and proteins within the downstream synthetic terpenoid synthesis pathway can be modulated to optimize terpenoid production. The synthetic downstream terpenoid synthesis pathway involves recombinant expression of terpenoid synthase enzymes and GGPPS enzymes. Any of the terpenoid synthase enzymes as discussed above can be expressed along with GGPPS depending on the downstream product being produced. For example, taxadiene synthase is used for the production of taxadiene. Recombinant expression of taxadiene synthase enzyme and GGPPS enzyme can be regulated independently or together. In some embodiments, two enzymes are modulated together in a modular fashion. For example, the two enzymes can be expressed in any order in the operon (GGPPS-TS referred to as "GT", or TS-GGPPS referred to as "TG").

Modulation of expression of genes and/or proteins, including modules such as the dxs-idi-ispDF operon, and the TS-GGPPS operon, can be accomplished via methods known in the art. For example, expression of a gene or operon can be regulated through the selection of promoters, such as inducible promoters with different strengths. Some non-limiting examples of promoters include Trc, T5 and T7. Additionally, expression of a gene or operon may be regulated through copy number manipulation of the gene or operon in the cell. For example, in certain embodiments, a strain containing an additional copy of the dxs-idi-ispDF operon on its chromosome under the control of the Trc promoter produces increased amounts of taxadiene compared to a strain that overexpresses only the synthetic downstream pathway. produce In some embodiments, expression of a gene or operon may be regulated through modulation of the order of genes within a module. For example, in certain embodiments, altering the order of genes in a downstream synthetic operon from GT to TG results in a 2-3 fold increase in taxadiene production. In some embodiments, expression of a gene or operon is regulated through integration of one or more genes or operon into a chromosome. For example, in certain embodiments, integration of the upstream dxs-idi-ispDF operon into the chromosome of the cell increases taxadiene production.

It should be understood that genes associated with the present invention may be obtained from a variety of sources. In some embodiments, the gene in the MEP pathway is a bacterial gene, such as an Escherichia coli gene. In some embodiments, the gene encoding the GGPPS enzyme is a plant gene. For example, the gene encoding GGPPS is a taxus, such as Taxus canadensis (<i>T. </i><i>canadensis</i>) can be derived from In some embodiments, the gene encoding taxadiene synthase is a plant gene. For example, a gene encoding taxadiene synthase may be derived from a species of Taxus, such as Taxus brevifolia (<i>T. </i><i>brevifolia</i>) can be derived from tea. Canadensis GGPPS and T. Representative GenBank accession numbers for Brevifolia taxadiene synthase are AF081514 and U48796, the sequences of which are incorporated herein by reference in their entirety.

As one of ordinary skill in the art will recognize, homologous genes for use in methods associated with the present invention can be obtained from other species and can be obtained by homology searches, for example, by the National Center for Biotechnology Information (NCBI). ) can be identified through a protein BLAST search available from the website (www.ncbi.nlm.nih.gov). Genes and/or operons associated with the present invention may be cloned from DNA from any DNA source containing a given gene, for example by PCR amplification and/or restriction digestion. In some embodiments, genes and/or operons associated with the invention are synthetic. Any means of obtaining genes and/or operons associated with the present invention are suitable for the present invention.

In some embodiments, further optimization of terpenoid production is achieved by modifying the gene prior to being recombinantly expressed in the cell. In some embodiments, the GGPPS enzyme has one or more of the following mutations: A162V, G140C, L182M, F218Y, D160G, C184S, K367R, A151T, M185I, D264Y, E368D, C184R, L331I, G262V, R365S, A114D, S239C, G295D, I276V, K343N, P183S, I172T, D267G, I149V, T234I, E153D and T259A. In some embodiments, the GGPPS enzyme has a mutation at residue S239 and/or residue G295. In certain embodiments, the GGPPS enzyme has the mutations S239C and/or G295D.

In some embodiments, modifying a gene prior to recombinantly expressed in a cell involves codon optimization for expression in a bacterial cell. Codon usage for various organisms can be found in the Codon Frequency Database (www.kazusa.or.jp/codon/). Codon optimization, including identification of optimal codons for various organisms, and methods of achieving codon optimization are well known to those skilled in the art and can be accomplished using standard methods.

In some embodiments, modifying a gene prior to recombinantly expressed in a cell involves making one or more mutations in the gene prior to recombinantly expressed in a cell. For example, mutations may involve substitutions or deletions of single nucleotides or multiple nucleotides. In some embodiments, a mutation of one or more nucleotides in a gene results in a mutation in a protein produced from the gene, such as a substitution or deletion of one or more amino acids.

In some embodiments, it may be advantageous to use cells optimized for the production of terpenoids. For example, in some embodiments, cells overexpressing one or more components of the non-mevalonate (MEP) pathway amplify the substrates of GGPPS, isopentyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). used at least in part for In some embodiments, overexpression of one or more components of the non-mevalonate (MEP) pathway is achieved by increasing the copy number of one or more components of the non-mevalonate (MEP) pathway. For example, components of rate limiting steps in the MEP pathway, such as (<i>dxs</i>,<i></i><i>ispD</i>,<i> ispF</i>,<i></i><i>idi</i>) can be amplified, for example, by further episomal expression.

In some embodiments, "rational design" is concerned when introducing certain mutations in a protein, such as an enzyme. As used herein, "rational design" refers to knowledge of an enzyme, or related enzyme, such as its three-dimensional structure, its active site(s), its substrate(s), and/or interactions between the enzyme and the substrate. means to include into the design of a particular mutation. Based on a rational design approach, mutations can be created in the enzyme and then screened for increased production of terpenoids compared to control levels. In some embodiments, mutations can be rationally designed based on homology modeling. As used herein, "homology modeling" refers to the process of constructing an atomic resolution model of a protein from its amino acid sequence and the three-dimensional structure of the related homologous protein.

In some embodiments, random mutations can be made in a gene, such as a gene encoding an enzyme, and the mutations can be screened for increased production of terpenoids compared to control levels. For example, screening for mutations in components of the MEP pathway, or components of other pathways leading to enhanced production of terpenoids, can be performed through random mutagenesis screening, or screening of known mutations. In some embodiments, shotgun cloning of genomic fragments will be used to identify genomic regions that increase production of terpenoids through screening of cells or organisms having genomic fragments for increased production of terpenoids. can In some cases, one or more mutations may be combined in the same cell or organism.

In some embodiments, the production of terpenoids in a cell may be increased through engineering of enzymes acting in the same pathway as the enzymes associated with the present invention. For example, in some embodiments, it may be advantageous to increase the expression of a target enzyme, such as an enzyme or other factor acting upstream of an enzyme associated with the present invention. This can be achieved by overexpressing the upstream factor using any standard method.

In addition, optimization of protein expression can be achieved through selection of appropriate promoters and ribosome binding sites. In some embodiments, this may comprise the selection of high copy number plasmids, or low or medium copy number plasmids. In addition, transcription termination steps can be targeted for regulation of gene expression through the introduction or removal of stem-loop-like structures.

Aspects of the invention relate to expression of a recombinant gene in a cell. The present invention includes any type of cell that recombinantly expresses a gene associated with the present invention, including prokaryotic and eukaryotic cells. In some embodiments, the cell is a bacterial cell, such as Escherichia spp., Streptomyces (<i>Streptomyces</i>) species, zymonas (<i>Zymonas</i>) species, Acetobacter (<i>Acetobacter</i>) species, Citrobacter (<i>Citrobacter</i>) species, synechocystis (<i>Synechocystis</i>) species, rhizobium (<i>Rhizobium</i>) species, Clostridium (<i>Clostridium</i>) species, Corynebacterium (<i>Corynebacterium</i>) species, Streptococcus (<i>Streptococcus</i>) species, Xanthomonas (<i>Xanthomonas</i>) species, Lactobacillus (<i>Lactobacillus</i>) species, Lactococcus (<i>Lactococcus</i>) species, Bacillus species, Alkaligenes (<i>Alcaligenes</i>) species, Pseudomonas (<i>Pseudomonas</i>) species, Aeromonas (<i>Aeromonas</i>) species, Azotobacter (<i>Azotobacter</i>) species, comamonas (<i>Comamonas</i>) species, Mycobacterium (<i>Mycobacterium</i>) species, Rhodococcus (<i>Rhodococcus</i>) species, gluconobacter (<i>Gluconobacter</i>) species, Ralstonia (<i>Ralstonia</i>) species, Acidithiobacillus (<i>Acidithiobacillus</i>) species, Microlunatus (<i>Microlunatus</i>) species, Geobacter (<i>Geobacter</i>) species, Geobacillus (<i>Geobacillus</i>) species, Artrobacter (<i>Arthrobacter</i>) species, Flavobacterium (<i>Flavobacterium</i>) species, Serratia (<i>Serratia</i>) species, Saccharopolispora (<i>Saccaropolyspora</i>) species, Thermus (<i>Thermus</i>) species, Stenotrophomonas (<i>Stenotrophomonas</i>) species, Chromobacterium (<i>Chromobacterium</i>) species, sinorizobium (<i>Sinorhizobium</i>) species, Saccharopolispora (<i>Saccaropolyspora</i>) species, Agrobacterium (<i>Agrobacterium</i>) species and Pantoea (<i>Pantoea</i>) is a species bacterial cell. The cell may be a Gram-negative cell, such as an Escherichia coli (E. coli) cell, or a Gram-positive cell, such as a species of Bacillus. In other embodiments, the cell is a fungal cell, such as a yeast cell, e.g., Saccharomyces spp., Schizozocaromyces (<i>Schizosaccharomyces</i>) species, Pichia (<i>Pichia</i>) bell, papia (<i>Paffia</i>) species, Kluyveromyces (<i>Kluyveromyces</i>) species, Candida (<i>Candida</i>) species, Thalalomyces (<i>Talaromyces</i>) species, Bretanomyces (<i>Brettanomyces</i>) species, pachysolen (<i>Pachysolen</i>) species, Devariomyces (<i>Debaryomyces</i>) species, Yarrowia species, and industrial polyploid yeast strains. Preferably the yeast strain is S. cerevisiae (<i>S. </i><i>cerevisiae</i>) strain or Yarrowia species strain. Another example of a fungus is Aspergillus (<i>Aspergillus</i>) species, Penicillium (<i>Pennicilium</i>) species, Fusarium (<i>Fusarium</i>) species, Rhizopus (<i>Rhizopus</i>) species, Acremonium (<i>Acremonium</i>) species, Neurospora (<i>Neurospora</i>) species, Sordaria (<i>Sordaria</i>) bell, Magnaforte (<i>Magnaporthe</i>) species, Allomyces (<i>Allomyces</i>) species, Ustilago (<i>Ustilago</i>) species, botrytis (<i>Botrytis</i>) species, and Trichoderma (<i>Trichoderma</i>) species. In other embodiments, the cell is an algal cell, or a plant cell. It should be understood that some cells suitable for the present invention may express endogenous and recombinant copies of one or more genes associated with the present invention. In some embodiments, if the cell has an endogenous copy of one or more genes associated with the invention, the method will not necessarily require the addition of a recombinant copy of the endogenously expressed gene(s). In some embodiments, the cell is capable of endogenously expressing one or more enzymes from a pathway described herein and recombinantly expressing one or more other enzymes from a pathway described herein for efficient production of terpenoids. .

A further aspect of the invention relates to the screening of bacterial cells or strains exhibiting optimized terpenoid production. As noted above, the methods associated with the present invention involve the generation of cells that overexpress one or more genes within the MEP pathway. Terpenoid production is measured from a culture of said cells and compared to control cells, wherein cells exhibiting higher amounts of terpenoid production compared to control cells are selected as first improved cells. Cells can be further modified by recombinant expression of a terpenoid synthase enzyme and a GGPPS enzyme. The expression level of one or more components of the non-mevalonate (MEP) pathway, a terpenoid synthase enzyme and/or a GGPPS enzyme in the cell can then be manipulated, and terpenoid production is measured again to obtain a first improvement A second improved cell that produces a greater amount of terpenoid than the old cell can be selected. In some embodiments, the terpenoid synthase enzyme is a taxadiene synthase enzyme.

A further aspect of the invention is the bacterial E. Identification and characterization (via GC-MS) of previously unknown metabolites in E. coli cells ( FIGS. 3 and 6 ). The accumulation level of the newly identified metabolite, indole, can be controlled by genetically engineering the microbial pathway by overexpression, downregulation or mutation of isoprenoid pathway genes. The metabolite indole shows an anticorrelation as a direct variable on taxadiene production in the engineered strain ( FIGS. 3 , 6 and 15 ). Further control of the accumulation of indoles to improve flux towards terpenoid biosynthesis in bacterial systems (specifically in cells, such as E. coli cells) or other cells may lead to upstream non-mevalonate isoprenoid pathways. This can be achieved by balancing downstream product synthesis pathways or by modifying or modulating the indole pathway. At this time, one of ordinary skill in the art can reduce or control the accumulation of indole, whereby taxadiene and other terpenoids derived from the described pathways, such as monoterpenoids, sesquiterpenoids (including amorphadiene) , diterpenoids (including levopimaradien), triterpenes, and tetraterpenes. Another method for reducing or controlling the accumulation of indole involves removing the accumulated indole from fermentation through chemical methods, such as by using absorbents, scavengers, and the like.

In another embodiment, a method is provided comprising determining the amount or concentration of an indole in a cell that produces one or more terpenoids or in a culture of a cell that produces one or more terpenoids. The amount or concentration of indole can be determined one, or two or more appropriate times using methods known in the art and described herein. The method can be used, for example, to guide a process for producing one or more terpenoids in process improvement. The method can be used to guide strain construction, for example for strain improvement.

The identification of means to achieve this balancing improved the overproduction of terpenoids, such as taxadiene, expressed in the heterologous taxadiene biosynthetic pathway by a factor of 15,000 compared to wild-type bacterial cells. Production was further increased through a modified fermentation method that produced a concentration of about 2 g/L, which is 1500-fold higher than any previously reported taxadiene production. As demonstrated herein, E. By genetically engineering the non-mevalonate isoprenoid pathway in E. coli, the accumulation of this metabolite is prevented by bacterial E. In E. coli cells, the flux can be controlled to regulate isoprenoid biosynthesis. Also exemplified herein is further directing of taxadiene production to the next important precursor to taxol, taxadien-5α-ol, which is achieved through manipulation of oxidation chemistry for taxol biosynthesis. Example 5 presents the first successful extension of the synthetic route from taxadiene to taxadiene-5α-ol. Similar to most other terpenoids, Taxol biosynthesis is an integrated mode of a "two-phase" biosynthetic process, i.e. (i) a "cyclase group" of linear coupling of prenyl precursors (IPP and DMAPP) to GGPP; This is followed by molecular cyclization and rearrangement to the important precursor taxadiene (Fig. 6, VIII-IX).^57,58. Following the important precursors, (ii) the cyclic olefin taxadiene core structure is converted to two acetate groups and a benzoate group by an acyl and aroyl CoA-dependent transferase, a keto group by a keto-oxidase, and an epoxidase An "oxidizing group" functionalized by seven cytochrome P450 oxygenases along with its redox partner decorated with an epoxide group by (Fig. 6, X-XIII)¹⁵. The approximate sequential order of the initial oxidative reactions is predicted, but the exact timing/sequence of some hydroxylation, acylation and benzoylation reactions is uncertain. However, cytochrome p450 enzymes with a high putative similarity (&gt;70%) to each other but limited similarity (&lt;30%) to other plant p450s, starting from cytochrome p450 mediated hydroxylation of the taxadiene core at the C5 position, with an initial branching It is clear that it leads to downstream hydroxylation using the homology family of⁴¹^,59. Additionally, structural and functional diversity by possible evolutionary analysis implies that the taxadien-5α-ol gene may be the parent sequence from which other hydroxylase genes in the taxol biosynthetic pathway are evolved, which is the reflect the order¹⁵.

A further aspect of the invention relates to a chimeric P450 enzyme. Functional expression of plant cytochrome P450 was considered problematic by the inherent limitations of bacterial platforms, such as the translational incompatibility of the membrane signaling module of the P450 enzyme by the electron transport mechanism, the absence of cytochrome P450 reductase, and the lack of the endoplasmic reticulum. .

In some embodiments, the taxadiene-5α-hydroxylase associated with the methods of the invention is optimized through N-terminal transmembrane manipulation and/or generation of a chimeric enzyme via translational fusion with a CPR redox partner. In some embodiments, the CPR redox partner is a taxus cytochrome P450 reductase (TCPR; FIG. 5B ). In certain embodiments, the cytochrome P450 taxadiene-5α-hydroxylase (T5αOH) is selected from Taxus cuspidate (<i>Taxus</i><i></i><i>cuspidate</i>) (GenBank Accession No. AY289209, the sequence of which is incorporated herein by reference). In some embodiments, the NADPH:cytochrome P450 reductase (TCPR) is obtained from Taxus cuspidate (GenBank Accession No. AY571340, the sequence of which is incorporated herein by reference).

The taxadiene 5α-hydroxylase and TCPR may be linked by a linker such as GSTGS (SEQ ID NO: 50). In some embodiments, the taxadiene 5α-hydroxylase and/or TCPR is truncated to remove all or a portion of the transmembrane region of one or both proteins. For example, in some embodiments the taxadiene 5α-hydroxylase is truncated to remove 8, 24, or 42 N-terminal amino acids. In some embodiments, the N-terminal 74 amino acids of the TCPR are truncated. Additionally, additional peptides may be fused to taxadiene 5α-hydroxylase. For example, one or more amino acids from bovine 17α hydroxylase can be added to the taxadiene 5α-hydroxylase. In certain embodiments, the peptide MALLLAVF (SEQ ID NO: 51) is added to the taxadiene 5α-hydroxylase. A non-limiting example of a polypeptide comprising a taxadiene 5α-hydroxylase fused to TCPR is At24T5αOH-tTCPR.

In some embodiments, the chimeric enzyme is capable of performing a first oxidation step wherein the conversion of taxadiene to taxadien-5α-ol and the byproduct 5(12)-oxa-3(11)-cyclotaxane is greater than 10%. . For example, the percentage conversion of taxadiene to taxadien-5α-ol and the byproduct 5(12)-oxa-3(11)-cyclotaxane is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, about 99% or about 100%.

In certain embodiments, the chimeric enzyme comprises a first oxidation with greater than 98% conversion of taxadiene to taxadien-5α-ol and the byproduct 5(12)-oxa-3(11)-cyclotaxane (OCT; FIG. 9A ). At245αOH-tTCPR, which was found to be capable of performing the steps. It has been found that manipulation of the taxadien-5α-ol production step is important for the production of taxol and limits previous efforts to establish this pathway in yeast. The engineered construct developed herein demonstrated greater than 98% conversion of taxadiene in vivo, a 2400-fold improvement over previous heterologous expression in yeast. Thus, in addition to the synthesis of significantly higher amounts of the important Taxol intermediate, this study also provides a basis for the synthesis of subsequent metabolites in the pathway by similar P450 chemistry.

As used herein, the terms "protein" and "polypeptide" are used interchangeably, and thus the term polypeptide may be used to refer to a full-length polypeptide, and may also be used to refer to a fragment of a full-length polypeptide. As used herein with reference to a polypeptide, protein, or fragment thereof, "isolated" means separated from its natural environment and present in an amount sufficient to permit its identification or use. When referring to a protein or polypeptide, isolated means, for example, (i) selectively produced by expression cloning or (ii) purified by chromatography or electrophoresis. An isolated protein or polypeptide may, but need not be, substantially pure. The term "substantially pure" means that the protein or polypeptide is essentially free of other substances that may be found in production, nature, or in vivo systems to the extent practical and appropriate for its intended use. Substantially pure polypeptides can be obtained naturally or produced using the methods described herein, and purified by techniques known in the art. Because an isolated protein may be mixed with other ingredients in the formulation, the protein may constitute only a small weight percentage of the formulation. Nevertheless, proteins are isolated in the sense that they are isolated from substances with which they can be associated in living systems, ie from other proteins.

The invention also encompasses nucleic acids encoding any of the polypeptides described herein, libraries containing any of the nucleic acids and/or polypeptides described herein, and compositions containing any of the nucleic acids and/or polypeptides described herein. do.

In some embodiments, one or more genes associated with the invention are expressed in a recombinant expression vector. As used herein, a "vector" means any number of any number into which the sequence(s) required for transport between different genetic environments or for expression in a host cell can be inserted by restriction and ligation into it. It may be a nucleic acid. Vectors typically consist of DNA, although RNA vectors are also available. Vectors include, but are not limited to, plasmids, fosmids, phagemids, viral genomes and artificial chromosomes.

A cloning vector can replicate autonomously or integrate into the genome within the host cell, and the DNA sequences required for the vector to be cleaved in a determinable manner and for the new recombinant vector to retain its ability to replicate in the host cell are lysed into it. It is further characterized by one or more endonuclease restriction sites that can be gated. In the case of a plasmid, replication of the desired sequence may occur multiple times with increasing copy number of the plasmid in a host cell, such as a host bacterium, or only once per host before the host replicates mitotically. In the case of phage, replication can occur actively during the lysis phase or passively during the latency phase.

An expression vector is one that can be inserted into it by restriction and ligation such that the desired DNA sequence is operably linked to regulatory sequences and expressed as an RNA transcript. The vector may further contain one or more marker sequences suitable for use in the identification of cells that have not been transformed or transfected with the vector. A marker is, for example, a gene encoding a protein that increases or decreases resistance or susceptibility to an antibiotic or other compound, an enzyme whose activity is detectable by standard assays known in the art (eg, β-galacto sidase, luciferase or alkaline phosphatase), and genes that visually affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (eg, green fluorescent protein). Preferred vectors are those capable of autonomous replication and expression of structural gene products present in operably linked DNA segments.

As used herein, a coding sequence and a regulatory sequence are said to be "operably" linked when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequence. When a coding sequence is required to be translated into a functional protein, the two DNA sequences are determined that induction of a promoter within the 5' regulatory sequence causes transcription of the coding sequence and that the linkage properties between the two DNA sequences (1) frame shift (frame). -shift) is operably linked if it does not cause introduction of a mutation, (2) inhibition of the promoter region's ability to direct transcription of the coding sequence, or (3) inhibition of the ability of the corresponding RNA transcript to be translated into protein. is mentioned Thus, a promoter region is operably linked to a coding sequence if the promoter region is capable of effecting transcription of a DNA sequence such that the resulting transcript can be translated into the desired protein or polypeptide.

When a nucleic acid molecule encoding any of the enzymes of the claimed invention is expressed in a cell, various transcriptional control sequences (eg, promoter/enhancer sequences) can be used to direct its expression. A promoter may be a native promoter, ie, a promoter of a gene within its endogenous background that provides for normal regulation of gene expression. In some embodiments, a promoter may be constitutive, ie, the promoter is unregulated, allowing continuous transcription of its associated gene. A variety of conditional promoters may also be used, such as those controlled in the presence or absence of the molecule.

The exact nature of the regulatory sequences required for gene expression may vary depending on the species or cell type, but generally, if necessary, the 5' non-transcribed and 5' non-translated sequences involved in the initiation of transcription and translation, respectively, such as TATA boxes, capping sequences, CAAT sequences, and the like. In particular, said 5' non-transcriptional regulatory sequence will comprise a promoter region comprising a promoter sequence for transcriptional control of an operably linked gene. In addition, regulatory sequences may also include enhancer sequences or upstream activator sequences, if desired. The vector of the invention may optionally comprise a 5' leader or signal sequence. The selection and design of an appropriate vector is within the ability and judgment of one of ordinary skill in the art.

Expression vectors containing all elements necessary for expression are commercially available and known to those skilled in the art. See, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by introducing heterologous DNA (RNA) into the cell. Heterologous DNA (RNA) is placed under the operable control of a transcription element that allows expression of the heterologous DNA in a host cell. Heterologous expression of genes relevant to the present invention for the production of terpenoids such as taxadiene can be achieved in E. It is exemplified in the Examples section using E. coli. In addition, novel methods for terpenoid production can also be expressed in other bacterial cells, fungi (including yeast cells), plant cells, and the like.

Nucleic acid molecules encoding enzymes associated with the present invention can be introduced into a cell or cells using standard methods and techniques in the art. For example, nucleic acid molecules can be introduced by standard protocols such as transformation, eg, chemical transformation and electroporation, transduction, particle bombardment, and the like. Expression of a nucleic acid molecule encoding an enzyme of the claimed invention can be achieved by integrating the nucleic acid molecule into the genome.

In some embodiments, one or more genes associated with the invention are recombinantly expressed in a bacterial cell. Bacterial cells according to the present invention can be cultured in any type (abundant or minimal) medium and in any composition. As will be appreciated by those skilled in the art, routine optimization allows the use of different types of media. The selected medium may be supplemented with a variety of additional ingredients. Non-limiting examples of some of the supplemental ingredients include glucose, antibiotics, IPTG for gene induction, ATCC Trace Mineral Supplement, and glycolate. Similarly, other aspects of the medium, and growth conditions, of the cells of the invention can be optimized through routine experimentation. For example, pH and temperature are non-limiting examples of factors that can be optimized. In some embodiments, factors such as selection of medium, medium supplementation, and temperature may affect the level of production of terpenoids, such as taxadiene. In some embodiments, concentrations and amounts of supplemental ingredients may be optimized. In some embodiments, the frequency with which the medium is replenished with one or more supplemental components, and the amount of time the medium is cultured prior to collection of terpenoids, such as taxadiene, are optimized.

According to an aspect of the present invention, high titers of terpenoids, such as taxadiene, are produced through recombinant expression of genes associated with the present invention in cells. As used herein, "high titer" means milligrams/liter (mg L^-1) means the potency of the scale. The titer produced for a given product will be influenced by several factors, including the choice of medium. In some embodiments, the total taxadiene titer is at least 1 mg L^-1am. In some embodiments, the total taxadiene titer is at least 10 mg L^-1am. In some embodiments, the total taxadiene titer is at least 250 mg L^-1am. For example, the total taxadiene titer is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900 mg L^-1or 900 mg L^-1 may be greater than, inclusive of any intermediate value. In some embodiments, the total taxadiene titer is at least 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 , 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 g L^-1or 5.0 g L^-1 may be greater than, inclusive of any intermediate value.

In some embodiments, the total taxadien 5α-ol titer is at least 1 mg L^-1am. In some embodiments, the total taxadien 5α-ol titer is at least 10 mg L^-1am. In some embodiments, the total taxadien 5α-ol titer is at least 50 mg L^-1am. For example, the total taxadiene 5α-ol titer is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 , 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 , 70 mg L^-1or 70 mg L^-1 may be greater than, inclusive of any intermediate value.

The liquid culture medium used to grow the cells associated with the present invention may be contained in any culture vessel known and used in the art. In some embodiments, large-scale production in aeration reaction vessels, such as stirred tank reactors, can be used to produce large amounts of terpenoids, such as taxadiene, that can be recovered from cell culture. In some embodiments, terpenoids are recovered from the gas phase of the cell culture, for example, by adding an organic layer, such as dodecane, to the cell culture and recovering the terpenoids from the organic layer.

Terpenoids, such as taxadiene, produced via the methods described herein include pharmaceuticals such as paclitaxel (Taxol), artemisinin, gingcolide, eluterobin and pseudoterosine, and many other potential pharmaceutical compounds. It has a wide range of applications. Further applications include compounds used in spices and cosmetics, such as geraniol, farnesol, geranylgeraniol, linalool, limonene, phenene, cineol and isoprene. Further applications include compounds for use as biofuels, such as alcohols 5, 10, and 15 carbon atoms in length. Note that the compound is currently produced as extracts of various plants. Plant extract-based methods are tedious, produce small amounts, and are limited on the actual molecules that can be obtained thereby, i.e., this method allows for the easy production of derivatives that may retain properties far superior to the original compounds. I never do that.

<b><u>Example</u></b>

Way

<b>Strains, plasmids, oligonucleotides and genes</b>

this. E. coli K12 MG1655 strain was used as the host strain for all taxadiene strain constructions. this. E. coli K12MG1655Δ(recA,endA) and E. The E. coli K12MG1655Δ(recA,endA)ED3 strain was provided by Professor Kristala Prather's laboratory at MIT (Cambridge, Massachusetts, USA). Details of all plasmids constructed for the study are presented in Table 2. All oligonucleotides used in this study are presented in Table 3.

Geranylgeranyl pyrophosphate synthase (<i>GGPPS</i>)⁵⁰, taxadiene synthase (<i>ts</i>)⁵¹, cytochrome P450 taxadiene 5α-hydroxylase (<i>T5</i><i>α</i><i>OH</i>) and Taxus NADPH:cytochrome P450 reductase (<i>TCPR</i>)⁴⁶The sequences of were obtained from Taxus canadensis, Taxus brevifolia, Taxus cuspidate (GenBank accession codes: AF081514, U48796, AY289209 and AY571340). The gene is this. For introduction of E. coli translation codons and removal of restriction sites for cloning, see Kodumal et al.⁵² (Supplementary details Appendix 1)] was custom synthesized using the reported plasmid and protocol. Nucleotides corresponding to 98 and 60 N-terminal amino acids of GGPPS and TS (chromosome transport peptide) were removed, and the translational insertion sequence Met was inserted¹⁷.

<b>MEP</b><b> Route (</b><b>dxs</b><b>-</b><b>idi</b><b>-</b><b>idpDF</b><b></b><b>operon</b><b>) of the construction.</b>

The dxs-idi-ispDF operon was constructed using primers dxs(s), dxs(a), idi(s), idi(a), ispDF(s) and ispDFI(a) under pET21C+ plasmid (p20T7MEP) with T7 promoter. this. It was initially constructed by cloning each gene from the genome of E. coli K12 MG1655.⁵³. Using primers dxsidiispDFNcoI (s) and dxsidiispDFKpnI (a), the dxs-idi-ispDF operon was subcloned into pTrcHis2B (Invitrogen) plasmid digested with NcoI and KpnI to generate pTrcMEP plasmid (pTrcMEP plasmid). The p20TrcMEP plasmid was digested with MluI and Pmel and cloned into the pACYC184-melA (P2A) plasmid digested with MluI and Pmel to construct the p10TrcMEP plasmid. The pTrcMEP plasmid was digested with BstZ17I and ScaI and cloned into the PvuII digested pCL1920 plasmid to construct the p5TrcMEP plasmid. To construct the p20T5MEP plasmid, the dxs-idi-ispDF operon was initially cloned into the pQE plasmid with the T5 promoter (pQE-MEP) using primers dxsidiispDFNcoI(s) and dxsidiispDFXhoI(a). A fraction of the operon DNA with the T5 promoter was amplified using primers T5AgeI(s) and T5NheI(a) from the pQEMEP plasmid. The DNA fragment was digested with AgeI/NheI and cloned into p20T7MEP plasmid digested with SGrAI/NheI enzymes.

<b>taxadiene</b><b> Route (</b><b>GT</b><b> and </b><b>TG</b><b></b><b>operon</b><b>) of the construction.</b>

The downstream taxadiene pathways (GT and TG operons) are the primers GGPPSNcoI(s), GGPPSEcoRI(a), TSEcoRI(s), TSsalI(a), TSNcoI(s), TSEcoRI(a), GGPPSEcoRI(s) and GGPPSSalI(a). ) was used to create p20TrcGT and p20TrcTG by cloning the PCR fragments of GGPS and TS into the Nocl-EcoRI and EcoRI-SalI sites of the pTrcHIS2B plasmid. To construct the p20T5GT, the operon was initially amplified with primers GGPPSNcoI(s) and TSXhoI(a) and cloned into the pQE plasmid under the T5 promoter digested with NcoI/XhoI. Additional sequences were digested with XbaI and XhoI and cloned into the amplified pTrc plasmid backbone using primers pTrcSal(s) and pTrcXba(a). p10T7TG was constructed by subcloning the NcoI/SalI digested TG operon from p20TrcTG into the NcoI/SalI digested pACYC-DUET1 plasmid. p5T7TG was constructed by cloning the BspEI/XbaI digested fragment into XbaI/BspEI digested DNA amplified from the pCL1920 plasmid using pCLBspEI(s) and pCLXbaI(a) primers.

<b>chromosomal integration </b><b>MEP</b><b> Construction of pathway plasmids</b>

To construct a plasmid with the FRP-Km-FRP cassette to amplify the sequence for integration, p20T7MEP and p20T5MEP were digested with XhoI/ScaI. The FRP-Km-FRP cassette was amplified from the Km cassette with the FRP sequence from the pkD13 plasmid using primers KmFRPXhoI(s) and KmFRPScaI(a). The amplified DNA was digested with XhoI/ScaI and cloned into XhoI/ScaI digested p20T7MEP and p20T5MEP plasmids (p20T7MEPKmFRP and p20T5MEPKmFRP). Similarly, the p20TrcMEP plasmid was digested with SacI/ScaI, the amplified DNA was digested using primers KmFRPSacI(s) and KmFRPScaI(a) and cloned into the p20TrcMEP plasmid (p20TrcMEPKm-FRP).

<b>MEP</b><b> path cassette (</b><b>LacIq</b><b>-</b><b>MEP</b><b>-</b><b>FRP</b><b>-</b><b>km</b><b>-</b><b>FRP</b><b>) of chromosomal integration</b>

The MEP pathway, built under promoters T7, T5 and Trc, was localized in the region of the ara operon in the chromosome with the Kan marker. PCR fragments were amplified from p20T7MEPKmFRP, p20T5MEPKmFRP and p20TrcMEPKm-FRP using primers IntT7T5(s), IntTrc(s), and Int(a), followed by E. red. coli MG1655 recA-end- and E. coli. Electroporated into E. coli MG1655 recA-end-EDE3 cells⁵⁴. Site-specific localization was confirmed, and the Km marker was removed through the action of FLP recombinase after successful gene integration.

<b>taxadiene</b><b> Construction of the 5α-ol pathway</b>

The transmembrane regions (TM) of taxadiene 5α-ol hydroxylase (T5αOH) and taxus cytochrome P450 reductase (TCPR) were identified using PredictProtein software (www.predictprotein.org).⁵⁵. For transmembrane manipulation, selective truncation was performed at 8, 24 and 42 amino acid residues on the N-terminal transmembrane region of taxadien 5α-ol hydroxylase (T5αOH) and at the 74 amino acid region in TCPR. Removal of 8, 24 and 42 residues N-terminal amino acids of taxadiene 5α-ol hydroxylase (T5αOH), 1 amino acid substituted bovine 17α hydroxylase N-terminal 8 residues peptide MALLLAVF (SEQ ID NO:51) N-terminal truncated T5αOH sequence of⁴⁴ and integration into the GSTGS peptide linker was performed using primers CYP17At8AANdeI(s), CYP17At24AANdeI(s), CYP17At42AANdeI(s) and CYPLinkBamHI(a). Each of the modified DNAs was amplified using the above primers, digested with NdeI/BamHI, and cloned into NdeI/BamHI digested pACYC DUET1 plasmids to construct p10At8T5αOH, p10At24T5αOH and p10At42T5αOH plasmids. A 74 amino acid truncated TCPR (tTCPR) sequence was amplified using primers CPRBamHI(s) and CPRSalI(a). The amplified tTCPR sequence and plasmids p10At8T5αOH, p10At24T5αOH and p10At42T5αOH were digested with BamHI/SalI and cloned to construct plasmids p10At8T5αOH-tTCPR, p10At24T5αOH-tTCPR and p10At42T5αOH-tTCPR5αOH-tTCPR.

<b>Taxadiene</b><b> culture growth for screening and </b><b>taxadiene</b><b>-5α-ol analysis</b>

Pre-engineered E. coli with appropriate plasmids in which the upstream (MEP), downstream taxadiene pathway and taxadien 5α-ol are present. Single transformants of E. coli strains were placed in Luria-Bertani (LB) medium (appropriate antibiotics, 100 mg/mL carbenicillin, 34 mg/mL chloramphenicol, 25 mg/L kanamycin or 50 mg/L spec. supplemented with tinomycin) at 30° C. for 18 h. For small-scale cultures to screen engineered strains, a starting A of 0.1 was used using the above preinoculum.₆₀₀fresh 2-mL enrichment medium (5 g/L yeast extract, 10 g/L tryptone, 15 g/L glucose, 10 g/L NaCl, 100 mM HEPS, 3 mL/L Antifoam B, pH 7.6 , 100 μg/mL carbenicillin and 34 μg/mL chloramphenicol). Cultures were maintained at 22° C. for 5 days with appropriate antibiotics and 100 mM IPTG for gene induction.

<b>taxadiene</b><b> Bioreactor experiments for 5α-ol producing strains.</b>

A 3-L Bioflo bioreactor (New Brunswick) was assembled according to the manufacturer's instructions. Strain 26-At24T5αOH-tTCPR grown in LB medium containing the same concentration of antibiotics (100 mg/mL carbenicillin, 34 mg/mL chloramphenicol) in 1 liter of enriched medium with 1% glycerol (v/v) 50 mL of 8 h culture (approximately 2.2 A₆₀₀) was inoculated. 1 L-bioreactor with two-phase liquid-liquid fermentation with 20% v/v dodecane. Oxygen was supplied as filtered air at 0.5 v/v/m and agitation was adjusted to maintain dissolved oxygen levels above 50%. The pH of the culture was controlled to 7.0 using 10% NaOH. The temperature of the culture broth in the fermenter was controlled at 30° C. until the cells grew to an optical density (OD600) of about 0.8 measured at a wavelength of 600 nm. The temperature of the fermenter was reduced to 22° C. and cells were induced with 0.1 mM IPTG. Dodecane was added aseptically at 20% (v/v) of the medium volume. During the fermentation process, the concentration of glycerol and the accumulation of acetate were monitored at regular time intervals. During fermentation, glycerol (3 g/L) was introduced into the bioreactor as the glycerol concentration was depleted below 0.5 g/L.

Defined feed medium containing 0.5% yeast extract and 20% (v/v) dodecane (13.3 g/L KH₂PO₄, 4 g/L (NH₄)₂HPO₄, 1.7 g/L citric acid, 0.0084 g/L EDTA, 0.0025 g/L CoCl₂, 0.015 g/L MnCl₂, 0.0015 g/L CuCl₂, 0.003 g/LH₃BO₃, 0.0025 g/L Na₂MoO₄, 0.008 g/L Zn(CH₃COO)₂, 0.06 g/L Fe(III) citrate, 0.0045 g/L Thiamine, 1.3 g/L MgSO₄, 10 g/L glycerol, 5 g/L yeast extract, pH 7.0) was used to further optimize fermentation. The same medium composition was used for fermentation of strains 17 and 26 with the appropriate antibiotics (strain 17: 100 μg/mL carbenicillin and 50 μg/mL spectinomycin; strain 26: 50 μg/mL spectinomycin).

For the taxadien-5α-ol producing strain, strain 26 grown in LB medium containing 50 μg/mL spectinomycin and 34 μg/mL chloramphenicol in 1 liter of complex medium with 1% glycerol (v/v) -At24T5αOH-tTCPR was inoculated with 50 mL of an 8h culture (OD of about 2.2). Oxygen was supplied as filtered air at 0.5 (vvm) and agitation was adjusted to maintain dissolved oxygen levels above 30%. The pH of the culture was controlled to 7.0 using 10% NaOH. The temperature of the culture broth in the fermenter is controlled at 30° C. until the cells grow to an optical density (OD600) of about 0.8 measured at a wavelength of 600 nm. The temperature of the fermenter was reduced to 22° C. and the route was induced with 0.1 mM IPTG. Dodecane was added aseptically at 20% (v/v) of the medium volume. During the fermentation process, the concentration of glycerol and the accumulation of acetate were monitored at regular time intervals. During fermentation, 3 g/L of glycerol was introduced into the bioreactor as the glycerol concentration was depleted to 0.5-1 g/L.

<b>taxadiene</b><b> and </b><b>taxadiene</b><b>of -5α-ol </b><b>GC</b><b>-</b><b>MS</b><b> analysis</b>

For analysis of taxadiene accumulation from small-scale cultures, 1.5 mL of culture was vortexed with 1 mL hexane for 30 min. The mixture was centrifuged to separate the organic layer. For the bioreactor, 1 μl of the dodecane layer was diluted to 200 μl with hexane. 1 μl of the hexane layer was analyzed by GC-MS (Varian saturn 3800 GC connected to a Varian 2000 MS). Samples were injected into a HP5ms column (30 mx 250 uM x 0.25 uM thick) (Agilent Technologies USA). Helium (ultra-pure) was used as carrier gas at a flow rate of 1.0 ml/min. The oven temperature was first held at 50° C. for 1 min, then increased to 220° C. in increments of 10° C./min, and finally held at this temperature for 10 min. The injector and pipeline temperatures were set at 200°C and 250°C, respectively.

Standard compounds from biological or synthetic sources for taxadiene and taxadien 5α-ol were not commercially available. Therefore, we produced taxadiene in a 2 L bioreactor to extract pure material E. Fermentation of E. coli was carried out. After the taxadiene was extracted by solvent extraction using hexane, silica column chromatography was performed multiple times to obtain a pure material for preparing a standard curve for GC-MS analysis. The present inventors compared the GC and MS profiles of pure taxadiene with the reported literature to confirm the reliability of the compound.⁶⁰. To check the purity, we performed 1 HNMR of taxadiene. Since the accumulation of taxadien-5α-ol is very low, we used taxadiene as a measure to quantify the production of this molecule and the reliable mass spectral fragmentation properties from previous reports.⁴².

<b>For transcriptional analysis of engineered strains </b><b>qPCR</b><b> measurement</b>

Transcriptional gene expression levels of each gene were detected by qPCR on mRNA isolated from the appropriate strain. To prevent degradation, RNA was stabilized prior to cell lysis using RNAprotect bacterial reagent (Qiagen). Subsequently, total RNA was isolated using the RNeasy mini kit (Qiagen) in combination with nuclease removal of genomic DNA contaminants. cDNA was amplified using the iScript cDNA synthesis kit (Biorad). qPCR was performed on a Bio-Rad iCycler using an iQ SYBR Green Supermix (BioRad). not applied to variable expression<i>rrsA</i> Expression levels of genes were used for normalization of qPCR values.⁵⁶. Table 3 shows the primers used for qPCR. For each primer pair, E. A standard curve was created using the mRNA of E. coli.

<b>Example</b><b> 1: </b><b>taxadiene</b><b> accumulation is upstream </b><b>MEP</b><b> and downstream synthesis </b><b>taxadiene</b><b> It shows a strong non-linear dependence on the relative strength of the path.</b>

1B shows various methods of combining promoter and gene copy number to modulate the relative flux (or intensity) through the upstream and downstream pathways of taxadiene synthesis. A total of 16 strains were constructed to solve the bottleneck of the MEP pathway and optimally balance this pathway with the downstream taxadiene pathway. Figure 2a,b summarizes the results of taxadiene accumulation in each of the above strains, Figure 2a highlights the dependence of taxadiene accumulation on the upstream pathway for a constant value of the downstream pathway, and Figure 2b shows the constant upstream pathway intensity. (See also Table 1 for calculation of upstream and downstream pathway expression from reported promoter strengths and plasmid copy numbers.)³³^-36). Clearly, there are visible maxima for both upstream and downstream pathway expression. For constant downstream pathway expression ( FIG. 2A ), as upstream pathway expression increases from very low levels, taxadiene production initially increases due to increased supply of precursors to the entire pathway. However, after the median, further upstream pathway increases cannot be accommodated by the capacity of the downstream pathway. This pathway imbalance results in the accumulation of intermediates (see below) that may be inhibitory to the cell or simply represent flux diversion to competing pathways that ultimately lead to reduced taxadiene accumulation.

For constant upstream pathway expression ( FIG. 2B ), maxima are similarly observed for downstream pathway expression levels. This is due to the initial limitation of taxadiene production by the low expression level of the downstream pathway, i.e. the rate limitation for taxadiene production. At high levels of downstream pathway expression, we are likely to see a negative effect of high copy number on cell physiology, and thus a maximum exists for downstream pathway expression. These results demonstrate that dramatic changes in taxadiene accumulation can be obtained from within a narrow range of changes in expression levels for the upstream and downstream pathways. For example, strains containing an additional copy of the upstream pathway on their chromosomes under the control of the Trc promoter (strain 8, FIG. 2A ) have 2000-fold more taxa than those overexpressing only the synthetic downstream pathway (strain 1, FIG. 2A ). diene was produced. Also, changing the sequence of genes in the downstream synthetic operon from GT (GPPS-TS) to TG (TS-GPPS) resulted in a 2-3 fold increase in production (strains 1-4 compared to strains 5, 8, 11 and 14). ). The observed results show that the key to taxadiene overproduction is sufficient downstream pathway capacity and careful balancing between the upstream precursor pathway and the downstream synthetic taxadiene pathway. Altogether, engineered strains established that MEP pathway fluxes could be significant if broad expression levels for endogenous upstream and synthetic downstream pathways were simultaneously searched for.

<b>Example</b><b> 2: Chromosomal integration and fine-tuning of upstream and downstream pathways </b><b>taxadiene</b><b> further improve production.</b>

To provide sufficient downstream pathway strength while minimizing the metabolic load by the plasmid³⁷, 2 new sets of 4 strains in which the downstream pathway is under the control of a strong promoter (T7), respectively, while maintaining relatively low copy numbers of 5 and 10, respectively (strains 25-28 and 29-32) were engineered. It can be seen that while maintaining the taxadiene maximum at high downstream intensities (strains 21-24), a monotonic response is obtained at low downstream path intensities (strains 17-20, FIG. 2c) ( FIG. 2c ). Because of these observations, two additional sets of four strains (strains 25-28 and 29-32, Figure 2d) were constructed, each maintaining the same level of downstream pathway strength as before but expressing very low levels of the upstream pathway. . In addition, the operon of the upstream pathway of the latter set of strains was integrated into the chromosome. It can be seen that not only does the taxadiene maximum recover, but much higher taxadiene maximums (300 mg/L) are obtained despite the very low upstream pathway level. We believe that this significant increase may be due to a decrease in the metabolic load of the cells. This was achieved by 1) elimination of plasmid dependence through pathway integration into chromosomes and 2) obtaining a good balance between upstream and downstream pathway expression.

With 32 recombinant constructs, we were able to properly investigate the modular pathway expression space and improve taxadiene production by about 15000 fold. This is reported. It is by far the highest terpenoid production from the E. coli MEP isoprenoid pathway ( FIG. 3A ). Additionally, the observed fold improvement in terpenoid production is significantly higher than that of the reported combinatorial metabolic engineering methods that search the extensive gene space containing billions of combinatorial modifications of the isoprenoid pathway.³⁰. This suggests that pathway optimization is much more dependent on good balancing of pathway module expression than multi-source combinatorial gene optimization. The multiple maxima seen in the phenotypic context of Figure 1 underscores the importance of examining the expression space with sufficient analysis to identify regions of optimal overall pathway performance. 7 shows fold improvement in taxadiene production from a modular pathway expression search.

<b>Example</b><b> 3: </b><b>metabolites are</b><b></b><b>taxadiene</b><b> production and </b><b>of metabolites</b><b> confirmation </b><b>inverse proportion</b><b> show the relationship</b>

Metabolic analysis of previously engineered strains identified hitherto unknown metabolite byproducts with strong correlations with pathway expression levels and taxadiene production ( FIGS. 3 and 8 ). Although the chemical class of the metabolite is unknown, we hypothesized that it is an isoprenoid by-product produced by pathway conversion and exhibits an anti-correlation as a direct variable for taxadiene production from the engineered strain (Fig. 3 and Fig. 8). A key feature of our optimal strain is a good balancing act that alleviates the accumulation of the metabolites, leading to more taxadiene production. This balancing can be adjusted at different levels from chromosomes showing significantly different taxadiene accumulation, or from plasmids of different copy numbers using different promoters.

Subsequently, the corresponding peaks in the gas chromatography-mass spectrometry (GC-MS) chromatogram are GC-MS, ¹H and ¹³C was identified as an indole in nuclear magnetic resonance (NMR) spectroscopy studies (Fig. 16). We found that taxadiene synthesis by strain 26 was significantly inhibited by exogenous indole at indole levels higher than about 100 mg/L ( FIG. 15b ). In addition, further increasing the indole concentration inhibited cell growth, and the level of inhibition was highly strain dependent ( FIG. 15c ). Although the biochemical mechanism for the isoprenoid pathway and indole interaction is currently unclear, the results in Figure 15 suggest a possible synergistic effect between indole and terpenoid compounds of the isoprenoid pathway when inhibiting cell growth. Although the specific mechanism is not known, strain 26 appears to ameliorate the effects of indoles, and we have pursued further studies.

<b>Example</b><b> 4: Cultivation of engineered strains.</b>

To investigate the taxadiene production potential under controlled conditions for the engineered strains, the three highest taxadiene accumulating strains (about 60 mg/L from strain 22; about 125 mg/L from strain 17; of about 300 mg/L) of fed-batch culture was performed in a 1 L-bioreactor ( FIG. 17 ). Fed-batch culture studies were performed as liquid-liquid two-phase fermentations using a 20% (v/v) dodecane overlay. An organic solvent was introduced to prevent air stripping of secreted taxadiene from the fermentation medium as indicated by preliminary findings. In defined media with controlled glycerol feed, taxadiene productivity was 174 ± 5 mg/L (SD), 210 ± 7 mg/L (SD), and 1020 ± 80 mg/L (SD) for strains 22, 17 and 26, respectively. SD) (Fig. 17a). Additionally, taxadiene production had a significant effect on growth phenotype, acetate accumulation and glycerol consumption ( FIGS. 17B-17D ).

Figure 17c shows that initially acetate accumulates in all strains, but after about 60 hrs acetate decreases in strains 17 and 26 while continuing to increase in strain 22. This phenomenon highlights the difference in central carbon metabolism between high MEP flux strains (26 and 17) and low MEP flux strains (22). Additionally, this observation is another example of good physiology characterizing well-balanced functional strains. Acetic acid as a product of hypermetabolism is initially produced by all strains because of the high initial glycerol concentrations and correspondingly high glycerol pathway fluxes used for the fermentation. The flux is sufficient to supply the MEP pathway, and other metabolic pathways, in the cell.

At about 48 hrs, the initial glycerol is depleted and the culture is switched to fed-batch, during which a low but constant glycerol level is maintained. This results in a low total glycerol flux that is mostly redirected to the MEP pathway with minimal over-metabolism for strains with high MEP flux (strains 26 and 17). As a result, acetic acid production is reduced or even eliminated entirely. For the decrease in acetic acid concentration, some degree of acetic acid assimilation may have occurred, but this was not further investigated from a flux analysis point of view. Some evaporation and dilution with glycerol feed further contributes to the observed decrease in acetic acid concentration. In contrast, for the strain with low MEP flux (strain 22), the flux conversion to the MEP pathway is not very significant, so the glycerol flux continues to supply all necessary carbon and energy requirements. Excessive metabolism continues to occur, resulting in acetate secretion.

Obviously, the high productivity and more robust growth of strain 26 allowed for very high taxadiene accumulation. Further improvements will be possible through optimization of conditions in the bioreactor, balancing of nutrients in the growth medium, and optimization of carbon transfer.

<b>Example</b><b> 5: Upstream and downstream pathway expression levels and cell growth reveal fundamental complexity.</b>

For a more detailed understanding of the engineered balance of pathway expression, we present dxs (upstream pathway) and Transcriptional gene expression levels of TS (downstream pathway) were quantified (Fig. 4a,b). As we hypothesized, expression of the upstream pathway was achieved using the native promoter, Trc, T5, T7, and promoter strength and copy number for MEP vectors from 10 and 20 copy plasmids as observed for DXS expression. monotonically increased (Fig. 4a). Thus, we found that dxs expression levels were highly correlated with upstream pathway strength. Similar associations with other genes in the upstream pathway<i>idi</i>,<i></i><i>ispD</i><i></i>and<i></i><i>ispF</i>was found for (Fig. 14a, b). In downstream gene expression, about 2-fold improvement was quantified after passing the pathway from 5 to 10 copy plasmids (25-28 series and 29-32 series) ( FIG. 4B ).

Although promoter and copy number effects affected gene expression, secondary effects on expression of other pathways were also significant. Figure 4a shows that dxs expression was increased by increasing the copy number of the TS plasmid from 5 to 10 for the same dxs expression cassette. Interestingly, the 5 copy TS plasmid (strain 25-28 series) showed substantially higher taxadiene yield (Fig. 2d) and smaller growth (Fig. 4c,d) than the 10 copy TS plasmid. The control plasmid that did not contain the taxadiene heterologous pathway grew to a 2-fold higher density, meaning that growth inhibition in the strain 25-28 series was directly related to the taxadiene metabolic pathway and the accumulation of taxadiene and its direct intermediates ( Fig. 4c). However, strain 29-32 series showed moderately increased growth rate when the empty control plasmid was compared with the taxadiene expressing strain ( FIG. 4d ). This interaction between growth, taxadiene production, and expression levels can be observed in plasmid-based upstream expression vectors (strains 17 and 22). Growth inhibition was much greater in 10 copies of high taxadiene producing strain (strain 17) compared to 20 copies of lower taxadiene producing strain (strain 22) ( FIG. 4d ). Thus, product toxicity and carbon conversion into heterologous pathways are likely to impede growth rather than plasmid-maintenance.

In addition, a profound effect of the upstream expression vector on downstream expression was not expected. Figure 4b will have two straight lines if there is no crosstalk between the paths. However, about a fold change in TS expression was observed for the different MEP expression vectors. This appears to be due to significant competition for resources (raw materials and energy) extracted from host metabolism for overexpression of both four upstream and two downstream genes.³⁸. Compared to control strain 25c, a 4-fold growth inhibition was observed in strain 25, indicating that high overexpression of the synthetic taxadiene pathway induced toxicity that alters the growth phenotype compared to overexpression of the native pathway (Fig. 4c). ). However, as upstream expression increased, downstream expression decreased to the desired level in our study to minimize growth inhibition by balancing the upstream and downstream pathways (strain 26).

Upon extreme protein overexpression, the T7 promoter-driven MEP pathway resulted in severe growth inhibition due to high levels of synthesis of four proteins (strains 28 and 32). Expression of the TS gene by T7 does not appear to have a drastic effect on its own. A high rate of protein synthesis by T7-induced expression (Fig. 4ab) can lead to downregulation of protein synthesis machinery, including components of housekeeping genes from the early growth phase, impairing cell growth and lowering the increase in biomass.³⁹^,40. We hypothesized that the complex growth phenotype observed by us was a cumulative effect of (1) toxicity induced by activation of isoprenoid/taxadiene metabolism, and (2) the effect of high recombinant protein expression. Altogether, our multivariate-modular pathway engineering method resulted in unexpected diversity of terpenoid metabolism and its association with pathway expression and cellular physiology. Rational design of microorganisms for secondary metabolite production will require an understanding of pathway expression that goes beyond a linear/independent understanding of promoter strength and copy number. However, a simple multivariate method as used herein (1) discovers high producers and (2) provides a context for systematic investigation of dominant but underestimated higher-order effects in metabolic pathway manipulation. can be introduced.

<b>Example</b><b> 6: E. </b><b>in coli</b><b></b><b>Taxol</b><b></b><b>P450</b><b>Manipulation of Systemic Oxidation Chemistry</b>

The most important feature of the biosynthesis of Taxol is oxygenation at multiple sites in the taxane core structure, a reaction thought to be mediated by cytochrome P450-dependent monooxygenase.⁴¹. After completion of the critical cyclization step of this pathway, the parent olefin, taxa-4(5),11(12)-diene, is then hydroxylated at the C5 position by the cytochrome P450 enzyme, which is 1 of 8 oxygenation steps (for the core) is shown (Figure 6)⁴². Thus, a key step for the manipulation of taxol-producing microorganisms is the development of P450-based oxidation chemistries in vivo. The first oxygenation step is catalyzed by cytochrome P450, taxadiene 5α-hydroxylase, a rare monooxygenase that catalyzes the hydroxylation reaction with double bond migration in the diterpene precursor taxadiene ( FIG. 5A ). We report for the first time the successful extension of the synthetic pathway from taxadiene to taxadiene-5α-ol, and E. We present the first example of in vivo production of any functionalized Taxol intermediate in E. coli.

In general, functional expression of plant cytochrome P450 is problematic by the intrinsic limitations of bacterial platforms, such as the translational incompatibility of the membrane signaling module of the P450 enzyme by the electron transport machinery, the absence of cytochrome P450 reductase, and the lack of the endoplasmic reticulum.⁴³. Recently, through transmembrane (TM) manipulation and generation of chimeric enzymes of P450 and CPR reductase, P450 in some plants has been converted into E. coli for the biosynthesis of functional molecules. expressed in E. coli²²^,44. In addition, all plant cytochrome p450s are unique in their transmembrane signal sequence and electron transport properties from their reductase counterparts.⁴⁵. Our initial study was the optimization of codon-optimized synthetic taxadiene 5α-hydroxylase expression by N-terminal transmembrane manipulation and the CPR redox partner from Taxus species, Taxus cytochrome P450 reductase (TCPR). We focused on the generation of chimeric enzymes via translational fusion with (Fig. 5b)^42,44,46. One of the resulting chimeric enzymes, At24T5αOH-tTCPR, was very efficient in carrying out the first oxidation step, including the taxadien-5α-ol of taxadiene and the by-product 5(12)-oxa-3(11)-cyclotaxane (OCT). ) was greater than 98% (Fig. 9a).

Compared to other chimeric P450s, At24T5αOH-tTCPR produced a 2-fold higher (21 mg/L) taxadien-5α-ol. In addition, the weaker active At8T5αOH-tTCPR and At24T5αOH-tTCPR have a complex structural reorganization of the recently characterized byproduct, the cyclic ether 5(12)-oxa-3(11)-cyclotaxane (OCT) of taxadiene. Accumulation of the array was induced ( FIG. 9 ).⁴⁷. The by-product accumulated in approximately the same amount as the desired product taxadien-5α-ol. OCT formation was mediated by an unprecedented sequence of Taxus cytochrome P450 reactions involving oxidation and subsequent cyclization.⁴⁷. Thus, by protein engineering of taxadiene 5α-hydroxylase, termination of the reaction prior to cyclization will prevent the accumulation of this undesirable by-product, and conversion of the flux towards taxadien-5α-ol may be achieved. seems to be

The productivity of strain 26-At24T5αOH-tTCPR was significantly reduced compared to taxadiene production by the parent strain 26 (ca. 300 mg/L), while at the same time the accumulation of previously described uncharacterized metabolites increased. No taxadiene accumulation was observed. Obviously, the introduction of an additional intermediate copy number plasmid (10 copies, p10T7) carrying the At24T5αOH-tTCPR construct disrupted the carefully engineered balance of the upstream and downstream pathways in strain 26. A small scale fermentation was performed in a bioreactor to quantify alcohol production by strain 26-At24T5αOH-tTCPR. The time course profile of taxadien-5α-ol accumulation ( FIG. 5D ) shows alcohol production up to 58 ± 3 mg/L and production of equivalent OCT byproducts. The observed alcohol production was S. about 2400 times higher than the previous production in cerevisiae¹⁷. Further increases in taxadien-5α-ol production are likely possible through pathway optimization and protein engineering.

A multivariate-module approach of pathway optimization has resulted in strains that produce very high amounts of the key Taxol precursor. In addition, recombinant constructs can be used to direct fluxes in the direction of synthesis of other complex pharmaceutical compounds engineered from the same route, such as mono-, sesqui- and di-terpenes (geraniol, linalool, amorphadiene and levopimaradiene). was equally effective when turned on (unpublished results). Thus, our pathway engineering presents a new means for biosynthesis of natural products, particularly in terms of terpenoids derived from microorganisms for use as fuels and chemicals from renewable sources. By focusing on the universal terpenoid precursors IPP and DMAPP, we first define key pathway modules, then modulate expression, such as optimizing the balance of pathway modules for seamless precursor conversion and minimal intermediate accumulation. it was possible This approach appears to be more effective than combinatorial searches of large gene spaces and does not rely on high-throughput screening.

The MEP-pathway is effectively balanced and thus is overall more efficient in the conversion of glucose or glycerol to isoprenoids. However, over the past decade, carotenoids²⁸^,47, sesquiterpenoids²³ and diterpenoids⁶¹ To increase the supply of important precursors IPP and DMAPP for overproduction E. Many attempts to manipulate the MEP-pathway in E. coli have had limited success. This inefficiency is It was due to an unknown regulatory effect specifically associated with the expression of the MEP-pathway in E. coli.²³. Here, we provide evidence that inhibition of isoprenoid pathway activity correlates with accumulation of metabolite indole by non-optimal expression of the pathway. Taxadiene overproduction (under conditions of inhibition of indole formation) established the MEP-pathway as a highly efficient pathway for the biosynthesis of pharmaceutical and chemical products of the isoprenoid family. This simply requires careful balancing of the modular paths as suggested by our multivariate-module path manipulation approach.

For the successful microbial production of Taxol, it is essential to demonstrate the chemical decoration of the taxadiene core by P450-based oxidation chemistry.⁴¹. Cytochrome P450 monooxygenase constitutes about one-half of the 19 distinct enzymatic steps in the Taxol biosynthetic pathway. Characteristically, the genes show unusual high sequence similarity (&gt;70%) to each other, but low similarity (&lt;30%) to other plant P450s.¹⁴. Because of the apparent similarity between taxol monooxygenases, expressing the appropriate activity to carry out specific P450 oxidation chemistries has been particularly problematic. Through TM engineering and construction of an artificial chimeric enzyme with a redox partner (TCPR), taxol cytochrome P450, taxadiene 5α-hydroxylase, was produced in E. It is functionally expressed in E. coli and has been shown to efficiently convert taxadiene to the corresponding alcohol product in vivo. Previous in vitro studies have elucidated the mechanism of conversion of taxadiene to taxadien-5α-ol by the native taxadiene 5α-hydroxylase enzyme, but have not discussed the same conversion in vivo.⁴². This oxygenation and rearrangement reaction involves the formation of an allyl radical intermediate by hydrogen extraction from the C20 position of taxadiene, followed by the production of an alcohol derivative by positional and stereospecific insertion at the C5 position (Fig. 5a). Moderate abundance of enzymes observed in Taxus cells, and low<i>k</i>_cat Values suggest that the 5α-hydroxylation step of Taxol biosynthesis is slow compared to downstream oxygenation and acylation of the Taxol pathway⁴¹. Thus, manipulation of this step is particularly important for prokaryotic hosts, such as E. It is important for Taxol synthesis in terms of functional manipulation of Taxol P450 in E. coli. Additionally, this step has been a limitation of previous efforts to establish pathways in yeast.¹⁷. The engineered construct in this study showed &gt;98% conversion of taxadiene in vivo with product accumulation of about 60 mg/L, a 2400-fold improvement over previous heterologous expression in yeast. This study therefore not only succeeded in the synthesis of significantly higher amounts of the important Taxol intermediate, but also provides a basis for the synthesis of subsequent metabolites in a similar P450 chemistry pathway.

Previous studies of the structure-activity relationship for Taxol have shown that alteration by removal or addition of some of its functional groups does not substantially alter the activity of Taxol.^1,48. However, these studies have been limited due to their limited ability to introduce changes by chemical synthesis. The availability of microbial pathways for Taxol synthesis will greatly expand the chemical modification space that can be investigated, increasing the probability of identifying more potent drug candidates. This offers exciting new opportunities for drug development, especially considering that the drug candidates will also be associated with efficient production pathways.

Over the past few decades, Taxol has attracted more attention to the scientific community and the general public than any other natural product drug candidate.¹⁰. A large supply crisis is foreseen in light of the expected increase in the expected use of Taxol or Taxol analogs for cancer chemotherapy, which necessitates the manipulation of new production pathways, such as the Taxol biosynthetic machinery in microorganisms.⁸. Although several endogenous fungi of the Taxus species have been isolated that are capable of producing Taxol in nature, the microbial system must demonstrate suitability for sustained production of drugs.⁴⁹. The results reported herein represent a disruptive step towards microbial derived Taxol or Taxol precursors by removing bottlenecks in critical precursor pathways. In addition, the combination of synthetic pathways opens up new possibilities for custom production of taxol analogs by selectively manipulating the pathways to alter the taxane structure. These advances allow optimization of microbial pathways for cost-effective production of Taxol or suitable Taxol precursors.

Table 1 Assessment of upstream and downstream pathway expression in arbitrary units (au). MEP pathway and GGPP synthase/taxadiene synthase pathway expression levels were estimated using published values of promoter strength and copy number. Promoter strength is determined by Brosius et al. and Brunner et al.^33,34was calculated as trc=1, T5=1.96, and T7=4.97 based on . The gene copy number was assigned the published copy number for the origin of replication of the different plasmids used, and one copy was used for integration.³⁵^-37. Total expression was calculated as the product of promoter strength and gene copy number. A value of 1 was arbitrarily assigned to native expression of the MEP pathway, and alteration of the operon sequence of GGPP synthase and taxadiene was hypothesized to affect taxadiene synthase expression by 20%.³⁵. These total expression estimates guided the manipulation effort. E - E. with two deletions ΔrecAΔendA. coli K12 MG1655; EDE3 - K12 MG1655 ΔrecAΔendA with integrated T7 RNA polymerase (DE3); MEP - dxs-idi-ispDF operon; GT - GPPS-TS operon; TG - TS-GPPS operon; Ch1 - 1 copy within the chromosome; Trc - the trc promoter; T5 - T5 promoter; T7 - T7 promoter; p5 - ~5 copy plasmid (pSC101); p10 - ˜10 copy plasmid (p15A); and p20 - -20 copy plasmid (pBR322).

<tables num="1"><img file="KR20120101445A_D0001.tif" /></tables>

<img file="KR20120101445A_D0002.tif" />

&lt;Table 2&gt; Details of all plasmids constructed for the study

<img file="KR20120101445A_D0003.tif" />

&lt;Table 3&gt; Details of primers used for cloning of plasmids, chromosomal transfer of MEP pathway, and qPCR measurement

<img file="KR20120101445A_D0004.tif" />

<img file="KR20120101445A_D0005.tif" />

<img file="KR20120101445A_D0006.tif" />

&lt;Table 4&gt; Protein and codon optimized nucleotide sequences.

<img file="KR20120101445A_D0007.tif" />

<img file="KR20120101445A_D0008.tif" />

<img file="KR20120101445A_D0009.tif" />

<img file="KR20120101445A_D0010.tif" />

<b>references</b>

<img file="KR20120101445A_D0011.tif" />

<img file="KR20120101445A_D0012.tif" />

<img file="KR20120101445A_D0013.tif" />

<img file="KR20120101445A_D0014.tif" />

Having described several aspects of at least one embodiment of the present invention, it should be understood that various changes, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure and are intended to be within the spirit and scope of the present invention. Accordingly, the above description and drawings are presented by way of example only. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following claims.

All references disclosed herein are incorporated by reference in their entirety for the specific purpose set forth herein.