BR122017000806A2 - acyl acp reductase (aar) variant polypeptide, recombinant host cell, cell culture, method of producing fatty alcohol composition having increased titer - Google Patents

Abstract

acyl acp reductase (aar) variant polypeptide, recombinant host cell, cell culture, production method of fatty alcohol composition having increased titer ”description refers to the acyl acp reductase (aar) enzyme variants that result in the production of improved fatty aldehyde and improved fatty alcohol when expressed in recombinant host cells. The description relates to methods for preparing and using such aar variants for the production of fatty alcohol compositions having particular characteristics.

Description

POLYPEPTIDE OF THE VARIANT ACIL-ACP REDUCTASE (AAR), RECOMBINANT HOST CELL, CELL CULTURE, PRODUCTION METHOD OF FAT ALCOHOL COMPOSITION WITH INCREASED TITLE ”

Divided from BR1120150170056, deposited on 1/16/2014.

[001] This application claims the benefit of US Provisional Application No. 61 / 753,273, filed on January 16, 2013, the contents of which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING [002] The patent application contains a sequence listing, which was submitted electronically in ASCII format and is incorporated by reference in its

wholeness. Referred copy ASCII, created in 13 of January 2014, is named LS00046PCT SL.txt and is 155731 bytes in size. FIELD OF INVENTION [003] The description refers to the variants gives enzyme

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

BACKGROUND OF THE INVENTION [004] Fatty alcohols denote an important category of industrial biochemistry. For example, annual worldwide sales of fatty alcohols and their derivatives are in excess of $ 1 billion. These molecules and their derivatives have numerous applications, including use as surfactants,

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2/137 lubricants, plasticizers, solvents, emulsifiers, emollients, thickening agents, flavorings, fragrances, and fuels. Due to their amphiphilic nature, fatty alcohols behave as non-ionic surfactants, which are useful in personal care and home care products, for example, detergents. Short-chain fatty alcohols are used in the cosmetic and food industries as emulsifiers, emollients and thickeners.

[005] In nature, fatty alcohols are made by enzymes that are capable of reducing various molecules of acyl-ACP or acyl-CoA 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 herein by reference). However, current technologies involve mainly inorganic catalyst-mediated reduction of fatty acids 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 seed oil, fat and lard, or by chemical hydration of alpha-olefins produced from petrochemical raw materials. Fatty alcohols derived from natural sources have different chain lengths, which are relevant and specific to specific applications. Dehydration of fatty alcohols to alpha-olefins can be accomplished by chemical catalysis.

[006] Fatty aldehydes can be used to produce exclusive industrial chemicals. Per

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3/137 example, aldehydes are commonly used to produce polymers, resins, dyes, flavorings, plasticizers, perfumes and pharmaceuticals.

Aldehydes can also be used as solvents, preservatives and disinfectants.

Certain natural and synthetic compounds, such as vitamins and hormones, are aldehydes, and many sugars contain aldehyde groups. Fatty aldehydes can be converted to fatty alcohols by chemical or enzymatic reduction.

[007] A greener and cleaner alternative for the production of fatty aldehydes and fatty alcohols is through fermentable sugars and / or biomass. However, 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 efficient recovery of the final product. The present disclosure addresses this need by providing compositions and methods to improve the production of fatty aldehydes and fatty alcohols, using manipulated host cells as biocatalysts.

SUMMARY OF THE INVENTION [008] The present invention provides photosynthetic and heterotrophic host cells, which directly produce fatty aldehydes and / or fatty alcohols of specific chain lengths such that the catalytic conversion of purified fatty acids is not necessary. This biological route provides a higher quality product, a significant cost reduction and a lower impact on the environment. More specifically, the present invention provides new variants of enzyme

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4/137 acyl-ACP reductase (AAR) that produce fatty aldehydes and / or fatty alcohols and their compositions. Also provided are nucleic acid and specific sequences of the AAR variant protein, as well as new recombinant host cells and cell cultures covering such enzyme variants

Manipulated AAR. The invention also provides methods of using host cells expressing recombinant AAR variants to prepare fatty aldehyde and / or fatty alcohol compositions with particular characteristics.

[009]

One aspect of the description provides polypeptides of the acyl-ACP reductase (AAR) variant that catalyze the conversion of an acyl-ACP to a fatty aldehyde, wherein the AAR polypeptide has at least 90% sequence identity with the corresponding polypeptide sequence of

gives AAR like wild presented as SEQ ID AT THE: 28, SEQ ID AT THE: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID AT THE: 36, SEQ ID AT THE: 38, SEQ ID NO: 40, SEQ ID NO: 42 or SEQ ID NO : 44, and

methods for expressing the variant polypeptides

AAR in a recombinant host cell resulting in a higher fatty aldehyde and / or fatty alcohol title composition compared to the fatty aldehyde title and / or fatty alcohol composition produced by expression of a wild-type AAR polypeptide in a corresponding host cell.

In one embodiment, the genetically engineered variant of the AAR polypeptide has at least 90% sequence identity with the corresponding wild-type AAR polypeptide sequence shown as SEQ ID NO:

28; and the expression of the variant

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5/137 of the AAR polypeptide in a recombinant host cell results in a higher titer of the fatty aldehyde and / or fatty alcohol composition or a higher titer of C12, C14 or C16 fatty alcohols compared to the titer produced by expression of a wild-type AAR polypeptide in a corresponding host cell.

[010] In one aspect, the AAR variant 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 AAR variant polypeptide has an S18W mutation. In another preferred embodiment, the genetically engineered AAR variant polypeptide has an S18W mutation and further comprises a mutation such as M21L, D24E, D24Y, L31V, W34F, W35F, D43E,

A50Q, C63A, C63G, C63Y, S86G, A112R, S113K, Q116G, R118Q, T120S, A135S, T148C, T148E, T148V, 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 1 L338W.

[011] In another aspect, the AAR variant polypeptide which has at least 90% sequence identity with the corresponding wild type AAR polypeptide sequence shown as SEQ ID NO: 34; and expression of the AAR variant polypeptide in a recombinant host cell results in a higher titer of fatty aldehyde and / or fatty alcohol or a higher titer of C12 fatty alcohol compared to the titer produced

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6/137 by expression of a wild type AAR polypeptide in a corresponding host cell. The polypeptide of the AAR variant has a mutation at the amino acid position including 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 polypeptide of the AAR variant has a mutation in the position of the amino acid Q40V, G52V, S58V, D61E,

G273E, K303G, K339L, H340P, L344A, L344D, L344S, L344T,

L345R, V346P, V346G, and / or S588V.

[012] Another aspect of the description provides a recombinant host cell that has one or more mutations, as described above, and where when the host cell is engineered to express an AAR polypeptide of SEQ ID NO: 28 or SEQ ID NO : 34. This recombinant host cell produces a fatty alcohol and / or fatty aldehyde composition with a titer that is at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, or at least 30% higher than the title of a fatty aldehyde and / or fatty alcohol composition produced by a host cell expressing the corresponding wild type AAR polypeptide, when grown in medium containing a carbon source under conditions effective to express the polypeptide of the AAR variant. In one embodiment, the fatty aldehyde or fatty alcohol composition is produced with a concentration of 30 g / L to 250 g / L, for example, a titer of at least 100 mg / L. In another embodiment, the fatty alcohol composition is produced extracellularly.

[013] The disclosure further encompasses a cell culture that includes the recombinant host cell, as

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7/137 described above, wherein the fatty alcohol composition includes one or more C6, C8, C10, C12, C13, C14, C15, C16, C17 and C18 fatty alcohol, for example, a C10: 1 unsaturated fatty alcohol, C12: 1, C14: 1, C16: 1 or C18: 1. In yet another embodiment, the fatty alcohol composition comprises a saturated fatty alcohol.

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

[015] Another aspect of the invention provides an acyl-ACP reductase (AAR) variant polypeptide

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8/137 having at least 90% sequence identity with the

SEQ ID NO: 57, where the AAR variant polypeptide has another mutation in the 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 L33 8W. In a modality preferred, the polypeptide of the variant of AAR have a mutation M21L, a mutation C63G, an S113K mutation, a mutation T154A, is mutation A281L (SEQ ID NO: 58). In

another preferred embodiment, the polypeptide of the variant of

AAR has an L8A mutation, an M21L mutation, an

C63G, an S113K mutation, a T154A mutation, and a mutation

A281L (SEQ ID NO: 59). In another preferred embodiment, the AAR variant polypeptide has a D16L mutation, an M21L mutation, a C63G mutation, an S113K mutation, a T154A mutation, and an A281L mutation (SEQ ID NO: 60). In another preferred embodiment, the AAR variant polypeptide has an L8A mutation, a D24V mutation, a C63G mutation, an S113K mutation, a Q155L mutation, and an A281L mutation (SEQ ID NO: 61). In another preferred embodiment, the AAR variant polypeptide has a D24P mutation, an L31M mutation, a C63G mutation, an S113K mutation, a

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9/137 T154A mutation, and an A281L mutation (SEQ ID NO: 62). In another preferred embodiment, the AAR variant polypeptide has an L8A mutation, a D16L mutation, a D24V mutation, a C63G mutation, an S113K mutation, a T154A mutation, and an A281L mutation (SEQ ID NO: 63). In another preferred embodiment, the AAR variant polypeptide has a D24E mutation, a C63G mutation, an S113K mutation, a T154A mutation, and an A281L mutation (SEQ ID NO: 64).

[016] Another aspect of the present invention provides a recombinant host cell expressing the polypeptides of the AAR variants, as previously described (supra). In one embodiment, the recombinant host cell produces a fatty alcohol or fatty aldehyde composition with a titer that is at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, or at least 30 % greater than the title of a fatty aldehyde or fatty alcohol composition produced by a host cell expressing a corresponding wild-type AAR polypeptide, when grown in medium containing a carbon source under conditions effective to express the AAR variant polypeptide . In one embodiment, the fatty aldehyde or fatty alcohol composition produced by the recombinant host cell is produced at a concentration of about 30 g / L to about 250 g / L. In another embodiment, the fatty aldehyde or fatty alcohol composition is produced extracellularly.

[017] The description further contemplates a cell culture including the recombinant host cell that expresses the polypeptides of the AAR variants as previously described (supra). In one modality, the

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10/137 fatty alcohol composition includes saturated fatty alcohol and / or unsaturated fatty alcohol. In one embodiment, the cell culture includes a fatty alcohol composition that includes one or more of a C6, C8, C10, C12, C13, C14, C15, C16, C17 and C18 fatty alcohol. In another embodiment, the fatty alcohol composition includes one or more unsaturated fatty alcohol C10: 1, C12: 1 C14: 1, C16: 1, and a C18: 1. In yet another embodiment, the fatty alcohol composition includes a fatty alcohol having a double bond at position 7 in the carbon chain between C7 and C8 from the reduced end of the fatty alcohol.

[018] The disclosure further encompasses a method of producing a fatty alcohol composition having an increase in titer, including cultivating the host cell expressing the variant of AAR (as described above) with a carbon source; and collect a fatty alcohol composition. In one embodiment, the fatty alcohol titer is at least 20% to 30% greater than the fatty alcohol titer produced by a host cell expressing wild type AAR.

[019] Another aspect of the invention provides an acyl-ACP reductase (AAR) variant polypeptide having at least 90% sequence identity with SEQ ID NO: 65, wherein the polypeptide catalyzes the conversion of an acylACP to an aldehyde fatty. In one embodiment, expression of the AAR variant polypeptide in a recombinant host cell results in a higher titer of a fatty aldehyde or fatty alcohol composition when compared to a titer of a fatty aldehyde or fatty alcohol composition produced by the expression of a AAR polypeptide from

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11/137 wild type in a corresponding wild type host cell. In another embodiment, expression of the AAR variant polypeptide in a recombinant host cell results in a higher titer of a C12, C14 and / or C16 fatty alcohol composition, compared to a titer of a fatty aldehyde or alcohol composition fatty tissue produced by the expression of a wild-type AAR polypeptide in a corresponding wild-type host cell. In a particular aspect, the description provides an acylACP reductase variant (AAR) polypeptide having at least 90% sequence identity with SEQ ID NO: 65, wherein the polypeptide has a mutation at amino acid position 61. In one preferred embodiment, the mutation is D61E.

[020] The disclosure further encompasses a polypeptide of the acyl-ACP reductase (AAR) variant having at least 90% sequence identity with SEQ ID NO: 34, wherein the expression of the AAR variant polypeptide in a host cell recombinant results in a higher titer of a fatty aldehyde or fatty alcohol composition or a higher titer of a fatty alcohol composition C12, C14 and / or C16, compared to a title of a fatty alcohol composition produced by expression of a wild-type AAR polypeptide in a corresponding wild-type host cell, and in which the AAR polypeptide has a mutation at the position of amino acid 40, 52, 273, 303, 340, 344, 345, or 346. In one embodiment , the polypeptide of the AAR variant has a selected mutation Q40V, G52V, G273E, K303G, H340P, L344A, L344D, L344S, L344T, L345R, V346P, and V346G. In a

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12/137 preferred embodiment, the AAR variant polypeptide has a mutation in V346P (SEQ ID NO: 66). In another preferred embodiment, the Q40V mutation polypeptide (SEQ ID NO: preferred, the A345R mutation polypeptide (SEQ ID NO: preferred, the L344S mutation polypeptide (SEQ ID NO: preferred, the V346G mutation polypeptide ( SEQ ID NO: preferred, the L344D mutation polypeptide (SEQ ID NO: preferred, the G52V mutation polypeptide (SEQ ID NO: preferred, the L344T mutation polypeptide (SEQ ID NO: preferred, the mutation polypeptide in K303G (SEQ ID NO: preferred, L344A mutation polypeptide (SEQ ID NO: preferred, H340P mutation polypeptide (SEQ ID NO: preferred, G273E mutation polypeptide (SEQ ID NO: 77 variant of AAR has an

67). In another variant modality of AAR it has a

68). In another variant modality of AAR it has a

69). In another variant modality of AAR it has a

70). In another variant modality of AAR it has a

71). In another variant modality of AAR it has a

72). In another variant modality of AAR it has a

73). In another variant modality of AAR it has a

74). In another variant modality of AAR it has a

75). In another variant modality of AAR it has a

76). In another variant modality of AAR it has a

).

[021] Yet another aspect of the description provides a recombinant host cell expressing the AAR variant polypeptide as previously described (supra). In one embodiment, the recombinant host cell produces a fatty alcohol or fatty aldehyde composition with a titre that is at least 10% higher,

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13/137 at least 15% greater, at least 20% greater, at least 25% greater, or at least 30% greater than the title of a fatty aldehyde or fatty alcohol composition produced by a host cell expressing an AAR polypeptide of the corresponding wild type, when grown in medium containing a carbon source under conditions effective to express the AAR variant polypeptide. In another embodiment, the fatty alcohol composition is produced with a concentration of about 30 g / L to about 250 g / L. In another embodiment, the fatty alcohol composition is produced extracellularly.

[022] The description further contemplates a cell culture with the recombinant host cell that expresses the AAR variant polypeptide as previously described (supra). In one embodiment, the fatty alcohol composition includes one or more of a C6, C8, C10, C12, C13, C14, C15, C16, C17 and C18 fatty alcohol. In another embodiment, the fatty alcohol composition includes unsaturated fatty alcohol 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, unsaturated fatty alcohol and a C18: 1. In another embodiment, the fatty alcohol composition includes a fatty alcohol having a double bond at position 7 in the carbon chain between C7 and C8 of the reduced end of the fatty alcohol.

[023] Another aspect of the invention provides a method of producing a fatty alcohol composition having an increase in titer, including cultivating the host cell expressing the AAR (as described above) with a carbon source; and collect a fatty alcohol composition.

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In one embodiment, fatty alcohol is at least 20% to 30% greater than the title of a fatty alcohol composition produced by a host cell expressing wild type AAR.

BRIEF DESCRIPTION OF THE DRAWINGS [024] The present invention is best understood when read in conjunction with the accompanying figures, which serve to illustrate the preferred embodiments. It is understood, however, that this disclosure is not limited to the specific modalities described in the figures.

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

[026] Figure 2 is a schematic overview of an exemplary fatty acid biosynthesis cycle, in which the stretching 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 β-keto-acyl-ACP is reduced by a β-ketoacyl-ACP reductase (fabG) NADPH to produce a β-hydroxy-acyl-ACP, which is dehydrated to a trans-2-enoyl-acyl-ACP by βhydroxyacyl-ACP dehydratase (fabA or fabZ). FabA can also isomerize trans-2-enoyl-acyl-ACP to cis-enoyl-acyl-ACP, which can bypass fabl and can be used by fabB (typically up to a length of C16 aliphatic chain) to produce β-keto-acyl-ACP . The last step in each cycle is catalyzed by enoyl-ACP reductase (fabI) dependent on

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NADH or NADHPH which converts trans-2-enoyl-acyl-ACP to acyl-ACP. In the methods described here, the termination of fatty acid synthesis occurs by removing the thioesterase from the acyl group from the acyl-ACP to release the free fatty acids (FFA). Thioesterases (e.g., tesA) hydrolyze thioester bonds, which occur between acyl and ACP chains through sulfhydryl bonds.

[027] Figure 3 illustrates the structure and function of the acetyl-CoA carboxylase enzyme complex (accABCD). Biotin carboxylase is encoded by the accC gene, while the biotin carboxyl carrier protein (BCCP) is encoded by the accB gene. The two subunits involved in carboxyl transferase activity are encoded by the accA and accD genes. BCCP's covalently bound biotin carries the carboxylate moiety. The birA gene biotinylates the holo-accB gene.

[028] Figure 4 presents a schematic overview of an exemplary biosynthetic pathway for the production of fatty alcohol starting with acyl-ACP, where the production of fatty aldehyde is catalyzed by the enzymatic activity of acyl-ACP reductase (AAR) or a 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).

[029] Figure 5 shows fatty alcohol production in E. coli DV2 expressing Synechococcus elongatus acyl-ACP reductase (AAR_7942) and coexpressing several cyanobacterial acyl transporter proteins (ACPs).

[030] Figure 6 shows the production of fatty alcohol

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16/137 in E. coli DV2 expressing Synechococcus elongatus acyl-ACP reductase (AAR_7942) (pLS9-185) and coexpressing the Corynebacterium glutamicum acetyl carboxylase complex (pSL9185-D +) (three individual strains are shown, see D + 1, D + 2 and D + 3).

[031] Figure 7 presents the results that illustrate improved fatty alcohol production from recombinant host cells that depend on AAR-mediated ifab and ifadR overexpression.

[032] Figure 8 shows results showing high levels of fatty alcohols in recombinant host cells that express AAR_7942 variants derived from combination libraries (AAR_Com 2a-d), which are co-expressed with ACP, AlrA (alcohol dehydrogenase (ADH )) and a synthetic acc operon in the Shu2 strain. A number of alcohol dehydrogenase polypeptides are useful according to the disclosure and include, but are not limited to, Alra de Acinetobacter sp. M-1 (SEQ ID NO: 52) and an Alra counterpart such as AlrAadpl (SEQ ID NO: 53).

[033] Figures 9A and 9B show the results from a fermentation tank of the AAR_7942 S18W variant that illustrate FALC title (9A) and glucose yield (9B) showing that the expression of the AAR_7942 S18W variant results in altered activity and distribution of chain length.

[034] Figure 10 presents the results that illustrate a change in chain length distribution for FALC from C16 to C14 when the D61E MED4_AAR variant is expressed in recombinant host cells.

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DETAILED DESCRIPTION OF THE INVENTION

Overview [035] One way to eliminate our dependence on petrochemicals is to produce derivatives of fatty acids, such as fatty aldehydes and fatty alcohols through ecological microorganisms that serve as miniature production hosts. Such cellular hosts (ie, recombinant host cells or production strains) have been manipulated to produce fatty aldehydes and / or fatty alcohols from renewable sources, as a renewable raw material (for example, fermentable sugars, carbohydrates, biomass, cellulose, glycerol, CO, CO ₂ , etc.). These fatty aldehydes and fatty alcohols are raw materials for many industrial products, including detergents and fuels.

[036] The present invention relates to variants of the enzyme acyl-ACP reductase (AAR) that result in improved titer, yield and / or productivity of the fatty aldehyde and / or fatty alcohol compositions when expressed in recombinant host cells. Here, the improved biosynthesis of fatty aldehyde and / or fatty alcohol is carried out by transforming host cells in such a way that they express an acyl-ACP reductase (AAR) variant protein, which catalyzes the reaction of an acyl-ACP to a fatty aldehyde and / or a fatty alcohol. The description further refers to recombinant host cells or production strains that express the AAR enzyme variants.

Definitions [037] As used in this report and the appended claims, the singular forms one, one,

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18/137 a and o include plural equivalents unless the context clearly dictates otherwise. Thus, for example, the reference to a host cell includes two or more of such host cells, the reference to a fatty ester includes one or more fatty acid esters or mixtures of esters, the reference to a nucleic acid sequence includes one or more more nucleic acid sequences, the reference to an enzyme includes one or more enzymes, and the like.

[038] Access numbers throughout this description were obtained from databases provided by NCBI (National Center for Biotechnology Information) maintained by National Institutes of Health - National Institutes of Health, USA ( which are identified here as NCBI Accession Numbers - NCBI Access Numbers or, alternatively, as GenBank Accession Numbers - GenBank Access Numbers), and from UniProt Knowledgebase (UniProtKB) and Swiss-Prot databases provided by the Swiss Institute of Bioinformatics - Swiss Institute of Bioinformatics (which are identified here as UniProtKB Accession Numbers UniProtKB Access Numbers).

[039] Enzyme classification numbers (EC) are established by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB), description of which is available on the IUBMB Enzyme website Nomenclature Nomenclature of IUBMB Enzymes on the World Wide Web. EC numbers classify enzymes according to the reaction they

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19/137 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-protein transporter] reductase) of AAR's functionality and prokaryotes from a long chain or EC 1.2. 1.42. The one conserved in most species for the other. Thus, different microbial species can carry the same AAR enzyme activity that is classified under E.C.

1.2.1.80 or E.C. 1.2.1.42.

[040]

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. 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 the analogs of the occurrence bases natural or unnatural are also included. Naturally occurring sugar is deoxyribose pentose (five-carbon sugar) (which constitutes DNA) or ribose (which forms RNA), although it should be understood that the analogs of naturally occurring or non-naturally occurring sugars are also included. Nucleic acids are generally linked via phosphate bonds to form nucleic acids or polynucleotides, although many other bonds are known in the art (for example, phosphorothioates, boranophosphates, and the like).

[041] The term polynucleotide refers to a polymer of ribonucleotides (RNA) or deoxyribonucleotides (DNA), which can be stranded

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20/137 single or double stranded and which may 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, or RNA, or DNA. These terms refer to the primary structure of the molecule and therefore include double and single stranded DNA, and single and double stranded RNA. The terms include, as equivalents, RNA or DNA analogs or made from manipulated nucleotide and polynucleotide analogs, such as, but not limited to, methylated and / or encapsulated polynucleotides. The polynucleotide can be in any form, including, but not limited to, plasmid, viral, chromosome, EST, cDNA, mRNA, and rRNA.

[042] As used herein, the terms polypeptide and protein are used interchangeably to refer to a polymer of amino acid residues. The term recombinant polypeptide refers to a polypeptide that is produced by recombinant techniques, in which, generally, the DNA or RNA encoding the expressed protein is inserted into a suitable expression vector, which is in turn used to transform a host cell into produce the polypeptide. Likewise, the terms recombinant polynucleotide or recombinant nucleic acid or recombinant DNA are produced by recombinant techniques that are known to those skilled in the art.

[043] As used herein, the terms homologous and homologous refer to a polynucleotide or

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21/137 a polypeptide comprising a sequence that is at least about 50 percent (%) identical to the corresponding polynucleotide or polypeptide sequence. Preferably, homologous polynucleotides or polypeptides have polynucleotide sequences or amino acid sequences that have 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% of the homology with the corresponding amino acid sequence or a polynucleotide sequence. As used herein, the terms of the homology sequence and the identity sequence are used interchangeably. One skilled in the art is well aware of methods for determining homology between two or more sequences. Briefly, homology calculations between two sequences can be performed as follows. The sequences are aligned for the purpose of optimal comparison (for example, gaps can be inserted into one or both of a first and an amino acid or nucleic acid sequence for the second 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 least 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

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22/137 amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions of the first sequence and the second sequence are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical in 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 gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and the determination of the percentage of homology between two sequences can be performed using a mathematical algorithm, such as the BLAST programs (Altschul et al. (1990) J. Mol Biol. 215 (3): 403-410). The percentage of homology between two amino acid sequences can also be determined using the Needleman and Wunsch algorithm, which was incorporated into the GAP program in the GCG software package, using a Blossum 62 matrix or a PAM250 matrix, and a weight gap of 16, 14, 12, 10, 8, 6 or 4 and a weight of the length of

1, 2,

3, 4, 5, or (Needleman

Wunsch (1970) J. Mol

Biol.

percentage of homology between two nucleotide sequences can also be determined using GAP program (gap) in the GCG software package, using an NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a weight of length of 1, 2, 3, 4, 5, or 6. An ordinary person skilled in the art can perform initial homology calculations and adjust the parameters of the

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23/137 agreement. A preferred set of parameters (and one that should be used if a practitioner is uncertain about which parameters should be used to determine if a molecule is within a homology limitation of the claims) is a Blossum 62 scoring matrix with a penalty of gap of 12, an extended gap penalty of 4, and a gap gap penalty of reading frame 5. Additional methods of sequence alignment are known in biotechnology techniques (see, for example, Rosenberg (2005) BMC Bioinformatics 6 : 278; Altschul et al. (2005) FEBS J. 272 (2 (20)): 5101-5109).

[044] The term hybridizes under conditions of low stringency, medium stringency, high stringency, or very high stringency describes hybridization and washing conditions. Guidelines for conducting hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 - 6.3.6. Aqueous and non-aqueous methods are described in this reference and any method can be used. Specific hybridization conditions referred to here are as follows: (1) low stringency hybridization conditions - 6X sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by two washes in 0.2X SSC, SDS a 0.1% at least at 50 ° C (the wash temperature can be increased to 55 ° C during conditions of low rigor); (2) medium stringency hybridization conditions - 6X SSC at about 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 about 45 ° C, followed by one or more washes in 0.2.X SSC, 0.1% SDS at 65 ° C; and (4) very stringent hybridization conditions

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24/137 high phosphate - 0.5 M sodium phosphate, 7% SDS at 65 ° C, followed by one or more washes at 0.2X SSC, 1% SDS at 65 ° C. Very high stringency conditions (4) are the preferred conditions, unless otherwise specified.

[045] An endogenous polypeptide refers to a polypeptide encoded by the genome of the parental cell (or host cell). An exogenous polypeptide refers to a polypeptide that is not encoded by the parent cell's genome. A variant or mutant polypeptide is an example of an exogenous polypeptide. Thus, a nucleic acid molecule that does not occur naturally is considered to be exogenous to a cell, once introduced into the cell. A naturally occurring nucleic acid molecule can also be exogenous to a particular cell. For example, an entire coding sequence isolated from X cells is an exogenous nucleic acid relative to cell Y, since the coding sequence is introduced into cell Y, even though X and Y are of the same cell type.

[046] The term overexpressed means that a gene is caused to be transcribed at a high rate compared to the rate of endogenous transcription for that gene. In some instances, overexpression additionally includes a high rate of translation of the gene compared to the rate of endogenous translation for that gene. Assay methods for overexpression are well known in the art, for example, the transcribed RNA levels can be assessed using rtPCR and the protein levels can be assessed using SDS in page gel analysis.

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25/137 [047] The term heterologous means derived from a different organism, different cell type, or different species. As used herein, this refers to the nucleotide, polynucleotide, polypeptide or protein sequence, not naturally present in a given organism. For example, a polynucleotide sequence that is native to cyanobacteria can be introduced into a host cell in E. coli by recombinant methods, and the polynucleotide from cyanobacteria is then heterologous to the E. coli cell (for example , recombinant cells). The term heterologous can also be used with reference to a nucleotide, polynucleotide, polypeptide, or protein sequence that is present in a recombinant host cell in a non-native state. For example, a heterologous nucleotide, polynucleotide, polypeptide or protein sequence can be manipulated in relation to the wild type sequence naturally present in the corresponding wild type host cell, for example, a change in the level of expression or in the sequence of a nucleotide , polynucleotide, polypeptide or protein.

[048] As used herein, the term polypeptide fragment refers to a smaller portion of a full-length polypeptide or protein that varies in size from two amino acid residues with the entire amino acid sequence minus one residue amino acid. In certain embodiments of the present description, a fragment refers to the entire amino acid sequence of a domain of a polypeptide or protein (for example, a

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26/137 substrate binding domain or a catalytic domain).

[049] 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 noncoding nucleic acid sequences that result in engineered protein activity.

[050] A mutation, as used herein, refers to a permanent change in a nucleic acid position of a gene or an amino acid position of a polypeptide or protein. Mutations include substitutions, additions, insertions and deletions. For example, a mutation in the 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 replaced by an alanine (A), a lysine (L) can be substituted with a T (Threonine), etc.). As such, a polypeptide or protein can have one or more mutations in which one amino acid is replaced by another amino acid.

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

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27/137 expresses the variant of AAR (for example, a recombinant cell). In one embodiment, the title and / or yield of a fatty alcohol produced by a cell that expresses the variant of AAR is at least twice that of a corresponding wild-type cell (ie, a corresponding cell that does not express the variant AAR). In a heterologous host, such as Escherichia coli, fatty aldehydes can be converted to fatty alcohols by endogenous alcohol dehydrogenases. In another modality, the title and / or the yield of a fatty alcohol produced by a cell that expresses the variant of AAR is at least

fence in 1 turn, fur any less fence in 2 times, fur any less fence in 3 times, fur any less fence in 4 times, fur any less fence in 5 times, fur any less fence in 6 times, fur any less fence in 7 times, fur any less fence in 8 times, fur any less fence in 9 times, or at least about in 10 times greater than

than a corresponding wild-type cell. In one embodiment, the title and / or yield of a fatty alcohol produced by a cell that expresses the variant of AAR is at least about 1 percent, at least about 2 percent, at least about 3 percent, at least about 4 percent, at least about 5 percent, at least about 6 percent, at least about 7 percent, at least about 8 percent, at least about 9 percent, or about 10 percent higher than that of a corresponding wild-type cell. In another embodiment, the titer and / or yield of fatty alcohols produced in a recombinant cell due to the expression of an AAR variant is at least about 20 percent to at least about 100 percent higher than that of

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28/137 a wild type cell. In particular embodiments, the titer and / or yield of a fatty alcohol produced by a cell is at least about 20 percent, at least about 25 percent, at least about 30 percent, at least about 35 percent percent, at least about 40 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.

[052] 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 RNA or protein ( for example, such sequences include but are not limited to promoter sequences or enhancer sequences) or operably linked nucleic acid sequences that encode sequences that affect RNA or protein expression (for example, such sequences include, but are not limited to, sites binding ribosomes or translational control sequences).

[053] Expression control sequences are known in the art and include, for example, promoters, enhancers, polyadenylation signals, terminators of

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29/137 transcription, internal ribosome entry sites (IRES), and the like, which provide expression of the polynucleotide sequence in a host cell. Expression control sequences interact specifically with cellular proteins involved in transcription (Maniatis et al. (1987) Science 236: 12371245). Examples of expression control sequences are described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (nineteen ninety) . In the methods of the disclosure, an expression control sequence is operably linked to a polynucleotide sequence. By operably linked means that a polynucleotide sequence and an expression control sequence are linked in such a way as to allow expression of the gene when the appropriate molecules (for example, transcription-activating proteins) are linked to the expression control sequence. Operatively linked promoters are located upstream of the selected polynucleotide sequence in terms of the direction of transcription and translation. Operatively linked intensifiers can be located upstream, in or downstream of the selected polynucleotide.

[054] As used herein, the term vector refers to a nucleic acid molecule capable of transporting another nucleic acid, that is, a polynucleotide sequence, to which it has been attached. A useful type of vector is an episome (that is, a nucleic acid capable of extrachromosomal replication). Useful vectors are those capable of autonomous replication and / or acid expression

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30/137 nucleic to which they are linked. Vectors capable of directing the expression of genes to which they are operationally linked are referred to here as expression vectors. In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids, which generally refer to circular double-stranded DNA loops that, in their vector form, are not linked to the chromosome. Other useful expression vectors are provided in linear form. Also included are any other forms of expression vectors that serve as equivalent functions and that are subsequently known in the art here. In some embodiments, a recombinant vector further includes a promoter operably linked to the polynucleotide sequence. In some embodiments, the promoter is an elaborately 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 with 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 secretion sequence operably coupled to the polynucleotide sequence; and a targeting sequence operably coupled to the polynucleotide sequence. In certain modalities, the

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31/137 nucleotide sequence is stably incorporated into the host cell's genomic DNA; and the expression of the nucleotide sequence is under the control of an adjustable 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 factors such as the choice of host cell to be transformed, desired polypeptide expression level, etc.

The expression vectors described herein can be introduced into host cells for the production of 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 often performed with vectors containing constitutive or inducible promoters directing 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 terminal amino or carboxy of the recombinant polypeptide. Such fusion vectors typically serve one or more of the following three purposes, including to increase the expression of the recombinant polypeptide; to increase the solubility of the recombinant polypeptide; and to assist in the purification of the recombinant polypeptide by acting as a linker in affinity purification. Often, in fusion expression vectors, a cleavage site

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32/137 proteolytic is introduced at the junction of the fusion portion 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 T5 bacteriophage.

[055] 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 the yeast S. cerevisiae include pYepSec1 (Baldari et al. (1987) EMBO J. 6: 229-234); pMFa (Kurjan et al. (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 baculovirus expression vector. Baculovirus vectors available for protein expression in cultured insect cells (eg, Sf9 cells) include, for example, the pAc series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and pVL series (Lucklow et al. (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 expression systems suitable for prokaryotic cells and eukaryotic cells are well known in the art; see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, (1989).

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33/137 [056] As used herein, the term CoA or acyl-CoA reductase refers to an acyl thioester formed between the carbonyl carbon of the alkyl chain and the sulfhydryl group of the 4'-phospopantethyl radical of coenzyme A (CoA ), which has the formula RC (O) S-CoA, where R is an alkyl group having at least 4 carbon atoms.

[057] The term ACP means acyl carrier protein. ACP is a highly conserved transporter of acyl intermediates during fatty acid biosynthesis, where the growing chain is linked during synthesis as a thiol ester in the distal thiol of a portion of the 4'-phospopantetein. The protein exists in two forms, that is, apo-ACP (inactive in the biosynthesis of fatty acids) and ACP or holo-ACP (active in the biosynthesis of fatty acids). The terms ACP and holo-ACP are used interchangeably here and refer to the active form of the protein. An enzyme called phospopantheteinyltransferase is involved in the conversion of inactive apo-ACP to active holo-ACP. More specifically, ACP is expressed in the inactive apo-ACP form and a 4'-phospopantetein radical must be post-translationally bound to a serine residue conserved in ACP by the action of the holoacyl carrier protein (ACP) synthase, a phospopantheteinyltransferase, in order to produce holo-ACP.

[058] 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 phospopanteteinyl portion of an acyl carrier protein (ACP). In some embodiments, an ACP is an intermediary in the synthesis of fully saturated acyl-ACPs.

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34/137

In other modalities, an ACP is an intermediary in the synthesis of unsaturated acyl-ACPs. In some embodiments, the carbon chain will have about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, or 26 carbon atoms.

[059] As used herein, the term fatty acid derivative means a fatty acid or a fatty acid derivative, which can be referred to as a fatty acid or its derivative. 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 made, in part, from the fatty acid biosynthetic pathway of the production host organism. Fatty acid derivatives include products prepared, in part, from ACP, acyl-ACP or derivatives of acyl-ACP. Examples of fatty acid derivatives include, for example, acyl-CoA, fatty acids, fatty aldehydes, short and long chain alcohols, fatty alcohols, hydrocarbons, esters (e.g. waxes, fatty acid esters or fatty esters), olefins terminals, internal olefins, and ketones.

[060] As used herein, the term fatty acid biosynthetic pathway means a biosynthetic pathway that produces fatty acids and their derivatives. The fatty acid biosynthetic pathway may include additional enzymes for the production of fatty acid derivatives that have desired characteristics.

[061] As used herein, fatty aldehyde

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35/137 means an aldehyde having the formula RCHO characterized by a carbonyl group (C

O) .

In some embodiments, the fatty aldehyde is any aldehyde in a fatty alcohol.

In certain modalities, group R is, fur any less 5, fur any less 6, fur any less 7, at any less 8, fur any less 9, fur any less 10, fur any less 11, at any less 12, fur any less 13, fur any less 14, fur any less 15, at any less 16, fur any less 17, fur

carbon of fewer atoms of

18, or at least length.

Alternatively, or in addition, the group or less, 19 or less, 18 or less, 17 or less, or less, or less, 14 or less, or less, or less, or less, or less, 9 or less, 8 or less, 7 or less, or less carbon atoms in length.

So, group

R can have a group R delimited by any of the two end points above. For example, the group

R can be 6-16 carbon atoms in length,

10-14 carbon atoms in length, or 1218 carbon atoms in length. In some embodiments, the fatty aldehyde is a C6 fatty aldehyde,

C7, C8,

C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22,

C9, C10, C11,

^C 23 ^, C24, C25, or C26. In certain modalities, the fatty aldehyde it is a aldehyde fatty C6, C8, C10, C12, C13, C14, C15, C16, C17, or C18. [062] As here used, fatty alcohol

means an alcohol with the formula ROH. In some embodiments, the group R is at least 5, at least 6, at least 7, at least 8, at least 10, at least 11, at least 12, at least 13, at least 14, at least at least 15, at least 16, at least 17, at least 18, or at least 19 carbon atoms in length. In

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36/137 alternative, or in addition, the group R is 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 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 carbon atoms in length. Thus, the group R can have a group R bounded by any of the two end points above. For example, the group R can be 6-16 carbon atoms in length, 10-14 carbon atoms in length, or 12-18 carbon atoms in length. In some embodiments, fatty alcohol is a C6, C7 ^{, C} 9 ^, C10, C11, ^C 12 ^{, C} 13 ^{, C} 15 ^{, C} 16 ^{, C} 17 ^{, C} 18 ^{, C} 19 ^{, C} 20 fatty alcohol ^,

C ₂₁ , C ₂₂ , C ₂₃ , C ₂₄ , C ₂₅ , or C ₂₆ . In certain embodiments, fatty alcohol is a C ₆ , C ₈ , C ₁₀ , C ₁₂ , C ₁₃ , C ₁₄ , C ₁₅ , C16, C17, or C18 fatty alcohol.

[063] 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 fatty acid derivative compositions may comprise, for example, a mixture of fatty alcohols with different chain lengths and saturation or branching characteristics. In still other cases, the fatty alcohol composition comprises a mixture of both more than one type of product and products with different chain lengths and saturation or branching characteristics.

[064] A host cell engineered to produce a fatty aldehyde will typically convert some of the fatty aldehyde to a fatty alcohol. In a modality

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37/137 exemplary, 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 that encodes a fatty alcohol biosynthetic polypeptide is expressed or overexpressed in the host cell. In certain embodiments, the fatty alcohol biosynthetic polypeptide has aldehyde reductase activity or alcohol dehydrogenase activity.

[065] Examples of the alcohol dehydrogenase polypeptides useful according to the invention include, but are not limited to, AlrA of Acinetobacter sp. M1 (SEQ ID NO:

52) or AlrA counterparts, such as AlrAadp1 (SEQ ID NO:

53) and endogenous E. coli, alcohol dehydrogenases like YjgB,

(AAC77226), DkgA (NP_417485), DkgB (NP_414743), YdjL (AAC74846), YdjJ (NP_416288), AdhP (NP_415995), YhdH (NP_417719), Yahk (NP_414859) , YphC (AAC75598), YqhD (446856) and YbbO [ AAC73595.1]. Examples additional They are

described in International Patent Application Publication Nos. WO 2007/136762, WO2008 / 119082 and WO 2010/062480. In certain embodiments, the fatty alcohol biosynthetic polypeptide has aldehyde reductase or alcohol dehydrogenase activity (EC 1.1.1.1).

[066] The R group of a 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, branched fatty acid, branched fatty aldehyde, or branched fatty alcohol is an acid

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38/137 branched fatty, branched fatty aldehyde, or branched fatty alcohol C6, C7, C8, C9, C10,

C11, C12, C13, C14, C15, C16,

C17, C18, C19, C20, C21, C22, ^C 23 ^{, C} 24 ^{, C} 25 ^{, or C} 26 ^{. In} particular embodiments, branched fatty acid, branched fatty aldehyde, or branched fatty alcohol is branched fatty acid, branched fatty aldehyde, or branched fatty alcohol C6, C8,

C10, ^C 12 ^, C13 ^,

C14, C15, C16, C17, or C18. In certain embodiments, the hydroxyl group of branched fatty acid, branched fatty aldehyde, or branched fatty alcohol is in the primary position (C ₁ ).

[067]

In certain embodiments, branched fatty acid, branched fatty aldehyde, or branched fatty alcohol an iso-fatty acid, iso-fatty aldehyde or iso-fatty alcohol, or a priorio-fatty acid, a priorio-fatty aldehyde, or before-alcohol fatty. In exemplary embodiments, branched fatty acid, branched fatty aldehyde, or branched branched fatty alcohol is selected from branched fatty acid, branched fatty aldehyde or branched fatty alcohol iso-C _{7: 0} , isoC8: 0, iso-C9: 0, iso-C10: 0, iso-C11: 0, iso-C12: 0, iso-C13: 0, isoC14: 0, iso-C15: 0, iso-C16: 0, iso-C17: 0, iso- C18: 0, iso-C19: 0, anteiso-C _{7: 0} , anteiso-C _{8: 0} , anteiso-C _{9: 0} , anteiso-C _{10: 0} , anteiso-C _{11: 0} , anteiso-C _{11: 0} , anteiso-C _{12: 0} , anteiso-C _{13: 0} , anteiso-C _{14: 0} , anteiso-C _{15: 0} , anteiso-C _{16: 0} , anteiso-C17: 0, anteiso-C18: 0, and anteiso-C _{19: 0} .

[068]

The group R 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 group R may have one or more than one point of unsaturation. In some

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39/137 modalities, 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, unsaturated fatty acid, unsaturated fatty aldehyde, or unsaturated fatty alcohol is a C6: 1, C7: 1, C8: 1, C9: 1, C10: 1, C11: 1, C12: 1, C13: 1, C14: 1, C15: 1, C16: 1, C17: 1, C18: 1, C19: 1, C20: 1, C21: 1, C22: 1, C23: 1, C24: 1, C25: 1, or a C26: 1 of unsaturated fatty acid, unsaturated fatty aldehyde, or unsaturated fatty alcohol. In certain preferred embodiments, 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 modalities, unsaturated fatty acid, unsaturated fatty aldehyde, or unsaturated fatty alcohol is unsaturated in the omega-7 position. In certain embodiments, unsaturated fatty acid, unsaturated fatty aldehyde, or unsaturated fatty alcohol comprises a cis double bond.

[069] As used herein, a recombinant host cell or host cell is a host cell, for example, a microorganism that has been manipulated in such a way 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 enzyme activity, in which the recombinant host cell produces a fatty alcohol composition, when grown in the presence of a carbon source under conditions effective to express polynucleotides.

[070] As used herein, the term clone

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40/137 typically refers to a cell or group of cells descended from and essentially genetically identical to a single common predecessor, for example, bacteria from a colony of cloned bacteria arising from a single bacterial cell.

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

[072] The terms culture or cultivation refer to the growth of a population of cells (for example, microbial cells) under suitable conditions in a liquid or solid medium. In specific modalities, culture 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 in sources commercials, per example, under the DIFCO and the means BBL. In one example not limiting, the means nutrient watery is one half rich

comprising complex sources of nitrogen, salts, and carbon, such as the YP medium, comprising 10 g / L of peptone and 10 g / L of yeast extract from such medium.

[073] The host cell can be further manipulated to assimilate carbon efficiently and use cellulosic materials as carbon sources according to the methods described, for example, in US Patents

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41/137

5,000,000; 5,028,539; 5,424,202; 5,482,846; 5,602,030 and WO2010127318. In addition, the host cell can be engineered to express an invertase so that sucrose can be used as a carbon source.

[074] As used herein, the term under conditions effective for expressing said heterologous nucleotide sequences means all conditions that allow a host cell to produce a desired fatty aldehyde or desired fatty alcohol. Suitable conditions include, for example, fermentation conditions.

[075] As used herein, modified or an altered level of activity of a protein, for example, an enzyme, in a recombinant host cell refers to a difference of one or more characteristics in the determined activity in relation to the original host cell or native. Typically, differences in activity are determined between a recombinant host cell, having modified activity, and the corresponding wild-type host cell (e.g., comparing a culture of a recombinant host cell against the wild-type host cell). The modified activities can be the result of, for example, modified amounts of protein expressed by a recombinant host cell (for example, as the result of an increased or reduced number of copies of DNA sequences encoding the protein, an increased or reduced number of mRNA transcripts encoding the protein, and / or increased or reduced amounts of protein translation from mRNA); changes in the structure of

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42/137 protein (for example, changes to the primary structure, such as changes in the sequence encoding the protein that result in changes in substrate specificity, changes in observed kinetic parameters); and changes in protein stability (for example, increased or decreased protein degradation). In some embodiments, the polypeptide is a mutant or variant of any of the polypeptides described herein. In certain cases, the coding sequence for the polypeptides as described herein are codons optimized for expression in a particular host cell. For example, for expression in E. coli, one or more codons can be optimized (Grosjean et al. (1982) Gene 18: 199-209).

[076] The term regulatory sequence, as used herein, typically refers to a sequence of DNA bases, operably linked to the 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 translation regulation sequences (such as ribosome binding sites (e.g., Shine-Dalgarno sequences in prokaryotes or Kozak sequences in eukaryotes), initiation codons, termination codons).

[077] As used herein, the phrase the expression of said nucleotide sequence is manipulated in relation to the nucleotide sequence of the type

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43/137 wild, means an increase or a reduction in the level of expression and / or activity of an endogenous nucleotide sequence, or the expression and / or activity of a heterologous sequence or nucleotide sequence encoding non-native polypeptide.

[078] As used herein, the term expressed with respect to a polynucleotide is to induce its functioning. A polynucleotide that encodes a polypeptide (or protein), when expressed, will be transcribed and translated to produce that polypeptide (or protein). As used herein, the term overexpressing means expressing (or inducing expression) a cell polynucleotide or polypeptide at a higher concentration than is normally expressed in a corresponding wild-type cell, under the same conditions.

[079] The terms change in expression level and modified level of expression are used interchangeably and mean that a polynucleotide, polypeptide, or hydrocarbon is present at a different concentration in a engineered host cell, compared to its concentration in a corresponding cell wild type under the same conditions.

[080] As used herein, the term titer refers to the amount of fatty aldehyde or fatty alcohol produced per unit volume of host cell culture. In any aspect of the compositions and methods described herein, a fatty alcohol is produced in a titer of about 25 mg / L, about 50 mg / L, about 75 mg / L, about 100 mg / L, about 125 mg / L, about 150 mg / L, about 175 mg / L, about 200 mg / L, about 225 mg / L, about

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250 mg / L, fence in 275 mg / L, fence in 300 mg / L, fence in 325 mg / L, fence in 350 mg / L, fence in 375 mg / L, fence in 400 mg / L, fence in 425 mg / L, fence in 450 mg / L, fence in 475 mg / L, fence in 500 mg / L, fence in 525 mg / L, fence in 550 mg / L, fence in 575 mg / L, fence in 600 mg / L, fence in 625 mg / L, fence in 650 mg / L, fence in 675 mg / L, fence in 700 mg / L, fence in 725 mg / L, fence in 750 mg / L, fence in 775 mg / L, fence in 800 mg / L, fence in 825 mg / L, fence in 850 mg / L, fence in 875 mg / L, fence in 900 mg / L, fence in 925 mg / L, fence in 950 mg / L, fence in 975 mg / L, fence in 1000 mg / L, fence in 1050 mg / L, fence in 1075 mg / L, fence in 1100 mg / L, fence in 1125 mg / L, fence in 1150 mg / L, fence in 1175 mg / L, fence in 1200 mg / L, fence in 1225 mg / L, fence in 1250 mg / L, fence in 1275 mg / L fence 1300 mg / L, about

about 1325 mg / L, about 1350 mg / L, about 1375 mg / L, about 1400 mg / L, about 1425 mg / L, about 1450 mg / L, about

1475 mg / L, on 1500 mg / L, fence in 1525 mg / L, fence in 1550 mg / L, about in 1575 mg / L, fence in 1600 mg / L, fence in 1625 mg / L, about in 1650 mg / L, fence in 1675 mg / L, fence in 1700 mg / L, about in 1725 mg / L, fence in 1750 mg / L, fence in 1775 mg / L, about in 1800 mg / L, fence in 1825 mg / L, fence in 1850 mg / L, about in 1875 mg / L, fence in 1900 mg / L, fence in 1925 mg / L, about in 1950 mg / L, fence in 1975 mg / L, fence in 2000 mg / L (2 g / L) , 3g / L, 5 g / L, 10g / L, 20g / L, 30g / L , 40g / L,

g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100g / L or a range limited by any two of the preceding values. In other embodiments, a fatty alcohol or fatty aldehyde is produced with a concentration greater than 100 g / L, greater than 200 g / L, greater than 300 g / L, or greater, such as 500 g / L, 700 g / L, 1000 g / L, 1200 g / L, 1,500 g / L or 2000

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45/137 g / L. The preferred titer of fatty aldehyde or fatty alcohol produced by a recombinant host cell according to the methods of the present description is 5 g / L to 200 g / L, 10 g / L to 150 g / L, 20 g / L to 120 g / L and 30 g / L to 100g / L.

[081] As used herein, the term yield of fatty aldehyde or fatty alcohol produced by a host cell refers to the efficiency by which a carbon input source is converted to product (ie fatty alcohol or fatty aldehyde) in a host cell. Host cells engineered to produce fatty alcohols and / or fatty aldehydes according to the methods of the present description have a yield of at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, fur

any less 8%, fur any less 9% _,: at least 10%, fur any less 11%, fur any less 12%, fur any less 13%, fur any less 14%, fur any less 15%, fur any less 16%, fur any less 17%, fur any less 18%, fur any less 19%, fur any less 20%, fur any less 21%, fur any less 22%, fur any less 23%, fur any less 24%, fur any less 25%, fur any less 26%, fur any less 27%, fur minus 28% at least 29 %, or

at least 30% or a range limited by any two of the preceding values. In other modalities, a fatty alcohol or fatty aldehyde is produced with 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. Thus, the yield can be limited by either of the two end points above. For example, the yield of fatty alcohol or fatty aldehyde produced by the recombinant host cell according to the methods of the invention can be from 5% to 15%, 10%

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46/137 to 25%, 10% to 22%, 15% to 27%, 18% to 22%, 20% and 28%, or 20% to 30%. The preferred fatty alcohol yield produced by the recombinant host cell according to the methods of the present description is 10% to 30%.

[082] 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 here, the productivity of the fatty aldehyde or fatty alcohol produced

by a cell recombinant host is at least 100 mg / L / hour, fur any less 200 mg / L hour, fur any less 300 mg / L / hour, fur any less 400 mg / L / hour, fur any less 500 mg / L / hour, fur any less 600 mg / L / hour, fur any less 700 mg / L / hour, fur any less 800 mg / L / hour, fur any less 900 mg / L / hour, fur any less 1000 mg / L / hour, fur any less 1100 mg / L / hour, fur any less 1200 mg / L / hour, fur any less 1300 mg / L / hour, fur any less 1400 mg / L / hours, fur any less, 1500 mg / L / hour, fur any less 1600 mg / L / hour, fur any less 1700 mg / L / hour, fur any less 1800 mg / L / hour, fur any less 1900 mg / L / hour, fur any less, 2000 mg / L / hour, fur any less 2100 mg / L / hour, fur any less 2200 mg / L / hour, fur any less 2300 mg / L / hour, fur minus 2400 mg / L / hour, or fur any less, 2500

mg / L / hours. Alternatively, or in addition, productivity is

2500 mg / L / hour or less, mg / L / OD _S 00 or any less, 120 mg / L / hour or any less, 800 mg / L / hour or any less. Like this limited per any a D

2000 mg / L / OD600 or less, mg / L / hour or less, mg / L / hour or less, or, productivity two points of

1500

1000

600 can be end up. For example, productivity can be 3 to 30

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47/137 mg / L / hour, 6 to 20 mg / L / hour, or 15 to 30 mg / L / hour. The preferred productivity of a fatty alcohol or fatty aldehyde produced by a recombinant host cell according to the methods of the disclosure is selected from 500 mg / L / hour to 2500 mg / L / hour, or from 700 mg / L / hour at 2000 mg / L / hour.

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

[084] As used herein, the term glucose utilization rate means the amount of glucose used by the culture per unit time, reported as grams / liter / hour (g / L / h).

[085] As used herein, the term carbon source refers to a substrate or compound suitable for use as a carbon source for simple eukaryotic or prokaryotic cell growth. Carbon sources can take many forms, including, but not limited to, polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides and gases (e.g.

CO and CO2)

Examples of carbon sources include, but are not limited to, monosaccharides, such as glucose, fructose, mannose, galactose, xylose, and arabinose;

oligosaccharides, such as fructo-oligosaccharides and

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48/137 galacto-oligosaccharide; polysaccharides, such as starch, cellulose, pectin and xylan; disaccharides, such as sucrose, maltose, cellobiosis, and turanosis; cellulosic material and variants, such as hemicellulose, 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 gas mixture containing CO from flu gas. In another embodiment, the carbon source is a gas mixture containing CO from carbon reform containing a material, such as biomass, coal or natural gas. In other modalities, the carbon source is synthesis gas, methane or 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 embodiments, the carbon source is glycerol. In other preferred embodiments, the carbon source is sugar cane juice, sugar cane syrup or corn syrup. In other preferred embodiments, the carbon source is obtained from renewable raw materials, such as CO ₂ , CO, glucose, sucrose, xylose, arabinose, glycerol, mannose, or mixtures thereof. In other embodiments, the carbon source is obtained from renewable raw materials, including starches, cellulosic biomass, molasses, and other sources of carbohydrates, including mixtures of carbohydrates derived from the hydrolysis of cellulosic biomass, or materials

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49/137 residues derived from the processing of plant or natural oil.

[086] As used herein, the term biomass refers to any biological material from which it is derived from a carbon source. In some modalities, a biomass is transformed into a carbon source, which is suitable for bioconversion. In other embodiments, biomass does not require further processing from a carbon source. An exemplary source of biomass is the material of the plant or vegetation, such as corn, sugar cane, or switchgrass (fast-growing grass type). Another exemplary source of biomass is metabolic waste, such as animal matter (eg cow manure). Other examples of biomass sources include algae and other marine plants. Biomass also includes waste products from industry, agriculture, forestry and household products, including, but not limited to, glycerol, fermentation waste, silage, straw, sawn wood, sewage, garbage, urban cellulosic waste, and food waste (for example , soaps, oils and fatty acids). The term biomass can also refer to carbon sources, such as carbohydrates (for example, monosaccharides, disaccharides or polysaccharides).

[087] As used herein, the term isolated, with respect to products (such as fatty acids and their derivatives), refers to products that are separated from cellular components, cell culture media, or chemical precursors or synthetic. Fatty acids and their derivatives produced by the methods described here can be relatively immiscible in the broth of

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50/137 fermentation, as well as in the cytoplasm. Consequently, fatty acids and their derivatives can collect in an organic phase or intracellularly or extracellularly.

[088]

As used herein, the terms purify purified or purification mean the removal or isolation of a molecule from its environment, for example, isolation or separation. Substantially purified molecules are at least about

60% free (e.g. at least about 70% free, at least about 75% free, at least about 85% free, at least about 90% free, at least about 95% free, at least about 97% free, at least about 99% free) from other components with which they are associated. As used herein, these terms also refer to the removal of contaminants from a sample. For example, 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 removing proteins from the host cell or other materials from the host cells. After purification, the percentage of fatty acid derivative in the sample is increased. The terms purify, purified and purification are relative terms, which 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 components.

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51/137 cells (for example, nucleic acids, polypeptides, lipids, carbohydrates, or other hydrocarbons).

Increased Fatty Alcohol Production [089] The description provides for the production of a fatty alcohol composition that is improved as a result of altered 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 altered expression of another gene involved in the biosynthetic pathway that converts a fatty aldehyde to a fatty alcohol. Here, the invention provides recombinant host cells, which have been engineered to express a variant of AAR to provide greater fatty alcohol biosynthesis compared to wild-type or native or unmanaged host cells that express wild-type AAR or other polypeptides fatty alcohol biosynthetic 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 the identity of the related AAR absolute polynucleotide sequence is not necessary. For example, changes to a sequence of a specific polynucleotide can be made and the polypeptide activity. Such conservative changes and example mutations, through the coded traced to the, typically include silent mutations, such as, by codon optimization. The manipulated or mutated polynucleotides (i.e., mutant) and polypeptides of encoded variants can be screened

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52/137 for a desired function, such as an improved function compared to the original polypeptide, including, but not limited to, increased catalytic activity, increased stability, or reduced inhibition (eg, reduced inhibition of return ), using methods known in the art. The description identifies enzymatic activities involved in various stages (ie reactions) of the fatty acid biosynthesis pathways described here according to the enzyme classification number (EC), and provides exemplary polypeptides (enzymes) categorized by such EC numbers, and exemplary polynucleotides that code for such polypeptides. These exemplary polypeptides and polynucleotides, which are identified herein by the Accession Numbers and / or Sequence Identifying Numbers (SEQ ID NOs), are useful for the fatty acid pathways manipulated in original host cells to obtain the 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 exemplary sequences of polypeptide homologues described herein are available to those skilled in the art, using databases, such as, for example, the Entrez databases provided by National Center for Biotechnology Information (NCBI), the ExPasy databases provided by Swiss Institute of Bioinformatics, the BRENDA database provided by the Technical University of Braunschweig, and the KEGG database provided by Bioinformatics Center of Kyoto University and University of Tokyo, all are available on the World Wide Web.

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53/137 variety of different host cells can be manipulated to express fatty alcohol biosynthetic enzymes of the AAR variant, such as those described herein, resulting in recombinant host cells suitable for increasing the production of fatty alcohol compositions. It is understood that a variety of cells can provide sources of genetic material, including polynucleotide sequences that encode polypeptides suitable for

use in a recombinant host cell, such as described here. Polypeptides gives Acyl-ACP reductase (AAR) and at your variants [090] In one aspect, the description if at improved production derivatives of fatty acids, such how

fatty aldehydes and / or fatty alcohols for modifying a host cell to express a native or non-native acyl-ACP reductase (AAR) protein. AAR protein catalyzes the reduction of an acyl-ACP to a fatty aldehyde and can also catalyze the conversion of a fatty aldehyde to a fatty alcohol (see US Patent Publication US

At the.

20120282663, incorporated herein by reference).

polypeptide from

AAR or the polynucleotide sequence encoding the AAR polypeptide can be native (for example, endogenous) or non-native (for example, exogenous, heterologous, etc.), that is, it can be different from the wild type sequence and the its expression naturally present in the corresponding wild type host cell. Examples include a modification in the sequence of the AAR polynucleotide, AAR polypeptide or AAR protein, resulting in an AAR variant (for example,

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54/137 example, mutant) and / or its expression level. Disclosure includes AAR polypeptides, homologues and variants.

[091] In one embodiment, an AAR polypeptide for use in the practice of disclosure has at least 90% identity of the sequence to the polypeptide sequence of the

AAR's type wild SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID AT THE: 38, SEQ ID NO: 40, SEQ ID NO: 42 or SEQ ID NO: 44. In some modalities, the AAR is derived from leave in an species

Synechococcus or a Prochlorococcus species. In other embodiments, an AAR polypeptide for use in the practice of disclosure is at least 75% (for example, 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 for the sequence of

wild type AAR polypeptide from SEQ ID NO: 28, SEQ ID AT THE: 30, SEQ ID AT THE : 32, SEQ ID NO: 34, SEQ ID AT THE: 36, SEQ ID AT THE: 38, SEQ ID AT THE : 40, SEQ ID NO : 42 or SEQ ID AT THE: 44,

and may also include one or more substitutions, which result in useful characteristics and / or properties, as described herein. In one embodiment, the AAR polypeptide for use in the practice of the present invention has

fur any less 75% (per example, 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 fur any less 99%)

of the sequence identity to the 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% identity

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in sequence with the SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID AT THE: 32, SEQ ID NO: 34 , SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID AT THE: 40, SEQ ID NO: 42 or SEQ ID NO: 44. Still in others

modalities, the polypeptide sequence of the AAR or improved variant is derived from a species other than a Synechococcus species or Prochlorococcus species.

[092] In a related embodiment, the disclosure includes AAR polypeptides that have an amino acid sequence encoded by a nucleic acid sequence

what have at least 75% (per example, fur minus 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 and

99%) in string identity with SEQ ID AT THE: 27, SEQ ID AT THE: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID AT THE: 35, SEQ ID AT THE: 37, SEQ ID NO: 39, SEQ ID NO: 41 or SEQ ID NO: 43. In

in some embodiments, the nucleic acid sequence encoding an AAR variant with one or more substitutions, which results in improved characteristics and / or properties, as described herein. In yet another related embodiment, an AAR polypeptide for use in the practice of disclosure is encoded by a nucleotide sequence with 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 the AAR polypeptides that comprise an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions over substantially the entire length of a nucleic acid corresponding to SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:

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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.

[093] In a preferred embodiment, the description provides an AAR polypeptide for use in the practice of disclosure that has at least 90% identity of the sequence to the polypeptide sequence of the AAR variant of any of SEQ ID NO: 57 a SEQ ID NO: 78. In some embodiments, the AAR variant is derived from a Synechococcus species or a Prochlorococcus species. In other embodiments, an AAR polypeptide for use in the practice of disclosure is at least 75% (for example, 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 AAR variant polypeptide sequence of any of SEQ ID NO: 57 through SEQ ID NO: 78. The AAR variant polypeptide may include one or more substitutions that result in useful characteristics and / or properties, such as as described here. In another preferred embodiment, the

polypeptide from AAR to use at practice of disclosure has at least 75% (for example, at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85% , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 9 7%, 98% or at least 9 9%) of identity in sequence with The sequence of variant polypeptide of the SEQ ID AAR AT THE : 57, SEQ ID NO: 58, SEQ ID NO: 59 or SEQ ID NO: 65 . In others

embodiments, an AAR polypeptide for use in the practice of disclosure has 100% sequence identity of any of SEQ ID NO: 57 to SEQ ID NO: 78. In still other embodiments, the variant polypeptide sequence

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57/137 of AAR or improved is derived from a species other than Synechococcus or Prochlorococcus.

[094] The inventors built an error-prone library of acyl-ACP reductase from Synechococcus PCC7942 elongatiis (AAR_7942) in order to track variants that have improvements over the wild type AAR_7942 (see Example 3, below). The improvements are classified as either an improvement in the total fatty alcohol titer or an increase in the fraction of fatty alcohols C10, C12, C14 or C16, without significantly affecting the titer. The error-prone library identified several amino acid positions including position of amino acid 18. Saturation and combination of libraries were prepared to further test these positions. SEQ ID NO: 57 codes for the amino acid sequence for an AAR variant (mutant) that has 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, below). More specifically, the S18W mutation leads to a 227 percent increase in total fatty alcohol production (FALC) and a 324 percent increase in C14 fatty alcohols compared to the wild type AAR used as a control (see Tables 4A and 4B, infra).

[095] The inventors built the saturation libraries based on the S18W mutation in order to identify the variants (mutants) that still increased the total FALC titer or the fraction of C12 fatty alcohols (see Example 4 and Table 5, below). Combination libraries that used the S18W mutation (SEQ ID NO: 57) as a standard produced 7

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58/137 combination mutants that showed additional significant increases in the total FALC titer and / or C12 fatty alcohol production (see Table 6B, below). The 7 combination mutants include A18 with S18W mutation (SEQ ID NO: 57); AAR with M21L, C63G, S113K, T154A, A281L mutations (SEQ ID NO: 58); AAR with L8A, M21L, C63G, A77A mutations (mutation in the silent codon GCC to GCA), S113K, T154A, A281L (SEQ ID NO: 59); AAR with D16L, M21L, C63G, S113K, T154A, A281L mutations (SEQ ID NO: 60); AAR with L8A, D24V, C63G, S113K, Q155L, A281L mutations (SEQ ID NO: 61); AAR with D24P, L31M, C63G, S113K, T154A, A281L mutations (SEQ ID NO: 62); AAR with L8A, D16L, D24V, C63G, S113K, T154A, A281L mutations (SEQ ID NO: 63); and AAR with D24E, C63G, S113K, T154A, A281L mutations (SEQ ID NO: 64). Notably, SEQ ID NO: 58 showed the highest fraction of C12 fatty alcohols, while SEQ ID NO: 59 showed the highest titer of the combination mutants (see Table 6B, below).

[096] The inventors also built a total saturation library of acyl-ACP reductase from Prochlorococcus marinus MED4_AAR, in order to track variants that show improvements over wild-type MED4_AAR (see Example 7, below). AAR variants were selected based on the production of more fatty alcohols than the wild type AAR enzyme or the ability to produce fatty alcohols with an altered profile length chain, for example, an increase in the fraction of C12 fatty alcohols, C14 or C16. Table 8 (see Example 7, below) shows data representative of the 16 AAR variants that produced the largest FALC titers ranging from 1.4 times to 2.2 times compared to

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MED4_AAR of the wild type. The inventors have also screened AAR variants that have a modified chain length profile, in which a greater proportion of FALC species with a shorter chain length than C16 is of interest. Two clones of the variants that lead to a 2-3-fold increase in the amount of FALC are shown in Figure 10. The expression of one of these variants of the AAR, that is, the mutant D61E (SEQ ID NO: 65) in a recombinant host cell, inclined to the distribution of the length of the chain of fatty alcohol species to shorter carbon chains. All variants led to a greater amount of FALC as the titer increased, but only SEQ ID NO: 65 had an increase in the C14 fraction (and higher titer). Table 9 (see Example 7, below) illustrates the FALC chain length distribution produced by recombinant host cells that express the MED4_AAR D61E variant compared to the wild type MED4_AAR (WT) and the MED4_AAR V346P variant that do not produce products with altered chain lengths.

Acil Transport Protein (ACP) [097] There are conflicting reports in the literature as to the factors that may limit the biosynthesis of fatty acids in host cells, such as Escherichia coli (E. coli). Although acyl-bearing proteins (ACP) are conserved to some extent 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, limitations

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60/137 may exist, such as in the recognition, affinity and / or rotation of the enzyme from the recombinant pathway towards fatty acyl ACPs (see Suh et al. (1999) The Plant Journal 17 (6): 679-688; Salas et al (2002) Archives of Biochemistry and Biophysics 403: 25-34). One suggestion is that a limitation of the main precursors for the biosynthesis of acids

fatty, per example, acetyl-CoA and malonyl-CoA can to result at decrease of synthesis in acid derivatives fatty. An approach to increase the flow through

fatty acid biosynthesis is to manipulate various enzymes in the pathway (see Figures 1-3). The supply of acyl-ACPs from acetyl-CoA through the acetyl-CoA carboxylase complex (acc) and the fatty acid biosynthesis (fab) pathway can impact the rate of production of fatty acid derivatives (see Figure 2 ). As detailed in Examples (infra), the effect of ACP overexpression on the production of fatty alcohols was evaluated for the purposes of illustration.

[098] A host cell that is manipulated to express an ACP may exhibit an increase in the titer 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%, fur any less 11%, fur any less 12%, fur minus 13%, fur any less 14%, fur any less 15%, fur any less 16%, fur any less 17%, fur any less 18%, fur any less 19%, fur any less 20%, fur any less 21%, fur any less 22%, fur any less 23%, fur minus 24%, fur any less 25%, fur any less 26%, fur any less 27%, fur any less 28%, fur any less 29%, or at least 30 % higher than

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61/137 that the title of the composition of fatty aldehyde and / or fatty alcohol produced by a corresponding host cell that does not express ACP, when grown under the same conditions. In one aspect, the description refers to the improvement of fatty aldehyde production and / or fatty alcohol composition by modifying a host cell to express a native (for example, endogenous) or non-native (for example, exogenous, ACP protein) heterologous). The sequence of the ACP polypeptide or sequence of the ACP polynucleotide that encodes the ACP polypeptide may be non-native, that is, it may be different from the wild-type sequence naturally present in the wild-type host cell. corresponding nucleotide. The expression level examples either in the polypeptide or protein. Disclosure includes polypeptides from ACP and their counterparts.

[099]

In one embodiment, an ACP polypeptide for use in the practice of disclosure has at least 70%

gives identity in sequence with SEQ ID AT THE: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID AT THE: 10. In some modalities, The ACP is derivative in Marinobacter

hydrocarbonoclasticus or E.

coli. In other embodiments, an ACP polypeptide for use in the practice of disclosure is at least 75% (for example, 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 identity of the sequence with the sequence of the wild-type ACP polypeptide 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, which

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62/137 result in useful characteristics and / or properties, as described herein. In one aspect, an ACP polypeptide for use in the practice of disclosure has 100% sequence identity with 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 polypeptide sequence of the ACP or improved variant is derived from a different species of M. hydrocarbonoclasticus or E. coli. In a related aspect, an ACP polypeptide for use in the practice of disclosure is encoded by a nucleotide sequence having 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. In another related aspect, the disclosure relates to ACP polypeptides that comprise an amino acid sequence encoded by a nucleic acid sequence that is at least 75% (for example, at least minus 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 the sequence identity with to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9. In some embodiments, the nucleic acid sequence encodes an ACP variant with one or more substitutions that result in improved characteristics and / or properties, as described herein. In other embodiments, the nucleic acid sequence of the ACP or enhanced variant is derived from a species other than the species Marinobacter or E. coli. In another aspect, the disclosure refers to ACP polypeptides that have an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions over

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63/137 substantially the entire 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 [100] In some embodiments, the AAR or DA ACP polypeptide is a mutant or a variant of any of the polypeptides described herein. A variant or mutant polypeptide, as used herein, refers to a polypeptide having 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, isoleucine, and, with another aliphatic amino acid; replacement of a serine with a threonine; replacement of a threonine with a serine; replacement of an acidic residue, such as aspartic acid and glutamic acid, with another acid residue;

replacing a residue supporting an amide group, such as asparagine and glutamine, with another residue supporting an amide group; exchange of a basic residue, such as lysine and arginine, for another basic residue; and replacing an aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue. In some embodiments, the variant or mutant polypeptide 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 function as a variant or mutant retain some or

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64/137 the total biological function (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 about 100% of the biological function of the corresponding wild-type polypeptide. Guidance for determining which amino acid residues can be replaced, inserted or deleted without affecting biological activity can be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR, Inc., Madison, WI). In still other embodiments, a fragment exhibits increased biological function compared to a corresponding wild-type polypeptide. For example, a fragment can exhibit at least 10%, at least 25%, at least 50%, at least 75%, or at least 90% improvement in enzyme activity compared to the corresponding wild-type polypeptide. In other embodiments, the fragment exhibits at least 100%, at least 200%, or at least 500% of the improvement in enzymatic activity compared to the corresponding wild-type polypeptide.

[101] It is understood that the polypeptides described herein may have additional conservative or non-essential amino acid substitutions that do not have a substantial effect on the polypeptide function. Whether or not a particular substitution will be tolerated (that is, it will not adversely affect the desired biological function, such as

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65/137 an acyl-ACP reductase activity), can be determined as known in the art (see Bowie et al. (1990) Science, 247: 1306-1310). A conservative amino acid substitution is one in which the amino acid residue is replaced by an amino acid residue having 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 (for example, lysine, arginine, histidine), acidic side chains (for example, acid, aspartic, glutamic acid), uncharged polar side chains (for example, glycine, asparagine, glutamine, serine , threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine).

[102] Variants can be naturally occurring or created in vitro. In particular, these variants can be created using genetic engineering techniques, such as site-directed mutagenesis, random chemical mutagenesis, Exonuclease III deletion procedures, or conventional cloning techniques. Alternatively, such variants, mutants, fragments, analogs, or derivatives can be created using chemical synthesis or modification procedures. Methods of preparing variants are well known in the art. For example, variants can be prepared using random mutagenesis and can be targeted to the site. THE

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66/137 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 achieved using error-prone PCR (see, for example, Leung et al. (1989) Technique 1: 11-15; and Caldwell et al. (1992) PCR Methods Applic. 2: 2833). In error-prone PCR, real PCR is performed under conditions, where the fidelity of the copy of the DNA polymerase is low, such that a high rate of point mutations is obtained over the entire length of the PCR product. Briefly, in such procedures, the nucleic acids to be mutagenized (for example, a polynucleotide sequence encoding an AAR enzyme) are mixed with PCR primers, reaction buffer, MgCl2, MnCl2, Taq polymerase, and an adequate concentration of dNTPs to achieve a high rate of point mutation over the entire length of the PCR product. For example, the reaction can be performed using 20 fmol of nucleic acid to be mutated, 30 pmol of each PCR primer, a reaction buffer comprising 50 mMKCl, 10 mM Tris HCl (pH 8.3), 0.01% gelatin, 7 mM MgCl2, 0.5 mM MnCl2, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP and 1 mM dTTP. PCR can be performed 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 where 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. Mutagenesis directed to the

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67/137 can be achieved 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: 5357). Briefly, in such processes a plurality of double-stranded oligonucleotides supporting one or more mutations to be introduced into the cloned DNA is synthesized and inserted into the cloned DNA to be mutated (for example, a polynucleotide sequence encoding an AAR polypeptide). The clones containing the mutated DNA are recovered, and the activities of the polypeptides they encode are evaluated.

[103] Another method for generating variants is assembly PCR. Assembly PCR involves assembling a PCR product from a mixture of small fragments of DNA. A large number of different PCR reactions occur in parallel in the same vial, with the products of one reaction initiating the products of another reaction (see US Patent 5,965,408). Yet another method of generating variants is sexual PCR mutagenesis. Sexual PCR mutagenesis, forced homologous recombination occurs between DNA molecules from 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 fixing the crossing by extension of the primer in a PCR reaction. Sexual PCR mutagenesis is described in publications known in the art (see, for example, Stemmer (1994) Proc. Natl. Acad. Sci. U.S.A. 91: 10747-10751). Variants can also be

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68/137 created through in vivo mutagenesis. In some embodiments, random mutations in the nucleic acid sequence are generated by the propagation sequence of a bacterial strain, such as an E. coli strain, which 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. Propagation of a DNA sequence (for example, a polynucleotide sequence that encodes an AAR polypeptide) in one of these strains will eventually generate random mutations within the DNA. Mutant strains suitable for use in in vivo mutagenesis are described in the publication in the art (see, for example, International Patent Application Publication No. WO 1991/016427). Variants can also be generated using cassette mutagenesis. In cassette mutagenesis, a small region of a double-stranded DNA molecule is replaced by a synthetic oligonucleotide cassette, which differs from the native sequence. The oligonucleotide often contains a completely and / or partially randomized native sequence. Mutagenesis of the recursive assembly can also be used to generate the variants. Recursive assembly mutagenesis is an algorithm for protein engineering (ie, protein mutagenesis) developed to produce diverse populations of phenotypically related mutants whose members differ in the amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis (see, for example, Arkin et al. (1992) Proc. Natl. Acad. Sci., U.S.A. 89: 7811-7815). In some

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69/137 modalities, the variants are created using exponential assembly mutagenesis. Exponential assembly mutagenesis is a process for the generation of combinatorial libraries with a high percentage of unique and functional mutants, in which small groups of residues are randomized in parallel to identify, in each altered position, amino acids that lead to functional proteins (see, for example, Delegrave et al. (1993) Biotech. Res. 11: 15481552). In some embodiments, variants are created using shuffling procedures, in which portions of a plurality of nucleic acids encoding distinct polypeptides are fused together to create chimeric nucleic acid sequences that encode chimeric polypeptides (as described, for example, in US Patent Nos. 5,965,408 and 5,939,250).

The production of fatty aldehydes and fatty alcohols [104] A native or recombinant host cell may comprise a polynucleotide that encodes an enzyme having fatty aldehyde biosynthesis activity (also referred to herein as a fatty aldehyde biosynthetic polypeptide or a polypeptide or biosynthetic enzyme of fatty aldehyde). 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 (AAR) polypeptide in a recombinant host cell can result in the production of a fatty aldehyde by the recombinant host cell. In one embodiment, the recombinant host cell produces a fatty aldehyde. In some embodiments, the fatty aldehyde produced by the recombinant host cell is converted

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70/137 in a fatty alcohol. In some embodiments, the biosynthetic polypeptides of native (endogenous) fatty aldehydes, such as aldehyde reductases, are present in the host cell (eg, E. coli) and are effective for converting fatty aldehydes to fatty alcohols. In other embodiments, a native fatty aldehyde (endogenous) biosynthetic polypeptide is overexpressed. In still other embodiments, an exogenous fatty aldehyde biosynthetic polypeptide is introduced into a recombinant fatty aldehyde host cell that can be either expressed or overexpressed. One produced by expressing or over expressing, in the recombinant host cell, a polynucleotide that encodes a fatty aldehyde biosynthetic polypeptide, such as a polypeptide having acyl-ACP reductase (AAR) activity. Expression of AAR in a recombinant host cell results in the production of fatty aldehydes and fatty alcohols (Figure 4). Examples of AAR polypeptides are described here and in the PCT Publication

We. WO2009 / 140695 and WO / 2009/140696, both of which are expressly incorporated herein by reference.

[105] A composition comprising fatty aldehydes (a fatty aldehyde composition) is produced by cultivating a 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 engineered 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

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71/137 is recovered from the extracellular environment of the recombinant host cell, i.e., the cell culture medium. In some embodiments, the recombinant host cell includes a polynucleotide that encodes a polypeptide (an enzyme) having a 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 fatty alcohols (i.e., a fatty alcohol composition) can be produced by cultivating the recombinant host cell in the presence of a carbon source under conditions effective to express a fatty alcohol biosynthetic enzyme. The native (for example, endogenous) aldehyde reductases present in a recombinant host cell (for example, E. coli), will convert fatty aldehydes into fatty alcohols. In some embodiments, the biosynthetic polypeptides of native fatty aldehydes (eg endogenous), 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 that encodes a polypeptide biosynthetic activity having fatty alcohol that converts a fatty aldehyde to a fatty alcohol. For example, an alcohol dehydrogenase (also referred to herein as an aldehyde reductase, for example, EC 1.1.1.1), may be useful in the practice of disclosure. As used herein, the term alcohol dehydrogenase refers to a polypeptide capable of catalyzing the conversion of an aldehyde

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72/137 fatty to a fatty alcohol. One of ordinary skill in the art will appreciate that certain alcohols dehydrogenases are capable of catalyzing other reactions as well, and these non-specific alcohols dehydrogenases are also encompassed by the term alcohol dehydrogenase. Examples of alcohol dehydrogenase polypeptides useful according to the invention include, but are not limited to, Alinet de Acinetobacter sp. M-1 (SEQ ID NO: 52) or Alra homologs, such as AlrAadp1 (SEQ ID NO: 53) and E. coli endogenous alcohol dehydrogenases such as YjgB, (AAC77226) (SEQ ID NO: 5), DkgA (NP_417485) , DkgB (NP_414743), YdjL (AAC74846), YdjJ (NP_416288), AdhP (NP_415995), YhdH (NP_417719), YahK (NP_414859), YphC (AAC75598), yqhD (44) and yqhD (44) Additional examples are described in International Patent Application Publication Nos. WO2007 / 136762, WO2008 / 119082 and WO2010 / 062480, each of which is expressly incorporated herein by reference. 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 fatty alcohol biosynthetic polypeptide (e.g., endogenous) is overexpressed and in other embodiments, an exogenous fatty alcohol biosynthetic polypeptide is introduced into a recombinant and expressed or overexpressed host cell.

[106] Fatty alcohols can be produced via an acyl-CoA dependent pathway using fatty acyl-ACP and fatty acyl-CoA intermediates and an acyl-CoA independent pathway using fatty acyl-ACP intermediates, but not an acyl intermediate -CoA fatty. In

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73/137 particular modalities, the enzyme encoded by the overexpressed gene is selected from a fatty acid synthase, an acyl-ACP thioesterase, a fatty acyl-CoA synthase and an acetyl-CoA carboxylase. Fatty alcohols are also prepared in nature by enzymes that are capable of reducing to various molecules of acyl-ACP or acyl-CoA for the corresponding primary alcohols (see US Patent Publication US Nos. 20100105963 and 20110206630, and American Patent US No. 8,097,439, hereby expressly incorporated by reference here). A fatty alcohol composition often includes fatty alcohols together with other fatty acid derivatives, for example, fatty aldehydes and / or fatty acids. Typically, the fatty alcohol composition is recovered from the extracellular environment of the recombinant host cell, i.e., the cell culture medium. In certain embodiments, an enzyme the expression of a polypeptide, for example, directly or indirectly involved in fatty acid biosynthesis is modulated (for example, expressed, over-expressed or attenuated), in which such modulation results in a higher yield , higher titer or higher productivity of a fatty acid derivative of interest, such as a fatty alcohol. The enzyme can be encoded by a fatty acid biosynthesis polynucleotide that is exogenous or heterologous (for example, a polypeptide originating from an organism except the original host cell, or, a variant of a polypeptide native to the original microbial cell) or an endogenous polypeptide (for example, a polypeptide native to the original host cell), in which the

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74/137 endogenous polypeptide is overexpressed in the recombinant host cell. Table 1 provides a listing of examples of proteins that can be expressed in recombinant host cells to facilitate the production of certain fatty alcohol compositions.

Table 1: Gene designations

Gene designation Body Source Enzyme Name No. of Access Number EC Exemplary Use Increased Fatty Acid Production / Increased Product Production accA E. coli, Lactococci acetyl-CoA carboxylase, subunit A (alpha carboxyltransferase) AAC7329, NP_414727 6.4.1.2 Increased production of Malonil-CoA accB E. coli, Lactococci acetyl-CoA carboxylase, subunit B (BCCP: biotin carboxyl carrier protein) NP_417721 6.4.1.2 Increased production of Malonil-CoA accC E. coli, Lactococci acetyl-CoA carboxylase, subunit C (biotin carboxylase) NP_417722 6.4.1.2, 6.3.4.14 Increased production of Malonil-CoA accD E. coli, Lactococci acetyl-CoA carboxylase, subunit D (carboxyltransferase beta) NP_416819 6.4.1.2, Increased production of Malonil-CoA fadD E. coli W3110 acylCoA synthase AP_002424 2.3.1.86, 6.2.1.3 Production increased by

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fatty acid fabA E. coli K12 B- NP_415474 4.2.1.60 Production hydroxidecanylthioest increased by erdehydratase / isomer acil-ACP / CoA if fatty fabB E. coli 3-oxoacyl- [protein BAA16180 2.3.1.41 Production loader increased by acyl] synthase I acil-ACP / CoA fatty fabD E. coli K12 [carrier protein AAC74176 2.3.1.39 Production acil] S- increased by malonyltransferase acil-ACP / CoA fatty fabF E. coli K12 3-oxoacyl- [protein AAC74179 2.3.1.179 Production loader increased by acyl] synthase II acil-ACP / CoA fatty fabG E. coli K12 3-oxoacyl- [protein AAC74177 1.1.1.100 Production loader increased by acyl] reductase acil-ACP / CoA fatty fabH E. coli K12 3-oxoacyl- [protein AAC74175 2.3.1.180 Production loader increased by acyl] synthaseIII acil-ACP / CoA fatty fabl E. coli K12 enoil- [protein NP-415804 1.3.1.9 Production loader increased by acyl] reductase acil-ACP / CoA fatty fabR E. coli K12 Repressor NP_418398 none Modular

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transcriptional gra acid production unsaturated from xo fabV Vibrio Enoil- [protein YP_001217283 1.3.1.9 Production Cholerae loader increased in acyl] reductase acil-ACP / CoA fatty fabZ AND. coli K12 Carrier protein NO_414722 4.2.1- Production (3R) -hydroximyristol increased in acyl dehydratase acil-ACP / CoA fatty fabE AND. coli K13 Acil-CoA AAC73325 1.3.99.3, Degradation in dehydrogenase 1.3.99.- fatty acid reduced fabR AND. coli Regulatory protein NP_415705 none Degradation in transcriptional fatty acid reverse or blocked Chain Length Control tesA (with or AND. coli thioesterase - P0ADA1 3.1.2.- Length in without leader string is , 3.1.1.5 chain C18 sequence amino acids 1-26 leader) tesA (with or AND. coli thioesterase AAC73596, 3.1.2.-, Length in without NP_415027 3.1.1.5 string C18: 1 sequence leader tesA AND. coli thioesterase L109P 3.1.2.-, Length in (mutant in 3.1.1.5 string <C18 E. coli

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thioesterase Complexed with octanoic acid) fatBl Umbellulari thioesterase Q41635 3.1.2.14 Length in a CA string C12: 0 lifornica fatB2 Cupheahooke thioesterase AAC49269 3.1.2.14 Length in riana string C8: 0 - C10: 0 fatB3 Cupheahooke thioesterase AAC72881 3.1.2.14 Length in riana string C14: 0 - C16: 0 fatB cinnamomumc thioesterase Q39473 3.1.2.14 Length in amphora string C14: 0 fatB Arabidopsis thioesterase CAA85388 3.1.2.14 Length in thaliana chain C16: 1 fa tA1 Helianthus thioesterase AAL79361 3.1.2.14 Length in annuus string C18: 1 atfata Arabidopsis thioesterase NP_189147, 3.1.2.14 Length in thaliana NP_193041 string C18: 1 fatA Brassica thioesterase CAC39106 3.1.2.14 Length in juncea string C18: 1 fatA Cupheahooke thioesterase AAC72883 3.1.2.14 Length in riana string C18: 1 tesA Photbacteri thioesterase YP_130990 3.1.2.14 Length in one jail profundum tesB E. Coli thioesterase NP_414986 3.1.2.14 Length in jail

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fadM AND. Coli thioesterase NP_414977 3.1.2.14 Length jail in yciA AND. Coli thioesterase NP_415769 3.1.2.14 Length in jail ybgC AND. Coli thioesterase NP_415264 3.1.2.14 Length in jail Saturation Level Control * Sfa AND. Coli FabA suppressor AAN79592, none Increase in AAC44390 fatty acids monounsaturated fabA AND. Coli K12 B- NP_415474 4.2.1.60 Production in hydroxidecanylthioest fatty acids erdehydratase / isomer unsaturated if GnsA AND. Coli Suppressors gives ABD18647.1 none Increase in null secG mutation esters in fatty acids unsaturated GnsB AND. Coli Suppressors gives AAC74076.1 none Increase in null secG mutation esters in fatty acids unsaturated fabB AND. Coli 3-oxoacyl- [protein BAA16180 2.3.1.41 Modulation gives loader in production in acyl] synthase I fatty acids unsaturated des Bacillus D5 acyl desaturase O34653 1,14,19 Modulation gives subtilis fatty production in fatty acids unsaturated

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Product Yield: Ester Production AT3G51970 Arabidopsis thaliana Long-chain O-fatty alcohol acetyltransferase NP_190765 2.3.1.26 Production of wax ELO1 Pichiaangus OK Elongase fatty acid BAD98251 2.3.1.- Production of very long chain fatty acids plsC Saccharomyc es cerevisiae acyltransferase AAA16514 2.3.1.51 Production of wax DAGAT / DGAT Arabidopsis thaliana diacylglycerolaciltra nsferase AAF19262 2.3.1.20 Production of wax hWS Homo sapiens Acid wax alcohol CoA acyltransferase AAX48018 2.3.1.20 Production of wax Aftl Acinetobact er sp. ADP1 Bifunctional / acyl wax ester synthase CoA: diacilglycerolaci ltransferase AAO17391 2.3.1.20 Production of wax ES9 Marinobacte r hydrocarbon oclasticus Ester synthase of wax ABO21021 2.3.1.20 Production of wax mWS Simmondsiac hinensis Ester synthase of wax AAD38041 2.3.1.- Production of wax Acrl Acinetobact er sp. ADP1 Acyl-CoA reductase YP_047869 1.2.1.42 Yield modified yqhD E. Coli K12 Alcohol dehydrogenase AP_003562 1.1.-.- Yield modified

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AAT Fragaria x ananassa Alcohol acetyl O- transferase AAG13130 2.3.1.84 Wax production in Product Yield: Fatty Alcohol Production Thioesterases (see Increase gives above) production in alcohol fatty / acid fatty BmFAR Bombyxmori FAR (fatty alcohol BAC79425 1.1.1.- Conversion of forming acyl-CoA acyl-CoA for reductase) fatty alcohol Acr1 Acinetobact acyl-CoA reductase YP_047869 1.2.1.42 Reduction in er sp. ADP1 acyl-CoA fatty for aldehydes fatty yqhD E. Coli alcohol dehydrogenase AP_003562 1.1.-.- Reduction in W3110 aldehydes fatty for alcohols fatty; increase production fatty alcohol alrA Acinetobact alcohol dehydrogenase CAG70252 1.1.-.- Reduction in er sp. ADP1 aldehydes fatty for alcohols fatty BmFAR Bombyxmori FAR (fatty alcohol BAC79425 1.1.1.- Reduction in forming acyl-CoA acyl-CoA fatty reductase) for alcohol fatty GTNG_1865 Geobacillus Chain aldehyde YP_001125970 1.2.1.3 Reduction in

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thermodenit rificans NG80-2 long dehydrogenase fatty aldehydes for fatty alcohols AAR Synechococc us elongatus Acil-ACP reductase YP_400611 1.2.1.80 1.2.1.42 Reduction of fatty ACP-CoA / CoA for fatty aldehydes carB Mycobacteri a smegmatis Protein reductase from carboxylic acid (CAR) YP_889972 6.2.1.3, 1.2.1.42 Reduction of fatty acids to fatty aldehydes FadD E. Coli K12 Acyl-CoA synthetase NP_416319 6.2.1.3 Activation of fatty acids for fatty acyl-CoAs atoB Erwiniacaro tovora acetyl-CoA acetyltransferase YP_049388 2.3.1.9 Production of butanol hbd Butyrivibri ofibrisolve ns Beta-hydroxybutyryl- CoA dehydrogenase BAD51424 1.1.1.1. 57 Production of butanol CPE0095 Clostridium perfringens Crotonasebutyryl-CoA dehydrogenase BAB79801 4.2.1.55 Production of butanol B C D Clostridium beijerincki i Butyryl-CoA dehydrogenase AAM14583 1.3.99.2 Production of butanol ALDH Clostridium beijerincki i Coenzyme A-acylation of aldehyde dehydrogenase AAT66436 1.2.1.3 Production of butanol AdhE E. Coli CFT073 Aldehyde-alcohol dehydrogenase AAN80172 1.1.1.1 1.2.1.10 Production of butanol

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Export product AtMRP5 Arabidopsis thaliana Multipharmaceutical associated with resistance to Arabidopsis thaliana NP_177908 none Product export quantity modification gives in AmiS2 Rhodococcus ABC transporter AmiS2 JC5491 none Modification gives sp. amount exported in product AtPGP1 Arabidopsis Arabidopsis thaliana NP_181558 none Modification gives thaliana p glycoprotein 1 amount exported in product AcrA CandidatusP AcrA protein CAF23274 none Modification gives rotochlamyd efflux transportation amount iaamo multi-drug exported in ebophila putative product UWE25 AcrB CandidatusP AcrB protein CAF23275 none Modification gives rotochlamyd efflux transportation amount iaamo multi-drug exported in ebophila likely product UWE25 TolC Francisella Membrane protein ABD59001 none Modification gives tularensis external [biogenesis of amount subsp. cell envelope] exported in novicide product Acre Shigellason Protein YP_312213 none Modification gives nei Ss046 transmembrane affects amount septum formation and exported in

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cell membrane product AcrF E. Coli Resistant F protein P24181 none Modification gives to acriflavin amount exported in product tll1619 Thermosynec Efflux transportation NP_682409.1 none Modification gives hococcuselo multi-drug amount ngatus [BP- exported in 1] product T110139 Thermosynec Efflux transportation NP_680930.1 none Modification gives hococcuselo multi-drug amount ngatus [BP- exported in 1] product Fermentation Genes of Increase gives replication efficiency gives from the point of production control umuD Shigellason DNA polymerase V, YP_310132 3.4.2.1 Increase gives nei Ss046 subunit efficiency gives production umuC E. Coli DNA polymerase V, ABC42261 2.7.7.7 Increase gives subunit efficiency gives production pntA, pntB Shigellafle NADH: NADPH P07001, 1.6.1.2 Increase gives xneri transhydrogenase P0AB70 efficiency gives (alpha and beta production subunits) Others fabK Streptococc trans-2-enoyl-ACP AAF98273 1.3.1.9 Contribution

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us pneumoniae reductase II for biosynthesis of fatty acid fabL Bacillus licheniform is DSM 13 Enoil- (protein carrying acyl) reductase AAU39821 1.3.1.9 Contribution to fatty acid biosynthesis fabM Streptococc us mutans Trans-2, cis-3- decenoil-ACP- isomerase DAA05501 4.2.1.17 Contribution to fatty acid biosynthesis

Recombinant Host Cells and Cell Cultures [107] Strategies for increasing the production of fatty aldehyde or fatty alcohol compositions by recombinant host cells include increasing flow through the fatty acid biosynthesis pathway by overexpression of biosynthetic genes from fatty aldehydes or native fatty alcohols and expression of biosynthetic genes fatty aldehydes and exogenous fatty alcohols from different organisms in the production host. As used herein, the term recombinant host cell or host cell refers to a host cell, whose genetic composition has been altered in relation to the corresponding wild-type host cell, for example, by the deliberate introduction of new genetic elements and / or modification deliberate selection of genetic elements naturally present in the host cell. The offspring of such recombinant host cells also contain these new genetic and / or manipulated elements. In any aspect of the description

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85/137 described herein, the host cell can be selected from a plant cell, insect cell, fungal cells (for example, a filamentous fungus, such as

Candida sp. , Or a germinating 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, Kluyveromyces, Pichia, Mucor Myceliophtora, 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 is a bacterial cell

Gram-negative. In some embodiments, the host cell is an E. coli cell. In other embodiments, the host cell is a Bacillus lentus cell, a Bacillus brevis cell, a Bacillus stearothermophilus cell, a Bacillus lichenoformis cell, a Bacillus alkalophilus cell, a Bacillus coagulans cell, a Bacillus circulans 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 other embodiments, the host cell is a Trichoderma koningii cell, a cell

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86/137 of Trichoderma viride, a cell of Trichoderma reesei, a cell of Trichoderma longibrachiatum, a cell of Aspergillus awamori, a cell of Aspergillus fumigates, a cell of Aspergillus foetidus, a cell of Aspergillus nidulans, a cell of an Aspergillus niger, Aspergillus oryzae cell, Humicola insolens cell, Humicola lanuginosis cell, Rhodococcusopacus cell, Rhizomucormiehei cell, or 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. In some embodiments, the host cell is a Saccharomyces cerevisiae cell.

[108] In other embodiments, the host cell is a eukaryotic plant cell, an algae cell, a cyanobacterial cell, a sulfur-green bacterial cell, a sulfur-free bacterial cell, a purple-sulfur bacterium cell, a sulfur-free purple bacterial cell, an extremophile cell, a yeast cell, a fungus cell, a cell manipulated with any of the species described here, or a synthetic organism. In some embodiments, the host cell is dependent on light or fixed carbon. 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 an Arabidopsis thaliana cell,

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87/137

Panicum virgatum, Miscanthus giganteus, Zea mays,

Botryococcuse braunii,

Chlamydomonas reinhardtii, Dunaliela salina, Synechococcus

Sp. PCC 7002, Synechococcus Sp. PCC

7942, Synechocystis

Sp. PCC 6803, Thermosynechococcus elongates

BP-1,

Chlorobhim tepidum,

Chlorojlexus auranticus,

Chromatiumm vinosum, Rhodospirillum rubrum,

Rhodobacter capsulatus,

Rhodopseudomonas palusris,

Clostridium ljungdahlii,

Clostridium thermocellum,

Penicillium chrysogenum,

Pichiapastoris,

Saccharomyces cerevisiae,

Schizosaccharomyces pombe,

Pseudomonas fluorescens, or Zymomonas mobilis.

Manipulated Host Cells [109]

In some embodiments, a polynucleotide sequence (or gene) is supplied to the host cell via a recombinant vector, which includes a promoter operably linked to the polynucleotide sequence. In certain embodiments, the promoter is a promoter developed in a developed manner, a specific organelle, a specific tissue, an inducible, a constitutive, cell-specific. In some embodiments, the recombinant vector includes at least one sequence selected from an expression control sequence operably coupled with 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 secretion sequence operably coupled to the polynucleotide sequence; and a sequence of

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88/137 targeting operatively coupled to the polynucleotide sequence. The expression vectors described herein include a 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 factors such as the choice of host cell to be transformed, the desired level of polypeptide expression, and the like. The expression vectors described herein host cells for a including polypeptides can be introduced into the production of polypeptides, fusion, encoded by the polynucleotide sequences, as described above (supra). Expression of genes encoding polypeptides in prokaryotes, for example, E. coli, is most often performed with vectors containing constitutive or inducible promoters directing the expression of any fusion or non-fusion polypeptide. Fusion vectors add a number of amino acids to a polypeptide encoded therein, generally to the amino or carboxy terminal of the recombinant polypeptide. Such fusion vectors typically serve one or more of the following three purposes, including to increase expression of the recombinant polypeptide; to increase the solubility of the recombinant polypeptide; and to assist in the purification of the recombinant polypeptide by acting as a linker in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion portion and the recombinant polypeptide. This allows for the separation of the polypeptide

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89/137 recombinant from the fusion portion after purification of the fusion polypeptide. Examples of such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, and enterokinase. Exemplary fusion expression vectors include pGEX vector (Pharmacia Biotech, Inc., Piscataway, NJ; Smith et al. (1988) Gene 67: 31-40), pMAL vector (New England Biolabs, Beverly, MA), and vector of pRITS (Pharmacia Biotech, Inc., Piscataway, NJ), which fuse glutathione-S-transferase (GST), maltose binding protein E, or protein A, respectively, to the recombinant target polypeptide.

[110] Examples of inducible and non-fusion E. coli expression vectors include pTrc vector (Amann et al. (1988) Gene 69: 301-315) and pET 11d 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 based on the transcription of host RNA polymerase from a hybrid trp-lac fusion promoter. Expression of the target gene from the pET 11d vector is based on transcription from a T7 gen10-lac fusion promoter mediated by a co-expressed viral RNA polymerase (T7 gn1). This viral polymerase is provided by host strains, such as BL21 (DE3) or HMS174 (DE3) cells, from a resident λ profago harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter. prokaryotic and eukaryotic cells are well known in the art (see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor.

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90/137

Laboratory). Examples of inducible and non-fusion E. coli expression vectors include pTrc vector (Amann et al. (1988) Gene 69: 301-315) and PET 11d 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 T5 bacteriophage. In one embodiment, the host cell is a yeast cell. In this modality, the expression vector is a yeast expression vector. Vectors can be introduced into prokaryotic cells or eukaryotic cells through a variety of techniques recognized in the art for introducing foreign nucleic acid (for example, DNA) into a host cell. Suitable methods for transforming or transfecting host cells can be found in, for example, Sambrook et al. (supra). For the stable transformation of bacterial cells, it is known that (depending on the expression vector and transformation technique used) a certain fraction of the cells will take and replicate the expression vector. In order to identify and select these transformants, a gene that encodes a selectable marker (for example, resistance to an antibiotic) can be introduced into host cells along with the gene of interest. Selectable markers include those that confer resistance to drugs, such as, but not limited to, ampicillin, kanamycin, chloramphenicol or tetracycline. Nucleic acids encoding a selection marker can be introduced into a host cell in the same vector that encodes a polypeptide described herein or can be

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91/137 introduced in 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 recombinant or engineered host cell as described herein (supra) is a cell used to produce a fatty acid derivative composition such as a fatty aldehyde or a fatty alcohol. In any of the aspects of the description described here, the host cell can be selected from a eukaryotic plant, bacteria, algae, cyanobacteria, sulfur-green bacteria, sulfur-free green bacteria, purple-sulfur bacteria, purple-sulfur-free bacteria extremophile, yeast, fungi, manipulated organisms thereof, or a synthetic organism. In some embodiments, the host cell is dependent on light or fixed carbon.

In some embodiments, the host cell has autotrophic activity. Various host cells can be used to produce fatty acid derivatives, as described herein.

[111]

The host or microorganism cells of the invention include host strains or host cells that are genetically engineered or modified to contain changes in order to test the effectiveness of specific mutations on enzyme activities (for example, recombinant cells or microorganisms).

Several optional genetic manipulations and changes can be used alternately from one host cell to another, depending on which native enzyme pathways are present in the original host cell. In one embodiment, a host strain can be

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92/137 used to test the expression of an AAR polypeptide in combination with other biosynthetic polypeptides (for example, enzymes). A host strain can encompass a series of genetic changes in order to test specific variables, including, but not limited to, culture conditions, including fermentation components, carbon source (eg raw materials), temperature, reduced pressure, contamination conditions of the crop, and oxygen levels.

[112] dehydrogenase

In one embodiment, a host strain includes an optional fadE and fhuA deletion. Acyl-CoA (fadE) is an enzyme that is important for the metabolism of fatty acids.

It catalyzes the second step in the use of fatty acids (beta-oxidation), which is the process of breaking down long chains of fatty acids (acyl-CoAs) into acetyl-CoA molecules. More specifically, the second stage of the fatty acid degradation β-oxidation cycle in bacteria is the oxidation of acyl-CoA to 2-enoyl-CoA, which is catalyzed by. When E. coli does not have FadE, it cannot grow on fatty acids as a carbon source, but it 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, mutant FadE strains where the FadE function is disrupted. The fadE gene can be optionally neutralized or attenuated to ensure that acyl-CoAs, which can be an intermediate pathway in a fatty acid derivative, can accumulate in the cell in such a way that all acyl-CoAs can be efficiently converted into derivatives of fatty acid. However, fadE attenuation is

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93/137 optional when sugar is used as a carbon source, as long as the condition expression of FadE is likely to be suppressed and FadE, therefore, may be present in small amounts and not be able to compete efficiently with ester synthase or other enzymes for acyl-CoA substrates. 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, sugar is consumed first by suppressing the fad regulon (see D. Clark, J Bacteriol. (1981) 148 (2): 521-6)). In addition, the absence of sugars and the presence of fatty acids induces the expression FadE. Acyl-CoA intermediates could be lost to the beta oxidation pathway since the proteins expressed by the fad regulon (including FADE) are upwardly regulated and will compete efficiently for acyl-CoAs. Thus, it may be advantageous to have the fadE gene neutralized or attenuated. Since most carbon sources are based mainly on sugar, it is optional to mitigate FadE. The fhuA gene codes for the TonA protein, which is an energy-coupled carrier and receptor on the outer membrane of E. coli (V. Braun (2009) J Bacteriol. 191 (11): 3431-3436). Its elimination is optional. The fhuA deletion allows the cell to become more resistant to phage attack which can be beneficial in certain fermentation conditions. Thus, it may be desirable to delete fhuA in a host cell that is likely to be subject to potential contamination during fermentation operations.

[113] In another embodiment, the host strain (supra) also includes optional overexpression of one or more

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94/137 more of the following genes including fadR, fabA, fabD, fabG,

fabH, fabV, and / or fabF. Examples in such genes are fadR a from Escherichia coli, fabA starting in Salmonella typhimurium (NP_460041 ), fabD The leave in Salmonella typhimurium (NP_460164 ), fabG The leave in Salmonella typhimurium (NP_460165 ), fabH The leave in Salmonella typhimurium (NP_460163) , fabV a from Vibrio cholera (YP_001217283), and fabF a from Clostridium

acetobutylicum (NP_350156). Overexpression of one or more of these genes, which code for enzymes and regulators in fatty acid biosynthesis, can serve to increase the titer of fatty acid-derived compounds including fatty aldehydes and fatty alcohols under different culture conditions.

[114] In another embodiment, E. coli strains are used as host cells for the production of fatty acid derivatives, such as fatty aldehydes and / or fatty alcohols. Likewise, these host cells provide optional overexpression of one or more biosynthesis genes (i.e. genes encoding fatty acid biosynthesis enzymes and regulators) that can further increase or enhance the titer of fatty acid-derived compounds, such as derivatives of fatty acids (e.g., 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 changes include fadR a

from Escherichia coli, fabA starting in Salmonella typhimurium (NP_ 460041), fabD a leave in Salmonella typhimurium (NP_ _460164), fabG a leave in Salmonella

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95/137 typhimurium (NP_460165), fabH from Salmonella typhimurium (NP_460163), fabV from Vibrio cholera (YP_001217283), and fabF from Clostridium acetobutylicum (NP_350156). In some embodiments, the synthetic operons that carry these biosynthetic genes can be manipulated and expressed in cells in order to test for overexpression of fatty aldehyde and / or fatty alcohol under various culture conditions and / or to further increase production of fatty aldehyde and / or alcohol fatty. Such synthetic operons contain one or more biosynthetic genes. The ifab138 operon, for example, is a manipulated operon that contains optional fatty acid biosynthesis genes, including fabV from Vibrio cholera, fabH from Salmonella typhimurium, fabD from S. typhimurium, fabG from S. typhimurium, fabA from S. typhimurium and / or fabF from Clostridium acetobutylicum which can be used to facilitate the overexpression of fatty acid derivatives in order to test specific crop conditions. An advantage of such synthetic operons is that the rate of production of the fatty acid derivatives can be increased or improved.

[115] In some embodiments, host cells or microorganisms that are used to express ACP and biosynthetic enzymes (eg TE, ES, CAR, AAR, ADC, etc.) will still express genes that encompass certain activities enzymes that can increase the production of one or more particular fatty acid derivatives (s), such as fatty alcohols, fatty aldehydes, fatty esters, weak amines, derivatives of bifunctional fatty acids, diacids and the like. In a

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96/137 modality, the host cell has thioesterase activity (E.C. 3.1.2. * Or E.C. 3.1.2.14 or E.C. 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 (E.C. 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 / or alcohol-dehydrogenase activity (EC 1.1.1.1.) And / or acyl-CoA reductase fatty alcohol (FAR) activity ) (EC 1.1.1. *) And / or carboxylic acid reductase (RCA) activity (EC 1.2.99.6) for the production of fatty alcohols. In another embodiment, the host cell has acyl-ACP reductase (AAR) activity (E.C. 1.2.1.80) for the production of fatty aldehydes. In another embodiment, the host cell has acyl-ACP reductase (AAR) activity (E.C. 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 ester synthase activity (EC 2.3.1.75), 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 acid esters. In another modality, the host cell has OleA activity for the production of ketones. In another modality, the host cell has OleBCD activity for the production of internal olefins. In another embodiment, the host cell has

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97/137 acyl-ACP reductase (AAR) activity (E.C. 1.2.1.80) and alcohol dehydrogenase activity (E.C. 1.1.1.1.) For the production of fatty alcohols. In another embodiment, the host cell has thioesterase activity (E.C. 3.1.2. * Or E.C.

3.1.2.14 or E.C. 3.1.1.5) and decarboxylase activity for the preparation of 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, host cells or microorganisms that are used to express ACP and other biosynthetic enzymes will include some activities of native enzymes that are up-regulated or over-expressed in order to produce one or more particular fatty acid derivatives (s) , such as fatty aldehydes and / or fatty alcohols. In one embodiment, the host cell has a native thioesterase activity (E.C. 3.1.2. * Or E.C. 3.1. 2.14 or E.C. 3.1.1.5) for the production of fatty acids that can be increased by overexpressing the thioesterase gene.

[116] The present invention includes host strains or microorganisms that express the genes encoding AAR and other biosynthetic enzymes (supra). Recombinant host cells produce derivatives of fatty acids, such as fatty aldehydes and fatty alcohols and compositions and mixtures thereof. Fatty acid derivatives are typically recovered from the culture medium and / or are isolated from the host cells. In a

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98/137 modality, the derivatives of fatty acids, such as fatty aldehydes and fatty alcohols are recovered from the culture medium (extracellular). In another embodiment, derivatives of fatty acids, such as fatty aldehydes and fatty alcohols are isolated from host cells (intracellular). In another embodiment, derivatives of fatty acids, such as fatty aldehydes and fatty alcohols are recovered from the culture medium and isolated from the host cells. The composition of derived fatty acids 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 certain fatty acid derivatives, as well as chain lengths and degree of saturation of the components of the fatty acid derivative composition such as a fatty aldehyde and fatty alcohol compositions.

[117] Examples of host cells that function as microorganisms (for example, microbial cells), include, but are not limited to cells from the genus Escherichia, Bacillus, Lactobacillus,

Zymomonas, Rhodococcus, Pseudomonas, Aspergillus,

Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor,

Kluyveromyces, Pichia, Mucor, Myceliophtora, 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 is a bacterial cell

Gram-negative. In some embodiments, the host cell is an E. coli cell. In some embodiments, the cell

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99/137 host is an E. coli B cell, an E. coli C cell, and an E. coli K cell, or an E. coli W cell. In other embodiments, the host cell is a Bacillus cell lentus, a Bacillus brevis cell, a Bacillus stearothermophilus cell, a Bacillus lichenoformis cell, a Bacillus alkalophilus cell, a Bacillus coagulans cell, a Bacillus circulans 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 yet other embodiments, the host cell is a Trichoderma koningii cell, a Trichoderma viride cell, a Trichoderma reesei cell, a Trichoderma longibrachiatum cell, an Aspergillus awamori cell, an Aspergillus fumigatus cell, an Aspergillus foetidus cell, Aspergillus nidulans cell, Aspergillus niger cell, Aspergillus oryzae cell, Humicola insolens cell, Humicola lanuginose cell, Rhodococcus opacus cell, Rhizomucor miehei cell, or Mucor michei 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. In some embodiments, the host cell is a Saccharomyces cerevisiae cell. In other embodiments, the host cell is a eukaryotic cell from a plant, algae, cyanobacteria, sulfur-green bacteria, sulfur-free bacteria, sulfur-purple bacteria, sulfur-free purple bacteria, extremophile, yeast,

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100/137 fungus, a manipulated organism of the same, or a synthetic organism.

In some embodiments, the host cell is dependent on light or fixed carbon.

In some embodiments, the host cell has autotrophic activity.

In some modalities, 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,

Panicum virgatum, Miscanthus giganteus, Zea mays,

Botryococcuse braunii,

Chlamydomonas reinhardtii, Dunaliela salina, Synechococcus

Sp. PCC 7002, Synechococcus Sp. PCC

7942, Synechocystis

Sp. PCC 6803, Thermosynechococcus elongates

BP-1,

Chlorobium tepidum,

Chlorojlexus auranticus,

Chromatiumm vinosum, Rhodospirillum rubrum,

Rhodobacter capsulatus,

Rhodopseudomonas palusris,

Clostridium ljungdahlii,

Clostridium thermocellum,

Penicillium chysogenum, Pichia pastoris,

Saccharomyces cerevisiae,

Schizosaccharomyces pombe,

Pseudomonas fluorescens, or Zymomonas mobilis.

In a particular embodiment, a microbial cell is based on a cyanobacterium, including, but not limited to,

Prochlorococcus,

Synechococcus, Synechocystis, Cyanothece, and Nostoc spot. In another embodiment, the microbial cell is from a specific species of cyanobacteria including, but not limited to, Synechococcus elongatus PCC7942, Synechocystis sp. PCC6803, and Synechococcus sp. PCC7001.

Recombinant host cell culture and Fermentation [118] As used herein, the term fermentation

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101/137 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 their derivatives, by propagating a culture of the recombinant host cells in a medium that comprises the carbon source.

Permissive conditions for production refer to any conditions that allow a host cell to produce a desired product, such as a fatty aldehyde or a fatty alcohol. Likewise, the condition or conditions under which the polynucleotide sequence of a vector is expressed means any 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 medium composition.

Each of these conditions, individually and in combination, allows the host cell to grow. Fermentation can be aerobic, anaerobic, or variations (such as microaerobic).

Examples of culture media include broths or gels.

Generally, the metabolized medium includes a carbon source that can be directly by a host cell. In addition, enzymes can be used in a medium to facilitate mobilization (for example, depolymerization of cellulose or starch into fermentable sugars) and subsequent metabolism of the carbon source.

[119] For small-scale production, engineered host cells can be grown in

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102/137 batches of, for example, about 100 pL, 200 pL, 300 pL, 400 pL, 500 pL, 1 ml, 5 ml, 10 ml, 15 ml, 25 ml, 50 ml, 75 ml, 100 ml, 500 mL, 1 L, 2 L, 5 L or 10 L; fermented; and induced to express a desired polynucleotide sequence, such as a polynucleotide sequence that encodes an ACP and / or biosynthetic polypeptide. For large-scale production, engineered host cells can be grown in batches of about 10 L, 100 L, 1000 L, 10000 L, 100000 L, and 1000000 L or greater; fermented; and induced to express a desired polynucleotide sequence. Alternatively, large-scale fermentation of fed batches (batch-fed) can be carried out. The described fatty acid derivative compositions 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, transported to the extracellular environment or passively transferred to the extracellular environment of the recombinant host cell culture. The fatty acid derivative is isolated from a culture of recombinant host cells, using routine methods known in the art.

Products derived from recombinant host cells [120] As used herein, the modem carbon fraction or fM has the same meaning as defined by the National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs4990B and 4990C, known as

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103/137 oxalic acid standards HOxI and HOxII, respectively. The fundamental definition refers to 0.95 times the ¹⁴ C / ¹² C ratio of HOxI isotopes (with reference to AD 1950). This is roughly equivalent to the wood of the pre-industrial revolution with decay correction. For the current living biosphere (plant material), fM is approximately 1.1. Bioproducts (for example, fatty acid derivatives including fatty aldehydes and / or fatty alcohols produced in accordance with the present disclosure) include biologically produced organic compounds. In particular, the fatty acid derivatives produced, using the fatty acid biosynthesis pathway here, were not produced from renewable sources and, as such, are new compositions of matter. These new bioproducts can be distinguished from carbon-derived organic compounds from the petrochemicals based on double carbon-isotopic fingerprints or dating from ¹⁴ C. In addition, the specific source of bio-carbon carbon (eg glucose vs. glycerol) can be determined by double carbon-isotopic fingerprints (see, for example, US Patent No. 7,169,588). The ability to distinguish bioproducts from petroleum-based organic compounds is beneficial in tracking these materials in commerce. For example, organic compounds or chemicals including both bio-based profiles and petroleum-based carbon isotope profiles can be distinguished from organic compounds and chemicals prepared only from petroleum-based materials. Thus, the bioproducts here can be tracked or tracked commercially based on their unique isotope profile of

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104/137 carbon. Bioproducts can be distinguished from petroleum-based organic compounds by comparing the stable carbon isotope ratio ( ¹³ C / ¹² C) in each sample. The ¹³ C / ¹² C ratio in a given bioproduct is a consequence of the ¹³ C / ¹² C ratio in atmospheric carbon dioxide at the time the carbon dioxide is fixed. It also reflects the precise metabolic pathway. Regional variations also occur. Petroleum, C3 plants (broad leaf), C4 plants (grams) and marine carbonates, all show significant differences at ¹³ C / ¹² C and the corresponding ô ¹³ C values. In addition, the lipid matter of plants C3 and C4 analyze differently from materials derived from the carbohydrate components of the same plants as a consequence of the metabolic pathway. Within the measurement accuracy, ¹³ C shows great variations due to the isotopic fractionation effects, the most important of which for bioproducts is the photosynthesis mechanism.

The main cause of the differences in the carbon isotope ratio in plants is closely associated with the differences in the photosynthetic carbon metabolism pathway in plants, in particular, the reaction that occurs during primary carboxylation (that is, the initial fixation of atmospheric CO2).

Two major classes of vegetation are those that incorporate the C3 photosynthetic cycle (or Calvin-Benson) and those that incorporate the C4 photosynthetic cycle (or HatchSlack). In C3 plants, the primary CO2 fixation 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,

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105/137 are dominant in temperate zones. In C4 plants, an additional carboxylation reaction involving another enzyme, phosphoenol-pyruvate carboxylase, is the primary carboxylation reaction. The first stable carbon compound is a 4-carbon acid that is subsequently decarboxylated. The released CO ₂ is then fixed by cycle C3. Examples of C4 plants are tropical grasses, corn and sugarcane. Both C4 and C3 plants exhibit a range of ¹³ C / ¹² C isotopic ratios, but typical values are 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 located in this latter range. The ¹³ C measurement scale was originally defined by a set of zero by Pee Dee Belemnite limestone (APO), where the values are given in parts per thousand deviations of this material. The Ô13C values are expressed in parts per thousand (per thousand),

abbreviated,% 0, and are calculated as if Follow: ô ¹³ C (% 0) = [( ¹³ C / ¹² C) sample- ( ¹³ C / ¹² C) standard] / ( ¹³ C / ¹² C) X 1000 standard [121] One · time the material of reference (RM) PDB has been exhausted, a Serie of RMs alternatives were

developed in cooperation with the IAEA, USGS, NIST, and other selected international isotope laboratories. Notations for the deviations of one thousand from PDB are ô ¹³ C. Measurements are made in CO ₂ by high precision stable ratio mass spectrometry (IRMS) in molecular ions of masses 44, 45 and 46. The compositions described here include bioproducts produced by any of the methods described herein, including, for example,

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106/137 products derived from fatty acids. Specifically, the bioproduct can have an ô ¹³ C 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 can have a ô ¹³ C of about -30 to about 15, of about -27 to about -19, of about -25 to about -21, of about -15 to about from -5, from about -13 to about -7, or from about -13 to about -10. In other cases, the bioproduct may have an ô ¹³ C of about -10, -11, -12, or -12.3. Bioproducts produced according to the disclosure here, can also be distinguished from petroleum-based organic compounds by comparing the amount of ¹⁴ C in each compound. Because ¹⁴ C has a nuclear half-life of 5730 years, petroleum-based fuels containing older carbon can be distinguished from bioproducts containing more recent carbons (see, for example, Currie, Source Apportionmment of Atmospheric Particles, Characterization of Environmental Particles , J. Buffle and HP van Leeuwen, Eds. 1 of Vol. I of the IUPAC Environmental Analytical Chemistry Series (Lewis Publishers, Inc.) 3-74, (1992)). The basic assumption in dating radiocarbon is that a constant ¹⁴ C concentration in the atmosphere leads to a ¹⁴ C constant in living organisms. However, because of nuclear tests in the atmosphere since 1950 and the burning of fossil fuels since 1850, ¹⁴ C has acquired a second characteristic of geochemical time. Its concentration in atmospheric CO2, and therefore in the living biosphere, approximately double at the peak of nuclear testing in the mid-1960s. Since then, it has been gradually returning to the reference isotope rate

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107/137 cosmogenic at steady state (atmospheric) ( ¹⁴ C / ¹² C) of about 1.2 x 10 ^-12 , with an approximate half-life relaxation of 7-10 years. This last half-life is not to be taken literally; Instead, one should use the atmospheric nuclear input / decay function Detailed to trace the variation of atmospheric and biospheric ¹⁴ C since the onset of the nuclear age. It is this last feature of the biospheric ¹⁴ C weather that keeps the promise of dating the recent carbon of the biosphere annually. ¹⁴ C can be measured by mass spectrometry accelerator (AMS), with results presented in units of modern carbon fraction (fM). FM is defined by Standard Reference Material (SRMs) 4990B 4990C - Reference Materials

National Standard of the Institute of Standards and Technology National Institute of Standards and Technology (NIST). As used here, the modern carbon fraction or fM has the same meaning as defined by Standard Reference Material (SRMs) 4990B 4990C - Standard Reference Materials

National Institute of Standards and Technology National Institute of Standards and Technology (NIST), known as oxalic acid standards HOxI and HOxII, respectively. The fundamental definition relates to 0.95 times the ¹⁴ C / ¹² C HOxI isotope ratio (with reference to AD 1950). This is roughly equivalent to decay-corrected pre-Industrial Revolution wood. For the current living biosphere (plant material), fM is approximately 1.1. The compositions described in the present invention include bioproducts that can have a fM ¹⁴ C of at least about 1. For example, the bioproducts of the disclosure can have a fM ¹⁴ C of at least about 1.01, a fM ¹⁴ C of

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fence in 1 The fence of 1.5, one fM ¹⁴ C of fence of 1.04 The fence in 1, 18 , or a ¹⁴ C fM in fence from 1,111 about in 1,124. [122] Another measurement in ¹⁴ C is known as being

the percentage of modern carbon (pMC). For an archeologist or geologist using dates from ¹⁴ C, AD 1950 equals zero years of age. This also represents 100 pMC. The carbon in the bomb in the atmosphere reached almost twice the normal level in 1963, at the peak of thermo-nuclear weapons. Its distribution within the atmosphere has been approximate since its appearance, showing values that are greater than 100 pMC for plants and animals that live since AD 1950. This has gradually decreased over time with today's value being close to 107.5 pMC. This means that a fresh biomass material, such as corn, would give a ¹⁴ C signature close to 107.5 pMC. Petroleum-based compounds will have a pMC value of zero. Combining fossil carbon with current-day carbon will result in a dilution of the current-day pMC content. Assuming 107.5 pMC represents the ¹⁴ C content of today's biomass materials and 0 pMC represents the ¹⁴ C content of petroleum-based products, the measured pMC value so that the material reflects the proportions of the two types of components . For example, a material derived 100% from soy today would give a radiocarbon signature close to 107.5 pMC. If this material was diluted to 50% with petroleum-based products, it would give a radiocarbon signature of approximately 54 pMC. A biologically based carbon content is derived by signing 100% equal to 107.5 pMC and 0% equal to 0 pMC. For example, a sample with 99 pMC

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109/137 will give an equivalent bio-based carbon content of 93%. This value is referred to as the average result of bio-based carbon and assumes all components within the analyzed material originating from or biological material of the present day or petroleum-based material. A bioproduct comprising one or more fatty acid derivatives as described herein 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

on here described can have a pMC of between about in 50 and about 100; fence in 60 and about 100; about 70 and fence in 100; about 80 and about 100; about 85 and fence in 100; about 87 and about 98; or about 90 and fence in

95. In still other examples, a fatty acid derivative described herein can have a pMC of about 90, 91, 92, 93, 94 or 94.2.

Compositions of fatty aldehyde and fatty alcohol and their use [123] Aldehydes are used to produce many special chemicals. For example, aldehydes are used to produce polymers, resins (for example, BAKELITE resin), dyes, flavorings, plasticizers, perfumes, pharmaceuticals and other chemicals, some of which can be used as solvents, preservatives or disinfectants. In addition, certain natural and synthetic compounds, such as vitamins and hormones, are aldehydes, and many sugars contain groups of aldehydes. Fatty aldehydes can be converted to fatty alcohols by chemical or enzymatic reduction. Fatty alcohols also have multiple commercial uses. Annual sales

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110/137 of fatty alcohols and their derivatives are in excess of US $ 1 billion. Short-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 non-ionic surfactants, which are useful in personal care and household products, such as, for example, detergents. In addition, fatty alcohols are used in waxes, gums, resins, ointments and pharmaceutical lotions, lubricating oil additives, textile antistatic agents and finishing agents, plasticizers, cosmetics, industrial solvents, and fat solvents.

[124] The invention 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 and used. For example, when the fatty alcohols described herein are used as a raw material for the production of surfactant or detergent, one of ordinary skill in the art will appreciate that the characteristics of the fatty alcohol raw material will affect the characteristics of the produced surfactant or detergent composition. Thus, the characteristics of the detergent or surfactant composition can be selected for the particular production of fatty alcohols for use as a raw material. A surfactant composition and / or a fatty alcohol based detergent described herein can be mixed with other

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111/137 surfactants and / or detergents well known in the art. In some embodiments, the mixture 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 limited by any two of the above mentioned values, by weight of fatty alcohol. In other examples, a detergent or surfactant composition can be prepared by including 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%, at least about 85%, at least about 90%, at least about 95%, or a range limited by any two of the values mentioned above, 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 surfactant or detergent compositions may also include at least one additive, such as a microemulsion or a surfactant or detergent from non-microbial sources, such as vegetable or petroleum oils, which may be present in an 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 limited by any of the above two values by weight fatty alcohol.

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EXAMPLES [125] The following specific examples are intended to illustrate the disclosure and should not be construed as limiting the scope of the claims.

Protocols and Methods

Scanning a library [126] All protocols described here depend on a 2 mL master-blocked 96-well plate system (Greiner Bio-One, Monroe, NC or Corning, Amsterdam, Netherlands) for growing cultures, and the plates (Costar, Inc.) for the extraction of fatty acid species from the culture broth. The protocols provided below are examples of fermentation conditions. Alternative protocols can be used to assess the

species production of fatty acid. Culture Protocol at 32 ° C (4NBT) [127] The 20pL culture of LB (from an culture in LB growth in plates in 96 wells) was used for inoculate 400 pL of media 2NBT (Table 2), what were in then incubated for about 16

hours at 32 ° C shaking. 20 pL of seeds overnight was used to inoculate 400 pl 4NBT with either 1 or 2 g / L nitrogen (NBT_1N or NBT_2N). After growing at 32 ° C for 6 hours, 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 detailed standard extraction protocol below.

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Table 2: Names and formulations of means

Name Middle of Formulation 1 X 5x Salt solution with NH4C1 1 g / L 100g / LNH4Cl 1 mg / L 10mg / mL Thiamine 1 III 1M MgSO4 2NBT 0.1 mM 1M CaC12 20 g / L 500g / L glucose 1 X 1000xTM2 10 mg / L 10g / LFe Citrate 100 gg / mL 100 mg / ml spectinomycin 100 mM 2M BisTris (pH7.0) 1 X 5x Salt solution with NH4C1 1 mg / L 10mg / mL Thiamine 1 mM lMMgSO4 0.1 mM 1M CaC12 4NBT_ 1N 40 g / L 500g / L glucose 1 X 1000xTM2 10 mg / L 10g / L Fe Citrate 100 pg / mL 100 mg / ml spectinomycin 100 mM 2M BisTris (pH7.0) 1 X 5χ Sol de Sal with NH4C1 1 g / L 100 g / LNH4Cl 1 mg / L 10mg / mL Thiamine 1 mM lMMgSO4 4NBT2N 0.1 mM 1M CaC12 40 g / L 500g / L glucose 1 X ΙΟΟΟχ TM2 10 mg / L 10g / L Fe Ci trato 100 μg / mL 100 mg / ml spectinomycin 100 mM 2M BisTris (pH7.0)

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Standard Protocol for Extraction of Fatty Acid Species [128] To each well to be extracted 40pL of 1M HCl, followed by 300pL of butyl acetate (with 500 mg / L of C11-FAME as internal standard) were added. The 96-well plates were then heat-sealed using a plate sealer (ALPS-300 heater; Abgene, ThermoScientific, Rockford, IL), and shaken for 15 minutes at 2000 rpm using a MIXMATE mixer (Eppendorf, Hamburg , Germany). After shaking, the plates were centrifuged for 10 minutes at 4500rpm at room temperature (Allegra X-15R, rotor SX4750A, 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, Netherlands), which was subsequently heat-sealed and stored at -20 ° C until evaluated by GCFID using the FALC_Broth.met method . The FALC_Broth.met method was performed as follows: 1 pL of sample was injected onto an analytical column (DB-1, 10 x 180 pm x 0.2 μΜ film thickness, available from JW 121101A) in an ultra device Agilent GC 7890A (Agilent, Santa Clara, CA) with a flame ionization detector (FID). The instrument was created to detect and quantify fatty alcohols C6 to C18.

The protocol detailed above represents standard conditions, which can be modified as needed, to optimize the analytical results.

Fatty acid species - Standard Nile Test Protocol

Red

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115/137 [129] After 24 hours of fermentation, a Nile Red test was performed by adding 70μL of fermentation broth to 130μL of 1.54μ9 / ι ^ of Nile Red in 84.6% water and 15 , 4% acetonitrile to a final assay concentration of 1 g / mL Nile Red on a Greiner MicrolonFluotrac 200 plate, and mixed by pipetting up and down. Relative fluorescence units were measured at 540 nm excitation and 630 nm emission using the SpectraMax M2 unit.

Construction Error-Prone Libraries [130] Conventional techniques known to those skilled in the art have been used to prepare error-prone libraries. In one example, the vector structure was prepared using restriction endonucleases in the vector, while the creation of diversity in the DNA insertion was generated by PCR amplification from a DNA template under conditions favoring the incorporation of unpaired nucleotides. In one approach, cloning of the vector skeleton and a DNA insertion with diversity was performed using INFUSION Cloning System - Infusion cloning system (Clontech Laboratories, Inc., Mountain View, CA), according to the manufacturer's protocol.

Construction Saturation Libraries [131] Conventional techniques known to those skilled in the art have been used to prepare saturation libraries. In one example, the vector backbone was prepared using restriction endonucleases in the vector, while creating diversity in the DNA insertion was generated using degenerate primers. In one approach, cloning the vector structure and inserting

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116/137 DNA with diversity was performed using INFUSION Cloning System - INFUSION cloning system (Clontech Laboratories, Inc., Mountain View, CA) according to the manufacturer's protocol.

Construction Combination Libraries [132] The mutations identified as beneficial have been combined to provide variants of AAR with other improvements in the production of fatty alcohol species. Standard techniques known to those skilled in the art were used to prepare the combination libraries. In one example, the vector structure was prepared using restriction endonucleases in the vector, while creating diversity at the insertion of DNA was generated using primers to introduce the desired mutations. As described above, in one approach, cloning of the vector structure and a DNA insertion with diversity was performed using the Cloning System INFUSION - Clontech Laboratories, Inc., Mountain View, CA), according to the manufacturer's protocol. Combination libraries can be generated using the PCR transfer protocol (tPCR) (Erijman et al.

(2011) J. Strutural Bio. 175: 171-177).

Library Tracking [133] Since the library diversity was generated in an error-prone saturation library or combination library, it was tracked using one of the methods described above. Two types of results were identified: (1) increased amount of fatty alcohol (FALC title); and / or (2) increasing the amount of medium-chain FALC, such as dodecanol (C12) or tetradecanol

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117/137 (C14). Hexadecanol (C16) and octadecanol (C18) were also identified. Mutations in the AAR variants within each result were identified by sequencing using conventional techniques normally employed by those skilled in the art. Tables 4, 5 and 6 list the mutations (results) identified as beneficial in saturation and combination libraries.

EXAMPLE 1: Improved fatty alcohol production using AAR_7942 by Acyl Transport Protein (ACP) mediated by increased flow through the Fatty Acid Synthesis Pathway [134] When terminal pathway enzymes from sources other than E. coli, are expressed in E. coli as a heterologous host to convert fatty acyl-ACPs to products, there may be limitations in the recognition, affinity and / or rotation of the recombinant pathway towards fatty E. coli ACP. Although ACP proteins are conserved to some extent in all organisms, their primary sequence may differ even within the same species. In order to test this hypothesis, the acp genes from various cyanobacteria were cloned downstream from Synechococcits elongatus PCC7942 acyl-ACP reductase (AAR_7942) present in plasmid pLS9-185, which is a derivative of pLC1920 (3-5 copies / cell ). In addition, the sfp gene (Accession No. X63158, SEQ ID NO: 11) from Bacillus subtilis, which encodes a phospopantheteinyl transferase with broad substrate specificity, was cloned downstream from the respective acp genes. This enzyme is involved in the conversion of inactive apo-ACP to active holo-ACP. The plasmids constructed

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118/137 are described in Table 3.

Table 3: Plasmids Coexpressing ACP Cyanobacteria with and without B. subtilis sfp downstream of S. elongatus PCC7942 AAR

Plasmid Base ACP font ACP - SEQ ID NO. (NA / AA *) Without sfp Without sfp pLS9-185 Synechococcus elongatus 7942 7/8 pDS168 pDS168S pLS9-185 Synechocystis sp. 6803 3/4 pDS169 not available pLS9-185 Prochlorococcus marinus MED4 5/6 pDS170 pDS170S pLS9-185 Nostoc spot 73102 1/2 pDS171 pDS171S pLS9-185 Nostocsp. 7120 9/10 pDS172 pDS172S

* NA = nucleic acid sequence; AA = amino acid sequence / polypeptide sequence [135] All acp genes were cloned with a synthetic RBS at the EcoRI site immediately downstream of the aar gene in pLS9-185 using INFUSION technology (infusion). The EcoRI site was reconstructed downstream from the acp gene. Likewise, the B. subtilis sfp gene was cloned by INFUSION (infusion) at this EcoRI site together with a synthetic RBS. All plasmids were transformed into E. coli MG1655 DV2. The control for these experiments was the expression of AAR alone (pLS9-185).

[136] Results from standard shake flask fermentation experiments are shown in Fig. 5. Significant improvement in fatty alcohol titers was observed in strains containing plasmids pDS171S pDS172S, pDS168 and pDS169, demonstrating that ACP overexpression may be beneficial for the production of fatty alcohol. While not wishing to be bound by theory, the hypothesis is suggested that ACP overexpression may be

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119/137 beneficial for the production of fatty alcohol, assisting in the recognition, affinity and / or rotation of acyl-ACPs by the enzyme of the heterologous terminal pathway (see Table 3 (supra) for the source of ACPs and the presence or absence of sfp).

Example 2: Improved fatty alcohol production using AAR_7942 by increased flow-mediated acetyl-CoA (ACC) through fatty acid synthesis [137] The main precursors for fatty acid biosynthesis are malonyl-CoA and acetyl-CoA. It has been suggested that these precursors limit the rate of biosynthesis of fatty acids in E. coli. In this example, a synthetic acetyl-CoA carboxylase, acc, operon [Corynebacterium glutamicum accDCAB + birA; SEQ ID NOs: 45 or 46, 48, and 50, also referred to as D +] was expressed along with Synechococcus elongatus PCC7942 acylACP reductase (AAR) (AAR_7942). The accD + operon was cloned downstream of the AAR_7942 gene in plasmid pLS9-185. The resulting plasmid and the 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, the coexpression of synthetic Corynebacterium glutamicum acc operon led to an increase in fatty alcohol production.

Example 3: Error-Prone Library, Limited Saturation and Combination Libraries prepared using AAR_7942 as a model

A. Error-Prone Library [138] An error-prone library of acyl-ACP reductase from Synechococcus elongatus PCC7942 (AAR_7942) was built and screened for variants that

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120/137 showed improvements over the wild type AAR_7942. The plasmid used to prepare the error prone library was designated pDS171S (see Table 3). The error-prone library was scanned using one of the standard protocols described above. The improvements were classified either as another improved titer or an increase in the fraction of fatty alcohols C10-C14 produced without significantly affecting the titer (results not shown).

B. The Combination and Limited Saturation Libraries [139] Conventional techniques known to those skilled in the art have been used to prepare combination libraries and saturation libraries based on positions 17, 18 and 19. The mutations tested in the combined libraries and the libraries of saturation (Tables 4A and 4B) were originally identified as error-prone library of AAR_7942 described above. The plasmids, strains and screening protocols used were the same as described in Example 1. The results from the error-prone library scan are shown in Tables 4A and 4B. Table 4A shows AAR_7942 mutations that led to an increase in fatty alcohol titers and Table 4B shows mutations that led to an increase in the C14 fatty alcohol fraction without significantly affecting the overall fatty alcohol titre.

Table 4A: Mutations from AAR 7942 Limited Saturation and Combination Libraries correlated with Enhanced Fatty Alcohol Titer

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Mutations Combo d library FALC title (total *) C14-FALC (% fraction) Normalized FALC (% under control) C14 Normalized l (% under control) S18T, C63Y, S96T, L177H, A281V 2473 48% 251% 294% S18T, C63Y, S96T, L177H, A281V E284K 2202 50% 200% 229% S18T, C63Y, S96T, L177H 2088 43% 189% 197% S18T, C63Y 1798 39% 182% 241% C63H, S967T, L177H, A281V 2045 53% 180% 237% S18T, C63Y, A281V, E283K 1696 45% 172% . 278% S18T, C63Y 1640 43% 166% 260% S18T, L177H, A281V, E283K 1618 44% 164% 270% S18T, L177H, A281V, E283K 1617 43% 164% 264% SI 8T, S96T, L177H, E283K 1814 43% 160% 194% S18T, C63H, L177H 1811 39% 160% 173% S18T, L177H, A281V, E283K 1503 39% 152% 237% L65F, S96T, A281V, E283K 1638 43% 144% 191% C63Y, A281V, A282T 1622 38% 143% 168% L11F, C63Y, S96T, L177H, A281V A282T, E283K 1579 41% 143% 186% L65F, S96T, L177H, A281V, E283K 1585 42% 140% 189% G22S, C63H, S96T, L177H, E283K 1229 39% 125% 237% C63H, L65F, S96T, A281V 1178 41% 119% 252% S18T, L65F, S96T, L177H, A281V 1136 33% 115% 203% C63R, L177H, A281V, E283K 1275 44% 112% 196%

Saturation Libraries Mutations Title falc ¹ C14-FALC Normalized FALC [% under control) Normalized C14 ((% under control) (total*) '(/ the fraction) S18M 2809 40% 243% 212% S18F 2564 54% 222% 285% S18Y 2550 45% 221% 236% S18W 2530 55% 227% 314% S18M 2495 41% 224% 231% S18Y 2039 44% 188% 252% S18Y 1997 44% 179% 251%

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Saturation Libraries S18T 1729 31% 155% 177% S18T 1690 31% 152% 176% S18T 1683 31% 151% 176% S18T 1599 30% 143% 172% S18L 1568 48% 141% 271% S18C 1381 28% 120% 149% S18C 1372 26% 123% 147% S18C 1368 28% 118% 148% S18L 1359 47% 122% 266% S18C 1355 26% 121% 148% S18V 1344 30% 121% 170% S18C 1340 26% 120% 147% S18L 1326 47% 119% 268% S18L 1315 47% 118% 266% S18C 1313 26% 118% 149% S18V 1306 30% 121% 170% S18C 1305 26% 117% 148% S18L 1302 47% 117% 267% V17L 1188 23% 110% 130% V17L 1185 33% 103% 173% V17L 1175 32% 102% 171% Β Combo Library Title FALC (total*) C14-FALC (% fraction) Normalized FALC [% under control) £ 14 Standardized '(% under control) / S18W, S96T, E283K, R286Q, A324V 3437 49% 246% 255% S18W 3286 53% 235% 275% S18W, S96T, E283K 3199 46% 229% 237% S18W, C63Y, S96T 3186 46% 228% 241% S18W, C68Y, E2834K 2949 50% 211% 260% C63Y, E283K 2680 36% 192% 189% S18W, C63Y 2675 54% 191% 280% C63Y, S96T 2583 38% 185% 196% C63Y, E283K, Q316K 2420 36% 173% 188% S96T 1605 23% 115% 121% E283K 1532 25% 110% 131%

Table 4B: Mutations from the Limited Saturation and Limited Combination Libraries AAR_7942 correlated with increased C14 Fatty Alcohol fraction

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Combo Library (í Title FALC (total*) C14-FALC (% fraction) | Normalized FALC [% under control) C14 Normalized [% under control) C63H, S96T, L177H, A281V 2045.36 53% 180% 237% G22S, C63H, F64V, S96T, L177H A281V 152.47 51% 14% 233% S18T, C63H, S96T, L177H, A281V E283K 2202.46 50% 200% 229% S18T, C63Y, S96T, L177H, A281V 2472.80 48% 251% 294% S18T, C63Y, A281V, E283K 1695.59 45% 172% 278% S18T, L177H, A281V, E283K 1618.20 44% 164% 270% C63R, L177H, A281V, E283K 1275.38 44% 112% 196% S18T, C63H, L65F, S96T, L177H A281V, E283K 912.50 44% 83% 199% S18T, S96T, L177II, Ε283Κ 1814.34 43% 160% 194% S18T, L179H, A281V, E283K 1616.51 43% 164% 264% S18T, C63H, S96T, L177H 2088.12 43% 189% 197% L65F, S96T, A281V, E283K 1638.28 43% 144% 191% C63H, L65F, A281V, E283K 632.66 43% 56% 190% S18T, C63Y 1640.33 43% 166% 260% L65F, S96T, LI 7711, A281V, E283K 1585.32 42% 140% 189% C63Y, L65F, L177H 639.46 41% 56% 184% C63H, L65F, S96T, A281V 1177.76 41% 119% 252% T.11F, C63Y, S96T, L177H, A281V A282T, E283K 1578.54 41% 143% 186% S18T, C63Y 1797.99 39% 182% 241% S18T, L178H, A282V, E284K 1502.59 39% 152% 237% G23S, C63H, S96T, L177H, E283K 1229.34 39% 125% 237% S18T, C63H, L177H 1811.40 39% 160% 173% C63Y, A281V, A282T 1622.23 38% 143% 168% S18T, C63Y, L65F, S96T, L177H A281V, E283K, M284I 454.42 35% 41% 162% S18T, L65F, S96T, L177H, A281V 1135.95 33% 115% 203% L65F, L178H, G180S, E283I < 151.40 33% 13% 146% L66F, L177H, G180S, E283K 143.83 31% 13% 138% S18T, C63H, L65F, L177H 103.21 30% 9% 133% Saturation Libraries Title FALC ¹ (total *) ¹ C14-FALC.% Fraction) Standardized IFALC [% under control) C14 Normalized under control) S18W 2530 55% 227% 314% S18F 2564 54% 222% 285% V17N 571 49% 49% 260% S18L 1568 48% 141% 271% S18L S18L 1024 1326 47% 47% 92% 119% 268% 268% S18L 1302 47% 117% 267% S18L 1106 47% 99% 267% S18L 1315 47% 118% 266% S18L 1359 47% 122% 266% S18Y 2550 45% 221% 236% S18Y 2039 44% 188% 252% S18Y 1997 44% 179% 251% S18M 2495 41% 224% 231% S18M 2809 40% 243% 212% VI7L 1127 33% 97% 175%

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V17L 1185 33% 103% 173% VI7L 1091 32% 94% 171% VI7L 1175 32% 102% 171% S18T 1729 31% 155% 177% S18T 1690 31% 152% 176% S18T 1683 31% 151% 176% S18T 1599 30% 143% 172% S18V 1344 30% 121% 170% S18V 1306 30% 121% 170% S18V 1015 30% 91% 169% S18C 1381 28% 120% 149% S18C 1368 28% 118% 148% R19H, R58S 873 27% 81% 152% VI7C 927 27% 80% 140% V17C 882 26% 76% 139% S18C 1313 26% 118% 149% S18C 1355 26% 121% 148% S18C 1305 26% 117% 148% S18C 1372 26% 123% 147% S18C 1340 26% 120% 147% V17W 849 25% 73% 134% V17W 815 25% 71% 133% VI7W 831 25% 72% 132% V17W 840 25% 73% 131% R19V 844 24% 78% 139% R19V 813 24% 75% 136% R19V 821 24% 76% 135% R19V 862 24% 80% 135% R19V 814 24% 75% 135% R19K, V117L 1188 23% 110% 130% R19S 803 22% 74% 128% R19T 735 22% 68% 127% R19S 783 22% 72% 127% R19S 764 22% 75% 129% IR 91 862 22% 80% 123% R19A 901 21% 83% 122% R19A 705 21% 65% 119% R19M 937 20% 86% 114% Β Combo Library Title FALC 1 (total *) C14-FALC (% fraction ^ Normalized FALC% under control) C14 Normalized ;% under control) 296% S18W 3184 57% 228% S18W, C63Y 2675 54% 191% 280% S18W, C63Y, S95T 2832 54% 203% 279% S18W, S96T, E283K 2874 54% 206% 278% S18W, C63Y, E283K 2915 51% 209% 263% S18W, S96T, E283K, R286Q, A324V 3437 49% 246% 255% C63Y, S96T 2583 38% 185% 196% C63Y, E283K 2176 38% 156% 195% C63Y, E283K, Q316K 2420 36% 173% 188% E283K 1474 25% 105% 132% S96T 1605 23% 115% 121% Combo Library 3 FALC title (total *) C14-FALC (% fraction) Normalized FALC% under control) C14 Normalized (% under control) S18W 2853 217% 51% 274% Y23N 954 72% 35% 191% Y23N, Q238H 892 68% 35% 190% G150D, I274N 766 62% 30% 162% I274N 1359 107% 29% 161% I274N, A285V 1317 103% 28% 155% P38T, I274N, A285V 587 46% 26% 143% Q238H, M291V 1383 109% 22% 119% M291V 1618 123% 22% 118% G151D, M291V 1302 105% 21% 114% T135M. M291V 1265 100% 18% 101%

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Example 4: Saturation library prepared using AAR (S18W) as a model [140] The total saturation library of an acyl-ACP reductase variant from Synechococcus elongatus PCC7942 (AAR (S18W) _7942), was built and tracked for the variants which showed improvements over AAR (S18W), identified as significantly improved AAR variant in the first round of screening (Examples 2 and 3). The selection criterion was an increase in the FALC titre or an increase in the percentage fraction of C12. Manipulation efforts have focused on easing AAR's reliance on ACP overexpression to produce high titles. Although not wishing to be limited by theory, the advantage observed in strains overexpressing ACP was raised the hypothesis of resulting from a higher concentration of fatty acid biosynthesis intemiedaries available for AAR cleavage. By screening saturation and combination libraries built on AAR in a strain without ACP overexpression (having a lower concentration of FAS intermediates), variants with higher affinities for FAS intermediates can be selected.

[141] The plasmid used to prepare the complete saturation library was designated pAAR-1. It is a derivative of pLS9-185 harboring the AAR gene encoding the variant (S18W) followed by the aldehyde reductase gene, AlrA, from Acinetobacter baylyi (SEQ ID NO: 54). Alra was added to totally reduce the fatty aldehyde intermediates generated by AAR and not fully reduced by reductase fatty aldehyde

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126/137 endogenous of E. coli. The total saturation library was screened in the Shu.002 strain. The Shu.002 strain is DV2 PT5-ifabl38 PT5_ifadR (Table 7, infra). For ifab138 see SEQ ID NO: 55 in Table 10. The libraries were screened using one of the standard protocols described above. The improvements were classified either as another improved titer or as an increase in the fraction of C12 fatty alcohols produced by acyl-ACP reductase without significantly affecting the titer. The results from the tracking saturation libraries are shown in Table 5 below.

Table 5: Mutations from a complete AAR Saturation Library (S18W) 7942 correlated with the increase in the Fatty Alcohol title and / or increase in the C12 Fatty Alcohol fraction

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AAR mutations (in addition to S18W) FALC Title * C12 Total* FIOC ** Fraction * FIOC ** T148V 747 1.21 7.9% 0.99 A159V 702 1.14 7.0% 0.87 T157V 674 1.10 7.2% 0.90 A135S 803 1.31 7.8% 0.98 A328S 712 1.16 8.1% 1.02 Q191A 780 1.27 7.7% 0.96 A285V, M291V 837 1.36 8.0% 1.00 Q277V 854 1.39 7.9% 0.99 Q155C 626 1.02 8.3% 1.04 E210Y 676 1.10 8.5% 1.06 T120S 623 1.01 8.1% 1.02 T236C 691 1.12 7.6% 0.95 Q335M 763 1.24 6.8% 0.86 C172L 700 1.14 7.7% 0.97 E283S 858 1.39 8.0% 1.00 L209R 837 1.36 7.6% 0.96 I153P 606 0.99 8.0% 1.00 A211W 797 1.30 6.9% 0.86 A324T 909 1.48 4.0% 0.50 W34F 817 1.22 7.5% 0.98 T187V 825 1.23 6.9% 0.91 D24E 737 1.10 7.1% 0.93 T188H 862 1.29 8.6% 1.13 V18A 798 1.19 6.4% 0.84 V18A, L338W 732 1.27 7.6% 0.97 T188V 775 1.34 7.4% 0.95 I168V 731 1.27 8.1% 1.03 W35F 666 1.15 7.5% 0.96 T148V 836 1.45 6.2% 0.80 Q155L 740 1.28 8.5% 1.08 T148C 731 1.26 8.6% 1.10 T148B 694 1.20 8.3% 1.06 A50Q, L337V 800 1.38 7.2% 0.92 R118Q 724 1.25 7.3% 0.94 T, 3 1 V 901 1.56 6.8% 0.87 A135S 775 1.34 8.7% 1.12 D24Y 890 0.93 19.1% 2.05 C63A 895 0.94 16.1% 1.73 S113K 812 0.85 13.3% 1.43 B31V 645 0.68 15.7% 1.69 E283G 917 0.96 14.3% 1.54 A112R 9J 8 0.96 10.7% 1.15 D43E 1002 1.05 13.0% 1.39 Q116G 894 0.94 14.0% 1.50 S86G 849 0.89 13.4% 1.44

* Average from 4 repetitions ** FIOC or times increased under AAR control (S18W)

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Example 5: Combination Libraries prepared using AAR (S18W) as a model [142] Conventional techniques known to those skilled in the art have been used to prepare combination libraries. The mutations tested in combination libraries (Tables 6A, 6B and 6C) were initially identified in the complete saturation library (Example 4). The combined libraries were constructed on the same plasmid and screened on the same strain, as described in Example 3. The libraries were screened using one of the standard protocols described above. The improvements were classified either as another improved titer or an increase in the fraction of C12 fatty alcohols produced by acyl-ACP reductase without significantly affecting the titer. The results of screening AAR combination libraries are shown in Tables 6A, 6B and 6C below.

Table 6A: Mutations from the Combination Library from I ^to AAR (S18W) 7942 correlated with the increase in the titer of

Fatty Alcohol

AAR mutations (in addition to S18W) FALC Title * C12 mg / L * FIOC ** Fraction* FIOC ** A281L 1478 1.29 14.1% 1.11 A281Y 1527 1.28 12.1% 0.96 T154A, A281L 1550 1.28 14.3% 1.13 T154A. A281Y 1384 1.19 13.3% 1.05 C63G, A281F 1472 1.24 12.9% 1.02 C63G, A281L 1432 1.19 18.2% 1.44 N83G, Α28 ΓΥ 1428 1.23 10.7% 0.84 C63G, A281Y 1419 1.21 15.6% 1.24 S10G, A281L 1419 1.20 12.6% 1.00 S113K, Q155G 1305 1.15 12.3% 0.97 D43E, A50Q, A281L 1517 1.32 9.1% 0.72 M21L, N83G, A281Y 1513 1.29 10.2% 0.81 M21L, S113K, A281L 1392 1.20 20.4% 1.61 C63G, T154A, A281F 1369 1.24 12.4% 0.98 C63G, N83G, A281Y 1330 1.32 14.6% 1.16 Q155G, A281L, E283G 1327 1.16 22.2% 1.76 A50Q, C63G, A281F 1286 1.15 17.9% 1.41 S18W, E) 43E, A50Q, A281L 1635 1.32 9.4% 0.77 M21L, C63G, S113K, T154A A281L 1518 1.24 28.3% 2.23 M21L, L31V, N83G, Q116G A281L 1353 1.18 15.3% 1.21

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129/137 * Average of 4 repetitions ** FIOC = increase times over control (S18W)

Table 6B: Mutations from the 2nd ^to AAR Combination Library (S18W) _7942 correlated with the increase in the Fatty Alcohol title

AAR mutations (in addition to S18W) Title FALC * C12 mg / L * FIOC ** Fraction* FIOC ** S18W (control) 1034 1.00 10.0% 1.00 M21L, C63G, S113K, T154A, A281L 1179 1.14 29.4% 2.94 D16L, M21L, C63G, S113K, T154A, A281L 1221 1.18 19.4% 1.94 L8A, D24V, C63G, S113K, Q155L, A281L 1414 1.37 10.4% 1.04 L8A, M21L, C63G, A77A ***, S113K, T154A A281L 1310 1.27 14.2% 1.42 D24P, L31M, C63G, S113K, T154A, A281L 1437 1.39 13.0% 1.30 L8A, D16L, D24V, C63G, S113K, T154A A281L 1342 1.30 12.0% 1.20 D24E, C63G, S113K, T154A, A281L 1425 1.38 14.3% 1.43

* Average of 4 repetitions ** FIOC = increase times over control (S18W) *** A77A mutation is a silent codon mutation gcc a

GCA

Table 6C: Mutations of the AAR Combination Libraries (S 18W) _7942 correlated with increased alcohol fraction

Fatty Cl2

AAR mutations (in addition to S18W) C12 Title FALC * Fraction* FIOC ** FIOC ** S18AV (control) 11.0% 1.00 1.00 M21L, C63G, S113K, T154A, A281L 28.3% 2.57 1.24 M21L, Q155G, A281L 24.8% 2.25 1.10 Q155G, A281L, E283G 22.2% 2.02 1.16 M21L, S113K, A281L 20.4% 1.85 1.20 C63G, A281L 18.1% 1.65 1.18 A50Q, C63G, A281F 17.9% 1.63 1.15 C63G 17.7% 1.61 1.00 S10G, C63G, Q155G, S253N, A281F 16.1% 1.46 1.03 D43E, C63G, S113K, A281L 16.0% 1.46 0.98 A281L 15.6% 1.42 1.20 M21L, L31V, N83G, Q116G, A281L 15.3% 1.39 1.18 C63G, N83G, A281Y 14.6% 1.33 1.32 A281L] 14.1% 1.28 1.29

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130/137 * Average of 4 repetitions ** FIOC = increase times over control (S18W)

Example 6: Increased flow through the Fatty Acid Synthesis Pathway - iFab and iFadR mediated by the production of Fatty Alcohol Using AAR [143] In this example, improved fatty alcohol production using AAR was demonstrated by increased flow through the pathway fatty acid biosynthesis mediated by the overexpression of a synthetic operon comprising several FAB proteins (ifabl38) and / or overexpression of the FadR protein (ifad), a regulator of fatty acid metabolism. ÍFAB138 (SEQ ID NO: 55) includes in the following order the fabV genes from Vibrio cholerae, FabH, fabD, fabG and fabA from Salmonella typhimurhim, and FabF from Clostridium acetobutylicum, and is integrated as a synthetic operon controlled by or PlacUV5 promoter or PT5 promoter. iFadR includes the f adR gene from Escherichia coli (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 ÍFAB138 or fadR

Components DV2 BD061 BD064 Shu002 i (PlacUV5-fabl38) + “L · - i (PT5-fabl38) - + i (PT5-fadR) - - + +

[144] AAR (S18W) was expressed from plasmid pDS311, a variant of plasmid pDS171S, where codon AAR 18 specified a tryptophan instead of a serine (PCL-AAR (S18W) + ACP-sfp) and the gene of aldehyde reductase, alrA, from Acinetobacter baylyi (SEQ ID NO:

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54) was cloned downstream of the sfp gene from B. subtilis. pDS311 was transformed into strains DV2, BD061, BD064 and Shu002. Strains were evaluated using the 4NBT_2N protocol (see above). As shown in Figure 7, fatty alcohol production increased significantly as a result of the presence of ifabl38 and ifadR, with the strain Shu002 showing the highest fatty alcohol titer.

Example 7: Improved AAR variants in a strain with increased flow through the fatty acid synthesis pathway [145] In this example, improved fatty alcohol production in recombinant host cells transformed with AAR variants from combination libraries has been demonstrated in Shu2, an E. coli strain with increased flow through the fatty acid biosynthesis pathway mediated by overexpression of ifabl38 and overexpression of FadR protein (supra). Four variants of AAR were evaluated from the second combination library (Table 6B). 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, C63G, A77A, S113K, T154A, A281L; Com2d: D24E, C63G, S113K, T154A, A281L (see also Figure 8). These variants were cloned into the structure of plasmid pJL104. pJL104 was created by cloning the synthetic accD + operon from C. glutamicum (as described in Example 2, supra), downstream of the alrA gene in pDS311. The resulting plasmids were transformed into the strain Shu002 and the strains were evaluated in the 4NBT_1N protocol (supra). As shown in Figure 8, high fatty alcohol production was observed in all strains. The BD064 strain houses the

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132/137 plasmid pDS311, which expresses the S18W variant of AAR_7942, was also evaluated in tank fermentations. Fatty alcohols produced per strain with a maximum titer of 42 g / L, a yield of 12.6% (in glucose) and a yield of 0.62 g / L / h, as shown in Figure 9. The length distribution of the fatty alcohol chain was as follows: 1.2% C8, 7.7% C10, 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 Chain Length Selectivity for C14 fatty alcohol production [146] AAR is one of the essential components for the cyanobacterial alkane biosynthesis, another essential component is an aldehyde decarbonylase (ADC). The inventors found that Prochlorococcus marinus MED4_AAR is catalytically inactive without the presence of MED4_ADC, that is, when only MED4_AAR is expressed in E. coli no product is detected, and when MED4_AAR and ADC are coexpressed in E. coli only the detected products are alkanes. The inventors also found that when MED4_AAR is coexpressed in E. coli with an apparently inactive catalytic variant of MED4_ADC, where histidine 156 is replaced by arginine (later referred to as MED4_ADC (H165R)), fatty alcohols, and are not detected alkanes (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 the system to identify variants of MED4_AAR with increased activity and / or specificity by

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133/137 substrate changed for FALC production purposes.

[147] MED4_ADC (H156R) was expressed in conjunction with a total saturation library of MED4_AAR. The MED4_AAR saturation library was prepared on a pLC1920-derived plasmid (pLS9-195) and introduced into a production strain carrying plasmid pGLAK-043 (which is the plasmid pACYC-Ptrc-MED4_ADC harboring the H156R mutation in an ADC gene) . The clones were induced and the AAR variants were selected based on the production of more fatty alcohol than the wild type AAR enzyme or the ability to produce fatty alcohols with an altered chain length profile, for example, an increase in the fraction of fatty alcohol C14. Selected clones were then retested in a validation round. All variants that showed consistent FALC titers between primary and secondary fermentations were recultivated, plasmid DNA was isolated, sequenced and reintroduced into the original production strain for further testing. These new transformants were then subjected to another analysis and confirmation fermentation. Table 8 below shows representative data from 16 AAR variants that produced the largest FALC titles (ordered from top to bottom in the descending activity). The variants ranged from 1.4 times to 2.2 times in relation to the wild type. These variants showed the ability to increase MED4_AAR activity using targeted evolution techniques and, in addition, formed the basis for further advances.

Table 8: FALC productivity of MED4 AAR mutants compared to wild-type MED4 AAR (WT)

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AAR mutations FALC increase times over Selvaaem type V346P 2.2 Q40V 2.2 A345R 2.1 L344S 2.1 D61E 2.1 V346G 2.0 L344D 1.9 G52V 1.9 A345 * 1.9 L344T 1.8 K303G 1.8 L344A 1.8 H340P 1.6 S588V 1.5 K339L 1.4 G273E 1.4

* Truncated variant missing the last two amino acids.

[148] The data set was also scanned for AAR variants that exhibited altered chain length profiles. The most common species produced by wild-type AAR have a C16 chain length. An increase in the proportion of 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 these clones revealed that they were identical D61E mutants with the same nucleotide codon sequence. The plasmid DNA for the D61E variant was reintroduced into the original strain containing H156R ADC. The results show that the expression of the D61E variant of AAR in a recombinant host cell distorts the

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135/137 chain length distribution of FALC species to shorter carbon chains. The Table below illustrates the chain length distribution

FALC produced by recombinant host cells that express the variant

D61E of

MED4 AAR compared to MED4_AAR of the wild type (WT) and the variant

V34 6P of MED4 AAR that did not produce any products with altered chain lengths.

Table 9: Chain length distribution by AAR and wild type AAR variants

MED4 AAR / ADC Alcohol Cl 4% Fatty Cl 6% Cl 8% AAR WT / ADC (H156R) 6 92 2 AAR (D61E) / ADC (H156R) 14 * 85 0 AAR (V346P) / ADC (H156R) 4 92 4 AAR WT / ADC WT ND ND ND AAR (D61E) / ADC WT NI) ND ND AAR (V346P) / ADC WT ND ND ND

ND ⁼ not detected * 1% variance attributable to deviations from the mean [149] These variants can still be recombined and tracked to improve the MED4_ADC foundation (H156R). The MED4_AAR variant (D61E) and its mutant progeny may be useful to decrease the average chain length of both fatty alcohols and alkanes. The MED4_AAR (D61E) variant demonstrates that the MED4_AAR chain length specificity is malleable and presents the possibility of further improving this activity through additional protein manipulation efforts. All described variants were sequenced from the progeny of pLS9-195 and contained codon mutations

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136/137 corresponding to the listed amino acid substitutions.

Table 10: Names related to the Sequence Listing

SEQ ID NO Type Name 1 acid sequence Nostoc spot PCC 73102 acp Access # γρ 001867863 2 sea.deaminoàcido Nostoc spot PCC 73102 acp Access # γρ 001867863 3 aconucleic sequence Synechocystis sp. PCC 6803 acp Access # NP 440632.1 4 seq. deaminoacid Synechocystis sp. PCC 6803 acp Access # NP 440632.1 5 nucleic acid sequence Prochlorococcus marintis subsp. pastoris str. CCMP1986_acp Acessoi # NP 893725.1 6 sea-deaminoacid Prochlorococcus marinus subsp. pastoris str. CCMP1986_acp Access # NP 893725.1 7 sequential acid mnrl pí nr '. l- Synechococcus elongatus PCC 7942 acp Access # γρ 399555 8 seq.deamino acid Synechococcus elongatus PCC 7942 acp Access # YP 399555 9 seq. from nuclpicn acid to Nostoc sp. PCC 7120 acp Access # NP 487382.1 10 seq.deamino acid Nostoc sp. PCC 7120 acp Access # NP 487382.1 11 acid sequence B. subtilis sfp (synthesized) as in access # X63158.1 12 seq.deamino acid B. subtilis SÍp (synthesized) as in access # X63158.1 13 seq. initiator 168IFF 14 iseq. initiator 168IFR 15 seq. initiator 169IFF 16 seq. initiator 169IFR 17 iseq. initiator 170IFF 18 seq. initiator 170IFR 19 sea. initiator I171IFF 20 seq. initiator 171IFR 21 seq. initiator 172IFF 22 seq. initiator 172IFR 23 seq. initiator 168SIFF 24 seq. initiator 170S1FF 25 seq. initiator 171SIFF 26 seq. initiator 168SIFR 27 seq. nucleic acid Synechococcus elongatus PCC7942 YP 400611 (Synpcc7942 1594) Acyl-CoA Reductase (AAR) 28 seq. nucleic acid Synechococcus elongatus PCC7942 YP 400611 (Synpcc7942 1594) Acyl-CoA Reductase (AAR) 29 seq. d.e nucleic acid Synechocystis sp. PCC6803 S110209 (NP 442146) AAR 30 s and q. arri i η o c i do Synechocystis sp. PCC6803 S110209 (NP 442146) AAR 31 seq. d.e nucleic acid 1 Cyanothece sp. ATCC51142 cce 1430 (YP 001802846) AAR 32 ---------1 seq.deamino acid Cyanothece sp. ATCC51142 cce 1430 (YP 001802846) AAR 33 seq. nucleic acid Prochlorococcus marinus CCMP 1986 PMM0533 (NP 892651) AAR 34 seq. acidic Prochlorococcus marintis CCMP 1986 PMM0533 (NP 892651) AAR 35 seq. nucleic acid Gloeobacter violaceus PCC7421 NP 96091 (gll3145) AAR 36 seq.deamino acid Gloeobacter violaceus PCC7421 NP 96091 (gll3145) AAR 37 seq. nucleic acid Nostocpunctiforme PCC73102 ZP 00108837 (Npun02004176) AAR 38 seq. acidic Nostoc spot PCC73102 ZP 00108837 (Npun02004176) AAR 39 seq. nucleic acid Anabaena variabilis ATCC29413 YP 323044 (Ava 2534) AAR 40 seq.deaminoàcido Anabaena variabilis ATCC29413 YP 323044 (Ava 2534) AAR 41 seq. nucleic acid Synechococcus elongatus PCC6301 YP 170761 (syc0051 d) AAR 42 seq. acidic Synechococcus elongatus PCC6301 YP 170761 (syc0051 d) AAR 43 seq. cte nucleic acid Nostoc sp. PCC7120 alr5284 (NP 489324) AAR 44 seq.deaminoàcido Nostoc sp. PCC7120 alr5284 (NP 489324) AAR - 45 seq. nucleic acid _x birA ~ ã l Corynebacterium glutamicum (YP 224991) 46 Synthetic DNA birA of _t Corynebacterium glutamicum (YP 224991) 47 seq.deaminoàcido birA 4θ Corynebacterium glutamicum (YP 224991) 48 seq. d.e nucleic acid ± accDAl (dtsR} 4θ Corynebacterium glutamicum (YP 224991) 49 seq.deaminoàcido accDAl (<7 / .vR) from Corynebacterium glutamicum (YP 224991) 50 seq. d.e acid 1 nucleic «CcCB of Corynebacterium glutamicum (YP 224991) 51 seq.deaminoàcido occCB: 'de. Corynebacterium glutamicum (YP 224991) 52 seq.deamino acid AlrA Acinetobacter sp. M-l 53 amino acid sequence AlrAadpl 54 seq.deamino acid alrAadpl Acinetobacter baylyi ADP1-WT Protein 55 seq. cfe nucleic acid IFAB138 56 seq. nucleic acid E.coli MG1655 FadR (NP 415705) 57 seq.deamino acid AAR mutant with S18W mutation (prepared from SynechoCOCCllS elongatUS PCC7942 YP 400611 (Synpcc7942 1594) Acyl-CoA Reductase (AAR)) |

Petition 870170002722, of 13/01/2017, p. 141/161

137/137

[150] According It's evident for an expert at various modifications and variations of aspects and above modalities can be made without get away of scope and scope of this description. Such modifications and variations are within the scope of this disclosure.

Petition 870170002722, of 13/01/2017, p. 142/161

Claims (12)

1. Acyl-ACP reductase (AAR) variant polypeptide, CHARACTERIZED by the fact that it comprises at least 90% sequence identity for the amino acid sequence of SEQ ID NO: 65, wherein said AAR variant polypeptide comprises a mutation at the position of amino acid 61, where the mutation is D61E, and where said AAR polypeptide catalyzes the conversion of an acyl-ACP to a fatty aldehyde. 2. AAR variant polypeptide according to claim 1, CHARACTERIZED by the fact that expression of the AAR variant polypeptide in a recombinant host cell results in a higher titer of a fatty aldehyde or fatty alcohol composition when compared with a title of a fatty aldehyde or fatty alcohol composition produced by the expression of a wild type AAR polypeptide in a corresponding wild type host cell. 3. AAR variant polypeptide according to claim 1 or 2, CHARACTERIZED by the fact that said fatty alcohol composition is a C12, C14 or C16 fatty alcohol composition, or a combination thereof. 4. Recombinant host cell, CHARACTERIZED by the fact that it expresses the polypeptide of the AAR variant, as defined in any one of claims 1 to 3. 5. Recombinant host cell according to claim 4, CHARACTERIZED by the fact that the recombinant host cell produces a fatty aldehyde or fatty alcohol composition with a titre that is at least 10% greater, at least 15% greater, at least 20% bigger, at least 25% bigger, or at least 30% bigger than the title Petition 870170002722, of 13/01/2017, p. 143/161 2/3 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 for express the variant AAR polypeptide. in wake up with The 6. Host cell recombinant, claim 5, CHARACTERIZED by fact in what The fatty alcohol composition is produced with a title in about 30 g / L to about 250 g / L. 7. Host cell recombinant, in wake up with The claim 5 or 6, CHARACTERIZED by the fact that the fatty alcohol composition is released from the cell. 8. Cell culture, CHARACTERIZED by the fact that it comprises the recombinant host cell, as defined in any of claims 4 to 7. 9. Cell culture according to claim 8, CHARACTERIZED by the fact that the fatty alcohol composition comprises one or more of a C6, C8, C10, C12, C13, C14, C15, C16, C17 and C18 fatty alcohol. 10. Cell culture according to claim 9, CHARACTERIZED by the fact that the fatty alcohol composition comprises one or more of an unsaturated fatty alcohol C10: 1, C12: 1, C14: 1, C16: 1 and C18: 1 . 11. Cell culture, according to claim 9, CHARACTERIZED by the fact that the fatty alcohol composition comprises an unsaturated fatty alcohol. 12. Cell culture according to claim 9, CHARACTERIZED by the fact that the fatty alcohol composition comprises a fatty alcohol having a double bond at position 7 in the carbon chain between C7 and C8 from Petition 870170002722, of 13/01/2017, p. 144/161 3/3 reduced end of fatty alcohol. 13. Cell culture according to claim 9, CHARACTERIZED by the fact that the fatty alcohol composition comprises a saturated fatty alcohol. 14. Method of producing a fatty alcohol composition with an increase in the title, FEATURED by the fact that it comprises: i. cultivating the host cell, as defined in claim 4, with a carbon source; and ii. collect a fatty alcohol composition. 15. Method, in wake up with the claim 14, CHARACTERIZED BY fact that the title of fatty alcohol is at least 20% a 30% bigger than the title of an composition of alcohol fatty produced by a cell hostess expressing wild type AAR. 16. Method, in wake up with the claim 14, CHARACTERIZED by the fact that the said fatty alcohol composition has an increased fraction of C14 fatty alcohols.