Acyl-acp reductase with improved properties.

(54) Title: ACIL-ACP REDUCED WITH IMPROVED PROPERTIES.

(54) Title: ACYL-ACP REDUCTASE WITH IMPROVED PROPERTIES.

(57) Summary

The present invention relates to variants of the acyl-ACP reductase (AAR) enzyme that result in improved fatty aldehyde and fatty alcohol production when expressed in recombinant host cells. The description further relates to methods for making and using such AAR variants for the production of fatty alcohol compositions having particular characteristics.

(57) Abstract

The disclosure relates to acyl-ACP reducíase (AAR) enzyme variants that result in improved fatty aldehyde and fatty alcohol production when expressed in recombinant host cells. The disclosure further relates to methods of making and using such AAR variants for the production of fatty alcohol compositions having particular characteristics.

Institute Π · Ρ ”Ί ^ · Mexican Q Γ of Property Q [jiJH · Industrial B

PATENT TITLE NO. 346830

Owner (s): REG LIFE SCIENCES, LLC

Address: 600 Gateway Blvd. South, San Francisco, California, 94080, USA

Name: ACIL-ACP REDUCTASA WITH IMPROVED PROPERTIES.

Classification: lnt.CI.8: C12N15 / 53: C12N9 / 02; C12P7 / 04.

Inventor (s): MATHEW RUDE; NA TRINH; ANDREAS SCHIRMER; JACOB WIN

II

PRIORITY "

January 1, 2013 January 2034

Date:

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Country:

US patent of reference granted Industrial extension, effective

Number:

MX / a / 2015/009138 «a» · · ίΐ »· e Law of 05/17/1999, 3^or fraction V reformed the Organic tute of the Mexican Institute of Industrial Property (DOF 12/27/1999, amended 10/10/2002, 07/29/2004, 08/04/2004 and 09/13/2007); 1^or, 3rd and 5^or subsection a) of the Agreement that delegates powers to the Deputy General Directors, Coordinator, Divisional Directors, Holders of the

Filing date within January 2014

Validity: Twenty years

Due date:

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ass 23 to pe title

Office 5/01/20 nes I

8/07/20

We conform to the tempted from the rights

Who subscribes the Industrial property (D * 'Ό1 / 2004, 06/16/2005

Regional Offices, Divisional Deputy Directors, Departmental Coordinators and other subordinates of the Mexican Institute of Industrial Property. (DOF 12/15/1999, amended on 02/04/2000, 07/29/2004, 08/04/2004 and 09/13/2007).

Issue Date: April 3, 2017

THE DIVISIONAL DIRECTOR OF PATENTS

<img file="MX346830B_D0003.tif" />

NAHANNY CANAL REYES

Arenal No. 550. Floor 1 Coi. Santa María Tepepan Town, Xochimdco. CP 13020.

Mexico City Tel · (55j 53 34 07 00 wwwjmpi aob.mx

11111111111111

MX / 2017/29542

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ACIL-ACP REDUCED WITH IMPROVED PROPERTIES

FIELD OF THE INVENTION

The description refers to variants of the enzyme acylACP reductase (AAR) that result in improved production of fatty aldehyde and / or fatty alcohol when expressed in recombinant host cells. The description further relates to methods for making and using such AAR variants for the production of fatty alcohol compositions having particular characteristics.

BACKGROUND OF THE INVENTION

Fatty alcohols indicate an important category of industrial biochemistry. For example, annual worldwide sales of fatty alcohols and their derivatives are more than $ 1 billion. These molecules and their derivatives have numerous applications, including use as surfactants, lubricants, plasticizers, solvents, emulsifiers, emollients, thickeners, flavors, fragrances, and fuels. Due to their amphiphilic nature, fatty alcohols behave as nonionic surfactants, which are useful in personal and household care products, eg detergents. The shorter chain fatty alcohols are used in the cosmetic and

<img file="MX346830B_D0005.tif" />

In nature, fatty alcohols are made by enzymes that are capable of reducing various acyl-ACP or acyl-CoA molecules to the corresponding primary alcohols (for example, US Patent Nos. 8,323,924; 8,268,599 and

8,097,439; and US Patent Publication Nos.

20120282663 and 20100105963, incorporated by reference herein). However, current technologies primarily involve an inorganic catalyst-mediated reduction of fatty alcohols to the corresponding primary alcohols. These fatty alcohols are produced through catalytic hydrogenation of fatty acids produced from natural sources, such as coconut oil, palm oil, palm kernel oil, tallow and lard, or by chemical hydration of alpha-olefins produced from raw materials. petrochemicals. Fatty alcohols derived from natural sources have varying chain lengths, which are relevant and specific to particular applications. Dehydration of alpha-olefin fatty alcohols can be achieved by chemical catalysis.

Fatty aldehydes can be used to produce special industrial chemicals. For example, aldehydes are commonly used to produce polymers, resins, dyes, flavorings, plasticizers, perfumes, and³

INSTITUTO MEXICANO pharmaceutical products. Aldehydes also as solvents, preservatives and desiQ. £ g ^ i ^ fiX ^^_;and=_c= £ Í £ rt © s ^^ Natural and synthetic compounds, such as vitamins, hormones, are aldehydes, and many sugars contain aldehyde groups. Fatty aldehydes can be converted to fatty alcohols by chemical or enzymatic reduction.

A greener and cleaner alternative to the production of fatty aldehydes and fatty alcohols is through sugars and / or fermentable biomass. However, in order for the production of fatty aldehydes and fatty alcohols from fermentable sugars or biomass to be commercially viable, industrial processes must be optimized for efficient conversion and recovery of the final product. The present disclosure addresses this need by providing compositions and methods for improved production of fatty aldehydes and fatty alcohols using host cells designed as biocatalysts.

SUMMARY OF THE INVENTION

The present disclosure provides photosynthetic and heterotrophic host cells that directly produce fatty aldehydes and / or fatty alcohols of specific chain lengths such that catalytic conversion of purified fatty acids is not necessary. The biological route

INSTITUTO MEXICANO DE LA PROPIEDAD provides a higher quality product, uft8^UST# M * ducfe ^ rr for significant cost and less impact "'Sft¹more specifically, the present disclosure provides variants of the acyl-ACP reductase (AAR) enzyme that produce fatty aldehydes and / or fatty alcohols and compositions thereof. Specific AAR variant protein and nucleic acid sequences are also provided as well as novel recombinant host cells and cell cultures encompassing such engineered AAR enzyme variants. The disclosure also provides methods for using host cells expressing the recombinant AAR variant to make fatty aldehyde and / or fatty alcohol compositions with particular characteristics.

One aspect of the disclosure provides variant acyl-ACP reductase (AAR) polypeptides that catalyze the conversion of an acyl-ACP to a fatty aldehyde, wherein the AAR polypeptide has at least 90% sequence identity to the AAR polypeptide sequence. corresponding wild type presented as SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO : 42 or SEQ ID NO: 44, and methods of expressing variant AAR polypeptides in a recombinant host cell resulting in a higher concentration of fatty aldehyde and / or fatty alcohol composition

INSTITUTO MEXICANO as it is compared with the concentration of fatty aldehyde and / or fatty alcohol nrndnrida ñor] a ουγ.γακ-.λγ. . of the wild-type AAR polypeptide in a corresponding host cell. In one embodiment, the genetically engineered variant AAR polypeptide has at least 90% sequence identity to the corresponding wild-type AAR polypeptide sequence reported as SEQ ID NO: 28 and expression of the variant AAR polypeptide in a recombinant host cell results in a higher concentration of fatty aldehyde and / or fatty alcohol composition or a higher concentration of fatty alcohols of 012, C14 or C16 as compared to the concentration produced by expression of a wild-type AAR polypeptide in a corresponding host cell.

In one aspect, the variant AAR polypeptide has a mutation at one or more amino acid positions of amino acids 18, 24, 31, 34, 35, 43, 50, 63, 86, 112, 113, 116, 118, 120, 135, 148, 153, 155, 157, 159, 168, 172,187,

188, 191, 209, 210, 211, 236, 277, 283, 285, 291, 324,328,

335, 337 and 338 of SEQ ID NO: 28. In a preferred embodiment, the genetically engineered variant AAR polypeptide has an S18W mutation. In another preferred embodiment, the genetically engineered variant AAR polypeptide has an S18W mutation and further comprises a mutation such as M21L, D24E, D24Y,

<img file="MX346830B_D0006.tif" />

L31V, W34F, W35F, D43E, A50Q, ​​C63A, C63G, C

S113K, Q116G, R118Q, T120S, A135S, T

I153P, Q155C, Q155L, T157V, A159V, I168V, C172L, T187V,

T188H, T188V, Q191A, L209R, E210Y, A211W, T236C, Q277V,

E283G, E283S, A285V, M291V, A324T, A328S, Q335N, L337V and / or L338W.

In another aspect, the variant AAR polypeptide has at least 90% sequence identity to the corresponding wild-type AAR polypeptide sequence presented as SEQ ID NO: 3. 4 and expression of the variant AAR polypeptide in a recombinant host cell results in a higher concentration of fatty aldehyde and / or fatty alcohol or a higher concentration of C12 fatty alcohol as compared to the concentration produced by expression of a type AAR polypeptide natural in a corresponding host cell. The variant AAR polypeptide has a mutation at an amino acid position that includes amino acids 40, 52, 58, 61, 273, 303, 339, 340, 344, 345, 346, and 588 of SEQ ID NO: 34. In a preferred embodiment , the variant AAR polypeptide has a mutation at amino acid position Q40V, G52V, S58V, D61E, G273E, K303G, K339L, H340P, L344A, L344D, L344S, L344T, L345R, V346P, V346G, and / or S588V.

Another aspect of the disclosure provides a recombinant host cell having one or more mutations as described ⁷ IΜ PI Mexican institute OF PROPERTY describes in the above and where the cell hoá ^^ ff ^ ta ctraTíclo is designed expresses a variant AAR polypeptide of ¹ ¿0 TC'WT * 28 or SEQ ID NO: 34. This recombinant host cell produces a fatty aldehyde and / or fatty alcohol composition with a concentration that is at least 10% higher, at least 15% higher, at least 20% higher, at least 25% higher, or at least 30% higher. than the concentration of a fatty aldehyde and / or fatty alcohol composition produced by a host cell expressing the corresponding wild-type AAR polypeptide, when grown in a medium containing a carbon source under conditions effective to express the variant 7XAR polypeptide. In one embodiment, the fatty aldehyde and / or fatty alcohol composition is produced at a concentration of 30g / L to 250g / L, eg, a concentration of at least 100 mg / L. In another embodiment, the fatty alcohol composition is produced extracellularly.

The description further encompasses a cell culture that includes the recombinant host cell as described above, wherein the fatty alcohol composition includes one or more of a C6, C8, CIO, C12, C13, C14, C15, fatty alcohol, C16, C17, and C18, for example, a C10: 1, C12: 1, C14: 1, C16: 1, or C18: 1 unsaturated fatty alcohol. In still another embodiment, the fatty alcohol composition comprises a saturated fatty alcohol.

<img file="MX346830B_D0007.tif" />

<img file="MX346830B_D0008.tif" />

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

Another aspect of the disclosure provides a variant acyl-ACP reductase (AAR) polypeptide having at least 90% sequence identity to SEQ ID NO: 57, wherein the AAR polypeptide catalyzes the conversion of an acyl-ACP to a fatty aldehyde. . In one embodiment, expression of the variant AAR polypeptide in a recombinant host cell results in a higher concentration of a fatty aldehyde or fatty alcohol composition as compared to a concentration of a fatty aldehyde or fatty alcohol composition produced by expression of a wild-type AAR polypeptide from a corresponding wild-type host cell. In another embodiment, expression of the variant AAR polypeptide in a recombinant host cell results in a higher concentration of a fatty aldehyde or fatty alcohol composition that is a C12, C14 and / or C16 fatty alcohol composition as compared to a concentration of a fatty aldehyde or fatty alcohol composition produced by expression of a wild-type AAR polypeptide in a corresponding wild-type host cell. In another embodiment, the variant AAR polypeptide has a mutation at amino acid position 18, where the mutation is S18W.

Another aspect of the disclosure provides a variant acyl-ACP reductase polypeptide (AAR) having at

<img file="MX346830B_D0009.tif" />

IMPI mrrm'TO Mexican ⁵¹ of LA TRONLDAD minus 90% sequence identity to SEQ ID NO? ® * 57, the variant AAR polypeptide has another iiiULJ¿lnjn with an amino acid position at amino acid 8, 16, 21, 24, 31, 34, 35, 43, 50, 63, 86, 112, 113, 116, 118, 120, 135, 148, 153, 154, 155, 157, 159, 168, 172, 187, 188, 191, 209, 210, 211, 236, 277, 281, 283, 285, 291, 324, 328, 335, 337 and / or 338. In one embodiment, the mutation is selected from L8A, D16L, M21L, D24E, D24Y, D24V, D24P, L31V, L31M, W34F, W35F, D43E, A50Q, ​​C63A, C63G, C63Y, S86G, A112R, S113K, Q116G,

R118Q, T120S, A135S, T148C, T148E, T148V, I153P, T154A,

Q155C, Q155L, T157V, A159V, I168V, C172L, T187V, T188H,

T188V, Q191A, L209R, E210Y, A211W, T236C, Q277V, A281L,

E283G, E283S, A285V, M291V, A324T, A328S, Q335N, L337V and / or L338W. In a preferred embodiment, the variant AAR polypeptide has an M21L mutation, a C63G mutation, a S113K, T154A mutation, and an A281L mutation (SEQ ID NO: 58).

In another preferred embodiment, the variant AAR polypeptide has an L8A mutation, an M21L mutation, a C63G mutation, a S113K mutation, a T154A mutation, and an A281L mutation (SEQ ID NO: 59). In another preferred embodiment, the variant AAR polypeptide has a D16L mutation, an M21L mutation, a C63G mutation, a S113K, T154A mutation, and a

A281L (SEQ ID NO: 60). In another preferred embodiment, the variant AAR polypeptide has an L8A mutation, a

<img file="MX346830B_D0010.tif" />

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL VROVIETY

D24V, a C63G mutation, a S113K mutation, a Q155L mutation, and an A281L mutation (SEQ ID NO: 61). In another preferred embodiment, the variant AAR polypeptide has a D24P mutation, a L31M mutation, a C63G mutation, a S113K mutation, a T154A mutation, and an A281L mutation (SEQ ID NO: 62). In another preferred embodiment, the variant AAR polypeptide has an L8A mutation, a D16L mutation, a D24V mutation, a C63G mutation, a S113K mutation, a T154A mutation, and an A281L mutation (SEQ ID NO: 63). In another preferred embodiment, the variant AAR polypeptide has a D24E mutation, a C63G mutation, a S113K mutation, a T154A mutation, and an A281L mutation (SEQ ID NO: 64).

Another aspect of the present disclosure provides a recombinant host cell that expresses variant AAR polypeptides as described above (supra). In one embodiment, the recombinant host cell produces a fatty aldehyde or fatty alcohol composition with a concentration that is at least 10% higher, at least 15% higher, at least 20% higher, at least 25% higher, or at least 30% higher. % greater than the concentration of a fatty aldehyde or fatty alcohol composition produced by a host cell expressing a corresponding wild-type AAR polypeptide, when grown in a medium containing a carbon source under conditions effective to express the AAR polypeptide

<img file="MX346830B_D0011.tif" />

variant. In one embodiment, the fatty aldehyde or fatty alcohol composition produced by the recombinant host cell is produced at a concentration of from about 30g / L to about 250g / L. In another embodiment, the fatty aldehyde or fatty alcohol composition is produced extracellularly.

The disclosure further contemplates a cell culture that includes the recombinant host cell expressing variant AAR polypeptides as described above (supra). In one embodiment, the fatty alcohol composition includes a saturated and / or unsaturated fatty alcohol. In one embodiment, the cell culture includes a fatty alcohol composition that includes one or more than a C6, C8, CIO, C12, C13, C14, C15, C16, C17, and C18 fatty alcohol. In another embodiment, the fatty alcohol composition includes one or more of a C10: 1, C12: 1, C14: 1, C16: 1, and C18: 1 unsaturated fatty alcohol. In yet another embodiment, the fatty alcohol composition includes a fatty alcohol having a double bond at the 7-position on the carbon chain between C7 and C8 of the reduced end of the fatty alcohol.

The description further encompasses a method of producing a fatty alcohol composition having an increase in concentration, including culturing the variant AAR-expressing host cell (as described above) with

<img file="MX346830B_D0012.tif" />

_1? IMPI INDUSTRIAL a carbon source; and collect a composition of - 1. · - ^ • '' • '-i · *

c. - J.— 'fatty alcohol. In one embodiment, the concentration of the fatty alcohol is at least 20% to 30% greater than the concentration of a fatty alcohol composition produced by a host cell expressing wild-type AARs.

Another aspect of the disclosure provides a variant acyl-ACP reductase (AAR) polypeptide having at least 90% sequence identity to SEQ ID NO: 65, wherein the polypeptide catalyzes the conversion of an acyl-ACP to a fatty aldehyde. In one embodiment, expression of the variant AAR polypeptide in a recombinant host cell results in a higher concentration of a fatty aldehyde or fatty alcohol composition as compared to a concentration of a fatty aldehyde or fatty alcohol composition produced by expression of a wild-type AAR polypeptide in a corresponding wild-type host cell. In another embodiment, expression of the variant AAR polypeptide in a recombinant host cell results in a higher concentration of a C12, C14, and / or C16 fatty alcohol composition as compared to a concentration of a produced fatty aldehyde or fatty alcohol composition. by expression of a wild-type AAR polypeptide in a corresponding wild-type host cell. In a particular aspect, the disclosure provides an acyl-ACP reductase (AAR) polypeptide having at least 90% sequence identity at 65, wherein the polypeptide has a mutation at amino acid position 61. In a preferred embodiment the mutation is D61E.

The description further encompasses a variant acyl-ACP reductase polypeptide (AAR) having at least 90% sequence identity to SEQ ID NO: 34, wherein expression of the variant AAR polypeptide in a recombinant host cell results in a higher concentration of a fatty aldehyde or fatty alcohol composition or a higher concentration of a C12, C14 and / or C16 fatty alcohol composition as compared with a concentration of a fatty alcohol composition produced by the expression of a wild-type AAR polypeptide in a corresponding wild-type host cell, and wherein the AAR polypeptide has a mutation at amino acid position 40, 52, 273, 303, 340, 344, 345, or 346. In one embodiment, the variant AAR polypeptide has a mutation selected from Q40V, G52V,

G273E, K303G, H340P, L344A, L344D, L344S, L344T, L345R,

V346P, and V346G. In a preferred embodiment, the variant AAR polypeptide has a mutation in V346P (SEQ ID NO: 66). In another preferred embodiment, the variant AAR polypeptide has a mutation in Q40V (SEQ ID NO: 67). In another mode

IMPI

INSTITUTO MEXICANO preferred, variant AAR polypeptide has a mutation

A345R (SEQ ID NO: 68). In another preferred embodiment, the variant AAR polypeptide has a mutation at L344S (SEQ ID NO: 69). In another preferred embodiment, the variant AAR polypeptide has a mutation in V346G (SEQ ID NO: 70). In another preferred embodiment, the variant AAR polypeptide has a mutation in L344D (SEQ ID NO: 71). In another preferred embodiment, the variant AAR polypeptide has a mutation in G52V (SEQ ID NO: 72). In another preferred embodiment, the variant AAR polypeptide has a mutation in L344T (SEQ ID NO: 73). In another preferred embodiment, the variant AAR polypeptide has a mutation in K303G (SEQ ID NO: 74). In another preferred embodiment, the variant AAR polypeptide has a mutation in L344A (SEQ ID NO: 75). In another preferred embodiment, the variant AAR polypeptide has a mutation in H340P (SEQ ID NO: 76). In another preferred embodiment, the variant AAR polypeptide has a mutation in G273E (SEQ ID NO: 77).

Still another aspect of the disclosure provides a recombinant host cell expressing the variant ZXAR polypeptide as described above (supra). In one embodiment, the recombinant host cell produces a fatty aldehyde or fatty alcohol composition with a concentration that is at least 10% higher, at least 15% higher,

<img file="MX346830B_D0013.tif" />

ÍKSTITUTO MEXICANO, ..., ...&gt; the property at least 20% greater, at least 25% greater, or at nf ^ Y &amp; §^{TO THE}30 ^ higher than the concentration of a fatty alcohol or composition produced by a host cell expressing a corresponding wild-type AAR polypeptide, when cultured in a medium containing a carbon source under conditions effective to express the variant AAR polypeptide. In another embodiment, the fatty alcohol composition is produced in a concentration from about 30g / L to about 250g / L. In another embodiment, the fatty alcohol composition is produced extracellularly.

The disclosure further contemplates a cell culture with the recombinant host cell expressing the variant AAR polypeptide as described above (supra). In one embodiment, the fatty alcohol composition includes one or more than one C6, C8, CIO, C12, C13, C14, C15, C16, C17, and C18 fatty alcohol. In another embodiment, the fatty alcohol composition includes an unsaturated or saturated fatty alcohol. In another embodiment, the fatty alcohol composition includes one or more of a C10: 1, C12: 1, C14: 1, C16: 1, and C18: 1 unsaturated fatty alcohol. In another embodiment, the fatty alcohol composition includes a fatty alcohol that has a double bond at the 7-position on the carbon chain between C7 and C8 of the reduced end of the fatty alcohol.

Another aspect of the description provides a method

<img file="MX346830B_D0014.tif" />

«IMPI

INSTITUTO MEXICANO DE LAMI PIEDAD to produce a composition of fatty alcohol qu8^IDUF^rl^l^ n increase in concentration, including culturing the AAR-expressing host oSldld (as described above) with a carbon source; and collecting a fatty alcohol composition. In one embodiment, the fatty alcohol is at least 20% to 30% greater than the concentration of a fatty alcohol composition produced by a host cell expressing wild-type AARs.

BRIEF DESCRIPTION OF THE FIGURES

The present description is best understood when read in conjunction with the accompanying figures, which serve to illustrate the preferred embodiments. It is understood, however, that the description is not limited to the specific embodiments described in the figures.

Figure 1 is a schematic overview of an exemplary biosynthetic pathway for use in the production of acylCoA as a precursor for fatty acid derivatives in a recombinant host cell. The cycle is initiated by the condensation of malonyl-ACP and acetyl-CoA.

Figure 2 is a schematic overview of an exemplary fatty acid biosynthetic cycle, where the elongation cycles begin with the condensation of malonyl-ACP and an acyl-ACP catalyzed by β-ketoacyl-ACP synthase I (fabB) and β-ketoacyl-ACP synthase II (fabF) to produce a β-keto-acyl-ACP, then the β-keto-acyl-ACP is reduced by an NADPH-dependent β-ketoacyl-ACP reductase (fabG) to produce the β-hydroxy -acyl-ACP, which is dehydrated to a trans2-enoyl-acyl-ACP by β-hydroxyacyl-ACP dehydratase (fabA or fabZ). FabA can also isomerize trans-2-enoylacyl-ACP to cis-3-enoyl-acyl-ACP, which can prevent fabl and can be used by fabB (typically by up to a C16 aliphatic chain length) to produce β-ketoacyl-ACP. The final stage in each cycle is catalyzed by a NADH- or NADHPH-dependent enoyl-ACP reductase (fabl) that converts trans-2-enoyl-acyl-ACP to acyl-ACP. In the methods described herein, termination of fatty acid synthesis occurs by removal of the thioesterase from the acyl-ACP acyl group to release free fatty acids (FFA). Thioesterases (eg, tesA) hydrolyze thioester linkages, which occur between acyl and ACP chains via sulfhydryl linkages.

Figure 3 illustrates the structure and function of the acetyl-CoA carboxylase enzyme complex (accABCD). Biotine carboxylase is encoded by the accC gene, while biotin-carboxyl carrier protein (BCCP) is encoded by the accB gene. The two subunits involved in the activity

<img file="MX346830B_D0015.tif" />

of the carboxyl-transferase are encoded by the accA and accD genes. Biotin covalently bound to -BCCP carries the carboxylate portion. The gene birA biotinin the holo-accB.

Figure 4 presents a schematic overview of an exemplary biosynthetic pathway for fatty alcohol production beginning with acyl-ACP, where fatty aldehyde production is catalyzed by the enzymatic activity of acyl-ACP reductase (AAR) or thioesterase. and carboxylic acid reductase (Car). Fatty aldehyde is converted to fatty alcohol by aldehyde reductase (also referred to as alcohol dehydrogenase). This pathway does not include fatty acyl-CoA synthetase (fadD).

Figure 5 shows fatty alcohol production in Synechococcuselongatus acyl-ACP reductase that expresses E. coli DV2 (AAR_7942) and co-expresses various cyanobacterial acyl carrier proteins (ACPs).

Figure 6 shows the production of fatty alcohol in Synechococcuselongatusacil acyl-ACP reductase expressing E. coli DV2 (AAR_7942) (pLS9-185) and co-expressing the Corynebacteriumglutamicum acetyl-carboxylase complex (pSL9-185- D +) (three individual strains are shown, see D + l, D + 2 and D + 3).

Figure 7 presents the results illustrating the improved production of fatty alcohol from recombinant host cells that depend on the

<img file="MX346830B_D0016.tif" />

overexpression of ifab and ifadR.

Figure 8 presents the results showing the elevated levels of fatty alcohol in the recombinant host cells expressing the AAR_7942 variants derived from the combination libraries (AAR_Com 2a-d) that are co-expressed with ACP, AlrA (alcohol dehydrogenase ( ADH)) and a synthetic acc operon in strain Shu2. A variety of alcohol dehydrogenase polypeptides are useful according to the description and include, but are not limited to, AlrA from Acinetobacter sp. Ml (SEQ ID NO: 52) and an AlrA homolog such as AlrAadpl (SEQ ID NO: 53)

Figures 9A and 9B present the results of a tank fermentation of the variant AAR_7942 S18W illustrating the FALO concentration (Fig. 9A) and the glucose yield (Fig. 9B) showing that the expression of the variant AAR_7942 S18W gives resulting in altered activity and chain length distribution.

Figure 10 presents the results illustrating a change in chain length distribution for EALC from C16 to C14 when the D61E variant of MED4 AAR was expressed in the recombinant host cells.

IMPI

DETAILED DESCRIPTION OF THE INVENCIÓFT ^ .p »^? ^ General view _

One way to eliminate dependence on petrochemicals is to produce derivatives of fatty acids such as fatty aldehydes and fatty alcohols through environmentally friendly microorganisms that serve as miniature production hosts. Such cell hosts (i.e., recombinant host cells or production strains) have been designed to produce fatty aldehydes and / or fatty alcohols from renewable sources such as renewable feedstock (e.g., fermentable sugars, carbohydrates, biomass, cellulose, glycerol, CO, CO₂, etc.). These fatty aldehydes and fatty alcohols are the raw materials for many industrial products including detergents and fuels.

The present disclosure relates to acyl-ACP reductase (AAR) enzyme variants that result in improved concentration, yield, and / or productivity of fatty aldehyde and / or fatty alcohol compositions when expressed in recombinant host cells. At this point, improved fatty aldehyde and / or fatty alcohol biosynthesis is achieved by transforming host cells such that they express an acyl-ACP reductase (AAR) protein, which catalyzes the reaction of an acyl-ACP to a fatty aldehyde and / or to alcohol

<img file="MX346830B_D0017.tif" />

IMPI ^

INSTITUTO MIXICANO DE LA MI PIEDAD fatty. The description further refers to lárS '^us3 ^ 5.u recombinant strains or strains of production of variants of the AAR enzyme.

Definitions

As used in this specification and in the appended claims, the singular forms a, an, the, and the include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to a host cell includes two or more host cells, reference to a fatty ester includes one or more fatty esters, or mixtures of esters, reference to a nucleic acid sequence includes one or more plus nucleic acid sequences, reference to an enzyme includes one or more enzymes, and the like.

Accession numbers for this entire description were obtained from databases provided by NCBI (National Center for Biotechnology Information) maintained by the National Institutes of Health, USA. (identified herein as NCBI Accession Numbers or alternatively as GenBank Accession Numbers), and from UniProt Knowledgebase (UniProtKB) and the SwissProt databases provided by the Swiss Institute of Bioinformatics (identified herein as Numbers Access

<img file="MX346830B_D0018.tif" />

UniProtKB).

IMPI

MEXICAN INSTITUTE

OF INDUSTRIAL PROPERTY

Enzyme Classification numbers. ,. (F.CL -. ^ Sa established by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB), the description of which is available on the IUBMB Enzyme Nomenclature website on the World Wide Web. EC numbers classify enzymes according to the reaction they catalyze. For example, the enzymatic activity of acyl-ACP reductase (AAR) is classified under EC 1.2.1.80 (also known as acyl- [acyl-carrier-protein] reductase) long chain or EC 1.2.1.42. The functionality of the AAR is conserved in most prokaryotes from one species to the next. In this way, different microbial species can carry the same AAR enzymatic activity that is classified under EC 1.2.1.80 or EC 1.2.1.42.

As used herein, the term "nucleotide" refers to a monomeric unit of a polynucleotide that consists of a heterocyclic base, a sugar, and one or more phosphate groups. The naturally occurring bases (guanine, (G), adenine, (A), cytosine, (C), thymine, (T), and uracil (U)) are typically derived from purine or pyrimidine, although it should be understood that they also non-naturally occurring and naturally occurring base analogs are included. The naturally occurring sugar which is deoxyribose pentose (five carbon sugar) (which

INSTITUTO MEXICANO DRLAPR PIEDAD INDUSTRIAL forms DNA) or ribose (which forms RNA), although it should be understood that naturally occurring and non-naturally occurring sugar analogs are also included. Nucleic acids are typically linked through phosphate linkages to form nucleic acids or polynucleotides, although many other linkages are known in the art (eg, phosphorothioates, boranophosphates, and the like).

The term "polynucleotide" refers to a polymer of ribonucleotides (RNA) or deoxyribonucleotides (DNA), which can be single-stranded or double-stranded and which can contain unnatural or altered nucleotides. The terms polynucleotide, nucleic acid sequence, and nucleotide sequence are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either RNA or DNA. These terms refer to the primary structure of the molecule, and thus include double-stranded and single-stranded DNA, and double-stranded and single-stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, but not limited to, methylated and / or terminated polynucleotides. The polynucleotide can be in any form including, but not limited to, plasmid, viral, chromosomal, EST, cDNA, mRNA, and rRNA.

A IvJL 11 JL INSTITUTO MEXICANO

As used herein, the terms protein are used interchangeably for, mean, polymer of amino acid residues. The term "recombinant polypeptide" refers to a polypeptide that is produced by recombinant techniques, wherein generally the DNA or RNA encoding the expressed protein is inserted into a suitable expression vector which in turn is used to transform a host cell to produce the polypeptide. Similarly, the terms recombinant polynucleotide or recombinant nucleic acid or recombinant DNA are produced by recombinant techniques that are known to those of skill in the art.

As used herein, the terms homologous, and homologues refer to a polynucleotide or a polypeptide that comprises a sequence that is at least about 50 percent (%) identical to the corresponding polynucleotide or polypeptide sequence. Polynucleotides or polypeptides preferably homologous have polynucleotide sequences or amino acid sequences that are at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% homology to the corresponding amino acid sequence or polynucleotide sequence. How the terms are used here

INSTITUTO MEXICANO M LA HUMEDAD INDUSTRIAL Sequence homology and sequence identity are used interchangeably. A person of ordinary experience in the field is aware of the methods for determining the homology between two or more sequences. Briefly, homology calculations between two sequences can be carried out as follows. Sequences are aligned for optimal comparison purposes (for example, spaces can be introduced into one or both of a first and second amino acid or nucleic acid sequences for optimal alignment and non-homologous sequences can be ignored for comparison purposes ). In a preferred embodiment, the length of a first sequence that is aligned for comparison purposes is at least about 30%, preferably at least about 40%, more preferably at least about 50%, even more preferably at less about 60%, and even more preferably at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or about 100% of the length of a second sequence. The amino acid or nucleotide residues at the corresponding amino acid positions or nucleotide positions of the first and second sequences are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percentage of homology between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of spaces and the length of each space, which need to be introduced for the optimal alignment of the two sequences. Comparison of the sequences and determination of the percentage of homology between the two sequences can be achieved using a mathematical algorithm, such as BLAST (Altschul et al. (1990) J. Mol. Biol. 215 (3): 403-410). The percent homology between the two amino acid sequences can also be determined using the Needleman and Wunsch algorithm that has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a space weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 (Needleman and Wunsch (1970) J. Mol. Biol. 48 : 444-453). The percent homology between two nucleotide sequences can also be determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a space weight of 40, 50, 60, 70, or 80 and a weight of length of 1, 2, 3, 4, 5, or 6. A person of ordinary experience

IMPI ^ in the field can perform initial 'Tromo'logy calculations and adjust the algorithm parameters accordingly. A preferred set of parameters (and the one to use if a practitioner is unsure about which parameters to apply to determine if a molecule is within a claim homology limitation) is a Blossum 62 scoring matrix with a space penalty of 12, an extended space penalty of 4, and a reading frame space penalty of 5. Additional methods of sequence alignment are known in the biotechnology arts (see, for example, Rosenberg (2005) BMC Bioinformatics 6: 278; Altschuly collaborators (2005) FEBS J. 272 ​​(20): 5101-5109).

The term hybridization under low stringency, medium stringency, high stringency, or high stringency conditions describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley &amp; Sons, NY (1989), 6.3.1 - 6.3.6. Aqueous and non-aqueous methods are described in that reference and either method can be used. The specific hybridization conditions referred to herein are as follows: (1) Low stringency hybridization conditions - 6X sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by two

<img file="MX346830B_D0019.tif" />

²⁸ IMPI

ΜΤΠΤϋΤΟ MlfcCICANO

DC LA KQTIIQAD washed in 0.2X SSC, 0.1% SDS at least afND &amp; gKM? washing temperatures can be increased in low severity conditions); (2) medium stringency hybridization conditions - 6X SSC at approximately 45 ° C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60 ° C; (3) high stringency hybridization conditions -6X SSC at approximately 45 ° C, followed by one or more washes in 0.2.X SSC, 0.1% SDS at 65 ° C; and (4) very high stringency hybridization condition - 0.5M sodium phosphate, 7% SDS at 65 ° C, followed by one or more washes at 0.2X SSC, 1% SDS at 65 ° C. Very high severity conditions (4) are the preferred conditions unless otherwise specified.

An endogenous polypeptide refers to a polypeptide encoded by the genome of the precursor cell (or host cell). An exogenous polypeptide refers to a polypeptide that is not encoded by the genome of the parent cell. A variant or mutant polypeptide is an example of an exogenous polypeptide. In this way, a non-naturally occurring nucleic acid molecule is considered to be exogenous to a cell once it is introduced into the cell. A nucleic acid molecule that is naturally occurring can also be exogenous to a particular cell. For example, a complete coding sequence isolated from the

<img file="MX346830B_D0020.tif" />

<img file="MX346830B_D0021.tif" />

INSTITUTO Mexicano, DE LA FROFIEDAI »cell X is an exogenous nucleic acid with resjFiJPCTW¹· T cell once the sequence of cuúlfitdUTún-ST introduces into cell Y even if X and Y are of the same cell type.

The term "overexpressed" means that a gene is caused to be transcribed at a high rate compared to the endogenous transcription rate for that gene. In some examples, the overexpression further includes a high rate of translation of the gene compared to the rate of endogenous translation for that gene. Methods for testing for overexpression are well known in the art, for example transcribed RNA levels can be assessed using rtPCR and protein levels can be assessed using SDS gene analysis.

The term "heterologous" means a derivative of a different organism, a different cell type, or a different species. As used herein, this refers to a sequence of nucleotides, polynucleotides, polypeptides, or proteins, not naturally present in a given organism. For example, a polynucleotide sequence that is native to the cyano bacterium can be introduced into an E. coli by recombinant methods, and the cyanobacterial polynucleotide is then heterologous to the E. coli cell (eg, recombinant cell). The heterologous term

<img file="MX346830B_D0022.tif" />

INDUSTRIAL can also be used with reference to a sequence of nucleotides, polynucleotides, polypeptides, or proteins that is present in a recombinant host cell in a non-native state. A heterologous nucleotide, polynucleotide, polypeptide or protein sequence can be modified relative to the naturally occurring type sequence in the corresponding wild type host cell, for example, a modification in the expression level or in the sequence of a nucleotide, polynucleotide, polypeptide, or protein.

As used herein, the term "fragment of a polypeptide" refers to a shorter portion of a full-length polypeptide or protein that ranges in size from two amino acid residues to the full amino acid sequence minus one amino acid residue. In certain embodiments of the disclosure, a "fragment" refers to the entire amino acid sequence of a domain of a polypeptide or protein (eg, a substrate-binding domain or a catalytic domain).

The term mutagenesis refers to a process by which the genetic information of an organism is changed in a stable way. Mutagenesis of a nucleic acid sequence encoding the protein produces a mutant protein. Mutagenesis also refers to changes in the

<img file="MX346830B_D0023.tif" />

Non-coding nucleic acid sequences qqe „give dpi______ result in modified activity of the protein.

A mutation, as used herein, refers to a permanent change in a nucleic acid position of a gene or in an amino acid position of a polypeptide or protein. Mutations include substitutions, additions, insertions, and deletions. For example, a mutation at an amino acid position can be a substitution of one type of amino acid with another type of amino acid (for example, a serine (S) can be substituted for an alanine (A); a lysine (L) can replace with a T (Threonine); etc.). As such, a polypeptide or a protein can have one or more mutations where one amino acid is substituted for another amino acid.

The terms acyl-ACP reductase variant (AAR) and variant acyl-ACP reductase (AAR) are used interchangeably herein and mean an AAR-related polypeptide or protein that has one or more mutations in its amino acid sequence. 7XAR refers to an enzyme that catalyzes the reduction of an acyl-ACP to a fatty aldehyde and / or fatty alcohol. The AAR variant can encompass a mutation in one or more amino acids of its polypeptide sequence. When a cell has been transformed with an AAR variant, it is a cell that expresses ¿INSTITUTO MEXICANO

OS INDUSTRIAL PROPERTY ^{a: T} .

the AAR variant (eg, a recombinant cell). In one embodiment, the concentration and / or yield of a fatty alcohol produced by a cell expressing the AAR variant is at least twice that of a corresponding wild-type cell {i.e., a corresponding cell that does not express the AAR variant) . In a heterologous host such as Escherichia coli, fatty aldehydes can be converted to fatty alcohols to endogenous alcohol dehydrogenases. In another embodiment, the concentration and / or yield of a fatty alcohol produced by a cell expressing the AAR variant is at least about 1 time, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times greater than that of a corresponding wild-type cell. In one embodiment, the concentration and / or yield of a fatty alcohol produced by a cell expressing the AAR variant is at least about 1 percent, at least about 2 percent, at least about 3 percent, at least about 4 percent. percent, at least about 5 percent, at least about 6 percent, at least about 7 percent

<img file="MX346830B_D0024.tif" />

IMPI INSTITUTE MWttCAHO cent, at least about 8 by about 9 percent, or about lQ.poqp. ^ IentO '-'- greater than that of a corresponding wild-type cell. In another embodiment, the concentration and / or yield of the fatty alcohols produced in a recombinant cell due to expression of an AAR variant is less about 20 percent to at least about 100 percent greater than that of a wild-type cell. In particular embodiments, the concentration and / or yield of a fatty alcohol produced by a cell is less about 20 percent, at least about 25 percent, at least about 30 percent, at least about 35 percent, at least about 40 percent. percent, at least about 45 percent, at least about 50 percent, at least about 55 percent, at least about 60 percent, at least about 65 percent, at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 97 percent, at least about 98 percent, or at least about 100 percent greater than that of the corresponding wild-type cell.

As used herein, the term "gene" refers to nucleic acid sequences that encode either an RNA product or a protein product, as well as operably linked nucleic acid sequences that affect the expression of the RNA or protein (e.g. , such sequences include but are not limited to promoter or enhancer sequences) or operably linked nucleic acid sequences that encode sequences that affect RNA or protein expression (e.g., such sequences include but are not limited to ribosome binding sites or translational control sequences).

Expression control sequences are known in the art and include, for example, promoters, enhancers, polyadenylation signals, transcription terminators, internal and ribosome entry sites (IRES), and the like, which provide for expression of the sequence. of polynucleotides in a host cell. Expression control sequences specifically interact with cellular proteins involved in transcription (Maniatis and collaborators (1987) Science 236: 1237-1245). Exemplary expression control sequences are described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990) . In the methods of the description, an expression control sequence is operably linked

<img file="MX346830B_D0025.tif" />

to a polynucleotide sequence. By "operably linked" it is meant that a polynucleotide sequence and an expression control sequence are connected in such a way to allow gene expression when appropriate molecules (eg, transcriptional activator proteins) are linked to the expression control sequence. The operably linked promoters are located upstream of the selected polynucleotide sequence in terms of the direction of transcription and translation. Operably linked enhancers can be positioned upstream, within, or downstream of the selected polynucleotide.

As used herein, the term "vector" refers to an amino acid molecule capable of transporting another nucleic acid, i.e., a polynucleotide sequence, to the lime it has been linked. One type of useful vector is an episome (ie, a nucleic acid capable of extrachromosomal replication). Useful vectors are those capable of autonomous replication and / or expression of the nucleic acids to which they are linked. Vectors capable of directing the expression of the genes to which they are operatively linked are referred to herein as expression vectors. In general, expression vectors of utility in recombinant DNA techniques³⁶ ΙΜΡΙ ^%

IWrnUTpMEXICANO Of the raonuMD are frequently in the form of plasmid s • ndustr ^ iu generally refer to circular US ”DNA loops which, in their vector form, do not bind to the chromosome. Other useful expression vectors are provided in a linear fashion. Also included are such other forms of expression vectors which serve equivalent functions and which have been known in the art subsequently thereto. In some embodiments, a recombinant vector further includes a promoter operably linked to the polynucleotide sequence. In some embodiments, the promoter is a highly regulated promoter, an organelle specific promoter, a tissue specific promoter, an inducible promoter, a constitutive promoter, or a cell specific promoter. The recombinant vector typically comprises at least one sequence selected from an expression control sequence operably coupled to the polynucleotide sequence; a selection marker operably coupled to the polynucleotide sequence; a marker sequence operably coupled to the polynucleotide sequence; a purification portion operably coupled to the polynucleotide sequence; a secretory sequence operably coupled to the polynucleotide sequence; and a target sequence operably coupled to the polynucleotide sequence. In certain embodiments, the nucleotide sequence stably occurs in the genomic DNA of the J4wup¿deTtT7 "cell, and expression of the nucleotide sequence is under the control of a regulated promoter region. Expression vectors as used herein include a particular polynucleotide sequence as described herein in a form suitable for the expression of the polynucleotide sequence in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector may depend on such factors as the choice of host cell to be transformed, the level of expression of the desired polypeptide, and the like. The expression vectors described herein can be introduced into host cells to produce polypeptides, including fusion polypeptides, encoded by the polynucleotide sequences as described herein. Expression of genes encoding polypeptides in prokaryotes, for example, E. coli, is most frequently carried out with vectors containing constitutive or inducible promoters that direct the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino or carboxy terminus of the recombinant polypeptide. Such vectors of

IMVHTUTO MEXICANA DELAPUOHEDAU fusion typically serve one or more of the '^ K ^ Wienvé ^' three purposes, including to increase ίϊ ^ χρΊ ^ Ιδή 'recombinant polypeptide. To increase the solubility of the recombinant polypeptide; and to aid in recombinant polypeptide purification by acting as a ligand in affinity purification. Frequently, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide. This allows separation of the recombinant polypeptide from the fusion portion after purification of the fusion polypeptide. In certain embodiments, a polynucleotide sequence of the disclosure is operably linked to a promoter derived from bacteriophage T5.

In certain embodiments, the host cell is a yeast cell, and the expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSecl (Baldariy co-workers (1987) EMBO J. 6: 229-234); pMFa (Kurjany co-workers (1982) Cell 30: 933-943); pJRY88 (Schultz et al. (1987) Gene 54: 113-123); pYES2 (Invitrogen Corp., San Diego, CA), and picZ (Invitrogen Corp.,

San Diego, CA). In other embodiments, the host cell is an insect cell, and the expression vector is a vector.

<img file="MX346830B_D0026.tif" />

³⁹ Baculovirus expression IMPI. Ιν®3® £ 1 vectors available for protein expression "^toin «4 ^« ~ -eÁluXa§ „of ,._#Cultured insects (eg, Sf9 cells) include, for example, the pAc series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklowy collaborators (1989) Virology 170: 31- 39). In yet another embodiment, the polynucleotide sequences described herein can be expressed in mammalian cells using a mammalian expression vector. Other suitable expression systems for both prokaryotic and eukaryotic cells are well known in the art see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Coid Spring Harbor Laboratory, (1989).

As used herein, the term CoA or acylCoA refers to an acyl thioester formed between the carbonyl carbon of the alkyl chain and the sulfhydryl group of the 4'-phosphopantethionyl portion of coezyme A (CoA), having the formula RC (0) S-CoA, where R is any alkyl group having at least 4 carbon atoms.

The term ACP means acyl carrier protein. ACP is a highly conserved carrier of acyl intermediate during fatty acid biosynthesis, where the growth chain is ligated during synthesis as a thiol ester on the distant thiol of a

<img file="MX346830B_D0027.tif" />

phosphopantetheine. The protein exists in two forms, ^ ~ Ts ie, apo-ACP (inactive in fatty acid biosynthesis) and ACP or holo-ACP (active in fatty acid biosynthesis). The terms ACP and holo-ACP are used interchangeably herein and refer to the active form of the protein. An enzyme called a phosphopantetheinyltransferase is involved in the conversion of inactive apo-ACP to holo-ACP. More specifically, ACP is expressed in the inactive apo-ACP form and a portion of 4'-phosphopantetheine must be linked post-translationally to a conserved serine residue in the

ACP by the action of the carrier protein holo-acyl synthase (ACPS), a phosphopantetheinyltransferase, to produce holo-ACP.

As used herein, the term "acyl-ACP" refers to an acyl thioester formed between the carbonyl carbon of an alkyl chain and the sulfhydryl group of the phosphopantethenyl portion of an acyl carrier protein (ACP). In some embodiments, an ACP is an intermediate in the synthesis of fully saturated acyl-ACPs. In other embodiments an ACP is an intermediate in the synthesis of unsaturated acyl-ACPs. In some modalities, the carbon chain will have approximately 5,

6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, • S ·· 41

22, 23, 24, 25, or 26 carbons.

IMPI (Mexican KSTITUTO delafromioad industrial

<img file="MX346830B_D0028.tif" />

As used herein, the term "Hprivarin Ha" fatty acid means a fatty acid or a fatty acid derivative, which may be referred to as a fatty acid derivative thereof. The term "fatty acid" means a carboxylic acid having the formula RCOOH. R represents an aliphatic group, preferably an alkyl group. R can include between about 4 and about 22 carbon atoms. Fatty acids can be saturated, monounsaturated, or polyunsaturated. A fatty acid derivative is a product manufactured in part of the fatty acid biosynthetic pathway of production by the living body. Fatty acid derivatives include products made in part from ACP, acyl-ACP, or acyl-ACP derivatives. Exemplary fatty acid derivatives include, for example, acyl-CoA, fatty acids, fatty aldehydes, long and short chain alcohols, fatty alcohols, hydrocarbons, esters (eg, waxes, fatty acid esters, or fatty esters), terminal olefins, internal olefins and ketones.

As used herein, the term "fatty acid biosynthetic pathway" means a biosynthetic pathway that produces fatty acids and derivatives thereof. The fatty acid biosynthetic pathway can include additional enzymes to produce fatty acid derivatives that have desired characteristics.

<img file="MX346830B_D0029.tif" />

Μτπυτο MKxiCANb DIlAnOPlITY industrial

<img file="MX346830B_D0030.tif" />

As used herein, a 1 dehi dd "t | TcrSü'51 ^ 11 ÍPti5ár an aldehyde having the formula RCHO characterized by a carbonyl group (C = O). In some embodiments, the fatty aldehyde is any aldehyde made from a fatty alcohol. In certain embodiments, the R group is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19, carbons in length. Alternatively, or in addition, the R group is 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, or 6 or less carbons in length. In this way, the R group can have an R group linked by either of the above end points. For example, the R group can be 6-16 carbons in length, 10-14 carbons in length, or 12-18 carbons in length. In some embodiments, the fatty aldehyde is a fatty aldehyde Cg, C7, Cg, Cg, Ciq, Cu, C12, C13, C14, C15, Ci6, C17, Cig, Ci9, C20Z C21, C22, C23, C24, C25, ° C26In certain embodiments, the fatty aldehyde is fatty aldehyde C₆, C₈, C_i0, 012, C13, C14, C15, C16, C17, OR C18

As used herein, "fatty alcohol" means an alcohol having the formula ROH. In some modalities, the

INJHTUTO MtXICANO S? Á »eSS

DI LA PR PTIDAD V'aatszií'B INDUSTRIAL Xl · ™.

group R is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16 , at least 17, at least 18, or at least 19, carbons in length. Alternatively, or in addition, the R group is 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, or 6 or less carbons in length. In this way, the R group can have an R group linked by either of the above end points. For example, the R group can be 6-16 carbons in length, 10-14 carbons in length, or 12-18 carbons in length. In some embodiments, the fatty alcohol is a fatty alcohol Ce, C7, Ce, C9, Cio, Cu, C12, C13, C14, C15, Cie, C17, Cis, C19, C20, C21, C22, C₂3, C₂4, C25, or C₂6- In certain modalities, fatty alcohol is a fatty alcohol C₆, C₈, Cio, C12, C13, C14, C15, C_i6, C17, or Ci8.

A fatty alcohol composition as referred to herein is produced by a recombinant host cell and typically comprises a mixture of fatty alcohols. In some cases, the mixture includes more than one type of product (for example, fatty alcohols and fatty acids). In other cases, the compositions derived from fatty acids may comprise a mixture of fatty alcohols with various⁴⁴ IMPI chain lengths and -5 branching characteristics. In still other cases, the fatty compo ^ giáfe-a ^^ rtfDnol comprises a mixture of both of more than one type of product and products with various chain lengths and saturation or branching characteristics.

A host cell designed to produce a fatty aldehyde will typically convert some of the fatty aldehyde to a fatty alcohol. In an exemplary embodiment, acyl-ACP is converted to a fatty aldehyde through the action of AAR. The conversion of fatty aldehydes to fatty alcohols can be further facilitated, for example, through a fatty alcohol biosynthetic polypeptide. In some embodiments, a gene encoding a fatty alcohol biosynthetic polypeptide is expressed or overexpressed in the host cell. In certain embodiments, the fatty alcohol biosynthetic polypeptide has the activity of aldehyde reductase or alcohol dehydrogenase.

Examples of useful alcohol dehydrogenase polypeptides according to the disclosure include, but are not limited to, AlrA from Acinetobacter sp. Ml (SEQ ID NO: 52) or AlrA homologs, such as AlrAadpl (SEQ ID NO: 53) and alcohol dehydrogenases from endogenous E. coli such as YjgB, (AAC77226), DkgA (NP_417485), DkgB (NP_414743), YdjL (AAC74846), YdjJ (NP_416288), AdhP (NP_415995), YhdH

<img file="MX346830B_D0031.tif" />

IMPI (NP_417719), YahK (NP_414859), YphC (AAC7559 and YbbO [AAC73595.1]. Additional examples are ~ descrjUack- ^ üx ^ * »· International Patent Application Publication Nos. WO 2007/136762, WO2008 / 119082 and WO 2010/062480. In certain embodiments, the fatty alcohol biosynthetic polypeptide has the activity of aldehyde reductase or alcohol dehydrogenase (EC 1.1.1.1).

The R group of fatty acid, fatty aldehyde, or fatty alcohol can be a straight chain or a branched chain. Branched chains can have more than one branch point and can include cyclic branches. In some embodiments, the branched fatty acid, fatty aldehyde, or branched fatty alcohol C₆, C₇, C₈, C₉, Cio, Cu, C_i2, C13, C14, C15, C16, Cn, C18, C19, C₂0r C₂1 C₂₂, C₂3, C₂4, C₂5, OC₂6 branched fatty aldehyde, or branched fatty alcohol. In particular embodiments, the branched fatty acid, branched fatty aldehyde, or branched fatty alcohol is a branched fatty acid C₆, C₈, Cio, C_i2, C_i3, C14, C_i5, C_i6, C17, or Ci₈, branched fatty aldehyde, or branched fatty alcohol. In certain embodiments, the hydroxyl group of the branched fatty acid, branched fatty aldehyde, or branched fatty alcohol is in the primary position (Ci).

In certain embodiments, the branched fatty acid, branched fatty aldehyde, or branched fatty alcohol is an iso-fatty acid, iso-fatty aldehyde, oiso-a

INO'JSTMAL '“' SfsE-Sun anteiso-fatty acid, an anteiso-fatty aldehyde, or anteisoi * i · i * ww * · .Adt. »Fatty alcohol. In exemplary embodiments, the branched fatty acid, branched fatty aldehyde, or branched fatty alcohol are selected from a branched fatty acid, branched fatty aldehyde, or iso-C branched fatty alcohol._7:0, iso-C_8:0, iso-Cg_:0, iso-Ci_0:0, iso-Cn_:0, iso-Ci_2:or, iso-Ci_3:0, isoC14: O, ISO-C15: 0, ISO-Ci_6;Q, ÍSO-Ci_7:0, ÍSO-Ci_8;0, ÍSO-Ci_9:0, anteiso-C_7:0, anteiso-C_8:0, anteiso-C_9:0, anteiso-Ci_0:0, anteisoC_{n; 0}, anteiso-Ci2: o, anteiso-Ci_3:o / anteiso-Ci_4:or, anteiso-Ci_5:o / anteiso-Ci_6:0, anteiso-Cn_:0, anteiso-Ci_8:0, and anteiso-Ci_9;0.

The R group of a branched or unbranched fatty acid, branched or unbranched fatty aldehyde, or branched or unbranched fatty alcohol can be saturated or unsaturated. If unsaturated, the R group may have one or more than one point of unsaturation. In some embodiments, the unsaturated fatty acid, unsaturated fatty aldehyde, or unsaturated fatty alcohol is a monounsaturated fatty acid, monounsaturated fatty aldehyde, or monounsaturated fatty alcohol. In certain embodiments, the unsaturated fatty acid, unsaturated fatty aldehyde, or unsaturated fatty alcohol is an unsaturated fatty acid, unsaturated fatty aldehyde, or unsaturated fatty alcohol C6: 1, C7: 1, C8: 1, C9: 1, C10: 1 , Cll: l, C12: l,

C13: l, C14: l, C15: l, C16: l, C17: l, C18: l, C19: l, C20: l,

C21: 1, C22: 1, 023: 1, 024: 1, C25: 1,

<img file="MX346830B_D0032.tif" />

Preferred embodiments, the unsaturated fatty acid, unsaturated fatty aldehyde, or unsaturated fatty alcohol is C10: 1, C12: 1, C14: 1, C16: 1, or C18: 1. In still other embodiments, the unsaturated fatty acid, unsaturated fatty aldehyde, or unsaturated fatty alcohol is unsaturated at the omega-7 position. In certain embodiments, the unsaturated fatty acid, unsaturated fatty aldehyde, or unsaturated fatty alcohol comprises a double cis bond.

As used herein, a recombinant host cell or engineered host cell is a host cell, eg, a microorganism that has been modified such that it produces fatty alcohols. In some embodiments, the recombinant host cell comprises one or more polynucleotides, each polynucleotide encoding a polypeptide having fatty aldehyde and / or fatty alcohol biosynthetic enzymatic activity, wherein the recombinant host cell produces a fatty alcohol composition when cultured in presence of a carbon source under conditions effective to express the polynucleotides.

As used herein, the term "clone" typically refers to a cell or group of cells that are descended from and are essentially genetically identical to a single common ancestor, for example, the bacterium of a

<img file="MX346830B_D0033.tif" />

As used herein, the term "culture" typically refers to a liquid medium comprising viable cells. In one embodiment, a culture comprises cells that reproduce in a predetermined culture medium under controlled conditions, for example, a culture of recombinant host cells grown in a liquid medium comprising a source of selected carbon and / or nitrogen.

The terms cultivation or cultivation refer to growing a population of cells (eg, microbial cells) under suitable conditions in a solid liquid medium. In particular embodiments, cultivar refers to the fermentative bioconversion of a substrate to a final product. Culture media are well known and the individual components of such culture media are available from commercial sources, for example under DIEGO medium and BBL medium. In a non-limiting example, the aqueous nutrient medium is a rich medium comprising complex sources of nitrogen, salts and carbon, such as YP medium, comprising 10 g / L of peptone and 10 g / L of yeast extract of such means, medium.

The host cell can be further designed to

<img file="MX346830B_D0034.tif" />

<img file="MX346830B_D0035.tif" />

IMPI efficiently simulates carbon and uses cellulosic materials as carbon sources of Sd according to the methods described, for example, in US Patents 5,000,000; 5,028,539; 5,424,202; 5,482,846; 5,602,030 and

WO2010127318. Furthermore, the host cell can be engineered to express an invertase so that sucrose can be used as a carbon source.

As used herein, the term "under conditions effective to express the heterologous nucleotide sequences" means any of the conditions that allow a host cell to produce a desired fatty aldehyde or fatty alcohol. Suitable conditions include, for example, fermentation conditions.

As used herein, modified or an altered level of activity of a protein, eg, an enzyme, in a recombinant host cell refers to a reference to one or more characteristics in the activity determined relative to the precursor host cell. native. Typically, differences in activity are determined between a recombinant host cell, which has modified activity, and the corresponding wild-type host cell (e.g., comparison of a culture of a recombinant host cell versus the host cell of type natural). The modified activities may be the result of, j 'modified amounts of proteins expressed by uiiu¹ tfCTtiTS: ^ recombinant host (for example, as the result of increased or decreased number of copies of DNA sequences encoding proteins, increased or decreased number of mRNA transcripts encoding protein, and / or increased or decreased amounts of protein translation of mRNA protein); changes in protein structure (eg, changes to the primary structure, such as, changes to the protein coding sequence resulting in changes in substrate specificity, changes in observed kinetic parameters); and changes in the stability of the protein (eg, increased or decreased degradation of the protein). In some embodiments, the polypeptide is a mutant or a variant of any of the polypeptides described herein. In certain cases, the coding sequence for the polypeptides as described herein are codon optimized for expression in a particular host cell. For example, for expression in E. coli, one or more codons can be optimized (Grosjeany collaborators (1982) Gene 18: 199-209).

The term "regulatory sequences" as used herein typically refers to a sequence of bases in DNA, operably linked to DNA sequences encoding a protein that ultimately controls the expression of the protein. Examples of regulatory sequences include, but are not limited to, RNA promoter sequences, transcription factor binding sequences, transcription termination sequences, transcription modulators such as enhancer elements), nucleotide sequences that affect RNA stability , and translational regulatory sequences (such as ribosome binding sites (eg, Shine-Dalgarno sequences in prokaryotic or Kozak sequences in eukaryotes), start codons, stop codons).

As used herein, the phrase "expression of the nucleotide sequence is modified relative to the wild-type nucleotide sequence," means an increase or decrease in the level of expression and / or activity of an endogenous nucleotide sequence, or the expression and / or activity of a nucleotide sequence that encodes the heterologous or non-native polypeptide.

As used herein, the term "express" with respect to a polynucleotide is "cause it to function." A polynucleotide that encodes a polypeptide (or protein), when expressed, will be transcribed and translated to produce that polypeptide (or protein). As used in the

<img file="MX346830B_D0036.tif" />

INSTITUTO MEXICANO DE LA PROPIEDAD NOUSTRIAL present, the term "overexpressed" means that it expresses (or causes to express) a polynucleotide or polypeptide in a cell at a higher concentration than is normally expressed in a corresponding wild-type cell under the same conditions.

The terms altered level of expression and modified level of expression are used interchangeably and mean that a polynucleotide, polypeptide, or hydrocarbon is present in a different concentration in a designated host cell as compared to its concentration in a corresponding wild-type cell under the terms. same conditions.

As used herein, the term "concentration" refers to the amount of fatty aldehyde or fatty alcohol produced per unit volume of the host cell culture. In any aspect of compositions and methods described herein, a fatty alcohol is produced at a concentration of about 25 mg / L, about 50

<td>mg / L, approximately</td><td>75 mg / L</td><td>, approximately</td><td> 100</td><td>mg / L,</td>

<td>about 125</td><td>mg / L,</td><td>approximately</td><td> 150</td><td>mg / L,</td>

<td>about 175</td><td>mg / L,</td><td>approximately</td><td> 200</td><td>mg / L,</td>

<td>about 225</td><td>mg / L,</td><td>approximately</td><td> 250</td><td>mg / L,</td>

<td>about 275</td><td>mg / L,</td><td>approximately</td><td> 300</td><td>mg / L,</td>

<td>about 325</td><td>mg / L,</td><td>approximately</td><td> 350</td><td>mg / L,</td>

⁵³ IMPI

<td>approximately</td><td> 375</td><td>mg / L,</td><td>. , OF approximately</td><td>LA PROMBDAD rME? J§r ^ AL</td><td></td>

<td>approximately</td><td> 425</td><td>mg / L,</td><td colspan="3">approximate merrte *¹* 450- / by -</td>

<td>approximately</td><td> 475</td><td>mg / L,</td><td>approximately</td><td> 500</td><td>mg / L,</td>

<td>approximately</td><td> 525</td><td>mg / L,</td><td>approximately</td><td> 550</td><td>mg / L,</td>

<td>approximately</td><td> 575</td><td>mg / L,</td><td>approximately</td><td> 600</td><td>mg / L,</td>

<td>approximately</td><td> 625</td><td>mg / L,</td><td>approximately</td><td> 650</td><td>mg / L,</td>

<td>approximately</td><td> 675</td><td>mg / L,</td><td>approximately</td><td> 700</td><td>mg / L,</td>

<td>approximately</td><td> 725</td><td>mg / L,</td><td>approximately</td><td> 750</td><td>mg / L,</td>

<td>approximately</td><td> 775</td><td>mg / L,</td><td>approximately</td><td> 800</td><td>mg / L,</td>

<td>approximately</td><td> 825</td><td>mg / L,</td><td>approximately</td><td> 850</td><td>mg / L,</td>

<td>approximately</td><td> 875</td><td>mg / L,</td><td>approximately</td><td> 900</td><td>mg / L,</td>

<td>approximately</td><td> 925</td><td>mg / L,</td><td>approximately</td><td> 950</td><td>mg / L,</td>

<td>approximately</td><td> 975</td><td>mg / L,</td><td>approximately</td><td> 1000</td><td>mg / L,</td>

<td>approximately</td><td> 1050</td><td>mg / L,</td><td>approximately</td><td> 1075</td><td>mg / L,</td>

<td>approximately</td><td> 1100</td><td>mg / L,</td><td>approximately</td><td> 1125</td><td>mg / L,</td>

<td>approximately</td><td> 1150</td><td>mg / L,</td><td>approximately</td><td> 1175</td><td>mg / L,</td>

<td>approximately</td><td> 1200</td><td>mg / L,</td><td>approximately</td><td> 1225</td><td>mg / L,</td>

<td>approximately</td><td> 1250</td><td>mg / L,</td><td>approximately</td><td> 1275</td><td>mg / L,</td>

<td>approximately</td><td> 1300</td><td>mg / L,</td><td>approximately</td><td> 1325</td><td>mg / L,</td>

<td>approximately</td><td> 1350</td><td>mg / L,</td><td>approximately</td><td> 1375</td><td>mg / L,</td>

<td>approximately</td><td> 1400</td><td>mg / L,</td><td>approximately</td><td> 1425</td><td>mg / L,</td>

<td>approximately</td><td> 1450</td><td>mg / L,</td><td>approximately</td><td> 1475</td><td>mg / L,</td>

<td>approximately</td><td> 1500</td><td>mg / L,</td><td>approximately</td><td> 1525</td><td>mg / L,</td>

<td>approximately</td><td> 1550</td><td>mg / L,</td><td>approximately</td><td> 1575</td><td>mg / L,</td>

<td rowspan="2">approximately approximately</td><td rowspan="2"> 1600 1650</td><td rowspan="2">mg / L, mg / L,</td><td colspan="3">⁵⁴approaches damentS^ISTDEii ^ S ^ DA »industrial -------</td>

<td>approximately</td><td> 1675_</td><td>mg / L,</td>

<td>approximately</td><td> 1700</td><td>mg / L,</td><td>approximately</td><td> 1725</td><td>mg / L,</td>

<td>approximately</td><td> 1750</td><td>mg / L,</td><td>approximately</td><td> 1775</td><td>mg / L,</td>

<td>approximately</td><td> 1800</td><td>mg / L,</td><td>approximately</td><td> 1825</td><td>mg / L,</td>

<td>approximately</td><td> 1850</td><td>mg / L,</td><td>approximately</td><td> 1875</td><td>mg / L,</td>

<td>approximately</td><td> 1900</td><td>mg / L,</td><td>approximately</td><td> 1925</td><td>mg / L,</td>

<td>approximately</td><td> 1950</td><td>mg / L,</td><td>approximately</td><td> 1975</td><td>mg / L,</td>

<td>approximately</td><td colspan="3">2000mg / L (2g / L), 3g / L, 5g / L,</td><td>lOg / L,</td><td>20g / L,</td>

30g / L, 40g / L, 50g / L, 60g / L, 70g / L, 80g / L, 90g / L, 100g / L or an interval linked by any of the two of the above values. In other embodiments, a fatty aldehyde or fatty alcohol is produced in a concentration of more than 100g / L, more than 200g / L, more than 300g / L, or higher, such as 500g / L, 700g / L, 1000 g / L, 1200 g / L, 1500 g / L, or 2000 g / L. The preferred concentration of fatty aldehyde or fatty alcohol produced by a recombinant host cell according to the methods of the description is 5g / L to 200g / L, 10g / L to 150g / L, 20g / L to 120g / L and 30g / L to lOOg / L.

As used herein, the term "yield of fatty aldehyde or fatty alcohol produced by a host cell" refers to the efficiency by which an input carbon source is converted to the product (i.e., fatty alcohol or fatty aldehyde) into a host cell. Host cells designed to produce alcone &amp; ^ '^ graSrrs' '' and / or fatty aldehydes according to the— description have a yield of at least 3%, at least 4%, at least 5%, at least 6%, at at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least less 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least minus 27%, at least 28%, at least 29%, or at least 30% or a range bound by either of the two previous values. In other embodiments, a fatty aldehyde or fatty alcohol is produced in a yield of more than 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. Alternatively, or in addition, the yield is about 30% or less, about 27% or less, about 25% or less, or about 22% or less. In this way, performance can be tied to either of the two endpoints above. For example, the yield of the fatty alcohol or fatty aldehyde produced by the recombinant host cell according to the methods of the description can be 5% to 15%, 10% to 25%, 10% to 22%, 15% to 27%. , 18% to 22%, 20% to 28%, or 20% to 30%. The preferred yield of the fatty alcohol produced by the recombinant host cell according to the methods of the disclosure is 10% to 30%.

As used herein, the term "productivity" refers to the amount of fatty aldehyde or fatty alcohol produced per unit volume of the host cell culture per unit time. In any aspect of the compositions and methods described herein, the productivity of the fatty aldehyde or fatty alcohol produced by a recombinant host cell is at least 100 mg / L / hour, at least 200 mg / L / hour, at least 300 mg. / L / hour, at least 400 mg / L / hour, at least 500 mg / L / hour, at least 600 mg / L / hour, at least 700 mg / L / hour, at least 800 mg / L / hour, at least 900 mg / L / hour, at least 1000 mg / L / hour, at least 1100 mg / L / hour, at least 1200 mg / L / hour, at least 1300 mg / L / hour, at least 1400 mg / L / hour, at least 1500 mg / L / hour, at least 1600 mg / L / hour, at least 1700 mg / L / hour, at least 1800 mg / L / hour, at least 1900 mg / L / hour, at least 2000 mg / L / hour, at least 2100 mg / L / hour, at least 2200 mg / L / hour, at least 2300 mg / L / hour, at least 2400 mg / L / hour, or at least 2500 mg / L / hour. Alternatively, or in addition, the productivity is 2500 mg / L / hour or less, 2000 mg / L / OD₆₀₀ or less, 1500 mg / L / OD₆oo or less, 120 mg / L / hour, or less, 1000 mg / L / hour or less, 800 mg / L / hour, or less, or 600 mg / L / hour or less. In this way, productivity can be linked by any of two or more of the above end points. For example, productivity can be 3 to 30 mg / L / hour, 6 to 20 mg / L / hour, or^{57 IM}XI at 30 mg / L / hour. Preferred productivity

<img file="MX346830B_D0037.tif" />

Fatty or fatty alcohol produced by a recombinant host cell according to the methods of the description, is selected from 500 mg / L / hour to 2500 mg / L / hour, or from 700 mg / L / hour to 2000 mg / L / time.

The terms total fatty species and total fatty acid product can be used interchangeably herein with reference to the total amount of fatty alcohols, fatty aldehydes, free fatty acids, and fatty esters present in a sample as evaluated by GC-FID as is described in International Patent Application Publication WO 2008/119082. Samples can contain one, two, three, or four of these compounds depending on the context.

As used herein, the term "glucose utilization rate" means the amount of glucose used by the culture per unit time, reported as grams / hour (g / L / hr).

As used herein, the term "carbon source" refers to a substrate or compound suitable for use as a carbon source for the growth of simple prokaryotic or eukaryotic cells. Carbon sources can be in various forms including, but not limited to, polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides,^OF ^ iN Say ^ example, CO and C0₂). Sources of include, but are not limited to, monosaccharides, such as glucose, fructose, mannose, galactose, xylose, and arabinose; oligosaccharides, such as fructo-oligosaccharide and galactooligosaccharide; polysaccharide ales such as starch, cellulose, pectin, and xylan; disaccharides, such as sucrose, maltose, cellobiose, and turanose; cellulosic material and variants such as hemicelluloses, methyl cellulose, and sodium carboxymethyl cellulose; saturated or unsaturated fatty acids, succinate, lactate, and acetate; alcohols, such as ethanol, methanol, and glycerol, or mixtures thereof. The carbon source can also be a product of photosynthesis, such as glucose. In certain embodiments, the carbon source is a CO-containing gas mixture from flue gas. In another embodiment, the carbon source is a CO-containing gas mixture that comes from reforming a carbon-containing material, such as biomass, coal, or natural gas. In other modalities, the carbon source is synthesis gas, methane, natural gas. In certain preferred embodiments, the carbon source is biomass. In other preferred embodiments, the carbon source is glucose. In other preferred embodiments, the carbon source is sucrose. In other modalities, the source of ⁵⁹ ΙΜΡΙ carbon is glycerol. In other modes upww ^^^^ s^D Industrial carbon source is sugarcane juice, cane syrup .._______- - - sugar, or corn syrup. In other preferred embodiments, the carbon source is derived from renewable raw materials, such as CO2, CO, glucose, sucrose, xylose, arabinose, glycerol, mannose, or mixtures thereof. In other modalities, the carbon source is derived from renewable raw materials including starches, cellulosic biomass,

<img file="MX346830B_D0038.tif" />

molasses, and other carbohydrate sources including carbohydrate mixtures derived from hydrolysis of cellulosic biomass, or waste materials derived from natural or vegetable oil processing.

As used herein, the term biomass refers to any biological material from which a carbon source is derived. In some embodiments, a biomass is processed into a carbon source, which is suitable for bioconversion. In other modalities, biomass does not require additional processing into a carbon source. An exemplary source of biomass is plant matter or vegetation, such as corn, sugar cane, or millet grasses. Another exemplary source of biomass is metabolic waste products, such as animal matter (eg cow manure). Additional exemplary sources of biomass include algae and other marine plants. Biomass also includes products

<img file="MX346830B_D0039.tif" />

IMPI

MEXICAN INFTHVTO ^v PE IA FROP1RTY

INDUSTRIAL residuals from industry, agriculture, forestry, and ——— afteiCWi - 111 gjja^lLrp - ^. TT ^ ¿t household, including, but not limited to, glycerol, fermentation residues, silage, straw, wood, sewage, garbage, cellulosic municipal waste, and food scraps (for example, soaps, oils and fatty acids). The term biomass also refers to carbon sources, such as carbohydrates (eg, monosaccharides, disaccharides, or polysaccharides).

As used herein, the term "isolated", with respect to products (such as fatty acids and derivatives thereof,) refers to products that are separated from cellular components, cell culture media, or chemical or synthetic precursors. . The fatty acids and derivatives thereof produced by the methods described herein can be relatively immiscible in the fermentation broth, as well as in the cytoplasm. Therefore, fatty acids and derivatives thereof can be collected in an organic phase either intracellularly or extracellularly.

As used herein, the terms purify, purified, or purification mean the removal or isolation of a molecule from its environment by, for example, isolation or. separation. The substantially purified molecules are at least approximately fNíTTTUTO MEXICANO,, Di LA FROPIIDA »

60% free (for example, at least about 7 0 ^^ 5 ^ 6 3 ^ - at least about 75% free, at least

85% free, at least about 90% free, at least about 95% free, at least about 97% free, at least about 99% free) of other components with which they associate. As used herein, these terms also refer to the removal of contaminants from a sample. For example, the removal of contaminants can result in an increase in the percentage of fatty acid derivatives such as fatty alcohols in a sample. For example, when a fatty acid derivative is produced in a recombinant host cell, the fatty acid derivative can be purified by removal of host cell proteins or other host cell materials. After purification, the percentage of the fatty acid derivative in the sample increases. The terms purify, purified, and purification are relative terms that do not require absolute purity. Thus, for example, when a fatty acid derivative is produced in recombinant host cells, a purified fatty acid derivative is a fatty acid derivative that is substantially separated from other cellular components (e.g., nucleic acids, polypeptides, lipids, carbohydrates, or other hydrocarbons).

IMPI ^, INSTITUTO MÍZICANO

Increased Fatty Alcohol Production

The disclosure provides for the production of a fatty alcohol composition that is enhanced as a result of modified expression of an acyl-ACP reductase (AAR) gene in a host cell. AAR is involved in a biosynthetic pathway for the production of fatty aldehydes and fatty alcohols. Variant AAR is used alone or in combination with the modified expression of another gene involved in the biosynthetic pathway that converts a fatty aldehyde to a fatty alcohol. At this point, the disclosure provides recombinant host cells, which have been engineered to express a variant AAR to provide improved fatty alcohol biosynthesis relative to non-engineered host cells that are native or wild-type expressing wild-type or other AARs. Fatty alcohol biosynthetic polypeptides with the same function as AAR. The disclosure identifies AAR-related polynucleotides and polypeptides useful in recombinant host cells. However, it will be recognized that absolute sequence identity to AAR-related polynucleotides is not necessary. For example, changes can be made to the polynucleotide sequence and the encoded polypeptide is classified for activity. Such changes typically include conservation mutations and silent mutations such as, by. -, η .... , nwnwroMiMCANQ example, through the optimization of modified or mutated polynucleotides (i.e. mutants) .. and the encoded variant polypeptides can be classified for a desired function, such as, improved function compared to the parent polypeptide, including but not limited to increased catalytic activity, increased stability, or decreased inhibition (eg, decreased feedback inhibition), using methods known in the art. The description identifies enzymatic activities involved in various steps (i.e., reactions) of the fatty acid biosynthetic pathways described herein according to the Enzyme Classification (EC) number, and provides exemplary polypeptides (enzymes) classified by such numbers. EC, and exemplary polynucleotides encoding such peptides. Such exemplary polypeptides and polynucleotides, which are identified herein by Accession Numbers and / or Sequence Identifier Numbers (SEQ ID NOs), are useful for designing fatty acid pathways in precursor host cells to obtain recombinant host cells. described herein. It is to be understood, however, that the polypeptides and polynucleotides described herein are exemplary, and thus not limiting. The sequences of the homologues of the exemplary polypeptides described in

IMPI

INSTITUTO MEXICANO Oí LA FRDFISDAO 'INDUSTRIAL present are available to those with experience in the art using databases, such as, for example, the Entrez databases provided by the National Center for Biotechnology Information (NCBI), the databases of ExPasy data provided by the Swiss Institute of Bioinformatics, the BRENDA database provided by the Technical University of Braunschweig, and the KEGG database provided by the Kyoto University Center for Bioinformatics and the University of Tokyo, all of which are available on the World Wide Web. A variety of different host cells can be engineered to express variant AAR fatty alcohol biosynthetic enzymes such as those described herein, resulting in recombinant host cells suitable for improved production of fatty alcohol compositions. It is understood that a variety of cells can provide sources of genetic material, including polynucleotide sequences encoding suitable polypeptides for use in a recombinant host cell as described herein.

Acyl-ACP Reductase Polypeptides (AAR) and Variants Thereof

In one aspect, the description refers to the improved production of fatty acid derivatives such as aldehydes. ⁶⁵ fatty and / or fatty alcohols when designing a ^p to express a native or non-native acyl-ACP reductase protein (AAR). The AAR protein catalyzes the reduction of an acylACP to a fatty aldehyde and can also catalyze the conversion of a fatty aldehyde to a fatty alcohol (see US Patent Publication No. 20120282663, incorporated herein by reference). The AAR polypeptide or polynucleotide sequence encoding the polypeptide

AAR may be native (eg endogenous) or non-native (eg exogenous, heterologous, etc.), that is, it may differ from the wild-type sequence and the expression thereof naturally present in the type host cell. corresponding natural. Examples include a modification in the sequence of the AAR polynucleotide, polypeptide, or protein resulting in a variant (eg, mutant) AAR and / or in the level of expression thereof. The description includes AAR polypeptides, homologues, and variants.

In one embodiment, an AAR Polypeptide for use in the practice of the disclosure has at least 90% sequence identity to the wild-type AAR polypeptide sequence of SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO. : 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42 or SEQ ID NO: 44. In some embodiments the AAR is derived from a species of Synechococcus or a species of Prochlorococcus. In

<img file="MX346830B_D0040.tif" />

ΙΜΤΠνΤΟ KXICANO BELAFSOm * AÍ&gt;

other embodiments, an AAR polypeptide for use Μ * ρra of the description has at least 75% (pal Gjéiñ ^ lu, at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83% , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% ) of sequence identity to the wild-type AAR polypeptide sequence SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42 or SEQ ID NO: 44, and may also include one or more substitutions that result in the useful characteristics and / or properties as described herein. In one embodiment, the AAR polypeptide for use in the practice of the present disclosure is at least 75% {eg, at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83 %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99 %) sequence identity to wild type AAR polypeptide sequence of SEQ ID

NO: 28 or SEQ ID NO: 34.In other embodiments, an AAR polypeptide for use in the practice of the disclosure has 100% sequence identity to SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42 or SEQ ID NO: 44. In still other embodiments, the variant improved AAR polypeptide sequence it is derived from a species other than a Synechococcus species or a Prochlorococcus species.

In a related embodiment AAR polypeptides that have a la

<img file="MX346830B_D0041.tif" />

sequence

<img file="MX346830B_D0042.tif" />

encoded by a nucleic acid sequence that has at least 75% (eg, at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or / and at least 99%) of sequence identity to SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 or SEQ ID NO: 43. In some embodiments the nucleic acid sequence encodes an AAR variant with one or more substitutions that result in the improved characteristics and / or properties as described herein. In yet another related embodiment, an AAR polypeptide for use in the practice of the disclosure is encoded by a nucleotide sequence that has 100% sequence identity to SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 or SEQ ID NO: 43. In another aspect, the description refers to AAR polypeptides comprising an amino acid sequence encoded by a nucleic acid that hybridizes under severe conditions over substantially the full length of a nucleic acid corresponding to SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35,

SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID N0: 41 or SEQ ID NO: 43.

In a preferred embodiment the

<img file="MX346830B_D0043.tif" />

Mexican mrmvro MlAPtOriMAD

<img file="MX346830B_D0044.tif" />

an AAR polypeptide for use in pTVitrea of ​​the disclosure that has at least 90% sequence identity to the variant AAR polypeptide sequence of any of SEQ ID NO: 57 through SEQ ID NO: 78. In some embodiments the AAR variant is derived from a species of Synechococcus or a species of Prochlorococcus. In other embodiments, an AAR polypeptide for use in the practice of the disclosure has at least 75% (eg, at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83% , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% ) of sequence identity to the variant AAR polypeptide sequence of any of SEQ ID NO: 57 through SEQ ID NO: 78. The variant AAR polypeptide can include one or more substitutions that result in useful characteristics and / or properties as described herein. In another preferred embodiment, the AAR polypeptide for use in the practice of the disclosure has at least 75% (eg, at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83 %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99 %) sequence identity to the variant AAR polypeptide sequence of SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 or SEQ ID NO: 65. In other embodiments, an AAR polypeptide for use in the description practice

<img file="MX346830B_D0045.tif" />

has 100% identity of

INSTITUTO MEXICANO otunoracAD sequence anyone © gT'BílQ

NO: 57 to SEQ ID NO: 78. In still others ¹ niudal^,lTM (5 @^,S *, "^T^ Improved or variant AAR polypeptide sequence is derived from a species other than a Synechococcus species or a Prochlorococcus species.

The inventors constructed an error-prone library of Synechococcuselongatus acyl-ACP reductase PCC7942 (AAR_7942) in order to classify variants that have improvements over wild-type AAR_7942 (see Example 3, infra). Improvements are classified as improving either the total fatty alcohol concentration or increasing the fraction of the CIO, C12, C14 or C16 fatty alcohols without significantly affecting the concentration. The error prone library identified several amino acid positions including amino acid position 18. The saturation and pooling libraries were prepared to test these positions SEQ ID NO: 57 encodes the amino acid sequence for an AAR variant (mutant) having a mutation at amino acid 18. The S18W mutation where serine is replaced with tryptophan results in a significant increase in fatty alcohol production when expressed in cells (see Examples 3 and 4, infra). More specifically, the S18W mutation leads to a 227 percent increase in alcohol production.⁷⁰

INSTITUTO MEXICANO DE LA PROPIEDAD total fat (FALC) and a 324 percent increase in C14 fatty alcohols compared to the AAR of natural origin used as a control (see Tables 4A and 4B, infra).

The inventors constructed saturation libraries based on the S18W mutation in order to identify the variants (mutants) that further increased the total FALC concentration or the fraction of C12 fatty alcohols (see Example 4 and Table 5, infra). Pooling libraries using the S18W mutation (SEQ ID NO: 57) as a template produced 7 pooling mutants that showed additional significant increases in total FALC concentration and / or C12 fatty alcohol production (see Table 6B, infra) . The 7 combination mutants include AARs with the S18W mutation (SEQ ID NO: 57); AAR with the mutations M21L, C63G, S113K, T154A, A281L (SEQ ID NO: 58); AAR with the mutations L8A, M21L, C63G, A77A (GCC to GCA silencing codon mutation), S113K,

T154A, A281L (SEQ ID NO: 59); AAR with the mutations D16L, M21L, C63G, S113K, T154A, A281L (SEQ ID NO: 60); AAR with the mutations L8A, D24V, C63G, S113K, Q155L, A281L (SEQ ID NO: 61); AAR with the mutations D24P, L31M, C63G, S113K, T154A, A281L (SEQ ID NO: 62); AAR with the mutations L8A, D16L, D24V, C63G, S113K, T154A, A281L (SEQ ID NO: 63); and AAR with the mutations D24E, C63G, S113K, T154A, A281L (SEQ ID NO:

<img file="MX346830B_D0046.tif" />

i IMPI

MEXICAN INSTITUTE

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64). Notably, SEQ ID NO: 58 showed the highest fraction of the C12 fatty alcohols while SEQ ID NO: 59 showed the highest concentration of the combination mutants (see Table 6B, infra).

The inventors also constructed a complete saturation library of Prochlorococcusmarinus MED4_AAR acyl-ACP reductase in order to classify variants that show improvements over wild type MED4_AAR (see Example 7, infra). The AAR variants were selected based on the production of more fatty alcohols than the wild-type AAR enzyme and the ability to produce fatty alcohols with an altered chain length profile, eg, an increased C12, C14, or C16 fraction. Table 8 (see Example 7, infra) shows representative data for the 16 AAR variants that produced the highest FALC concentrations ranging from 1.4 times to 2.2 times over the wild type MED4_AARs. The inventors also classified AAR variants having an altered chain profile, wherein an increased portion of the FALC species with a chain length shorter than C16 is of interest. Two variant clones leading to a 2-3 fold increase in the amount of FALC are shown in Figure 10. The expression of one of these AAR variants, that is, the D61E mutant (SEQ ID NO: 65) in a recombinant host cell, shifted the chain length distribution from the es ^ S ^ SIR ^ SoAalc ^^^ fatty towards shorter carbon chains,.,. .All. variants led to a higher amount of FALC as they had increased the concentration, but only SEQ ID NO: 65 had a C14 fraction (and higher concentration). Table 9 (see Example 7, infra) illustrates the FALC chain length distribution produced by recombinant host cells expressing MED4_AAR variant D61E compared to wild-type MED4_AAR (WT) and MED4_AAR variant V346P that did not produce products with altered chain lengths.

Acyl Carrier Protein (ACP)

There are reports of conflict in the literature regarding the factors that can limit fatty acid biosynthesis in host cells, such as Escherichia coli (E. coli). Although acyl carrier proteins (ACPs) are conserved to some degree in all organisms, their primary sequence may differ. It has been suggested that when terminal pathway enzymes from sources other than E. coli are expressed in E. coli in order to convert fatty acyl-ACPs to products, there may be limitations such as in the recognition, affinity and / or change of the recombinant pathway enzyme towards fatty acyl-ACPs (see Suh et al. (1999) The Plant Journal 17 (6): 679-688; Salas and

IMPI ^ INSTITUTO MEXICANO collaborators (2002) Archives of Biochemistry

403: 25-34). One suggestion is that a major precursors of fatty acid biosynthesis, for example, acetyl-CoA and malonyl-CoA may result in decreased synthesis of the fatty acid derivatives. One procedure to increase flux through fatty acid biosynthesis is to manipulate various enzymes in the pathway, (see Figures 1-3). The supply of acetyl-CoA acyl-ACPs through the acetyl-CoA carboxylase complex (acc) and the fatty acid biosynthetic pathway (fab) can impact the rate of fatty acid-derived production (see Figure 2). As detailed in the Examples (infraj, the effect of ACP overexpression on the production of fatty alcohols was evaluated for the purpose of illustration.

A host cell that is engineered to express ACP may show an increase in the concentration of a fatty aldehyde and / or fatty alcohol composition or a specific fatty aldehyde and / or fatty alcohol composition where the increase is at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%

<img file="MX346830B_D0047.tif" />

27%, at least 28%, at least 29%, or at menaean ^^ ffíSydr``than the concentration of the fatty aldehyde and / or fatty alcohol composition produced by a corresponding host cell that does not express ACP when grown under the same conditions. In one aspect, the disclosure refers to the improved production of a fatty aldehyde and / or fatty alcohol composition by engineering a host cell to express a native (eg, endogenous) or non-native (eg, exogenous, heterologous) ACP protein. ). The ACP polypeptide or the polynucleotide sequence encoding the ACP polynucleotide may be non-native, that is, it may differ from the wild-type sequence naturally present in the corresponding wild-type host cell. Examples include a modification in the level of expression in the sequence of nucleotides, polypeptides or proteins. The description includes ACP polypeptides and homologues thereof.

In one embodiment, an ACP Polypeptide for use in the practice of the disclosure has at least 70% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10. In some embodiments ACP is derived from Marinobacterhydrocarbonoclasticusor E. coli. In other embodiments, an ACP polypeptide for use in the practice of⁷⁵IMSTITUTO MBXICAN · DtlAMt MEBAD the description has at least 75% (for example, a'I ^ ienos / S%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 8 5%, 8 6%,

89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least

99%) sequence identity to the wild-type ACP polypeptide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10, and can also include one or more substitutions that result in useful characteristics and / or properties as described herein. In one aspect, an ACP polypeptide for use in the practice of the disclosure has 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID. NO: 10. In other embodiments, the improved or variant ACP polypeptide sequence is derived from a species other than M. hydrocarbonoclasticusor or E. coli. In a related aspect, an ACP polypeptide for use in the practice of the disclosure is encoded by a nucleotide sequence that has 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID N0: 5 , SEQ ID NO: 7, or SEQ ID NO: 9. In another related aspect, the description refers to ACP polypeptides comprising an amino acid sequence encoded by a nucleic acid sequence that has at least 75% (eg, at least 76%, 77%, 78%, 79%, 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or and at least 99%) identity of

<img file="MX346830B_D0048.tif" />

<img file="MX346830B_D0049.tif" />

Mexican ifwrrrvro sequence to SEQ ID N0: 1, SEQ ID NO: 3, SEQ W888, NO: 7, or SEQ ID NO: 9. In some modalities, iO nucleic acids encode an ACP variant with one or more substitutions that result in characteristics and / or improved properties as described herein. In other embodiments, the improved and variant ACP and nucleic acid sequence is derived from a species other than a Marinobacter or E. coli species. In another aspect, the description refers to ACP polypeptide having an amino acid sequence encoded by a nucleic acid that hybridizes under severe conditions over substantially the full length of a nucleic acid corresponding to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.

Variations and Mutations

In some embodiments, the AAR or ACP polypeptide is a mutant or a variant of any of the peptides described herein. A "variant or mutant polypeptide" as used herein refers to a polypeptide that has an amino acid sequence that differs from a wild-type polypeptide by at least one amino acid. For example, the mutant may have one or more of the following conservative amino acid substitutions, including but not limited to, the replacement of an aliphatic amino acid, such as alanine, valine, leucine, and

<img file="MX346830B_D0050.tif" />

aliphatic amino acid; the replacement of a fight with a threonine; replacement of a threonine with a serine; the replacement of an acidic residue, such as aspartic acid, and glutamic acid, with another acid residue, the replacement of a residue that carries an amide group, such as asparagine and glutamine, with another residue that carries an amide group; exchanging a basic residue, such as lysine and arginine, with another basic residue; and replacing an aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue. In some embodiments, the polypeptide variant or mutant has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acid substitutions, additions, insertions, or deletions. Some preferred fragments of a polypeptide that fuses as a variant or mutant retain some or all of the biological fusion (eg, enzymatic activity) of the corresponding wild-type polypeptide. In some embodiments, the fragment retains at least 75%, at least 80%, at least 90%, at least 95%, or at least 98% or more of the biological function of the corresponding wild-type polypeptide. In other embodiments, the fragment or mutant retains approximately 100% of the biological function of the corresponding wild-type polypeptide. The guide in determining which amino acid residues

<img file="MX346830B_D0051.tif" />

inserting, or deleting without affecting biological activity, can be found using computer programs well known in the art, eg, the LASERGENE software (DNASTAR, Inc., Madison, WI). In still other embodiments, a fragment exhibits increased biological function as compared to a corresponding wild-type polypeptide. For example, a fragment may express at least 10%, at least 25%, at least 50%, at least 75%, or at least 90% improvement in enzymatic activity as compared to the polypeptide of corresponding natural type. In other embodiments, the fragment expresses at least 100%, at least 200%, or at least 500% improvement in enzymatic activity as compared to the corresponding wild-type polypeptide.

It is understood that the polypeptides described herein may have additional non-essential or conservative amino acid substitutions that do not have a substantial effect on the function of the polypeptide. Whether or not a particular substitution is to be tolerated (that is, it will not adversely affect the desired biological function, such as acyl-ACP reductase activity), can be determined as is known in the art (see Bowie et al. (1990 ) Science, 247: 1306-1310). A substitution of

<img file="MX346830B_D0052.tif" />

₇₅ IMPI ' ^and Conservative amino acids industrial MtOHSDAP is one in which the amino acid residue is replaced with an amino acid residue that has a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cistern), non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta branched side chains (eg threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine).

Variants can be naturally occurring or created in vitro. In particular, such variants can be created using genetic engineering techniques, such as site-directed mutagenesis, random chemical mutagenesis, Exonuclease III deletion procedures, or standard cloning techniques. Alternatively, such variants, mutants, fragments, analogs, derivatives can be created using methods of chemical synthesis or modification. The methods for manufacturing variants are well known in the so IMPI ^^

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OF THE PROPERTY . z _____ γν _ __i___ η _ i _ - ·। _ i INDUSTRIAL _ '- ~~ r technique. For example, variants can be prepared!

Using random and site-directed mutagenesis Random and site-directed mutagenesis are generally known in the art (see, for example, Arnold (1993) Curr. Opin.Biotech. 4: 450-455). Random mutagenesis can be accomplished using error-prone PCR (see, eg, Leung et al. {1989) Technique 1: 11-15; and Caldwell et al. (1992) PCR Methods Applic. 2:28

33). In error-prone PCR, current PCR is carried out under conditions where the fidelity of DNA polymerase copying is low, such that a high rate of point kills is obtained throughout the entire length of the PCR product. . Briefly, in such procedures, the nucleic acids to be mutagenized (eg, a polynucleotide sequence encoding an AAR enzyme) are mixed with the PCR primers, the reaction buffer, MgCl₂, MnCl₂, Taqpolymerase, and an appropriate concentration of dNTPs to achieve a high rate of point mutation along the full length of the PCR product. For example, the reaction can be carried out using 20 fmoles of nucleic acid to be mutagenized, 30 pmol of each PCR primer, a reaction buffer comprising 50 mM KC1, 10 mM Tris HC1 (pH 8.3), 0.01% gelatin, MgCl₂ 7 mM,

<img file="MX346830B_D0053.tif" />

⁸¹ IMPI ΐΗΒΊΤΓϋΤΟ MIXICAN

MnCl₂ 0.5 mM, 5 units Taq polymerase, dGTP

0.2 mM, 1 mM dCTP, and 1 mM dTTP. PCR can be ^^ - ^ - ev ^^ '^,to^r~ SaEo 'for 30 cycles of 94 ° C for 1 min, 45 ° C for 1 min, and 72 ° C for 1 min. However, it will be appreciated by those skilled in the art that these parameters can be varied as. be appropriate. The mutagenized nucleic acids are then cloned into an appropriate vector, and the activities of the polypeptides encoded by the mutagenized nucleic acids are evaluated. Site-directed mutagenesis can be accomplished using oligonucleotide-directed mutagenesis to generate site-specific mutations in any cloned DNA of interest. Oligonucleotide mutagenesis is described in the art (see, for example, Reidhaar-Olson et al. (1988) Science 241: 53-57). Briefly, in such methods a plurality of double-stranded oligonucleotides carrying one or more mutations to be introduced into the cloned DNA are synthesized and inserted into the cloned DNA to be mutagenized (e.g., a polynucleotide sequence that encodes an AAR polypeptide). Clones containing the mutagenized DNA are recovered, and the activities of the polypeptides encoding them are evaluated.

Another method of generating the variants is assembly PCR. Assembly PCR involves the assembly of a PCR product from a mixture of fragments of

<img file="MX346830B_D0054.tif" />

A large number of different PCR reactions are presented in parallel in the same vial, with the products of one reaction priming the products of another reaction (see US Patent 5,965,408). Yet another method of generating variants is sexual PCR mutagenesis. In sexual PCR mutagenesis, forced homologous recombination occurs between DNA molecules of different, but highly related, DNA sequences in vitro as a result of random fragmentation of the DNA molecule based on sequence homology. This is followed by fixation of the crossover by primer extension in a PCR reaction. Sex PCR mutagenesis is described in publications known in the art (see, for example, Stemmer (1994) Proc. Nati. Acad. Sci. USA 91: 10747-10751). Variants can be created by in vivo mutagenesis. In some embodiments, random mutations in a nucleic acid sequence are generated by propagating the sequence in a bacterial strain, such as an E. coli strain, that carries mutations in one or more of the DNA repair pathways. Such mutant strains have a higher random mutation rate than that of a wild-type strain. The propagation of a DNA sequence (e.g., polynucleotide sequence encoding an AAR polypeptide) in one of

WICKED. _____ _ _ _,. INrmUTOMEXICAN these strains will eventually generate mu tations within DNA. Mutant strains suitable for, expensive use. ___________ mutagenesis in vivo is described in the publication in the art (see, eg, International Patent Application Publication No. WO1991 / 016427). Variants can also be generated using cassette mutagenesis. In cassette mutagenesis. A small region of a double-stranded DNA molecule is replaced with a cassette and synthetic oligonucleotide that differs from the native sequence.

The oligonucleotide frequently contains a complete and / or partially randomized native sequence. Recursive pool mutagenesis can also be used to generate variants. Recursive pool mutagenesis is an algorithm for protein engineering (ie, protein mutagenesis) developed to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combination cassette mutagenesis (see, for example, Arkin et al. (1992) Proc. Nati. Acad. Sci., USA 89: 7811-7815). In some embodiments, the variants are created using exponential set mutagenesis. Exponential ensemble mutagenesis is a process to generate combination libraries with a high

IMPI ^ percentage of unique and functional mutants, ene «^ M§ ^ A Rg 1¾¾¾ small residues are chosen randomly in parallel to identify, at each altered position, the amino acids that lead to functional proteins (see, for example, Delegravey co-workers (1993) Biotech. Res. 11: 1548-1552). In some embodiments, the variants are created using rearrangement procedures wherein portions of a plurality of nucleic acids encoding distinct polypeptides are fused to create chimeric nucleic acid sequences encoding chimeric polypeptides (as described in, for example, US Patents US Nos. 5,965,408 and 5,939,250).

Production of Fatty Aldehydes and Fatty Alcohols

A recombinant or native host cell may comprise a polynucleotide encoding an enzyme having the activity of fatty aldehyde biosynthesis (also referred to herein as a fatty aldehyde biosynthetic polypeptide or a fatty aldehyde biosynthetic enzyme or polypeptide). A fatty aldehyde is produced when the fatty aldehyde biosynthetic enzyme is expressed or overexpressed in the host cell. Expression or overexpression of an acyl-ACP reductase polypeptide (AAR) in a recombinant host cell can result in the production of a fatty aldehyde by the host cell.

IMPIOUS

Recombinant MEXICAN INSTITUTE. In one embodiment, the recombinant cell 1 produces a fatty aldehyde ——- E "- embodiments, the fatty aldehyde produced by the recombinant host cell is converted to a fatty alcohol. In some embodiments, native (endogenous) fatty aldehyde biosynthetic polypeptides, such as aldehyde reductases, are present in the host cell (eg, E. coli) and are effective in converting fatty aldehydes to fatty alcohols. In other embodiments, a native (endogenous) fatty aldehyde biosynthetic polypeptide is overexpressed. In still other embodiments, an exogenous fatty aldehyde biosynthetic polypeptide is introduced into a recombinant host cell and expressed or overexpressed. A fatty aldehyde can be produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a fatty aldehyde biosynthetic polypeptide such as a polypeptide having acyl-ACP reductase activity (AAR). Expression of AAR in a recombinant host cell results in the production of fatty aldehydes and fatty alcohols (Figure 4). Exemplary AAR polypeptides are described herein and in PCT Publication Nos. WO2009 / 140695 and WO / 2009/140696, both of which are expressly incorporated by reference herein.

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Ot THE PROWITY

A composition comprising aldehydes • JfS'OT'ds a fatty aldehyde composition) is produced — ai · cu 1L lV3T * Hj'ha host cell in the presence of a carbon source under conditions effective to express the fatty aldehyde biosynthetic enzyme, for example, AAR. A recombinant host cell designed to produce a fatty aldehyde will typically convert some of the fatty aldehyde to a fatty alcohol. In some embodiments, the fatty aldehyde composition comprises fatty aldehydes and fatty alcohols. Typically, the fatty aldehyde composition is recovered from the extracellular environment of the recombinant host cell, ie, the cell culture medium. In some embodiments, the recombinant host cell includes a polynucleotide encoding a polypeptide (an enzyme) that has fatty alcohol biosynthetic activity (also referred to herein as a fatty alcohol biosynthetic polypeptide or a fatty alcohol biosynthetic enzyme), and a Fatty alcohol is produced by the recombinant host cell. A composition that includes the fatty alcohols (ie, a fatty alcohol composition) can be produced by culturing the recombinant host cell in the presence of a carbon source under conditions effective to express a fatty alcohol biosynthetic enzyme. Native aldehyde reductases [eg, endogenous)

<img file="MX346830B_D0055.tif" />

present in a recombinant host cell (eg, E. cali), will convert fatty aldehydes to fatty alcohols. In some embodiments, native (eg, endogenous) fatty aldehyde biosynthetic polypeptides, such as aldehyde reductases present in the host cell, are sufficient to convert fatty aldehydes to fatty alcohols. However, in other embodiments, a fatty alcohol is produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a polypeptide having fatty alcohol biosynthetic activity that converts a fatty aldehyde to a fatty alcohol. For example, an alcohol dehydrogenase (also referred to herein as an aldehyde reductase, eg EC 1.1.1.1), may be useful in the practice of description. As used herein, the term "alcohol dehydrogenase" refers to a polypeptide capable of catalyzing the conversion of a fatty aldehyde to a fatty alcohol. One of ordinary skill in the art will appreciate that certain alcohol dehydrogenases are capable of catalyzing other reactions as well, and these non-specific alcohol dehydrogenases are also referred to by the term alcohol dehydrogenase. Examples of useful alcohol dehydrogenase polypeptides according to the disclosure include, but are not limited to, AlrA from Acinetobacter sp. Ml (SEQ ID

<img file="MX346830B_D0056.tif" />

NO: 52) or AlrA homologs such as AlrAac? Ír ^^ || ^^ 53) and alcohol dehydrogenases from endogenous E. coli such as ^ _____ YjgB, (AAC77226) (SEQ ID NO: 5), DkgA (NP_417485) , DkgB (NP_414743), YdjL (AAC74846), Yd j J (NP_416288), AdhP (NP_415995), YhdH (NP_417719), YahK (NP_414859), YphC (AAC75598), YqhD (4435bO56) and YAC7bO []. Additional examples are described in International Patent Application Publication Nos. WO2007 / 136762, WO2008 / 119082, and WO2010 / 062480, each of which is expressly incorporated by reference herein. In certain embodiments, the fatty alcohol biosynthetic polypeptide has aldehyde reductase or alcohol dehydrogenase activity (EC 1.1.1.1). In some embodiments, a native (eg, endogenous) fatty alcohol biosynthetic polypeptide is overexpressed and in other embodiments, an exogenous fatty alcohol biosynthetic polypeptide is introduced into a recombinant host cell and expressed or overexpressed.

Fatty alcohols can be produced via an acyl-CoA-dependent route using fatty acyl-ACP and fatty acyl-CoA intermediates and in an acyl-CoA independent route using fatty acyl-ACP intermediates but not the intermediate of fatty acyl-CoA. In particular embodiments, the enzyme encoded by the overexpressed gene ___. __ _ _. Instituto Mexicano is selected from a fatty acid synthase, thioesterase, a fatty acyl-CoA synthase and a 1 -CnA carboxylase. Fatty alcohols are also made in nature by enzymes that are capable of reducing various molecules of acyl-ACP or acyl-CoA to the corresponding primary alcohols (see US Patent Publication Nos. 20100105963 and 20110206630, and US Patent No. 8097439, expressly incorporated by reference herein). A fatty alcohol composition frequently includes fatty alcohols together with other derivatives of fatty acids, for example fatty aldehydes and / or fatty acids. Typically, the fatty alcohol composition is recovered from the extracellular environment of the recombinant host cell, ie, the cell culture medium. In certain embodiments, the expression of a polypeptide, eg, an enzyme involved directly or indirectly in fatty acid biosynthesis, is modulated (eg, expressed, overexpressed, or attenuated), where such modulation results in a yield higher, higher concentration, or higher productivity of a fatty acid derivative of interest, such as a fatty alcohol. The enzyme may be encoded by a fatty acid biosynthetic polynucleotide that is exogenous or heterologous (e.g., a polypeptide that originates from an organism

<img file="MX346830B_D0057.tif" />

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- • r ______... _ n, DELAFEOHÍDAD.

other than the precursor host cell, or, urwmreri of a polypeptide native to the microbial cell psecufaora) · «an endogenous polypeptide (eg, a polypeptide native to the precursor host cell) wherein the endogenous polypeptide is overexpressed in the recombinant host cell . Table 1 provides a listing of exemplary proteins that can be expressed in recombinant host cells to facilitate the production of particular fatty alcohol compositions.

Table 1: Gene Designations

<td>Designation of the Gen</td><td>Organism of origin</td><td>Enzyme Name</td><td>Access No.</td><td>EC number</td><td>Exemplary Use</td>

<td colspan="6">Increase in Fatty Acid Production / Increase in Product Production</td>

<td>accA</td><td>E. coli, Lactococci</td><td>acetyl-Coñ carboxylase, ñ subunit (alpha carboxytransphere)</td><td>AAC73296, NP_414727</td><td> 6.4.1.2</td><td>increased Malonil-CoA production</td>

<td>accB</td><td>E. coli, Lactococci</td><td>Acetyl-Coñ carboxylase, subunit B (BCCP: Carboxyl booty carrier protein)</td><td>NP_417721</td><td> 6.4.1.2</td><td>increased Malonil-CoA production</td>

<td>accC</td><td>E. coli, Lactococci</td><td>Acetyl-Coñ carboxylase, subunit C (booty carboxylase)</td><td>NP_417722</td><td> 6.4.1.2, 6.3.4.14</td><td>increase in production of Malonil-CoA</td>

<td>accD</td><td>E. coli, Lactococci</td><td>Acetyl-Coñ carboxylase, D-subunit (beta-carboxytransferase)</td><td>NP_416819</td><td> 6.4.1.2</td><td>increase in production of Malonil-CoA</td>

<td>fado</td><td>E. coli W3110</td><td>acyl-Coñ synthase</td><td>AP_002424</td><td> 2.3.1.86, 6.2.1.3</td><td>Increased fatty acid production</td>

<td>fabA</td><td>E. coli K12</td><td>β-hydroxydecanoylthioester dehydratase / isomerase</td><td>NP_415474</td><td> 4.2.1.60</td><td>Increased production of fatty acylACP / CoA</td>

<td>fabB</td><td>E. coli</td><td>3-oxoacyl- [carrier acylprotein]</td><td>BAA16180</td><td> 2.3.1.41</td><td>Increased acyl production</td>

rDMKdTPW »** ' ⁹¹ IMPI ιχττπυτο MEXICAN —-- C7 ——- 7 — TTTTTT -——

<td>Designation of the Gen</td><td>Organism of origin</td><td colspan="2">Enzyme Name</td><td>Access No.</td><td>EC number</td><td></td>

<td></td><td></td><td colspan="2">synthase I</td><td></td><td></td><td>pcp / r ^ A j .....</td>

<td>fabD</td><td>E. coli K12</td><td colspan="2">[acyl carrier protein] Smalonyltransferase</td><td>AAC74176</td><td> 2.3.1.39</td><td>Increased production of fatty acylACP / CoA</td>

<td>fabF</td><td colspan="2">E. coli K12</td><td>3-oxoacyl- [carrier acylprotein] synthase II</td><td>AAC74179</td><td> 2,3.1.179</td><td>Increased production of fatty acylACP / CoA</td>

<td>fabG</td><td colspan="2">E. coli K12</td><td>3-oxoacyl- [carrier acylprotein] reductase</td><td colspan="2">AAC74177 1.1.1.100</td><td>Increased production of fatty acylACP / CoA</td>

<td>fabH</td><td colspan="2">E. coli K12</td><td>3-oxoacyl- [carrier acylprotein] synthase III</td><td>AAC74175</td><td> 2.3.1.180</td><td>Increased production of fatty acylACP / CoA</td>

<td>fabl</td><td colspan="2">E. coli K12</td><td>Enoyl- [acyl-carrier protein] reductase</td><td colspan="2">NP_415804, 1.3.1.9</td><td>Increased production of fatty acylACP / CoA</td>

<td>fabR</td><td colspan="2">E. coli K12</td><td>Transcriptional repressor</td><td colspan="2">NP_418398 none</td><td>modulated unsaturated fatty acid production</td>

<td>fabv</td><td colspan="2">Vibrio cholerae</td><td>Enoyl- [acyl-carrier protein] reductase</td><td colspan="2">YP 001217283 1.3.1.9</td><td>Increased production of fatty acylACP / CoA</td>

<td>fabz</td><td colspan="2">E, coli K12</td><td>(3R) -hydroxymyristol acyl carrier protein dehydratase</td><td colspan="2">NP_414722 4.2.1.-</td><td>Increased production of fatty acylACP / CoA</td>

<td>fadE</td><td colspan="2">E. coli K13</td><td>acyl-CoA dehydrogenase</td><td>AAC73325</td><td> 1.3.99.3, 1.3.99.-</td><td>reduced fatty acid degradation</td>

<td>fadR</td><td colspan="2">E. coli</td><td>transcriptional regulatory protein</td><td>NP_415705</td><td>none</td><td>Blocking or reversing fatty acid breakdown</td>

<td colspan="7">Chain Length Control</td>

<td>tesA (with or without leader sequence)</td><td colspan="2">E. coli</td><td>thioesterase - the leader sequence is from amino acids 1-26</td><td>P0ADA1</td><td> 3.1.2.-, 3.1.1.5</td><td>Chain Length C18</td>

<td>tesA (no leader sequence)</td><td colspan="2">E. coli</td><td>thioesterase</td><td>AAC73596, NP_415027</td><td> 3.1.2.-, 3.1.1.5</td><td>Chain Length C18: l</td>

<td>tesA (E. coli thioesterase I mutant formed in</td><td colspan="2">E. coli</td><td>thioesterase</td><td>L109P</td><td> 3.1.2.-, 3.1.1.5</td><td>«Chain Length C18</td>

ΪΜΡΙ • «gas

<td>Designation of the Gen</td><td>Organism of origin</td><td colspan="2">Enzyme Name</td><td>Access No.</td><td>EC number ^ '</td><td>WJ »r</td><td rowspan="23"></td>

<td rowspan="2">complex with octanoic acid)</td><td colspan="2" rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td><td></td><td></td>

<td></td><td></td>

<td>fatBl</td><td colspan="2">Umbellulariacal ifornica</td><td>thioesterase</td><td>Q41635</td><td> 3.1.2.14</td><td>Chain Length C12: 0</td>

<td>fatB2</td><td colspan="2">Cupheahookerian to</td><td>thioesterase</td><td>AAC49269</td><td> 3.1.2.14</td><td>Chain Length C8: 0 - C10: 0</td>

<td>fatB3</td><td colspan="2">Cupheahookerian to</td><td>thioesterase</td><td>AAC72881</td><td> 3.1.2.14</td><td>Chain Length C14: 0 - C16: 0</td>

<td>fatB</td><td colspan="2">Ci nnamomumcamph ora</td><td>thioesterase</td><td>Q39473</td><td> 3.1.2.14</td><td>Chain Length C14: 0</td>

<td>fatB</td><td colspan="2">Arabidopsis thaliana</td><td>thioesterase</td><td>CAA85388</td><td> 3.1.2.14</td><td>Chain Length C16: l</td>

<td>fatal</td><td colspan="2">Helianthus annuus</td><td>thioesterase</td><td>AAL79361</td><td> 3.1.2.14</td><td>Chain Length C18: l</td>

<td>atfata</td><td colspan="2">Arabidopsis thaliana</td><td>thioesterase</td><td>NP_189147, NP_193041</td><td> 3.1.2.14</td><td>Chain Length C18: 1</td>

<td>fatA</td><td colspan="2">Brassica júncea</td><td>thioesterase</td><td>CAC39106</td><td> 3.1.2.14</td><td>Chain Length C18: l</td>

<td>fatA</td><td colspan="2">Cupheahookerian to</td><td>thioesterase</td><td>AAC72883</td><td> 3.1.2.14</td><td>Chain Length C18: l</td>

<td>tesA</td><td colspan="2">Photbacteriumpr ofundum</td><td>thioesterase</td><td>YP_130990</td><td> 3.1.2.14</td><td>Chain Length</td>

<td>tesB</td><td colspan="2">E. coli</td><td>thioesterase</td><td>NP_414986</td><td> 3.1.2.14</td><td>Chain Length</td>

<td>fadM</td><td colspan="2">E. coli</td><td>thioesterase</td><td>NP_414977</td><td> 3.1.2.14</td><td>Chain Length</td>

<td>yciA</td><td colspan="2">E. coli</td><td>thioesterase</td><td>NP_415769</td><td> 3.1.2.14</td><td>Chain Length</td>

<td>ybgc</td><td colspan="2">E. coli</td><td>thioesterase</td><td>NP_415264</td><td> 3.1.2.14</td><td>Chain Length</td>

<td colspan="7">Saturation Level Control *</td>

<td>Sfa</td><td colspan="2">E. coli</td><td>fabA suppressor</td><td>AAN79592, AAC44390</td><td>jiinguno</td><td>Increase in unsaturated fatty acids</td>

<td>fabA</td><td colspan="2">E. coli K12</td><td>β- hydroxydecanoylthioes terdehydratase / isom erasse</td><td>NP_415474</td><td> 4.2.1.60</td><td>produce unsaturated fatty acids</td>

<td>GnsA</td><td colspan="2">E. coli</td><td>null secG nutation suppressors</td><td>ABD18647.1</td><td>none</td><td>Increase in unsaturated fatty acid esters</td>

<td>GnsB</td><td colspan="2">E. coli</td><td>suppressors of the null secG mutation</td><td>AAC74076.1</td><td>none</td><td>Increase in unsaturated fatty acid esters</td>

<td>fabB</td><td colspan="2">E. coli</td><td>3-oxoacyl- (carrier acylprotein]</td><td>BAA16180</td><td> 2.3.1.41</td><td>unsaturated fatty acid production</td>

IMPI

<td>Designation of the Gen</td><td>Organism of origin</td><td colspan="2">Enzyme Name</td><td>Access No.</td><td>EC number^Gave</td><td></td>

<td></td><td colspan="2"></td><td>synthase I</td><td></td><td></td><td>modulated, .., .....,</td>

<td>des</td><td colspan="2">Bacillus subtilis</td><td>D5 acyl desaturase grease</td><td> 034653</td><td> 1.14.19</td><td>modulated unsaturated fatty acid production</td>

<td colspan="7">Product Yield: Ester Production</td>

<td>AT3G51970</td><td colspan="2">Arabidopsis thaliana</td><td>long chain fatty alcohol 0acyltransferase</td><td>NP190765</td><td> 2.3.1.26</td><td>wax production</td>

<td>ELO1</td><td colspan="2">Pichiaangusta</td><td>elongase fatty acid</td><td>BAD98251</td><td> 2.3.1.-</td><td>production of very long chain length fatty acids</td>

<td>plsC</td><td colspan="2">Saccharomyces cerevisiae</td><td>acyltransferase</td><td>AAA16514</td><td> 2.3.1.51</td><td>wax production</td>

<td>DAGAT / DGAT</td><td colspan="2">Arabidopsis thaliana</td><td>diacylglycerolacyltr ansferase</td><td>AAF19262</td><td> 2.3.1.20</td><td>wax production</td>

<td>b.WS</td><td colspan="2">Homo sapiens</td><td>acyl-CoA wax alcohol acyltransferase</td><td>ΆΑΧ48018</td><td> 2.3.1.20</td><td>wax production</td>

<td>aftl</td><td colspan="2">Acinetobacter sp. ADP1</td><td>bifunctional wax ester synthase / acyl3oA: diacylglycerol iltransfe rasa</td><td>ΆΑΟ17391</td><td> 2.3.1.20</td><td>wax production</td>

<td>ES9</td><td colspan="2">Marinobacterhid rocarbonoclasti cus</td><td>wax ester synthase</td><td>ΆΒΟ21021</td><td> 2.3.1.20</td><td>wax production</td>

<td>mWS</td><td colspan="2">Siwmondsiachine nsis</td><td>wax ester synthase</td><td>AAD38041</td><td> 2.3.1.—</td><td>wax production</td>

<td>acrl</td><td colspan="2">Acinetobacter sp. ADP1</td><td>acyl-CoA reductase</td><td>YP_047869</td><td> 1.2.1.42</td><td>performance modification</td>

<td>yqhD</td><td colspan="2">E. Coli K12</td><td>alcohol dehydrogenase</td><td>AP_003562</td><td> 1.1.—.—</td><td>modification of performance</td>

<td>AAT</td><td colspan="2">Fragaria x ananassa</td><td>alcohol 0- acetyltrans pherase</td><td>AAG13130</td><td> 2.3.1.84</td><td>modification of performance</td>

<td colspan="7">Product Output: Fatty Alcohol Output</td>

<td></td><td colspan="2"></td><td>thioesterases (see above)</td><td></td><td></td><td>increase of fatty acid / fatty alcohol production</td>

<td>BmFAR</td><td colspan="2">Bombyxmori</td><td>FAR (fatty alcohol-forming acyl-CoA reductase)</td><td>BAC79425</td><td> 1.1.1.—</td><td>convert acyl-CoA to fatty alcohol</td>

<img file="MX346830B_D0058.tif" />

<td>Designation of the Gen</td><td>Organism of origin</td><td colspan="2">Enzyme Name</td><td>Access No.</td><td>—-- XSTHVS Number ΕΟΪΙ</td><td></td>

<td>acrl</td><td colspan="2">Acinetobacter sp. ADP1</td><td>acyl-CoA reductase</td><td>YP_047869</td><td></td><td>fat to aldehyde fatty</td>

<td>yqhD</td><td colspan="2">E. coli W3110</td><td>alcohol dehydrogenase</td><td>AP_003562</td><td> 1.1.-.-</td><td>reduce fatty aldehyde to fatty alcohol; increase the production of fatty alcohol</td>

<td>a Go To</td><td colspan="2">Acinetobacter sp, ADP1</td><td>alcohol dehydrogenase</td><td>CAG70252</td><td> 1.1.-.^-</td><td>reduce fatty aldehyde to fatty alcohol</td>

<td>BmFAR</td><td colspan="2">Bombyxmori</td><td>FAR (fatty alcohol-taking acyl-CoA reductase)</td><td>BAC79425</td><td> 1.1.1.-</td><td>reduce fatty acyl-CoA to fatty alcohol</td>

<td>GmG_1865</td><td colspan="2">Geobacillusther modenitrificans NG80-2</td><td>long chain aldehyde dehydrogenase</td><td>YP_001125970</td><td> 1.2.1.3</td><td>reduce fatty aldehyde to fatty alcohol</td>

<td>MR</td><td colspan="2">Synechococcusel ongatus</td><td>acyl-ACP reductase</td><td>YP_400611</td><td> 1.2.1.80 1.2.1.42</td><td>reduce fatty acyl-ACP / CoA to fatty aldehyde</td>

<td>carB</td><td colspan="2">Mycobacteri um smegmatis</td><td>carboxilic acid reductase (CAR) protein</td><td>YP_889972</td><td> 6.2.1.3, 1.2.1.42</td><td>reduce fatty acid to fatty aldehyde</td>

<td>FadD</td><td colspan="2">E. coli K12</td><td>acyl-CoA synthetase</td><td>NP_416319</td><td> 6.2.1.3</td><td>activate fatty acid to fatty acyl-CoAs</td>

<td>atoB</td><td colspan="2">Erwiniacarotovo ra</td><td>acetyl-CoA acetyltransferase</td><td>YP_O49388</td><td> 2.3.1.9</td><td>butane production!</td>

<td>hbd</td><td colspan="2">Butyrivibriofib risolvens</td><td>oeta-hydroxybutyryl- CoA dehydrogenase</td><td>BAD51424</td><td> 1.1.1.157</td><td>butanol production</td>

<td>CPE0095</td><td colspan="2">Clostridium perfringens</td><td>crotonasabutyryl-CoA dehydrogenase</td><td>ΒΆΒ79801</td><td> 4.2.1.55</td><td>butanol production</td>

<td>B C D</td><td colspan="2">Clostridium beijerinckii</td><td>outiril-CoA dehydrogenase</td><td>14583</td><td> 1.3.99.2</td><td>butanol production</td>

<td>ALDH</td><td colspan="2">Clostridiwi beijerinckii</td><td>coenzyme A-acylating aldehyde dehydrogenase</td><td>AAT66436</td><td> 1.2.1.3</td><td>butanol production</td>

<td>AdhE</td><td colspan="2">E. coli CFT073</td><td>aldehyde-alcohol dehydrogenase</td><td>AAN80172</td><td> 1.1.1.1 1.2.1.10</td><td>butanol production</td>

<td colspan="7">Product Export</td>

<td>AtMRP5</td><td colspan="2">Arabidopsis thaliana</td><td>Arabidopsis thaliana associated with drug resistance</td><td>NP_171908</td><td>none</td><td>modify the export quantity of the product</td>

<td>AmiS2</td><td colspan="2">Rhodococcus sp.</td><td>ABC transporter</td><td>JC5491</td><td>none</td><td>modify the</td>

<img file="MX346830B_D0059.tif" />

<td>Designation of the Gen</td><td>Organism of origin</td><td colspan="2">Enzyme Name</td><td>Access No.</td><td>ECDI number</td><td></td>

<td></td><td colspan="2"></td><td>AmiS2</td><td></td><td></td><td>amount of _____ product export</td>

<td>AtPGPl</td><td colspan="2">Arabidopsis thaliana</td><td>Arabidopsis thaliana glycoprotein P 1</td><td>NP_181228</td><td>none</td><td>modify the export quantity of the product</td>

<td>AcrA</td><td colspan="2">CandidatusProto chlamydiaamoebo philaUWE25</td><td>putative multi-drug efflux transport protein acrA</td><td>CAF23274</td><td>none</td><td>modify the product export quantity</td>

<td>AcrB</td><td colspan="2">CandidatusProto chlamydiaamoebo phila UWE25</td><td>probable acrB multi-drug efflux transport protein</td><td>CAF23275</td><td>none</td><td>modify the export quantity of the product</td>

<td>TolC</td><td colspan="2">Francisellatula rensis subsp. novitiate</td><td>outer membrane protein [Biogenesis of the cell envelope,</td><td>ABD59001</td><td>none</td><td>modify the product export quantity</td>

<td>Acre</td><td colspan="2">Shigellasonnei Ss046</td><td>transmembrane protein affects septum formation and cell membrane permeability</td><td>YP_312213</td><td>none</td><td>modify the export quantity of the product</td>

<td>AcrF</td><td colspan="2">E. COli</td><td>acriflavin resistance protein F</td><td>P24181</td><td>none</td><td>modify the export quantity of the product</td>

<td>Í111619</td><td colspan="2">Thermosynechoco ccuselongatus [BP-1]</td><td>multi-drug efflux transporter</td><td>NP_682409.1</td><td>none</td><td>modify the export quantity of the product</td>

<td>tll0139</td><td colspan="2">Thermosynechoco ccuselongatus [BP-1]</td><td>multi-drug efflux transporter</td><td>NP_680930.1</td><td>none</td><td>modify the export quantity of the product</td>

<td colspan="7">Fermentation</td>

<td>Replication Checkpoint Genes</td><td colspan="2"></td><td></td><td></td><td></td><td>increase throughput efficiency</td>

<td>umuD</td><td colspan="2">Shigellasonnei Ss046</td><td>DNA polymerase V, subunit</td><td>YP_310132</td><td> 3.4.21,-</td><td>increase the efficiency of</td>

<img file="MX346830B_D0060.tif" />

IMPI

MEXICAN INSTTTUTE

OF THE INDUSTRIAL KRÜPIEDAP

<td>Designation of the Gen</td><td>Organism of origin</td><td colspan="2">Enzyme Name</td><td>Access No.</td><td>EC number</td><td>Exemplary Use</td>

<td></td><td colspan="2"></td><td></td><td></td><td></td><td>performance</td>

<td>umuC</td><td colspan="2">E. COli</td><td>DNA polyrrerasase V, subunit</td><td>ABC42261</td><td> 2.7.7.7</td><td>increase throughput efficiency</td>

<td>pntA, pntB</td><td colspan="2">Shigellaflexner í</td><td>NADH: NADPH transhydrogenase (alpha and beta subunits)</td><td>P07001, P0AB70</td><td> 1.6.1.2</td><td>increase throughput efficiency</td>

<td colspan="7">Others</td>

<td>fabK</td><td colspan="2">Streptococcus pneumonia</td><td>trans-2-enoyl-ACP reductase II</td><td>AAF98273</td><td> 1.3.1.9</td><td>Contributes to fatty acid biosynthesis</td>

<td>fabL</td><td colspan="2">Bacillus licheniformis DSM 13</td><td>enoyl- (acyl carrier protein) reductase</td><td>AAU39821</td><td> 1.3.1.9</td><td>Contributes to fatty acid biosynthesis</td>

<td>fabM</td><td colspan="2">Streptococcus mutans</td><td>trans-2, cis-3decenoyl-ACP isomerase</td><td>DAA05501</td><td> 4.2.1.17</td><td>Contributes to fatty acid biosynthesis</td>

Recombinant Host Cells and Cell Cultures

Strategies to increase the production of fatty aldehyde or fatty alcohol compositions by recombinant host cells include increased flux through the fatty acid biosynthetic pathway by overexpression of native fatty aldehyde or fatty alcohol biosynthetic genes and the expression of the Exogenous fatty aldehyde and fatty alcohol biosynthetic genes from different organisms in the production host. As used herein, the term "recombinant host cells or engineered host cells" refers to a host cell whose genetic makeup has been altered in

<img file="MX346830B_D0061.tif" />

corresponding, for example, by the introduction ^ deliberate. ^ · of new genetic elements and / or deliberate modification of genetic elements naturally present in the host cell. The offspring of such recombinant host cells also contain these new and / or modified genetic elements. In any of the aspects of the description described herein, the host cell can be selected from a plant cell, insect cell, fungal cell {eg, a filamentous fungus, such as Candida sp., Or a budding yeast , such as

Saccharomyces sp.), An algae cell and a bacterial cell. In a preferred embodiment, the recombinant host cells are recombinant microorganisms. Examples of host cells that are microorganisms include, but are not limited to, cells of the genus Escherichia, Bacillus, Lactobacillus, Zymomonas, Rhodococcus, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyverphtoces, Pichiocor, Mycelio , Penicillium, Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas,

Schizosaccharomyces, Yarrowia, or Streptomyces. In some embodiments, the host cell is a Gram positive bacterial cell. In other embodiments, the host cell is⁹⁸ IMPI

INSTITUTO MEXICANO „. . „. of the property .

a Gram negative bacterial cell. In some 'rfR9OT4ida ^ e ^ -r the host cell is an E - ^ - ^ s &amp; irir cell: —Εττ “όtras ·” ”embodiments, the host cell is a Bacillus lentus cell, a Bacillus brevis cell, a Bacillus brevis cell of Bacillus stearothermophilus, a cell of Bacillus lichenoformis, a cell of Bacillus alkalophilus, a cell of Bacillus coagulans, a cell of Bacillus circulans, a cell of Bacillus pumilis, a cell of Bacillus thuringiensis, a cell of Bacillus clausii, a Bacillus megaterium cell, a Bacillus subtilis cell, or a Bacillus amyloliquefaciens cell. In other embodiments, the host cell is a Trichodermakoningii cell, a Trichodermaviride cell, a Trichodermareesei cell, a Trichodermalongibrachiatum cell, an Aspergillus awamori cell, an Aspergillus fumigates cell, an Aspergillus foetpergillus cell, an Aspergillus foetpergillus cell nidulans, a cell of Aspergillus niger, a cell of Aspergillus oryzae, a cell of Humicolainsolens, a cell of Humicola lanuginose, a cell of Rhodococcusopacus, a Rhizomucormiehei cell, or a Mucormichei cell. In still other embodiments, the host cell is a Streptomyces lividans cell or a Streptomyces murinus cell. In still other embodiments, the host cell is an Actinomycetes cell. In⁹⁹ IMPI®,. ,,. ,,, _,, INSTITUTO MEXICANO some modalities, the host cell is^p

Saccharomyces cerevisiae. _____ _ ~ _____

In other embodiments, the host cell is a eukaryotic plant cell, algae cell, a cyanobacterial cell, a green sulfur bacterial cell, a green non-sulfur bacterial cell, a purple sulfur bacterial cell, a purple non-sulfur bacterial cell. , an Extremophilic cell, a yeast cell, a fungal cell, an engineered cell of any species described herein, or a synthetic organism. In some embodiments, the host cell is light dependent or carbon fixed. In some embodiments, the host has autotrophic activity. In some embodiments, the host cell has photoautotrophic activity, such as in the presence of light. In some embodiments the host cell is heterotrophic or myxotrophic in the absence of light. In certain embodiments, the host cell is a cell of Arabidopsis thaliana, Panicumvirgatum, Miscanthusgiganteus, Zea mays, Botryococcusebraunii, Chlamydomonasreinhardtii, Dunalielasalina, Synechococcus Sp. PCC 7002, Synechococcus Sp. PCCcyonchois BP 68042, Synemos Sp. PCchoCnemos 1Bpc. , Chlorobiumtepidum, Chlorojlexusauranticus, Chromatiummvinosuin, Rhodospirillumrubrum,

Rhodobactercapsulatus, Rhodopseudomonaspalusris,

Clostridium

100 INSTITUTO MEXICANO Vf'wg

OF THE PBOFIEDA®

INDUSTRIAL '

Ijungdahlii, Clostridium thermocellum,

Penicilliumchrysogenum, Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomycespombe, Pseudomonas fluorescens, or Zymomonasmobilis.

Host Cell Design

In some embodiments, a polynucleotide sequence (or genes) is provided to the host cell by means of a recombinant vector, which includes a promoter operably linked to the polynucleotide sequence. In certain embodiments, the promoter is a developmentally regulated, an organelle-specific, a tissue-specific, an inducible, a constitutive, or a cell-specific promoter. In some embodiments, the recombinant vector includes at least one sequence selected from an expression control sequence operably coupled to the polynucleotide sequence; a selection marker operably coupled to the polynucleotide sequence; a marker sequence operably coupled to the polynucleotide sequence; a purification portion operably coupled to the polynucleotide sequence; a secretory sequence operably coupled to the polynucleotide sequence; and a target sequence operably coupled to the polynucleotide sequence. The expression vectors described herein include a

MEXICAN INSTITUTE

DELA INDUSTRIAL PROPERTY polynucleotide sequence in a form suitable for the expression of the polynucleotide sequence in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector may depend on such factors as the choice of host cell to be transformed, the level of expression of the desired polypeptide, and the like. The expression vectors described herein can be introduced into host cells to produce polypeptides, including fusion polypeptides, encoded by the polynucleotide sequences as described above (supra). The expression of the genes encoding the polypeptides in prokaryotes, for example, E. coli, is most frequently carried out with vectors containing constitutive or inducible promoters that direct the expression of either fusion or non-fusion polypeptide. Fusion vectors add a number of amino acids to the polypeptide encoded therein, usually to the amino or carboxy terminus of the recombinant polypeptide. Such fusion vectors typically serve one or more of the following three purposes which include increasing the expression of the recombinant polypeptide;

increase the solubility of the recombinant polypeptide; and aiding in the purification of the recombinant polypeptide by acting as a ligand in affinity purification.

102

MEXICAN INSTITUTE

Often in expression vectors ^DS&lt;5 ^ o? StS ^ proteolytic cleavage site ^ ft ^ BK ^ mi-é »· ^ © -1-a ... fusion portion and the recombinant polypeptide are introduced. This allows separation of the recombinant polypeptide from the fusion portion after purification of the fusion polypeptide. Examples of such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, and enterosinase. Exemplary fusion expression vectors include the pGEX vector (Pharmacia Biotech, Inc., Piscataway, NJ; Smith et al. (1988) Gene 67: 31-40), the pMAL vector (New England Biolabs, Beverly, MA), and the vector pRITS (Pharmacia Biotech, Inc., Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose-binding protein E, or protein A, respectively, to the target recombinant polypeptide.

Examples of inducible, non-fusion E. coli expression vectors include the pTrb vector (Amannet et al. (1988) Gene 69: 301-315) and the pET lid vector (Studier et al., Gene Expression Technology: Methods in Enzymology 185 , Academic Press, San Diego, Calif. (1990) 60-89). Expression of the target gene from the pTrc vector is dependent on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. The expression of the target gene of the pET lid vector depends on the transcription of a

103 Co-expressed viral INDUSTRIAL-mediated T7 gn10-lac fusion promoter (T7 gnl). This viral polymerase _ _is _ supplied by host strains such as BL21 (DE3) or HMS174 (DE3) of a resident λ prophage that houses a T7 gnl gene under the transcriptional control of the lacUV 5 promoter. Suitable expression systems for both prokaryotic and eukaryotic cells are well known in the art (see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Coid Spring Harbor Laboratory). Examples of expression vectors of E. inducible, non-fusion, include the pTrc vector (Amann et al. (1988) Gene 69: 301-315) and the PET lid vector (Studier et al. (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego , CA, pp. 60-89). In certain embodiments, a polynucleotide sequence of the disclosure is operably linked to a promoter derived from a T5 bacteriophage. In one embodiment, the host cell is a yeast cell. In this embodiment, the expression vector is a yeast expression vector. Vectors can be introduced into prokaryotic or eukaryotic cells through a variety of field-recognized techniques to introduce foreign nucleic acid (eg, DNA) into a host cell. Appropriate methods for transforming or transfecting cells

104

IMPI

INSTITUTO MEXICANO DE LA PROPIEDAD INDUSTRIAL hosts can be found in, for example, Sambrooky collaborators (supra). For stable transformation of bacterial cells, it is known that (depending on the expression vector and the transformation technique used) a certain fraction of cells will take up and replicate the expression vector. In order to identify and select their transformants, the gene encoding a selectable marker (eg, resistance to an antibiotic) can be introduced into host cells along with the gene of interest. Selected markers include those that confer resistance to drugs such as, but not limited to, ampicillin, kanamycin, chloramphenicol, or tetracycline. Nucleic acids encoding a selectable marker can be introduced into a host cell or the vector itself, that encoding a polypeptide described herein or can be introduced into a separate vector. Cells stably transformed with the introduced nucleic acid can be identified by growth in the presence of an appropriate selection drug. The engineered or recombinant host cell as described herein (supra) is a cell used to produce a fatty acid-derived composition such as a fatty aldehyde or a fatty alcohol. In any of the aspects of the description described herein, the host cell is

<img file="MX346830B_D0062.tif" />

You can select from a eukaryotic plant, bacteria, algae, cyanobacteria, green sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, purple non-sulfur bacteria, Extremophile, yeast, fungus, organisms designed thereof, or an organism synthetic. In some embodiments, the host cell is light dependent or carbon fixed. In some embodiments, the host cell has autotrophic activity. Various host cells can be used to produce fatty acid derivatives, as described herein.

The host cells or microorganisms of the disclosure include host strains or host cells that are engineered or genetically modified to contain alterations in order to test the effectiveness of specific mutations in enzyme activities (ie, recombinant cells or microorganisms). Various optional genetic manipulations and alterations can be used interchangeably from one host cell to another, depending on what native enzymatic pathways are present in the original host cell. In one embodiment, a host cell can be used to test the expression of an AAR polypeptide in combination with other biosynthetic polypeptides (eg, enzymes). A host strain can encompass a number of genetic alterations in order to

106

<img file="MX346830B_D0063.tif" />

testing specific variables, including but not limited to, culture conditions, including fermentation components, carbon source (eg, raw material), temperature, pressure, reduced culture contamination conditions, and oxygen levels.

In one embodiment, a host strain encompasses an optional fadE and fhuñ deletion. Acyl-CoA dehydrogenase (FadE) is an enzyme that is important for metabolizing fatty acids. It catalyzes the second stage in the utilization of fatty acid (beta-oxidation), which is the process of breaking the long chains of fatty acids (acyl-CoAs) in acetyl-CoA molecules. More specifically, the second stage of the β-oxidation cycle of fatty acid degradation in bacteria is the oxidation of acyl-CoA to 2-enoylCoA, which is catalyzed by FadE. When E. coli lacks FadE, it cannot grow on fatty acids as a carbon source but can grow on acetate. The inability to use fatty acids of any chain length is consistent with the reported phenotype of fadE strains, that is, fadE mutant strains where FadE function is altered. The fadE gene can be optionally inactivated or attenuated to ensure that acyl-CoAs, which can be intermediates in a fatty acid-derived pathway, can accumulate in the cell such that all¹⁰⁷ IMPI INSTITUTO MEXICANO, OF THE PROPERTY acyl-CoAs can be sufficiently converted to 'HS ^^ Vados - from fatty acids. However, the attenuation of ^ fad &amp; ^ is ^ -bpflslo'ftáT 'when sugar is used as a source of carbon since under such a condition expression FadE is probably repressed and FadE may therefore only be present in small amounts and not being able to compete efficiently with ester synthase or other enzymes for acyl-CoA substrates. The FadE is repressed due to catabolic repression. E. coli and many other microbes prefer to consume sugar over fatty acids, so when both sources are available the sugar is consumed first by repressing the fad region (see D. Clark, J Bacteriol. (1981) 148 (2 ): 521-6)). On the other hand, the absence of sugars and the presence of fatty acids induces the expression of FadE. Acyl-CoA intermediaries could be lost to the beta oxidation pathway since proteins expressed by the fad region (including FadE] are upregulated and will compete efficiently for acyl-CoAs. Thus, it may be beneficial to have a inactivated or attenuated fadE gene Since most carbon sources are primarily sugar-based, it is optional to attenuate the FadE. The fhuA gene encodes the TonA protein, which is an energy-coupled transporter and receptor on the outer membrane of E. coli (V. Braun (2009) J Bacteriol.

₁₀₈ IMPIgg

ΓΝΓΠΤυΤΟ MEXICAN

Μ INDUSTRIAL PROPERTY 191 (11): 3431-3436). Its deletion is optional. Suppression of fhuA allows the cell to be more resistant to phage attack which may be beneficial under certain fermentation conditions. Thus, it may be desirable to suppress fhuA in a host cell that is likely subject to potential contamination during fermentation runs.

In another embodiment, the host strain (supra) also encompasses the optional overexpression of one or more of the following genes including fadR, fabA, fabD, fabG, fabH, fabV, and / or fabF. Examples of such genes are Escherichia coli fadR, Salmonella typhimurium fabA (NP_460041), Salmonella typhimurium fabD (NP_460164), Salmonella typhimurium fabG (NP_460165), Salmonella typhimurium fabH (NP_4600163), Salmonella typhimurium fabH2 (NP_rio460163) fabH002 Vibrio , and Clostridium acetobutylicum fabF (NP_350156). Overexpression of one or more of these genes, which encode enzymes and regulators in fatty acid biosynthesis, can serve to increase the tityl of fatty acid derived compounds including fatty aldehydes and fatty alcohols under various culture conditions.

In another embodiment, the E. coli strains are used as host cells for the production of fatty acid derivatives such as fatty aldehydes and / or fatty alcohols.

109

MEXICAN INS-TTnJTO DE LA EROHÍDAD industrial -

Similarly, these host cells provide for the optional overexpression of one or more biosynthetic genes (eg, genes encoding fatty acid biosynthesis enzymes and regulators) that can further increase or improve the titer of fatty acid-derived compounds such such as fatty acid derivatives (eg, fatty alcohols, fatty aldehydes, etc.) under various culture conditions including, but not limited to, fadR, fabA, fabD, fabG, fabH, fabV and / or fabF. Examples of genetic alterations include Escherichia coli fadR, Salmonella typhimurium fabA (NP_460041), Salmonella typhimurium fabD (NP_460164), Salmonella typhimurium fabG (NP_460165), Salmonella typhimurium fabH (NP_460163), Viblera typhimurium_00 (NP_P1760V163) cholera. , and Clostridium acetobutylicum fabF (NP_350156). In some embodiments, synthetic operons carrying these biosynthetic genes can be designed and expressed in cells in order to test for overexpression of fatty aldehyde and / or fatty alcohol under various culture conditions and / or further enhance aldehyde production. fatty and / or fatty alcohol. Such synthetic operons contain one or more biosynthetic genes. The ifabl38 operon, for example, is a designed fibula that contains optional fatty acid biosynthetic genes, including Vibrio cholera fabV,

<img file="MX346830B_D0064.tif" />

¹¹⁰ ΙΜΡΙ (ΝΤΓΠνΤΟ MíXICANO Salmonella typhimurium, S. typhimuri fabD ^ m¡nm ^ 9 ^ G typhimurium, S. typhimurium fabA and / or acetobutylicumt that can be used to facilitate overexpression of fatty acid derivatives in order to subject to test specific growing conditions An advantage of such synthetic operon is that the rate of production of fatty acid derivatives can be further increased or improved.

In some embodiments, the host cells or microorganisms that are used to express ACP and biosynthetic enzymes (eg, TE, ES, CAR, AAR, ADC, etc.) will additionally express genes that encompass certain enzymatic activities that can increase production. to one or more derivatives of particular fatty acids such as fatty alcohols, fatty aldehydes, fatty esters, fatty amines, derivatives of bifunctional fatty acids, diacids, and the like. In one embodiment, the host cell has thioesterase activity (EC 3.1.2. * Or EC 3.1. 2.14 or EC 3.1.1.5) for the production of fatty acids that can be increased by overexpressing the gene. In another embodiment, the host cell has ester synthase activity (EC 2.3.1.75) for the production of fatty esters. In another embodiment, the host cell has Acyl-ACP reductase (AAR) activity (EC 1.2.1.80) and / or¹¹¹

Mexican INSTrtVTO

OtLAFROflEOAD activity of alcohol dehydrogenase (EC 1.1.1.1 ^^^ / o urra * activity of acyl-CoA reductase of alcohol gra ^ str ^ 'í í'Akj'^l'(E (CT 1.1.1. *) And / or a carboxylic acid reductase (CAR) activity (EC 1.2.99.6) for the production of fatty alcohols. In another embodiment, the host cell has an Acyl-ACP activity reductase (AAR) (EC 1.2.1.80) for the production of fatty aldehydes. In another embodiment, the host cell has an Acyl-ACP reductase (AAR) activity (EC 1.2.1.80) and decarbonylase (ADC) activity for the production of alkanes and alkenes. In another embodiment, the host cell has acyl-CoA reductase activity (EC 1.2.1.50, acyl-CoA synthase (FadD) activity (EC 2.3.1.86), and thioesterase activity (EC 3.1.2. * Or EC 3.1. 2.14 or EC 3.1.1.5) for the production of fatty alcohols. In another embodiment, the host cell has an ester synthase activity (EC 2.3.1.75), acyl-CoA synthase activity (FadD) (EC 2.3.1.86 ), and thioesterase activity (EC 3.1.2. * or EC 3.1. 2.14 or EC 3.1.1.5) for the production of fatty esters. In another embodiment, the host cell has OleA activity for ketone production. In another embodiment, the host cell has OleBCD activity for the production of internal olefins. In another embodiment, the host cell has an activity of

Acyl-ACP reductase (AAR) (EC 1.2.1.80) and activity of

112

IMPI INSTITUTO MEXICANO Di LA MOHEDA !. ' ..Wg ^ jK alcohol dehydrogenase (EC 1.1.1.1.) For the preWdóióri^l&amp; quot; -efe ^ fatty alcohols. In another embodiment, it has thioesterase activity (EC 3.1.2. * Or EC 3.1. 2.14 or EC 3.1.1.5) and decarboxylase activity to manufacture terminal olefins. The expression of enzymatic activities in microorganisms and microbial cells is taught by US Patent Numbers 8,097,439; 8,110,093; 8,110,670; 8,183,028; 8,268,599; 8,283,143;

8,232,924; 8,372,610; and 8,530,221, which are incorporated herein by reference. In other embodiments, the host cells or microorganisms that are used to express ACP and other biosynthetic enzymes will include certain native enzyme activities that are upregulated or overexpressed to produce one or more derivatives of particular fatty acids such as aldehydes. fatty and / or fatty alcohols. In some embodiment, the host cell has native thioesterase activity (EC 3.1.2. * Or EC 3.1. 2.14 or EC 3.1.1.5) for the production of fatty acids that can be increased by overexpressing the thioesterase gene.

The present disclosure includes host strains or microorganisms that express genes encoding AAR and other biosynthetic enzymes (supra). Recombinant host cells produce fatty acid derivatives such as₁₁₃ IMPI ^

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OF THE PROPERTY CjteMdg _ _&lt; INDUSTRIAL fatty aldehydes and fatty alcohols and compositions and mixtures thereof. The fatty acid derivatives are typically recovered from the culture medium and / or isolated from the host cells. In one embodiment, fatty acid derivatives such as fatty aldehydes and fatty alcohols are recovered from the culture medium (extracellular). In another embodiment, fatty acid derivatives such as fatty aldehydes and fatty alcohols are isolated from host cells (intracellular). In another embodiment, fatty acid derivatives such as fatty aldehydes and fatty alcohols are recovered from the culture medium and isolated from the host cells. Compositions of fatty acid derivatives produced by a host cell can be analyzed using methods known in the art, for example, GC-FID, in order to determine the distribution of the particular fatty acid derivatives as well as the chain lengths and the degree of saturation of the components of the fatty acid derivative composition such as fatty aldehyde and fatty alcohol compositions.

Examples of host cells that function as microorganisms (eg, microbial cells) include but are not limited to cells of the genus Escherichia, Bacillus, Lactobacillus, Zymomonas, Rhodococcus, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, ¹¹⁴ IMPI

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Rhizomucor, Kluyveromyces, Pichia, Mucor,

Penicillium, Phanerochaete, Pleurot tfs * · —- ^{eleven r}'

Chrysosporium, Saccharomyces, Stenotrophamonas,

Schizosaccharomyces, Yarrowia, or Streptomyces. In some embodiments, the host cell is a Gram positive bacterial cell. In other embodiments, the host cell is a Gram negative bacterial cell. In some embodiments, the host cell is an E. coli cell. In some embodiments, the host cell is an E. coli B cell, an E. coli C cell, an E. coli K cell, or an E. coli W cell. In other embodiments, the host cell is a Bacillus lentuscell cell, a Bacillus brevis cell, a Bacillus stearothermophilus cell, a Bacillus lichenoformis cell, a Bacillus alkalophilus cell, a Bacillus coagulaos cell, a circulate Bacillus cell, a Bacillus pumilis cell, a Bacillus thuringiensis cell, a Bacillus clausii cell, a Bacillus megaterium cell, a Bacillus subtilis cell, or a Bacillus amyloliquefaciens cell. In still other embodiments, the host cell is a Trichodermakoningii cell, a Trichodermaviride cell, a Trichodermareesei cell, a Trichodermalongibrachiatum cell, a Trichodermaviride cell.

Aspergillus awamori, a cell of Aspergillus fumigates, a

115

IMPIOS cell of Aspergillus foetidus, a cell of^OFnidulans, a cell of Aspergillus niger,, a cell. de— Aspergillus oryzae, a Humicolainsolens cell, a Humicolalanuginose cell, a Rhodococcusopacus cell, a Rhizomucormiehei cell, or a Mucormichei cell. In still other embodiments, the host cell is a Streptomyceslividans cell or a Streptomyces murinus cell. In still other embodiments, the host cell is an Actinomycetes cell. In some embodiments, the host cell is a Saccharomyces cerevisiae cell. In other embodiments, the host cell is a cell from a eukaryotic plant, algae, cyanobacteria, green sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, purple non-sulfur bacteria, Extremophilic, yeast, fungus, and an organism. designed from the same, or a synthetic organism. In some embodiments, the host cell is light dependent or carbon fixed. In some embodiments, the host cell has autotrophic activity. In some embodiments, the host cell has photoautotrophic activity, such as in the presence of light. In some embodiments, the host cell is heterotrophic or myxotrophic in the absence of light. In certain embodiments, the host cell is a cell from Arabidopsis thaliana, Panicumvirgatum, Miscanthusgiganteus,¹¹⁶

INSTITUTO MEXICANO „„, .. ,,, OF THE ΜΙΟζΙϊΟΑΕ »Ogs» ¿3Bfc4 ^

Zea mays, Botryococcusebraunn, ChlamydomonasVQ ^ l $ WarcÍ &amp; ¡^ -p ^

Dunalielasalina, Synechococcus Sp. PCC 7002 ', · SyiiechocOe'tt ^^'¹^ 'Sp. PCC 7942, Synechocystis Sp. PCC 6803, Thermosynechococcus elongates BP-1, Chlorobiumtepidum, Chlorojlexusauranticus, Chromatiummvinosum, Rhodospirillumrubrum,

Rhodobactercapsulatus, Rhodopseudomonaspalusris, Clostridium Ijungdahlii, Clostridium thermocellum,

Penicilliumchrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomycespombe, Pseudomonas fluorescens, or Zymomonasmobilis. In a particular embodiment, the microbial cell is from a cyanobacterium including, but not limited to, Prochlorococcus, Synechococcus,

Synechocystis, Cyanothece, and Nostocpunctiforme. In another embodiment, the microbial cell is from a specific cyanobacterial species that includes, but is not limited to, Synechococcuselongatus PCC7942, Synechocystis sp. PCC6803, and Synechococcus sp. PCC7001.

Recombinant Host Cell Culture and Fermentation.

As used herein, the term fermentation broadly refers to the conversion of organic materials into target substances by host cells, for example, the conversion of a carbon source by recombinant host cells to fatty acids or derivatives thereof upon propagating a culture of the aSbAT host cells.

¹¹⁷ WICKED

MEXICAN INSTITUTE. . ,,,,_{Ί Λ} Recombinant MU PROPERTY in a medium comprising the source

Permissive conditions for production -ge— any of the conditions that allow the host cell to produce a desired product, such as a fatty aldehyde or fatty alcohol. Similarly, the condition (s) under which the polynucleotide conditions of a vector are expressed means any of the conditions that allow a host cell to synthesize a polypeptide. Suitable conditions include, for example, fermentation conditions. Fermentation conditions can include many parameters including, but not limited to, temperature ranges, aeration levels, feed rates, and average composition. Each of these conditions, individually and in combination, allows the host cell to develop. Fermentation can be aerobic, anaerobic, or variations thereof (such as micro aerobic). Exemplary culture media include broths or gels. Generally, the medium includes a carbon source that can be metabolized by a host cell directly. Furthermore, enzymes can be used in the medium to facilitate mobilization (eg, depolymerization of starch or cellulose to fermentable sugars) and subsequent metabolism of the carbon source.

i »IMPI ^

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For small-scale production, 1 ^^^ 00 lutáir 'designed hosts can be developed * in lbtés "^' 3 ^ r'po'f * 'example, approximately 100 pL, 200 pL, 300 pL, 400 pL, 500 pL, 1 mL, 5 mL, 10 mL, 15 mL, 2 5 mL, 50 mL, 7 5 mL, 100 mL, 500 mL, 1 L, 2 L, 5 L, or 10 L; are fermented; and induced to express a desired polynucleotide sequence, such as a polynucleotide sequence encoding an ACP and / or biosynthetic polypeptide. For large-scale production, engineered host cells can be grown in batches of approximately 10 L, 100 L, 1000 L, 10,000 L, 100,000 L, and 1,000,000 L or larger; they can be fermented; and inducing to express a desired polynucleotide sequence. Alternatively, large scale feedlot fermentation can be carried out. The fatty acid-derived compositions described herein are found in the extracellular environment of recombinant host cell culture and can be easily isolated from the culture medium. A fatty acid derivative such as a fatty aldehyde or fatty alcohol can be secreted by the recombinant host cell, by transporting into the extracellular environment or passively transferring into the extracellular environment of the recombinant host cell culture. The fatty acid derivative is isolated from a recombinant host cell culture using routine methods known in the art.

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Products Derived From Host Cells Recembinanters ^

As used herein, the modem or fM carbon fraction has the same meaning as defined by the National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs4990B and 4990C, known as HOxI and HOxII oxalic acid standards, respectively. The fundamental definition refers to 0.95 times the isotope ratio ¹⁴C /¹²C HOxI (referred to as AD 1950). This is roughly the equivalent of corrected weathered pre-Industrial Revolution Lumber. For the current living biosphere (plant material), fM is approximately 1.1. Bioproducts (eg, fatty acid derivatives including fatty aldehydes and / or fatty alcohols produced in accordance with the present disclosure) include biologically produced organic compounds. In particular, fatty acid derivatives produced using the fatty acid biosynthetic pathway herein have not been produced from renewable sources and as such are novel compositions of matter. These new bioproducts can be distinguished from petrochemical carbon derived organic compounds on the basis of dated or double carbon isotopic footprint¹⁴C. Additionally, the specific source of the bio-source carbon (eg, glucose versus glycerol) can be determined

120

INSTITUTO MEXICANO DE LA PROPERTY iwdushual for the double isotopic carbon footprint (see, for example,

US Patent No. 7,169,588). The ability to distinguish bioproducts from petroleum-based organic compounds is beneficial in tracing these materials in commerce. For example, organic compounds or clinical products that include so many biologically based and petroleum-based carbon isotope profiles can be distinguished from organic compounds and chemicals made only from petroleum-based materials. Consequently, the bioproducts herein can be tracked and tracked in commerce based on their unique carbon isotope profile. By-products can be distinguished from petroleum-based organic compounds by comparing the appropriate carbon isotope ratio (¹³C /¹²C) in each sample. The relationship¹³C /¹²C in a given bioproduct is a consequence of the relationship ¹³C /¹²C in atmospheric carbon dioxide at the time carbon dioxide is fixed. It also reflects the precise metabolic pathway. There are also regional variations. Oil, plant C3 (the widest leaf), plant C4 (grasses), and marine carbonates show significant differences in¹³C /¹²C and 5 values¹³Corresponding C. Additionally, the lipid matter of plants C3 and C4 analyzes differently than the materials derived from

121

INSTITUTO MEXICANO t »the mewtDAP carbohydrate components of the same plantá ^^ títao 'Orrao' consequence of the metabolic pathway. Inside 06 ^ 73 ^^ 7777 ^ 7017 ^^ 6 ^⁵^ measurement, ¹³C shows large variations due to isotopic fractionation effects, the most significant of which for byproducts is the photosynthetic mechanism. The main cause of differences in the carbon isotope ratio in plants is closely associated with differences in the pathway of photosynthetic metabolism in plants, particularly the reaction that occurs during primary carboxylation (i.e., initiated fixation of CO₂ atmospheric). Two large classes of vegetation are those that incorporate the C3 (or Calvin-Benson) photosynthetic cycle and those that incorporate the C4 (or Hatch-Slack) photosynthetic cycle. In C3 plants, CO fixation₂ The primary or carboxylation reaction involves the enzyme ribulose-1,5-diphosphate carboxylase, and the first stable product is a 3-carbon compound. C3 plants, such as hardwoods and conifers, are dominant in temperate climates. In C4 plants, an additional carboxylation reaction involving another enzyme, phosphoenol-pyruvate carboxylase, is the primary carboxylation reaction. The first non-stable carbon compound is a 4-carbon acid that is subsequently decarboxylated. The C0₂ freed in this way becomes

122 IMPI ^

INSTITUTO MEXICANO DE LA PROPERTY set by cycle C3. Examples of tropical plants C4 ffi ^ '^ astVsr, corn, and sugar cane. Both C4 and 3 '~ show a range of isotopic ratios¹³C /¹²C, but typical values ​​are from about -7 to about -13 per thousand for C4 plants and about -19 to about -27 per thousand for C3 plants (see, for example, Stuiver et al. (1977) Radiocarbon 19: 355). Coal and oil are generally in the latter range. The measurement scale¹³C was originally defined by a zero set for the Pee Dee Belemnite (PDB) limestone, where values ​​are provided in parts per thousands of shares of this material. The 513C values ​​are expressed in parts per thousand (per thousand), abbreviated, Vo, and are calculated as follows:

5^X3C (fe) = [(¹³C /¹²C) sample- (¹³C /¹²C) standard] / (¹³C /¹²C) standard x 1000

Since the PDB (RM) reference material has been exhausted, a number of alternative RMs have been developed in cooperation with the IAEA, USGS, NIST, and other selected international isotype laboratories. The annotations for the deviations per thousand of PDB is or¹³C. Measurements are made in CO2 by high precision stable ratio mass spectrometry (IRMS) on molecular ions at masses 44, 45, and 46. The compositions described in ¹²³ IMPI

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M THE PROPERTY present include bioproducts produced by which '^ TRÍ ^ ra the methods described herein,' mrciU ^ S'WóT ¹example, products derived from fatty acids. Specifically, the bioproduct may have one or¹³C of about -28 or greater, about -27 or greater, 20 or greater, -18 or greater, -15 or greater, -13 or greater, -10 or greater, or -8 or greater. For example, the bioproduct may have one or¹³C about -30 to about -15, about -27 to about -19, about -25 to about -21, about -15 to about -5, about -13 to about -7, or about - 13 to about -10. In other cases, the bioproduct may have one or¹³C of about 10, -11, -12, or -12.3. Bioproducts produced in accordance with the description herein can also be distinguished from petroleum-based organic compounds by comparing the amount of¹⁴C in each compound. Because the¹⁴C has a nuclear half-life of 5730 years, petroleum-based fuels containing old carbon can be distinguished from bioproducts containing more recent carbon (see, for example, Currie, Source Apportionment of Atmospheric Particles, Characterization of Environmental Particles, J. Buffle and HP van Leeuwen, Eds., 1 of Vol. I of the IUPAC Environmental

<img file="MX346830B_D0065.tif" />

MEXICAN INSTITUTE OF PROPERTY

Analytical Chemistry Series (Lewis Publishers, Inc ^ T ^ 'S-TÍT' (1992)). The basic assumption in radiocarbon dating is that the concentration constancy of¹⁴C in the atmosphere leads to the constancy of ¹⁴C in living organisms. However, due to atmospheric nuclear testing since 1950 and fossil fuel burning since 1850, the¹⁴C acquired a second geochemical time characteristic. Its concentration in atmospheric CO2, and therefore in the living biosphere, roughly doubled at the peak of the nuclear test in the mid-1960s. Since it has gradually returned to the state cosmogonic baseline isotope rate permanent (atmospheric) (¹⁴C /¹²C) about 1.2 x 10 "¹², with a relaxation half-life of approximately 7-10 years. This last half-life should not be taken literally; rather, the detailed atmospheric nuclear input / decomposition function should be used to track the variation of the¹⁴Atmospheric and biospheric C since the beginning of the nuclear age. This last time characteristic of¹⁴Biospheric C that holds the promise of recent biospheric carbon yearly dating. He¹⁴C can be measured by accelerating mass spectrometry (AMS), with results given in units of fraction of modern carbon (fM). The fM is defined by the National Institute of Standards and Technology (NIST) Standard Reference Materials

125 IMPI

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M THE PROPERTY (SRMs) 4990B and 4990C. As used herein, ieT ^ modern carbon induction or fM has the same meaningⁿ as defined by the National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs) 4990B and 4990C, known as HOxI and HOxII oxalic acid standards, respectively. The fundamental definition refers to 0.95 times the isotope ratio¹⁴C /¹²C HOxI (referred to as AD 1950). This is roughly equivalent to decay corrected pre-Industrial Revolution Wood. For the current living biosphere (plant material), the fM is approximately 1.1. The compositions described herein include bioproducts that may have an fM¹⁴C of at least about 1. For example, the byproduct of the description may have an fM¹⁴C of at least about 1.01, an fM¹⁴C from about 1 to about 1.5, an fM¹⁴C from about 1.04 to about 1.18, or an fM¹⁴C from about 1,111 to about 1,124.

Other measurement of ¹⁴C is known as the percent modern carbon (pMC). For an archaeologist or geologist using dates from¹⁴C, AD 1950 equal to zero years of age. This also represents 100 pMC. Carbon from the bomb in the atmosphere reached almost twice the normal level in 1963 at the peak of thermonuclear weapons. Its distribution within the atmosphere has been approximated since its appearance, showing values

126

1Í which are greater than 100 pMC for plants and since AD ​​1950. It has gradually decreased over time with the current value being close to 107.5 pMC. This means that a recent biomass material, such as corn, would provide a signature of¹⁴C near 107.5 pMC. Petroleum-based compounds will have a pMC value of zero. Combining fossil carbon with current carbon will result in a dilution of the daily pMC content present. Assuming that 107.5 pMC represents the content of¹⁴C of current biomass materials and 0 pMC represents the content of ¹⁴For petroleum-based products, the measured pMC value for the material will reflect the proportions of the two component types. For example, a current 100% soy-derived material would provide a radiocarbon signature near 107.5 pMC. If this material was diluted

50% with petroleum based products, would provide a radiocarbon signature of approximately 54 pMC. A biologically based carbon content is derived by assigning 100% equal to 107.5 pMC and 0% equal to 0 pMC. For example, a sample measuring 99 pMC will provide an equivalent biologically based carbon content of 93%. This value is referred to as the mean biologically based carbon result and assumes all components within the analyzed material originating from either current biological material or the

127

IΜΡΙ petroleum based material. One byproduct or more fatty acid derivatives as described in,. present can have a pMC of at least about 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100. In other cases, a fatty acid derivative described herein may have a pMC of between about 50 and about 100; about 60 and about 100; about 70 and about 100; from about 80 to about 100; about 85 and about 100; about 87 and about 98; or about 90 and about 95. In still other cases, a fatty acid derivative described herein may have a pMC of about 90, 91, 92, 93, 94, or 94.2.

Fatty Aldehyde and Fatty Alcohol Compositions and Their Uses.

Aldehydes are used to make many specialty chemicals. For example, aldehydes are used to produce polymers, resins (eg, BAKELITE resin), dyes, flavorings, plasticizers, perfumes, pharmaceuticals, and other chemicals, some of which can be used as solvents, preservatives, or disinfectants. Also, certain natural and synthetic compounds, such as vitamins and hormones, are aldehydes, and many sugars contain aldehyde groups. Fatty aldehydes can be

128 convert to fatty alcohols by reduction 'N ^ fMica⁴⁵ θ 'enzymatic. Fatty alcohols also have strong commercial uses. Annual worldwide sales of fatty alcohols and their derivatives are $ 1 billion. Shorter chain fatty alcohols are used in the cosmetic and food industries as emulsifiers, emollients and thickeners. Due to their amphiphilic nature, fatty alcohols behave as nonionic surfactants, which are useful in personal care and household products, such as, for example, detergents. Additionally, fatty alcohols are used in waxes, gums, resins, pharmaceutical balms and lotions, lubricating oil additives, antistatic and textile finishing agents, plasticizers, cosmetics, industrial solvents or grease solvents.

The disclosure also provides a surfactant composition or a detergent composition that includes a fatty alcohol produced by any of the methods described herein. One of ordinary skill in the art will appreciate that, depending on the intended purpose of the surfactant or detergent composition, different fatty alcohols can be produced or used. For example, when the fatty alcohols described herein are used as a raw material for a "Μ

129 production of surfactants or detergent

<img file="MX346830B_D0066.tif" />

Ordinary experience in the art will show .... The characteristics of the fatty alcohol raw material will affect the characteristics of the surfactant or detergent composition produced. Accordingly, the characteristics of the surfactant or detergent composition can be selected by providing particular fatty alcohols for use as a raw material. A fatty alcohol-based surfactant and / or detergent composition described herein may be mixed with other surfactants and / or detergents well known in the art. In some embodiments, the blend may include at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or a range bound by either of the two values ​​above, by weight of the fatty alcohol. In other examples, a surfactant or detergent composition can be made that includes at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, when ¹³⁰ IMPI ® Mexican institute minus approximately 85%, at least approximately ^^?

at least about 95%, or a ίηΐ · ο ^ · .1ί l¹'pnr any of the two previous values, by weight of a fatty alcohol that includes a carbon chain that is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , or 22 carbons in length. Such composition or surfactants or detergents may also include at least one additive, such as a microemulsion or a surfactant or detergent from non-microbial sources such as vegetable oils or petroleum, which may be present in the amount of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or a range bound by either of the two values ​​above, by weight of the fatty alcohol.

EXAMPLES

The following specific examples are intended to illustrate the description and are not to be construed as limiting the scope of the claims.

131

<img file="MX346830B_D0067.tif" />

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Protocols and Methods

Classification of a Biblio

All protocols described herein rely on a 96-well plate-master block-2mL system (Greiner Bio-One, Monroe, NC or Corning, Amsterdam, The Netherlands) to develop cultures, and plates (Costar, Inc. .) to extract fatty acid species from the culture broth. The protocols provided below are examples of fermentation conditions. Alternative protocols can be used to evaluate the production of fatty acid species.

Culture Protocol at 32 ° C (4NBT)

20pL of LB culture (from a LB culture growing in a 96-well plate) was used to inoculate 400pL of 2NBT medium (Table 2), which was then incubated for approximately 16 hours at 32 ° C with shaking. 20pL of the nocturnal seed was used to inoculate 400pL of 4NBT with either 1 or 2 g / L of nitrogen (NBT_1N or NBT_2N). After growth at 32 ° C for 6 hours, the cultures were induced with IPTG (final concentration 1 mM) (Table 2). The cultures were then incubated at 32 ° C with shaking for 18 hours, after which they were extracted following the standard extraction protocol detailed below.

<img file="MX346830B_D0068.tif" />

<img file="MX346830B_D0069.tif" />

132

Table 2: Names of Media and Formulations

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<td>Name of Means, medium</td><td colspan="3">Formulation</td>

<td rowspan="10">2NBT</td><td> 1</td><td>X</td><td>5x Saline with NH4C1</td>

<td> 1</td><td>g / L</td><td>100 g / L NH4C1</td>

<td> 1</td><td>mg / L</td><td>10mg / mL Thiamine</td>

<td> 1</td><td>mM</td><td>1M MgSO4</td>

<td> 0.1</td><td>mM</td><td>CaC12 1M</td>

<td> 20</td><td>g / L</td><td>500g / L glucose</td>

<td> 1</td><td>X</td><td>lOOOx of TM2</td>

<td> 10</td><td>mg / L</td><td>lOg / L Fe Citrate</td>

<td> 100</td><td>pg / mL</td><td>100 mg / ml spectinomycin</td>

<td> 100</td><td>mM</td><td>BisTris 2M (pH7.0)</td>

<td rowspan="9">4NBT_1N</td><td> 1</td><td>X</td><td>5x Saline with NH4C1</td>

<td> 1</td><td>mg / L</td><td>10mg / mL Thiamine</td>

<td> 1</td><td>mM</td><td>1M MgSO4</td>

<td> 0.1</td><td>mM</td><td>CaC12 1M</td>

<td> 40</td><td>g / L</td><td>500g / L glucose</td>

<td> 1</td><td>X</td><td>1000x of TM2</td>

<td> 10</td><td>mg / L</td><td>lOg / L Fe Citrate</td>

<td> 100</td><td>pg / mL</td><td>100 mg / ml spectinomycin</td>

<td> 100</td><td>mM</td><td>BisTris 2M (pH7.0)</td>

<td rowspan="10">4NBT2N</td><td> 1</td><td>X</td><td>5x Saline solution with NH4C1</td>

<td> 1</td><td>g / L</td><td>100 g / L NH4C1</td>

<td> 1</td><td>mg / L</td><td>10mg / mL Thiamine</td>

<td> 1</td><td>mM</td><td>1M MgSO4</td>

<td> 0.1</td><td>inM</td><td>CaC12 1M</td>

<td> 40</td><td>g / L</td><td>500g / L glucose</td>

<td> 1</td><td>X</td><td>lOOOx of TM2</td>

<td> 10</td><td>mg / L</td><td>lOg / L Fe Citrate</td>

<td> 100</td><td>pg / mL</td><td>100 mg / ml spectinomycin</td>

<td> 100</td><td>mM</td><td>BisTris 2M (pH7.0)</td>

Standard Extraction Protocol for Fatty Acid Species

40pL of 1M HC1 was added to each well that is extracted, followed by 300pL of butyl acetate (with 500mg / L of

<img file="MX346830B_D0070.tif" />

133

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Cll-FAME as an internal standard). The d ^^ ififiarer ^ cr plates were then heat sealed using a soLlad ^ g 'do' plQga'S ^ (ALPS-300 heater; Abgene, ThermoScientific, Rockford, IL), and stirred for 15 minutes at 2000rpm using a MIXMATE mixer. (Eppendorf, Hamburg, Germany). After shaking, the plates were centrifuged for 10 minutes at 4500rpm at room temperature (Allegra X-15R, SX4750A rotor, Beckman Coulter, Brea, CA) to separate the aqueous and organic layers. 50pL of the organic layer was transferred to a 96-well plate (polypropylene, Corning, Amsterdam, The Netherlands), which was subsequently heat sealed and stored at -20 ° C until evaluated by GC-FID using the FALC_Broth method. .met. R1 FALC Broth.met method was carried out as follows: IpL of the sample was injected onto an analytical column (DB-1, 10 m χ 180 pm χ 0.2 pM film thickness, available from JW 121-101A) in a Agilent 7890A GC Ultra device (Agilent, Santa Clara, CA) with a flame ionization detector (FID). The instrument was set up to detect and quantify C6 to C18 fatty alcohols. The protocol detailed in the above represents the standard conditions, which can be modified as necessary to utilize the analytical results.

IMPI ^

Fatty Acid Species - Ens Protocol

INDUSTRIAL

Standard

After 24 hours of fermentation, a Nile Red test was carried out by adding 70pL of fermentation broth to 130pL of 1.54pg / mL of Nile Red in 84.6% water and 15.4% acetonitrile solution for a test concentration final Ipg / mL of Nile Red on a Greiner MicrolonFluotrac 200 plate, and mixed by pipetting up and down. Relative fluorescence units were measured at 540nm excitation and 630nm emission using the SpectraMax M2 unit.

Building Error-Prone Libraries

Standard techniques known to those of skill in the field were used to prepare error prone libraries. In one example, the vector backbone was prepared using restriction endonucleases in the vector, while the creation of diversity in the DNA insert was generated for PCR amplification of a DNA template under conditions that favor incorporation of the mismatched nucleotides. In one procedure, cloning of the vector backbone and a diversity DNA insert was carried out using the INFUSION Cloning System (Clontech Laboratories, Inc., Mountain View, CA), according to

135 with the manufacturer's protocol.

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INSTITUTO MEXICANO Dt LA INDUSTRIAL PROPERTY

Construction of Saturation Libraries ._...

Standard techniques known to those of skill in the field were used to prepare the saturation libraries. In one example, the vector backbone was prepared using vector restriction endonucleases, while the creation of diversity in the DNA insert was generated using degenerate primers. In one procedure, cloning of the vector backbone and a diverse DNA insert was carried out using the INFUSION Cloning System (Clontech Laboratories, Inc., Mountain View, CA) according to the manufacturer's protocol. Construction of Combination Libraries

The mutations identified as beneficial were combined to provide AAR variants with further enhancements in the production of fatty alcohol species. Standard techniques known to those of skill in the field were used to prepare the combination libraries. In one example, the vector backbone was prepared using restriction endonucleases in the vector, while the diversity creation of the DNA insert was generated using primers to introduce the desired mutations. As described in the

136 Mexican iNSirruro

Μ THE PREVIOUS PROPERTY, in a procedure, the cloning d¿^NDUn '^TO'vector backbone and a corT ^ i ^ rsi ^ T ^ DNA insert were' 'carried out using the INFUSION Cloning System (Clontech Laboratories, Inc., Mountain View, CA), according to the manufacturer's protocol . Pooling libraries can be generated using the transfer PCR (tPCR) protocol (Erijmany collaborators (2011) J. Structural Bio. 175: 171-177 /.

Libraries Classification

Once the diversity of libraries was generated into a saturation library, error prone or combination library, it was classified using one of the methods described above. Two types of hits were identified: (1) increased amount of fatty alcohol (FALC concentration); and / or (2) increased amount of medium chain FALC such as dodecanol (C12) or tetradecanol (C14). Hexadecanol (C16) and octadecanol (C18) were also identified. Mutations in the AAR variants within each hit were identified by sequencing using standard techniques routinely employed by those of skill in the field. Tables 4, 5 and 6 list the mutations (hits) identified as beneficial in the saturation libraries and the combination libraries.

<img file="MX346830B_D0071.tif" />

«⁷ IMPI

INSTITUTO MEXICANO „. , - _,. . . .,. . BE THE MOMSDAD

Example 1: Enhanced Alcohol Production in AAR_7942 by Increased Flux Mediader-by ^ Acyl Carrier Protein (ACP) Through the Pathway of Fatty Acid Synthesis

When terminal pathway enzymes from sources other than E. coli are expressed in E. coli as the heterologous host to convert the fatty Acyl-ACPs to products, there may be limitations in recognition, affinity and / or change of the enzyme of the recombinant pathway to the fatty acyl-ACPs of E. coli. Although ACP proteins are conserved to some degree in all organisms, their primary sequence may differ even within a given species. In order to test this hypothesis, the acp genes of several cyanobacteria were cloned downstream of the Synechococcuselongatus PCC7942 AcylACP reductase (AAR_7942) present in the plasmid pLS9-185, which is a derivative pCL1920 (3-5 copies / cells). Furthermore, the sfp gene (Accession No. X63158; SEQ ID NO: 11) from Bacillus subtilis, which encodes a phosphopanteteinol transferase with broad substrate specificity, was cloned downstream of the respective acp genes. This enzyme is involved in the conversion of inactive apo-ACP to active holo-ACP. The constructed plasmids are described in Table 3.

<img file="MX346830B_D0072.tif" />

138

MEXICAN INSTITUTE

DK THE PROPERTY

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Table 3: Plasmids that Co-express Cyanobacterial ACP with and without sfp B. subtilis Down Chain of PCC7942 AAR S. elongatus

<td>Base Plasmid</td><td>ACP source</td><td>ACP - SEQ ID NO. (NA / AA *)</td><td>Without sfp</td><td>With sfp</td>

<td>pLS9-185</td><td>Synechococcuselongatus 7942</td><td> 7/8</td><td>pDS168</td><td>pDS168S</td>

<td>PLS9-185</td><td>Synechocystis sp. 6803</td><td> 3/4</td><td>pDS169</td><td>not available</td>

<td>pLS9-185</td><td>Prochlorococcusmarinus MED4</td><td> 5/6</td><td>pDS170</td><td>pDS170S</td>

<td>pLS9-185</td><td>Nostocpunctiform! 3102</td><td> 1/2</td><td>pDS171</td><td>pDS171S</td>

<td>pLS9-185</td><td>We toe sp. 7120</td><td> 9/10</td><td>pDS172</td><td>pDS172S</td>

* NA = nucleic acid sequence; AA = amino acid sequence / polypeptide sequence.

All acp genes were cloned with a synthetic RBS at the EcoRI site immediately downstream of the aar gene in pLS9-185 using NFUSION technology. The EcoRI site was reconstructed downstream of the acp gene. Similarly, the B. subtilis sfp gene was cloned by INFUSION at this EcoRI site together with a synthetic RBS. All plasmids were transformed into E. coli MG1655 SV2. The control for these experiments was expression of AAR alone (pLS9-185).

The results of standard shake matrix fermentation experiments are shown in Fig. 5. Significant improvement in fatty alcohol concentrations were observed in strains containing plasmids pDS171S, pDS172S, pDS168 and pDS169, showing that overexpression of ACP may be beneficial for the production of fatty alcohol. As long as you don't want to be limited by

139

MPI

M UICAN INSTITUTE OF INDUSTRIAL PROPERTY the theory, it is hypothesized that the overexpression of ACP may be beneficial for the production of fatty alcohol by helping in the recognition, affinity and / or change of AcilACPs by the enzyme of the terminal pathway heterologous (see Table 3 (supraj for the source of the ACPs and the presence or absence of sfp).

EXAMPLE 2: Enhanced Fatty Alcohol Production using AAR_7942 by Increased Flux Mediated by Aceitl-CoA Carboxylase (ACC) Through Fatty Acid Synthesis

The main precursors for fatty acid biosynthesis are malonyl-CoA and acetyl-CoA. These precursors have been suggested to limit the rate of fatty acid biosynthesis in E. coli. In this example, a synthetic acetyl-CoA carboxylase, acc, operon [CorynebacteriumglutamicumaccbCAB + 'birA; SEQ ID NOs: 45 or 4 6, 48, and 50 also referred to as D +] was expressed along the Acyl-ACP reductase (AAR) of Synechococcuselongatus PCC7942 (AAR_7942). The accD + operon was cloned downstream of the AAR_7942 gene in plasmid pLS9-185. The resulting plasmid and control plasmid pLS9-185 were transformed into E. coli DV2. Strains were evaluated for fatty alcohol production in a standard shake flask protocol. As shown in Figure 6, co140 expression of the synthetic Corynebacteriumg acc operon led to increased production of fatty alcohol.

EXAMPLE 3: Error-Prone Library, Combination and Limited Saturation Libraries Prepared Using AAR_7942 as a Template

A. Error-Prone Library

An error prone library of the Synechococcuselongatus Acyl-ACP reductase PCC7942 (AAR_7942) was constructed and classified for variants that showed improvements over the wild type AAR_7 942. The plasmid used to make the library prone to errors was designated pDS171S (see Table 3). The error prone library was classified using one of the standard protocols described above. The improvements were classified as either improving the titer or increasing the fraction of the C10-C14 fatty alcohols produced without significantly affecting the titer (results not shown).

B. Combination and Limited Saturation Libraries

Standard techniques known to those of skill in the field were used to prepare the combination libraries and saturation libraries based on positions 17, 18 and 19. The mutations ni

INSTITUTO MEXICANO DE LA PROPIEDAD industrial tested in the combination libraries and saturation libraries (Tables 4A and 4B) were originally identified in the AAR_7942 error-prone library described above. The plasmids, strains and classification protocols used were the same as described in Example 1.

The results of the classification of the error prone library are shown in Tables 4A and 4B. Table 4A shows the AAR_7942 mutations that led to increased fatty alcohol concentrations and Table 4B shows mutations that led to increased C14 fatty alcohol fraction without significantly affecting the total fatty alcohol concentration.

Table 4A: AAR 7942 Limited Saturation and Combination Library Mutations Correlated with Enhanced Fatty Alcohol Titer

<td rowspan="2">Mutations</td><td colspan="4">Combo a library</td>

<td>Concentration FALC (total *)</td><td>C14-FALC (% fraction)</td><td>FALC normalized (% of over control)</td><td>C14 normalized (% of over control)</td>

<td>S18T, C63Y, S96T, L177H, A281V</td><td> 2473</td><td> 48%</td><td> 251%</td><td> 294%</td>

<td>S18T, C63Y, S96T, L177H, A281V, E284K</td><td> 2202</td><td> 50%</td><td> 200%</td><td> 229%</td>

<td>S18T, C63Y, S96T, L177H</td><td> 2088</td><td> 43%</td><td> 189%</td><td> 197%</td>

<td>S18T, C63Y</td><td> 1798</td><td> 39%</td><td> 182%</td><td> 241%</td>

<td>C63H, S967T, L177H, A281V</td><td> 2045</td><td> 53%</td><td> 180%</td><td> 237%</td>

<td>S18T, C63Y, A281V, E283K</td><td> 1696</td><td> 45%</td><td> 172%</td><td> 278%</td>

<td>S18T, C63Y</td><td> 1640</td><td> 43%</td><td> 166%</td><td> 260%</td>

<td>S18T, L177H, A281V,</td><td> 1618</td><td> 44%</td><td> 164%</td><td> 270%</td>

MEXICAN INSTTTVrO OF XDUSTRIAL PROPERTY

<td>E283K</td><td></td><td></td><td></td><td></td><td rowspan="2"></td>

<td rowspan="2">S18T, L177H, A281V, E283K</td><td rowspan="2"> 1617</td><td rowspan="2"> 43%</td><td></td><td></td>

<td> 164%</td><td> 264%</td><td rowspan="12"></td>

<td>S18T, S96T, L177H, E283K</td><td> 1814</td><td> 43%</td><td> 160%</td><td> 194%</td>

<td>S18T, C63H, L177H</td><td> 1811</td><td> 39%</td><td> 160%</td><td> 173%</td>

<td>S18T, L177H, A281V, E283K</td><td> 1503</td><td> 39%</td><td> 152%</td><td> 237%</td>

<td>L65F, S96T, A281V, E283K</td><td> 1638</td><td> 43%</td><td> 144%</td><td> 191%</td>

<td>C63Y, A281V, A282T</td><td> 1622</td><td> 38%</td><td> 143%</td><td> 168%</td>

<td>L11F, C63Y, S96T, L177H, A281V, A282T, E283K</td><td> 1579</td><td> 41%</td><td> 143%</td><td> 186%</td>

<td>L65F, S96T, L177H, A281V, E283K</td><td> 1585</td><td> 42%</td><td> 140%</td><td> 189%</td>

<td>G22S, C63H, S96T, L177H, E283K</td><td> 1229</td><td> 39%</td><td> 125%</td><td> 237%</td>

<td>C63H, L65F, S96T, A281V</td><td> 1178</td><td> 41%</td><td> 119%</td><td> 252%</td>

<td>S18T, L65F, S96T, L177H, A281V</td><td> 1136</td><td> 33%</td><td> 115%</td><td> 203%</td>

<td>C63R, L177H, A281V, E283K</td><td> 1275</td><td> 44%</td><td> 112%</td><td> 196%</td>

<td colspan="5">Libraries Saturation</td>

<td>Mutations</td><td>Concentration FALC (total *)</td><td>C14-FALC (% fraction)</td><td>FALC normalized (% of over control)</td><td>C14 normalized (% of over control)</td>

<td>S18M</td><td> 2809</td><td> 40%</td><td> 243%</td><td> 212%</td>

<td>S18F</td><td> 2564</td><td> 54%</td><td> 222%</td><td> 285%</td>

<td>S18Y</td><td> 2550</td><td> 45%</td><td> 221%</td><td> 236%</td>

<td>S18W</td><td> 2530</td><td> 55%</td><td> 227%</td><td> 314%</td>

<td>S18M</td><td> 2495</td><td> 41%</td><td> 224%</td><td> 231%</td>

<td>S18Y</td><td> 2039</td><td> 44%</td><td> 188%</td><td> 252%</td>

<td>S18Y</td><td> 1997</td><td> 44%</td><td> 179%</td><td> 251%</td>

<td>S18T</td><td> 1729</td><td> 31%</td><td> 155%</td><td> 177%</td>

<td>S18T</td><td> 1690</td><td> 31%</td><td> 152%</td><td> 176%</td>

<td>S18T</td><td> 1683</td><td> 31%</td><td> 151%</td><td> 176%</td>

Vm-iamriMlB · 1 H | Bu, I, Bu ||

<img file="MX346830B_D0073.tif" />

<td>S18T</td><td> 1599</td><td colspan="2"> 30%</td><td colspan="2"> 143%</td><td colspan="2">x ^ troísnúÍAt</td><td rowspan="31"></td>

<td>S18L</td><td> 1568</td><td colspan="2"> 48%</td><td colspan="2"> 141%</td><td colspan="2"> 271%</td>

<td>S18C</td><td> 1381</td><td colspan="2"> 28%</td><td colspan="2"> 120% </td><td colspan="2">14 and%</td>

<td>S18C</td><td> 1372</td><td colspan="2"> 26%</td><td colspan="2"> 123%</td><td colspan="2"> 147%</td>

<td>S18C</td><td> 1368</td><td colspan="2"> 28%</td><td colspan="2"> 118%</td><td colspan="2"> 148%</td>

<td>S18L</td><td> 1359</td><td colspan="2"> 47%</td><td colspan="2"> 122%</td><td colspan="2"> 266%</td>

<td>S18C</td><td> 1355</td><td colspan="2"> 26%</td><td colspan="2"> 121%</td><td colspan="2"> 148%</td>

<td>S18V</td><td> 1344</td><td colspan="2"> 30%</td><td colspan="2"> 121%</td><td colspan="2"> 170%</td>

<td>S18C</td><td> 1340</td><td colspan="2"> 26%</td><td colspan="2"> 120%</td><td colspan="2"> 147%</td>

<td>S18L</td><td> 1326</td><td colspan="2"> 47%</td><td colspan="2"> 119%</td><td colspan="2"> 268%</td>

<td>S18L</td><td> 1315</td><td colspan="2"> 47%</td><td colspan="2"> 118%</td><td colspan="2"> 266%</td>

<td>S18C</td><td> 1313</td><td colspan="2"> 26%</td><td colspan="2"> 118%</td><td colspan="2"> 149%</td>

<td>S18V</td><td> 1306</td><td colspan="2"> 30%</td><td colspan="2"> 121%</td><td colspan="2"> 170%</td>

<td>S18C</td><td> 1305</td><td colspan="2"> 26%</td><td colspan="2"> 117%</td><td colspan="2"> 148%</td>

<td>S18L</td><td> 1302</td><td colspan="2"> 47%</td><td colspan="2"> 117%</td><td colspan="2"> 267%</td>

<td>V17L</td><td> 1188</td><td colspan="2"> 23%</td><td colspan="2"> 110%</td><td colspan="2"> 130%</td>

<td>V17L</td><td> 1185</td><td colspan="2"> 33%</td><td colspan="2"> 103%</td><td colspan="2"> 173%</td>

<td>V17L</td><td> 1175</td><td colspan="2"> 32%</td><td colspan="2"> 102%</td><td colspan="2"> 171%</td>

<td rowspan="2"></td><td colspan="7">Combo β Library</td>

<td colspan="2">Concentration FALC (total *)</td><td colspan="2">C14-FALC (% fraction)</td><td colspan="2">FALC normalized (% of over control)</td><td>C14 normalized (% of over control)</td>

<td>S18W, S96T, E283K, R286Q, A324V</td><td colspan="2"> 3437</td><td colspan="2"> 49%</td><td colspan="2"> 246%</td><td> 255%</td>

<td>S18W</td><td colspan="2"> 3286</td><td colspan="2"> 53%</td><td colspan="2"> 235%</td><td> 275%</td>

<td>S18W, S96T, E283K</td><td colspan="2"> 3199</td><td colspan="2"> 46%</td><td colspan="2"> 229%</td><td> 237%</td>

<td>S18W, C63Y, S96T</td><td colspan="2"> 3186</td><td colspan="2"> 46%</td><td colspan="2"> 228%</td><td> 241%</td>

<td>S18W, C68Y, E2834K</td><td colspan="2"> 2949</td><td colspan="2"> 50%</td><td colspan="2"> 211%</td><td> 260%</td>

<td>C63Y, E283K</td><td colspan="2"> 2680</td><td colspan="2"> 36%</td><td colspan="2"> 192%</td><td> 189%</td>

<td>S18W, C63Y</td><td colspan="2"> 2675</td><td colspan="2"> 54%</td><td colspan="2"> 191%</td><td> 280%</td>

<td>C63Y, S96T</td><td colspan="2"> 2583</td><td colspan="2"> 38%</td><td colspan="2"> 185%</td><td> 196%</td>

<td>C63Y, E283K, Q316K</td><td colspan="2"> 2420</td><td colspan="2"> 36%</td><td colspan="2"> 173%</td><td> 188%</td>

<td>S96T</td><td colspan="2"> 1605</td><td colspan="2"> 23%</td><td colspan="2"> 115%</td><td> 121%</td>

<td>E283K</td><td colspan="2"> 1532</td><td colspan="2"> 25%</td><td colspan="2"> 110%</td><td> 131%</td>

Table 4B:

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Mutations of

Libraries

<img file="MX346830B_D0074.tif" />

INSTITUTO MEXICANO DELAWGFIEDAD, by CcWFM ^ ci de Saturation Limitada AAR_7942

Run it eidlTcfttcrs · '' · '⁷· - Increased Fraction of C14 Fatty Alcohol

<td rowspan="2"></td><td colspan="4">Combo a library</td>

<td>Concentration FALC (total *)</td><td>C14-FALC (% fraction)</td><td>Normalized FALC (% of over control)</td><td>314 normalized (% of over control)</td>

<td>C63H, S96T, L177H, A281V</td><td> 2045.36</td><td> 53%</td><td> 180%</td><td> 237%</td>

<td>G22S, C63H, F64V, S96T, L177H, A281V</td><td> 152.47</td><td> 51%</td><td> 14%</td><td> 233%</td>

<td>S18T, C63H, S96T, L177H, A281V, E283K</td><td> 2202.46</td><td> 50%</td><td> 200%</td><td> 229%</td>

<td>S18T, C63Y, S96T, L177H, A281V</td><td> 2472.80</td><td> 48%</td><td> 251%</td><td> 294%</td>

<td>S18T, C63Y, A281V, E283K</td><td> 1695.59</td><td> 45%</td><td> 172%</td><td> 278%</td>

<td>S18T, L177H, A281V, E283K</td><td> 1618.20</td><td> 44%</td><td> 164%</td><td> 270%</td>

<td>C63R, L177H, A281V, E283K</td><td> 1275.38</td><td> 44%</td><td> 112%</td><td> 196%</td>

<td>S18T, C63H, L65F, S96T, L177H, A281V, E283K</td><td> 912.50</td><td> 44%</td><td> 83%</td><td> 199%</td>

<td>S18T, S96T, L177H, E283K</td><td> 1814.34</td><td> 43%</td><td> 160%</td><td> 194%</td>

<td>S18T, L179H, A281V, E283K</td><td> 1616.51</td><td> 43%</td><td> 164%</td><td> 264%</td>

<td>S18T, C63H, S96T, L177H</td><td> 2088.12</td><td> 43%</td><td> 189%</td><td> 197%</td>

<td>L65F, S96T, A281V, Ξ283Κ</td><td> 1638.28</td><td> 43%</td><td> 144%</td><td> 191%</td>

<td>C63H, L65F, A281V, E283K</td><td> 632.66</td><td> 43%</td><td> 56%</td><td> 190%</td>

<td>S18T, C63Y</td><td> 1640.33</td><td> 43%</td><td> 166%</td><td> 260%</td>

<td>L65F, S96T, L177H, A281V, E283K</td><td> 1585.32</td><td> 42%</td><td> 140%</td><td> 189%</td>

<td>C63Y, L65F, L177H</td><td> 639.46</td><td> 41%</td><td> 56%</td><td> 184%</td>

<td>C63H, L65F, S96T, A281V</td><td> 1177.76</td><td> 41%</td><td> 119%</td><td> 252%</td>

<td>L11F, C63Y, S96T, L177H, A281V, A282T, E283K</td><td> 1578.54</td><td> 41%</td><td> 143%</td><td> 186%</td>

<td>S18T, C63Y</td><td> 1797.99</td><td> 39%</td><td> 182%</td><td> 241%</td>

<td>S18T, L178H, A282V, E284K</td><td> 1502.59</td><td> 39%</td><td> 152%</td><td> 237%</td>

<td>G23S, C63H, S96T, L177H, E283K</td><td> 1229.34</td><td> 39%</td><td> 125%</td><td> 237%</td>

<td>S18T, C63H, L177H</td><td> 1811.40</td><td> 3 9%</td><td> 160%</td><td> 173%</td>

<td>C63Y, A281V, A282T</td><td> 1622.23</td><td> 38%</td><td> 143%</td><td> 168%</td>

<td>S18T, C63Y, L65F, S96T, L177H, A281V, E283K, M284I</td><td> 454.42</td><td> 35%</td><td> 41%</td><td> 162%</td>

<td>S18T, L65F, S96T, L177H, A281V</td><td> 1135.95</td><td> 33%</td><td> 115%</td><td> 203%</td>

<td>L65F, L178H, G180S, E283K</td><td> 151.40</td><td> 33%</td><td> 13%</td><td> 146%</td>

<td>L66F, L177H, G180S, E283K</td><td> 143.83</td><td> 31%</td><td> 13%</td><td> 138%</td>

145

<img file="MX346830B_D0075.tif" />

INSTITUTO MÍXICANO n * LA FRC? IEOAD idW ^ AL

103.21

30%

S18T, C63H, L65F, L177H

<td rowspan="2"></td><td colspan="4">Saturation Libraries . -------- · - - -</td>

<td>FALC concentration (total*)</td><td>C14-FALC (% fraction)</td><td>FALC ' normalized (% overcontrol)</td><td>C14 normalized (% of over control)</td>

<td>S18W</td><td> 2530</td><td> 55%</td><td> 227%</td><td> 314%</td>

<td>S18F</td><td> 2564</td><td> 54%</td><td> 222%</td><td> 285%</td>

<td>V17N</td><td> 571</td><td> 49%</td><td> 49%</td><td> 260%</td>

<td>S18L</td><td> 1568</td><td> 48%</td><td> 141%</td><td> 271%</td>

<td>S18L S18L</td><td> 1024 1326</td><td> 47% 47%</td><td> 92% 119%</td><td> 268% 268%</td>

<td>S18L</td><td> 1302</td><td> 47%</td><td> 117%</td><td> 267%</td>

<td>S18L</td><td> 1106</td><td> 47%</td><td> 99%</td><td> 267%</td>

<td>S18L</td><td> 1315</td><td> 47%</td><td> 118%</td><td> 266%</td>

<td>S18L</td><td> 1359</td><td> 47%</td><td> 122%</td><td> 266%</td>

<td>S18Y</td><td> 2550</td><td> 45%</td><td> 221%</td><td> 236%</td>

<td>S18Y</td><td> 2039</td><td> 44%</td><td> 188%</td><td> 252%</td>

<td>S18Y</td><td> 1997</td><td> 44%</td><td> 179%</td><td> 251%</td>

<td>S18M</td><td> 2495</td><td> 41%</td><td> 224%</td><td> 231%</td>

<td>S18M</td><td> 2809</td><td> 40%</td><td> 243%</td><td> 212%</td>

<td>V17L</td><td> 1127</td><td> 33%</td><td> 97%</td><td> 175%</td>

<td>V17L</td><td> 1185</td><td> 33%</td><td> 103%</td><td> 173%</td>

<td>V17L</td><td> 1091</td><td> 32%</td><td> 94%</td><td> 171%</td>

<td>V17L</td><td> 1175</td><td> 32%</td><td> 102%</td><td> 171%</td>

<td>S18T</td><td> 1729</td><td> 31%</td><td> 155%</td><td> 177%</td>

<td>S18T</td><td> 1690</td><td> 31%</td><td> 152%</td><td> 176%</td>

<td>S18T</td><td> 1683</td><td> 31%</td><td> 151%</td><td> 176%</td>

<td>S18T</td><td> 1599</td><td> 30%</td><td> 143%</td><td> 172%</td>

<td>S18V</td><td> 1344</td><td> 30%</td><td> 121%</td><td> 170%</td>

<td>S18V</td><td> 1306</td><td> 30%</td><td> 121%</td><td> 170%</td>

<td>S18V</td><td> 1015</td><td> 30%</td><td> 91%</td><td> 169%</td>

<td>S18C</td><td> 1381</td><td> 28%</td><td> 120%</td><td> 149%</td>

<td>S18C</td><td> 1368</td><td> 28%</td><td> 118%</td><td> 148%</td>

<td>R19H, R58S</td><td> 873</td><td> 27%</td><td> 81%</td><td> 152%</td>

<td>V17C</td><td> 927</td><td> 27%</td><td> 80%</td><td> 140%</td>

<td>U17C</td><td> 882</td><td> 26%</td><td> 76%</td><td> 139%</td>

<td>S18C</td><td> 1313</td><td> 26%</td><td> 118%</td><td> 149%</td>

<td>S18C</td><td> 1355</td><td> 26%</td><td> 121%</td><td> 148%</td>

<td>S18C</td><td> 1305</td><td> 26%</td><td> 117%</td><td> 148%</td>

<td>S18C</td><td> 1372</td><td> 26%</td><td> 123%</td><td> 147%</td>

<td>S18C</td><td> 1340</td><td> 26%</td><td> 120%</td><td> 147%</td>

<td>V17W</td><td> 849</td><td> 25%</td><td> 73%</td><td> 134%</td>

<td>V17W</td><td> 815</td><td> 25%</td><td> 71%</td><td> 133%</td>

<td>V17W</td><td> 831</td><td> 25%</td><td> 72%</td><td> 132%</td>

<td>V17W</td><td> 840</td><td> 25%</td><td> 73%</td><td> 131%</td>

<img file="MX346830B_D0076.tif" />

146

R19V 844 124%

<td>R19V</td><td> 813</td><td colspan="2"> 24%</td><td> 75%</td><td colspan="2"> 136%</td>

<td>R19V</td><td> 821</td><td colspan="2"> 24%</td><td> 76%</td><td colspan="2"> 135%</td>

<td>R19V</td><td> 862</td><td colspan="2"> 24%</td><td> 80%</td><td colspan="2"> 135%</td>

<td>R19V</td><td> 814</td><td colspan="2"> 24%</td><td> 75%</td><td colspan="2"> 135%</td>

<td>R19K, V117L</td><td> 1188</td><td colspan="2"> 23%</td><td> 110%</td><td colspan="2"> 130%</td>

<td>R19S</td><td> 803</td><td colspan="2"> 22%</td><td> 74%</td><td colspan="2"> 128%</td>

<td>R19T</td><td> 735</td><td colspan="2"> 22%</td><td> 68%</td><td colspan="2"> 127%</td>

<td>R19S</td><td> 783</td><td colspan="2"> 22%</td><td> 72%</td><td colspan="2"> 127%</td>

<td>R19S</td><td> 764</td><td colspan="2"> 22%</td><td> 75%</td><td colspan="2"> 129%</td>

<td>R19I</td><td> 862</td><td colspan="2"> 22%</td><td> 80%</td><td colspan="2"> 123%</td>

<td>R19A</td><td> 901</td><td colspan="2"> 21%</td><td> 83%</td><td colspan="2"> 122%</td>

<td>R19A</td><td> 705</td><td colspan="2"> 21%</td><td> 65%</td><td colspan="2"> 119%</td>

<td>R19M</td><td> 937</td><td colspan="2"> 20%</td><td> 86%</td><td colspan="2"> 114%</td>

<td rowspan="2"></td><td colspan="6">Combo β Library</td>

<td>FALC concentration (total*)</td><td>C14-FALC (% fraction)</td><td colspan="3">Normalized FALC (% of over control)</td><td>C14 normalized (% overcontrol)</td>

<td>S18W</td><td> 3184</td><td> 57%</td><td colspan="3"> 228%</td><td> 296%</td>

<td>S18W, C63Y</td><td> 2675</td><td> 54%</td><td colspan="3"> 191%</td><td> 280%</td>

<td>S10W, C63Y, S95T</td><td> 2832</td><td> 54%</td><td colspan="3"> 203%</td><td> 279%</td>

<td>S18W, S96T, E283K</td><td> 2874</td><td> 54%</td><td colspan="3"> 206%</td><td> 278%</td>

<td>S18W, C63Y, E283K</td><td> 2915</td><td> 51%</td><td colspan="3"> 209%</td><td> 263%</td>

<td>S18W, S96T, E283K, R286Q, A324V</td><td> 3437</td><td> 49%</td><td colspan="3"> 246%</td><td> 255%</td>

<td>C63Y, S96T</td><td> 2583</td><td> 38%</td><td colspan="3"> 185%</td><td> 196%</td>

<td>C63Y, E283K</td><td> 2176</td><td> 38%</td><td colspan="3"> 156%</td><td> 195%</td>

<td>C63Y, E283K, Q316K</td><td> 2420</td><td> 36%</td><td colspan="3"> 173%</td><td> 188%</td>

<td>E283K</td><td> 1474</td><td> 25%</td><td colspan="3"> 105%</td><td> 132%</td>

<td>S96T</td><td> 1605</td><td> 23%</td><td colspan="3"> 115%</td><td> 121%</td>

<td rowspan="2"></td><td colspan="6">Combo 3 Library</td>

<td>FALC concentration (total*)</td><td>C14-FALC (% fraction)</td><td colspan="3">Normalized FALC (% of over control)</td><td>C14 normalized (% overcontrol)</td>

<td>S18W</td><td> 2853</td><td> 217%</td><td colspan="3"> 51%</td><td> 274%</td>

<td>Y23N</td><td> 954</td><td> 72%</td><td colspan="3"> 35%</td><td> 191%</td>

<td>Y23N, Q238H</td><td> 8 92</td><td> 68%</td><td colspan="3"> 35%</td><td> 190%</td>

<td>G150D, I274N</td><td> 766</td><td> 62%</td><td colspan="3"> 30%</td><td> 162%</td>

<td>I274N</td><td> 1359</td><td> 107%</td><td colspan="3"> 29%</td><td> 161%</td>

<td>I274N, A285V</td><td> 1317</td><td> 103%</td><td colspan="3"> 28%</td><td> 155%</td>

<td>P38T, I274N, A285V</td><td> 587</td><td> 46%</td><td colspan="3"> 26%</td><td> 143%</td>

<td>Q238H, M291V</td><td> 1383</td><td> 109%</td><td colspan="3"> 22%</td><td> 119%</td>

<td>M291V</td><td> 1618</td><td> 123%</td><td colspan="3"> 22%</td><td> 118%</td>

<td>G151D, M291V</td><td> 1302</td><td> 105%</td><td colspan="3"> 21%</td><td> 114%</td>

<td>T135M, M291V</td><td> 1265</td><td> 100%</td><td colspan="3"> 18%</td><td> 101%</td>

ι «⁷ ΙΜΡΙ ^^ 3

MEXICAN INSTITUTE

DltA VM0PISDAD V ^ Vss ^ gS ^ sg

EXAMPLE 4: Preparaf ^^^ ancfcr-Ta AAR Saturation Library (S18W) as a Template ......... '.......

A complete saturation library of a Synechococcuselongatus PCC7942 Acyl-ACP reductase variant ('AAR (S18W) _7 942), was constructed and classified for variants that showed improvements over AAR (S18W), identified as an AAR variant significantly improved in the first qualifying round (Examples 2 and 3). The selection criteria were an increase in the FALC concentration or an increase in the percentage of C12 fraction. Engineering efforts focused on improving the dependence of AAR on ACP overexpression in order to produce high concentrations. Although not wishing to be bound by theory, the advantage observed in ACP overexpressing strains was hypothesized to result from a higher concentration of fatty acid biosynthetic intermediates available for cleavage by AAR. By sorting the saturation and pooling libraries built in the AAR into a strain lacking ACP overexpression (having a lower concentration of FAS intermediates), variants with higher affinities for FAS intermediates can be selected.

The plasmid that he used to make the complete saturation library was designated pAAR-1. It is a derivative of

<img file="MX346830B_D0077.tif" />

pLS9-185 harboring the AAR gene encoding the variant ^ .l¾l.β_rVU..followed by the aldehyde reductase gene, AlrA, from Acinetobacterbaylyi (SEQ ID NO: 54). AlrA was added to completely reduce fatty aldehyde intermediates generated by AAR and was not completely reduced by endogenous E. coli fatty aldehyde reductase. The complete saturation library was sorted into strain Shu.002. The Shu.002 strain is DV2 PT5-ifabl38 PT5_ifadR (Table 7, infra). For ifab! 38 see SEQ ID NO: 55 in Table 10. Libraries were sorted using one of the standard protocols described above. The improvements were classified as either improving the titer or increasing the fraction of the C12 fatty alcohols produced by the Acyl-ACP reductase without significantly affecting the concentration. The results of the classification of the saturation libraries are shown in Table 5 below.

Table 5: Mutations of a Full Saturation Library AAR (S18W) 7942 Correlated with Increased Titer of Fatty Alcohol and / or Increased Fraction of C12 Fatty Alcohol

<td rowspan="2">AAR mutations (in addition to S18W)</td><td colspan="2">FALC concentration *</td><td colspan="2">C12</td>

<td>Total*</td><td>FIOC **</td><td>Fraction*</td><td>FIOC **</td>

<td>T148V</td><td> 747</td><td> 1.21</td><td> 7.9%</td><td> 0.99</td>

<td>A159V</td><td> 702</td><td> 1.14</td><td> 7.0%</td><td> 0.87</td>

674

1.10

7.2%

0.90

T157V

<img file="MX346830B_D0078.tif" />

IMPI

MEXICAN INSTITUTE

M LA RBOTIEDAD

149

<td>A135S</td><td> 803</td><td> 1.31</td><td>------------------- INI 7.8%</td><td>HJSTBIAL ---- Z2B ---- 0.98</td>

<td rowspan="2">A328S</td><td rowspan="2"> 712</td><td rowspan="2"> 1.16</td><td> 8.1%</td><td> 1.02 . „</td>

<td></td><td></td>

<td>Q191A</td><td> 780</td><td> 1.27</td><td> 7.7%</td><td> 0.96</td>

<td>A285V, M291V</td><td> 837</td><td> 1.36</td><td> 8.0%</td><td> 1.00</td>

<td>Q277V</td><td> 854</td><td> 1.39</td><td> 7.9%</td><td> 0.99</td>

<td>Q155C</td><td> 626</td><td> 1.02</td><td> 8.3%</td><td> 1.04</td>

<td>E210Y</td><td> 676</td><td> 1.10</td><td> 8.5%</td><td> 1.06</td>

<td>T120S</td><td> 623</td><td> 1.01</td><td> 8.1%</td><td> 1.02</td>

<td>T236C</td><td> 691</td><td> 1.12</td><td> 7.6%</td><td> 0.95</td>

<td>Q335N</td><td> 7 63</td><td> 1.24</td><td> 6.8%</td><td> 0.86</td>

<td>C172L</td><td> 700</td><td> 1.14</td><td> 7.7%</td><td> 0.97</td>

<td>E283S</td><td> 858</td><td> 1.39</td><td> 8.0%</td><td> 1.00</td>

<td>L209R</td><td> 837</td><td> 1.36</td><td> 7.6%</td><td> 0.96</td>

<td>I153P</td><td> 606</td><td> 0.99</td><td> 8.0%</td><td> 1.00</td>

<td>A211W</td><td> 7 97</td><td> 1.30</td><td> 6.9%</td><td> 0.86</td>

<td>A324T</td><td> 909</td><td> 1.48</td><td> 4.0%</td><td> 0.50</td>

<td>W34F</td><td> 817</td><td> 1.22</td><td> 7.5%</td><td> 0.98</td>

<td>T187V</td><td> 825</td><td> 1.23</td><td> 6.9%</td><td> 0.91</td>

<td>D2 4E</td><td> 737</td><td> 1.10</td><td> 7.1%</td><td> 0.93</td>

<td>T188H</td><td> 8 62</td><td> 1.29</td><td> 8.6%</td><td> 1.13</td>

<td>V18A</td><td> 798</td><td> 1.19</td><td> 6.4%</td><td> 0.84</td>

<td>V18A, L338W</td><td> 732</td><td> 1.27</td><td> 7.6%</td><td> 0.97</td>

<td>T188V</td><td> 775</td><td> 1.34</td><td> 7.4%</td><td> 0.95</td>

<td>I168V</td><td> 731</td><td> 1.27</td><td> 8.1%</td><td> 1.03</td>

<td>W35F</td><td> 666</td><td> 1.15</td><td> 7.5%</td><td> 0.96</td>

<td>T148V</td><td> 836</td><td> 1.45</td><td> 6.2%</td><td> 0.80</td>

<td>Q155L</td><td> 740</td><td> 1.28</td><td> 8.5%</td><td> 1.08</td>

<td>T148C</td><td> 731</td><td> 1.26</td><td> 8.6%</td><td> 1.10</td>

<td>T148E</td><td> 694</td><td> 1.20</td><td> 8.3%</td><td> 1.06</td>

<td>A50Q, ​​L337V</td><td> 800</td><td> 1.38</td><td> 7.2%</td><td> 0.92</td>

<td>R118Q</td><td> 724</td><td> 1.25</td><td> 7.3%</td><td> 0.94</td>

<td>L31V</td><td> 901</td><td> 1.56</td><td> 6.8%</td><td> 0.87</td>

<td>A135S</td><td> 775</td><td> 1.34</td><td> 8.7%</td><td> 1.12</td>

<td>D24Y</td><td> 890</td><td> 0.93</td><td> 19.1%</td><td> 2.05</td>

<td>C63A</td><td> 895</td><td> 0.94</td><td> 16.1%</td><td> 1.73</td>

<td>S113K</td><td> 812</td><td> 0.85</td><td> 13.3%</td><td> 1.43</td>

<td>L31V</td><td> 645</td><td> 0.68</td><td> 15.7%</td><td> 1.69</td>

<td>E283G</td><td> 917</td><td> 0.96</td><td> 14.3%</td><td> 1.54</td>

<td>A112R</td><td> 918</td><td> 0.96</td><td> 10.7%</td><td> 1.15</td>

<td>D43E</td><td> 1002</td><td> 1.05</td><td> 13.0%</td><td> 1.39</td>

<td>Q116G</td><td> 894</td><td> 0.94</td><td> 14.0%</td><td> 1.50</td>

<img file="MX346830B_D0079.tif" />

** FIOC = times of increase over an AAR control (S18W)

Example 5: Combination Libraries Prepared Using AAR (S18W) as a Template

Standard techniques known to those of skill in the field were used to prepare the combination libraries. The mutations tested in the combination libraries (Tables 6A, 6B and 6C) were originally identified in a complete saturation library (Example 4). Combination libraries were built on the same plasmid and sorted into the same strain as described in Example 3. Libraries were sorted using one of the flagship protocols described above. The improvements were classified as either improving the titre increasing the fraction of C12 fatty alcohols produced by the Acyl-ACP reductase without significantly affecting the concentration. The results of sorting the AAR pooling libraries are shown in Tables 6A, 6B and 6C below.

<img file="MX346830B_D0080.tif" />

151 «Mwro mrmcamo DI LA MOHEDAD

Table 6A: Mutations of the First Library of ÍPSíffi ^ inac l^stAAR (S18W) 7 942 Correlated with

Increased Fatty Alcohol

<td rowspan="2">AAR mutations (in addition to S18W)</td><td colspan="2">FALC concentration *</td><td colspan="2">C12</td>

<td>mg / L *</td><td>FIOC **</td><td>Fraction*</td><td>FIOC **</td>

<td>A281L</td><td> 1478</td><td> 1.29</td><td> 14.1%</td><td> 1.11</td>

<td>A281Y</td><td> 1527</td><td> 1.28</td><td> 12.1%</td><td> 0.96</td>

<td>T154A, A281L</td><td> 1550</td><td> 1.28</td><td> 14.3%</td><td> 1.13</td>

<td>T154A, A281Y</td><td> 1384</td><td> 1.19</td><td> 13.3%</td><td> 1.05</td>

<td>C63G, A281F</td><td> 1472</td><td> 1.24</td><td> 12.9%</td><td> 1.02</td>

<td>C63G, A281L</td><td> 1432</td><td> 1.19</td><td> 18.2%</td><td> 1.44</td>

<td>N83G, A281Y</td><td> 1428</td><td> 1.23</td><td> 10.7%</td><td> 0.84</td>

<td>C63G, A281Y</td><td> 1419</td><td> 1.21</td><td> 15.6%</td><td> 1.24</td>

<td>S10G, A281L</td><td> 1419</td><td> 1.20</td><td> 12.6%</td><td> 1.00</td>

<td>S113K, Q155G</td><td> 1305</td><td> 1.15</td><td> 12.3%</td><td> 0.97</td>

<td>D43E, A50Q, ​​A281L</td><td> 1517</td><td> 1.32</td><td> 9.1%</td><td> 0.72</td>

<td>M21L, N83G, A281Y</td><td> 1513</td><td> 1.29</td><td> 10.2%</td><td> 0.81</td>

<td>M21L, S113K, A281L</td><td> 1392</td><td> 1.20</td><td> 20.4%</td><td> 1.61</td>

<td>C63G, T154A, A281F</td><td> 1369</td><td> 1.24</td><td> 12.4%</td><td> 0.98</td>

<td>C63G, N83G, A281Y</td><td> 1330</td><td> 1.32</td><td> 14.6%</td><td> 1.16</td>

<td>Q155G, A281L, E283G</td><td> 1327</td><td> 1.16</td><td> 22.2%</td><td> 1.76</td>

<td>A50Q, ​​C63G, A281F</td><td> 1286</td><td> 1.15</td><td> 17.9%</td><td> 1.41</td>

<td>S18W, D43E, A50Q, ​​A281L</td><td> 1635</td><td> 1.32</td><td> 9.4%</td><td> 0.77</td>

<td>M21L, C63G, S113K, T154A, A281L</td><td> 1518</td><td> 1.24</td><td> 28.3%</td><td> 2.23</td>

<td>M21L, L31V, N83G, Q116G, A281L</td><td> 1353</td><td> 1.18</td><td> 15.3%</td><td> 1.21</td>

* Average of 4 replicates ** FIOC = fold increase over AAR control (S18W)

Table 6B: Mutations of 2^to Combination Library

AAR (S18W) 7942 with Increased Alcohol concentration

Fatty

<td rowspan="2">AAR mutations (in addition to S18W)</td><td>Concentration</td><td>FALC *</td><td colspan="2">C12</td>

<td>mg / L *</td><td>FIOC **</td><td>Fraction*</td><td>FIOC **</td>

<td>S18W (control)</td><td> 1034</td><td> 1.00</td><td> 10.0%</td><td> 1.00</td>

<td>M21L, C63G, S113K, T154A, A281L</td><td> 1179</td><td> 1.14</td><td> 29.4%</td><td> 2.94</td>

152

<img file="MX346830B_D0081.tif" />

<td>D16L, M21L, C63G, S113K, T154A, A281L</td><td> 1221</td><td> 1,18</td><td> 19.4%</td><td> 1.94 _ _</td><td rowspan="6"></td>

<td>L8A, D24V, C63G, S113K, Q155L, A281L</td><td> 1414</td><td> 1.37</td><td> 10.4%</td><td> 1.04</td>

<td>L8A, M21L, C63G, Α77Ά ***, S113K, Τ154Ά, A281L</td><td> 1310</td><td> 1.27</td><td> 14.2%</td><td> 1.42</td>

<td>D24P, L31M, C63G, S113K, T154A, A281L</td><td> 1437</td><td> 1.39</td><td> 13.0%</td><td> 1.30</td>

<td>L8A, D16L, D24V, C63G, S113K, T154A, A281L</td><td> 1342</td><td> 1.30</td><td> 12.0%</td><td> 1.20</td>

<td>D24E, C63G, S113K, T154A, A281L</td><td> 1425</td><td> 1.38</td><td> 14.3%</td><td> 1.43</td>

* average of 4 replicates * * FIOC = fold increase over control (S18W) * ** A77A mutation is a gcca to gca silencing codon mutation

Table 6C: Mutations of the AAR (S18W) _7942 combination libraries correlated with the increased fraction of the

C12 Fatty Alcohol

<td rowspan="2">AAR mutations (in addition to S18W)</td><td colspan="2">C12</td><td>Concentration FALC *</td>

<td>Fraction*</td><td>FIOC **</td><td>FIOC **</td>

<td>S18W (control)</td><td> 11.0%</td><td> 1.00</td><td> 1.00</td>

<td>M21L, C63G, S113K, T154A, A281L</td><td> 28.3%</td><td> 2.57</td><td> 1.24</td>

<td>M21L, Q155G, A281L</td><td> 24.8%</td><td> 2.25</td><td> 1.10</td>

<td>Q155G, A281L, E283G</td><td> 22.2%</td><td> 2.02</td><td> 1.16</td>

<td>M21L, S113K, A281L</td><td> 20.4%</td><td> 1.85</td><td> 1.20</td>

<td>C63G, A281L</td><td> 18.1%</td><td> 1.65</td><td> 1.18</td>

<td>A50Q, ​​C63G, A281F</td><td> 17,9%</td><td> 1.63</td><td> 1.15</td>

<td>C63G</td><td> 17.7%</td><td> 1.61</td><td> 1.00</td>

<td>S10G, C63G, Q155G, S253N, A281F</td><td> 16.1%</td><td> 1.46</td><td> 1.03</td>

<td>D43E, C63G, S113K, A281L</td><td> 16.0%</td><td> 1.46</td><td> 0.98</td>

<td>A281L</td><td> 15.6%</td><td> 1.42</td><td> 1.20</td>

<td>M21L, L31V, N83G, Q116G, A281L</td><td> 15.3%</td><td> 1.39</td><td> 1.18</td>

<td>C63G, N83G, A281Y</td><td> 14.6%</td><td> 1.33</td><td> 1.32</td>

<td>A281L</td><td> 14.1%</td><td> 1.28</td><td> 1.29</td>

* Average of 4 replicates ★★ FIOC = fold increase over control (S18W) ¿

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INDUSTRIAL, '' • ass —-— Example 6: Increased Flow Through the Fatty Acid Synthesis Pathway- iFab and iFadR Mediated Fatty Alcohol Production Using AAR

In this example, improved fatty alcohol production using AAR was shown by increasing flux through the fatty acid biosynthesis pathway mediated by overexpression of a synthetic operon comprising several FAB proteins (ifabl38) and / or overexpression of the protein FadR (ifad), a regulator of fatty acid metabolism. ÍFAB138 (SEQ ID NO: 55) includes in the following order the genes fabV from Vibrio cholerae, FabH, fabD, fabG and fabA from Salmonella typhimurium, and FabF from Clostridium acetobutylicum, and is integrated as a synthetic operon controlled either by the promoter PlacUV5 or PT5. The iFadR includes the Eschsrichia coli fadR gene (SEQ ID NO: 56) controlled by the T5 promoter. The components present in the E. coli strains evaluated in this example are shown in Table 7, below.

Table 7: E. coli strains with IFAB138 or fadR

<td>Components</td><td>DV2</td><td>BD061</td><td>BD064</td><td>Shu002</td>

<td>i (PlacUV5-fabl38)</td><td> -</td><td> +</td><td> +</td><td> -</td>

<td>i (PT5-fabl38)</td><td> -</td><td></td><td></td><td> +</td>

<td>i (PT5-fadR)</td><td></td><td> -</td><td> +</td><td> +</td>

AAR (S18W) was expressed from plasmid pDS311, a variant of plasmid pDS171S, in which the codon of AAR 18

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INSTITUTO MEXICANO Di LA tIMPIEDAD INDUSTRIAL specified a tryptophan instead of a serine (pCLAAR (S18W) + ACP-sfp) and the aldehyde reductase gene, alrA, from Acinetobacterbaylyi (SEQ ID NO: 54) was cloned downstream of the sfp gene of B. subtilis. The pDS311 was transformed into the strains. 5 DV2, BD061, BD064 and Shu002. The strains were evaluated in the 4NBT_2N protocol (see above). As shown in Figure 7, fatty alcohol production was significantly increased as a result of the presence of ifabl38 and ifadR, with the Shu002 strain showing the highest fatty alcohol concentration.

Example 7: Enhanced AAR Variants in a Strain with Increased Flux Through the Fatty Acid Synthesis Pathway

In this example, enhanced fatty alcohol production in recombinant host cells transformed with AAR variants from the combination libraries were shown in Shu2, an E. coli strain with increased flux through the fatty acid biosynthesis pathway mediated by ifabl38 overexpression and protein overexpression

FadR (supra). Four AAR variants from the second pool library (Table 6B) were evaluated. These variants harbor the following mutations: Com2a: S18W, D24P, L31M, C63G, S113K, T154A, A281L; Comb2b; S18W, D16L, M21L, C63G, S113K, T154A, A281L; Com2c: S18W, L8A, M21L,₁₅₅ IMPI ^ Mexican institute wZGksxí DE LA PROPERTY ICV, TOF industrial

C63G, A77A, S113K, T154A, A281L; Com2d: D24E, C63G, S113K, T154A, A281L (see also Figure 8). These variants were cloned into the backbone of plasmid pJL104. PJL104 was created by cloning the C. glutamicum synthetic accD + operon (as described in Example 2, supra} downstream of the alrA gene in pDS311. The resulting plasmids were transformed into the Shu002 strain and the strains were evaluated in the protocol 4NBT_1N (supra}. As shown in Figure 8, elevated fatty alcohol production was observed in all strains. Strain BD064 harboring plasmid pDS311, which expresses the S18W variant of AAR_7942, was also evaluated in tank fermentations. The strain produced fatty alcohols with a maximum concentration of 42 g / L, a yield of 12.6% (in glucose) and a productivity of 0.62 g / L / h as shown in Figures 9A and 9B. The chain length distribution of the fatty alcohols was as follows: 1.2% C8, 7.7% CIO, 26.9% C12, 44.7% C14, 16.0% C16, and 1.9% C18. The fraction of saturated and unsaturated fatty alcohols was 72.6% and 27.4%, respectively.

Example 8: Increased Activity of MED4 Acyl Aldehyde Reductase (AAR) and Redistribution of Selectivity of

Chain Length for C14 Fatty Alcohol Production

AAR is one of the essential components for

<img file="MX346830B_D0082.tif" />

¹⁵⁶ IMPI. ,.^{, N}^ «O &lt;g bacterial cyano alkane biosynthesis, essential component is an aldehyde decarbonylase (ADt!). 'The inventors discovered that rochlorococcusmarinus MED4_AAR is catalytically inactive without the presence of MED4_ADC, that is, when only MED4 AAR is expressed in E. coli no products are detected and when MED4_AAR and ADC are co-expressed in E, coli, the only products detected are alkanes. The inventors also found that when MED4 AAR is co-expressed in E. coli with an evidently catalytically inactive variant of MED4_ADC in which histidine 156 is replaced by arginine (referred to herein as MED4_ADC (H165R)), the fatty alcohols, and no alkanes are detected (see Figure 10). It was concluded from these data that MED4_AAR requires physical interaction with MED4_ADC to be catalytically active. The inventors used this system to identify MED4_AAR variants with increased activity and / or altered substrate specificity for purposes of FALC production.

MED4_ADC (H156R) was expressed together with a complete saturation library of MED4_AAR. The MED4_AAR saturation library was prepared into a plasmid derivative pCL1920 (pLS9-195) and introduced into a production strain bearing the plasmid pGLAK-043 (which is the plasmid pACYCPtrc-MED4_ADC harboring the H156R mutation in the ADC gene ). The

157 1 iVÍ F 1 clones were induced and AAR variants were selected based on the production of more fatty alcohol than wild-type AAR enzyme or the ability to produce fatty alcohols with an altered chain length profile, for example, a fraction increased C14 fatty alcohol. The selected clones were then retested in a validation run. All variants that showed consistent FALC concentrations between primary and secondary fermentations were regrown, plasmid DNA isolated, sequenced, and reintroduced into the parent production strain for further testing. This new transformant then underwent another confirmatory fermentation and analysis. Table 8 below shows representative data for the 16 AAR variants that produced the highest FALC concentrations (ordered from bottom to top in descending activity). The variants ranged from 1.4 times to 2.2 times over the wild type. These variants showed the ability to increase MED4_AAR activity using directed evolution techniques and further form the basis for improvements.

<img file="MX346830B_D0083.tif" />

additional.

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Table 8: FALC Productivity of Mutants * B "^orPffi^?®4

Relationship to MED4 AAR Natural Type (Wh

<td>AAR mutation</td><td>FALC fold increase over the Natural Type</td>

<td>V346P</td><td> 2.2</td>

<td>Q40V</td><td> 2.2</td>

<td>A345R</td><td> 2.1</td>

<td>L344S</td><td> 2.1</td>

<td>D61E</td><td> 2.1</td>

<td>V346G</td><td> 2.0</td>

<td>L344D</td><td> 1.9</td>

<td>G52V</td><td> 1.9</td>

<td>A345 *</td><td> 1.9</td>

<td>L344T</td><td> 1.8</td>

<td>K303G</td><td> 1.8</td>

<td>L344A</td><td> 1.8</td>

<td>H340P</td><td> 1.6</td>

<td>S588V</td><td> 1.5</td>

<td>K339L</td><td> 1.4</td>

<td>G273E</td><td> 1.4</td>

* Truncated variant that loses the last two amino acids.

The data set was also scanned for AAR variants that showed altered chain length profiles. The most common species produced by wild-type AAR has a chain length of C16. One provides increased FALC species with chain lengths shorter than C16 is of interest. Two variant clones were identified that showed an approximately 3-fold increase in the amount of C14 FALC (Figure 10). Sequencing of these clones revealed that they were identical to the D61E mutants with the same codon nucleotide sequence. Plasmid DNA for the D61E variant was re-

159 IMPI fwmvro MEXICAN PROPERTY introduce into the precursor strain containing βϊ ^ ΫδβΚ aJC. The results show that the expression "I" the varTant? ^ T1 ~ É of AAR in a recombinant host cell skews the chain length distribution of the FALC species towards the shorter carbon chains. Table 9 below illustrates the FALC chain length distribution produced by recombinant host cells expressing the D61E variant of MED4_AAR compared to the wild type (WT) MED4_AAR and the V346P variant of MED4_AAR that did not produce any of the products. with altered chain lengths.

Table 9: Chain Length Distribution for the

Variants AAR and AAR Natural Type

<td rowspan="2">MED4 AAR / ADC</td><td colspan="2">Alcohol</td><td rowspan="2">fat% C16</td><td rowspan="2">of</td><td rowspan="2">% C18</td><td rowspan="2">of</td>

<td>% C14</td><td>of</td>

<td>MR WT / ADC (H156R)</td><td> 6</td><td></td><td> 92</td><td></td><td> 2</td><td></td>

<td>AAR (D61E) / ADC (H156R)</td><td> 14*</td><td></td><td> 85</td><td></td><td> 0</td><td></td>

<td>AAR (V346P) / ADC (H156R)</td><td> 4</td><td></td><td> 92</td><td></td><td> 4</td><td></td>

<td>AAR WT / ADC WT</td><td>ND</td><td></td><td>ND</td><td></td><td>ND</td><td></td>

<td>AAR (D61E) / ADC WT</td><td>ND</td><td></td><td>ND</td><td></td><td>ND</td><td></td>

<td>AAR (V346P) / ADC WT</td><td>ND</td><td></td><td>ND</td><td></td><td>ND</td><td></td>

ND = not detected * 1% variance attributed to deviations from the mean

These variants can be recombined and further classified for improvements in the MED4_ADC (H156R) background. The MED4_AAR (D61E) variant and additional mutated progeny may be helpful in decreasing chain length

<img file="MX346830B_D0085.tif" />

<img file="MX346830B_D0086.tif" />

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160 Average of both variant fatty alcohols MED4_AAR (D61E) shows that as alkanes. The MED4_AAR chain length specificity is malleable and presents the possibility for further enhancement of this activity through additional protein engineering efforts.

All of the described variants were sequenced from the progeny of pLS9-195 and contained codon mutations corresponding to the listed amino acid substitutions.

Table 10: Names Related to the List of

Sequence

<td>SEQ ID NO</td><td>Type</td><td>Name</td>

<td> 1</td><td>Nucleic acid sec</td><td>Nostocpunctiforme PCC 73102_acp Access # YP_001867863</td>

<td> 2</td><td>Seq of amino acid</td><td>Nostocpunctiforme PCC 73102_acp Access # YP_001867863</td>

<td> 3</td><td>Nucleic acid sec</td><td>Synechocystis sp. PCC 6803_acp Access # NP_440632.1</td>

<td> 4</td><td>Seq of amino acid</td><td>Synechocystis sp. PCC 6803_acp Access # NP_440632.1</td>

<td> 5</td><td>Nucleic acid sec</td><td>Prochlorococcusmarinus subsp. pastoris str. CCMP1986_acp Access # NP_893725.1</td>

<td> 6</td><td>Seq of amino acid</td><td>Prochlorococcusmarinus subsp. pastoris str. CCMP1986_acp Access # NP_893725.1</td>

<td> 7</td><td>Nucleic acid sec</td><td>Synechococcuselongatus PCC 7942_acp Access # YP_399555</td>

<td> 8</td><td>Seq of amino acid</td><td>Synechococcuselongatus PCC 7942_acp Access # YP_399555</td>

<td> 9</td><td>Nucleic acid sec</td><td>Nostoc sp. PCC 7120_acp Access # NP_487382.1</td>

<td> 10</td><td>Seq of amino acid</td><td>Nostoc sp. PCC 7120_acp Access # NP_487382.1</td>

<td> 11</td><td>Nucleic acid sec</td><td>B. subtilis sfp (synthesized) as in Accession # X63158.1</td>

<td> 12</td><td>Seq of amino acid</td><td>B. subtilis sfp (synthesized) as in Accession # X63158.1</td>

<td> 13</td><td>Primer sec.</td><td>168IFF</td>

<td> 14</td><td>Primer sec.</td><td>168IFR</td>

<img file="MX346830B_D0087.tif" />

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<td> 15</td><td>Primer sec.</td><td>169IFF _____</td>

<td> 16</td><td>Primer sec.</td><td>169IFR </td>

<td> 17</td><td>Primer sec.</td><td>170IFF</td>

<td> 18</td><td>Primer sec.</td><td>170IFR</td>

<td> 19</td><td>Primer sec.</td><td>171IFF</td>

<td> 20</td><td>Primer sec.</td><td>171IFR</td>

<td> 21</td><td>Primer sec.</td><td>172IFF</td>

<td> 22</td><td>Primer sec.</td><td>172IFR</td>

<td> 23</td><td>Primer sec.</td><td>168SIFF</td>

<td> 24</td><td>Primer sec.</td><td>170S1FF</td>

<td> 25</td><td>Primer sec.</td><td>171SIFF</td>

<td> 26</td><td>Primer sec.</td><td>168SIFR</td>

<td> 27</td><td>Nucleic acid sec</td><td>Synechococcuselongatus PCC7942 YP_400611 (Synpcc7942_1594) Acyl-CoA Reductase (AAR)</td>

<td> 28</td><td>Nucleic acid sec</td><td>Synechococcuselongatus PCC7942 YP_400611 (Synpcc7942_1594) Acyl-CoA Reductase (AAR)</td>

<td> 29</td><td>Nucleic acid sec</td><td>Synechocystissp. PCC6803 S110209 (NP_442146) AAR</td>

<td> 30</td><td>Seq of amino acid</td><td>Synechocystissp. PCC6803 S110209 (NP_442146) AAR</td>

<td> 31</td><td>Nucleic acid sec</td><td>Cyanot / iecesp. ATCC51142 cce_1430 (YP_001802846) AAR</td>

<td> 32</td><td>Seq of amino acid</td><td>Cyanothecesp. ATCC51142 cce_1430 (YP_001802846) AAR</td>

<td> 33</td><td>Nucleic acid sec</td><td>ProchlorococcusmarínusCCMP1986 PMM0533 (NP_892651) AAR</td>

<td> 34</td><td>Seq of amino acid</td><td>ProcMorococcusmarinusCCMP1986 PMM0533 (NP_892651) AAR</td>

<td> 35</td><td>Nucleic acid sec</td><td>Gloeobacter violaceous PCC7421 NP_96091 (gll3145) AAR</td>

<td> 36</td><td>Seq of amino acid</td><td>Gloeobacter violaceous PCC7421 NP_96091 (gll3145) AAR</td>

<td> 37</td><td>Nucleic acid sec</td><td>Nostocpunctifornie PCC73102 ZP_00108837 (Npun02004176) AAR</td>

<td> 38</td><td>Seq of amino acid</td><td>Nostocpunctifornie PCC73102 ZP_00108837 (Npun02004176) AAR</td>

<td> 39</td><td>Nucleic acid sec</td><td>Anabaena variabilis ATCC29413 YP_323044 (Ava_2534) AAR</td>

<td> 40</td><td>Seq of amino acid</td><td>Anabaena variabilis ATCC29413 YP_323044 (Ava__2534) AAR</td>

<td> 41</td><td>Nucleic acid sec</td><td>SynechococcuselongatusPCC6301 YP_170761 (syc0051_d) AAR</td>

<td> 42</td><td>Seq of amino acid</td><td>SynecíococcuselongatusPCC6301 YP_170761 (syc0051_d) AAR</td>

<td> 43</td><td>Nucleic acid sec</td><td>Nostocsp. PCC7120alr5284 (NP_489324) AAR</td>

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<td> 44</td><td>Seq of amino acid</td><td>Nostocsp. PCC7120alr5284 (NP_489324) AAR</td>

<td> 45</td><td>Nucleic acid sec</td><td>birAfrom Corynebacteriumglutamicum (YP_224991)</td>

<td> 46</td><td>Synthetic DNA</td><td>birAfrom Corynebacteriumglutamicum (YP_224991)</td>

<td> 47</td><td>Seq of amino acid</td><td>birA from Corynebacteriumglutamicum (YP_224991)</td>

<td> 48</td><td>Nucleic acid sec</td><td>accDAl (dtsR) from Corynebacteriumglutamicum (YP ^ 224991}</td>

<td> 49</td><td>Seq of amino acid</td><td>accOAl (dtsR) from Corynebacteriumglutamicum (YP_224991)</td>

<td> 50</td><td>Nucleic acid sec</td><td>accCB from Corynebacteriumglutamicum (YP_224991)</td>

<td> 51</td><td>Seq of amino acid</td><td>accCB from Corynebacteriumglutamicum (YP_224991)</td>

<td> 52</td><td>Seq of amino acid</td><td>AlrAAcinetobacter sp. Ml</td>

<td> 53</td><td>Seq of amino acid</td><td>AlrAadpl</td>

<td> 54</td><td>Seq of amino acid</td><td>ADP1-WT protein from alrAadpl Acinetobacterbaylyi</td>

<td> 55</td><td>Nucleic acid sec</td><td>IFAB138</td>

<td> 56</td><td>Nucleic acid sec</td><td>FadR of E.colí MG1655 (NP_415705)</td>

<td> 57</td><td>Seq of amino acid</td><td>Ñútante AAR with the S18W mutation (made from fromSynechococcuselongatus PCC7942 YP_400611 (Synpcc7942_1594) Acyl-CoA Reductase (AAR))</td>

As is apparent to a person skilled in the art, various modifications and variations of the above aspects and modalities can be made without departing from the spirit and scope of this description. Such modifications and variations are within the scope of this description.

163