WO2023178197A1

WO2023178197A1 - Recombinant microbes for production of trans-2 unsaturated fatty acids and derivatives thereof

Info

Publication number: WO2023178197A1
Application number: PCT/US2023/064465
Authority: WO
Inventors: Andreas W. Schirmer; David Ryan GEORGIANNA; Erin Frances PERRY
Original assignee: Genomatica, Inc.
Priority date: 2022-03-16
Filing date: 2023-03-15
Publication date: 2023-09-21

Abstract

Recombinant microbes comprising a novel pathway for producing trans-2 fatty acids and derivatives thereof are provided herein. The recombinant microbes comprise (1) either a combination of a 3-hydroxy acyl-ACP thioesterase and an acyl-CoA synthetase, or a 3-hydroxy acyl-ACP:CoA transacylase; (2) a heterologous R-3-hydroxy-acyl-CoA dehydratase or a heterologous R-specific enoyl-CoA hydratase; and, optionally (3) a fatty acid derivative enzyme, such as, for example, an ester synthase, an acyl-CoA reductase, or an acyl-CoA thioesterase. Methods of producing trans-2 fatty acids or derivatives thereof are also provided, in addition to cell cultures and fatty acid compositions produced by the recombinant microbes. The trans-2 fatty acids and derivatives thereof produced by the recombinant microbes may be used to produce fragrances, flavors, pheromones, pharmaceutical agents, nutraceuticals, or precursors thereof.

Description

RECOMBINANT MICROBES FOR PRODUCTION OF TRANS-2 UNSATURATED

FATTY ACIDS AND DERIVATIVES THEREOF

REFERENCE TO AN EEECTRONIC SEQUENCE EISTING

[0001] This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted concurrently herewith as the sequence listing xml file entitled “ST26_SL_15_Mar_2023.xml”, file size 48 KiloBytes (KB), created on 15 March 2023. The aforementioned sequence listing is hereby incorporated by reference in its entirety.

FIEED

[0002] The disclosure relates to the field of specialty chemicals and methods for their preparation. The disclosure provides recombinant microbes engineered to express:

(1) either a combination of a 3-hydroxy-acyl-ACP thioesterase and an acyl-CoA synthetase, or a 3 -hydroxy-acyl- ACP:CoA transacylase; and

(2) a heterologous R-3-hydroxy-acyl-CoA dehydratase or a heterologous //-specific enoyl- CoA hydratase. In some embodiments, the recombinant microbes further comprise:

(3) an ester synthase, an acyl-CoA reductase, or an acyl-CoA thioesterase.

The disclosure further provides pathways and methods for the biological production of various trans-2 unsaturated fatty acids and derivatives thereof, and compositions comprising the same.

BACKGROUND

[0003] Trans-2 fatty acids and derivatives thereof have a trans-double bond in the 2-position. Trans-2 fatty acids and derivatives thereof, such as trans-2 aldehydes (e.g., trans-2 decenal, trans- 2 nonenal, and trans-2 dodecenal) and trans-2 esters (e.g., methyl-2-nonenoate or methyl-2- decenoate), have various applications and are of particular use as components in flavors and fragrances (see e.g., Wright (2010) Perfumer and Flavorist). Interestingly, trans-2-decenoic acid ethyl ester has been reported to have neuroprotective pharmacological properties (see e.g. , Tanaka (2012) Int. J. Mol. Sci.). Additionally, 10-hydroxy-trans-2-decenoic acid, also known as queen bee acid, is the bioactive component of royal jelly, and is marketed as a dietary supplement (see e.g., Weiser (2017) Nutrients).

[0004] Trans-2 fatty acids and derivatives thereof are minor components of naturally produced lipids, despite the fact that thioesters of trans-2 fatty acids (i.e., trans-2-enoyl-ACP and trans-2- enoyl-CoA) are intermediates in fatty acid biosynthetic pathways. FIG. 1A shows the reductive cycle of acyl-ACP-dependent fatty acid biosynthesis. FIG. IB shows the reductive cycle of acyl- ACP-independent fatty acid biosynthesis. The acyl-ACP in these pathways can be elongated with malonyl-ACP, catalyzed by 3-keto-acyl-ACP synthase (FIG. 1A), or elongated with acetyl-CoA or malonyl-CoA by a thiolase or elongase (FIG. IB) (see, e.g., Zhang and Rock, (2008) Nature Rev. Microbiol, vol. 6: 222-233; Dellomonaco (2011) Nature, 476:355-9; Lynch, W02015/010103; Dittrich (1998) Eur. J. Biochem. 252, kh477-485).

[0005] The reductive cycle of fatty acid biosynthesis can be manipulated to direct synthesis of particular products. The “driving force” of the reductive cycle is the last step carried out by trans-2 enoyl-thioester reductase (i.e., trans-2-enoyl-ACP reductase or trans-2-enoyl-CoA reductase) (see e.g., Heath and Rock, J. Biol. Chem, (1995) 270:26538-26542). The reversible enzymatic reactions leading to the enoyl-thioester intermediates are catalyzed by 3-hydroxy-acyl- thioester dehydratases or enoyl-thioester hydratases. Formation of the 3-hydroxy-acyl-thioester intermediate is thermodynamically favored. Thus, trans-2 enoyl-thioester reductases efficiently convert the trans-2 enoyl thioester substrate to acyl-ACP or acyl-CoA, and drive the trans-2 enoyl thioester intermediates towards fully reduced acyl-thioesters, which are then elongated, rather than towards trans-2 fatty acids.

[0006] The activity of trans-2 enoyl thioester reductases pose a problem if the artisan seeks to produce high titers of trans-2 fatty acids or derivatives thereof from the fatty acid biosynthetic pathways. Thioesterases or acyl-transferases are necessary to produce trans-2 fatty acids or derivatives thereof. However, trans-2 enoyl thioester reductases have a greater affinity for the trans-2 enoyl thioester substrates than the thioesterases or acyl-transferases. However, attenuating trans-2 enoyl thioester reductase activity may lead to a reduced rate of fatty acid biosynthesis and chain elongation and/or to an accumulation of 3-hydroxy acyl thioester intermediates (i.e., 3- hydroxy-acyl-ACP or 3-hydroxy-acyl-CoA), leading to production of 3-hydroxy fatty acids rather than trans-2 fatty acids.

[0007] Currently, methods for producing short chain trans-2 carboxylic acid (C4 or C5 chain lengths) rely on a thiolase reaction to condense two molecules of acetyl-CoA with thiolase to produce crotonic acid, or to condense acetyl-CoA and propionyl-CoA to produce trans-2 pentenoic acid. A 3-hydroxy-acyl-CoA dehydrogenase and a dehydratase complete the reaction. This synthetic scheme occurs outside the fatty acid biochemical pathway. There remains a need to efficiently produce trans-2 fatty acids, particularly with a C6-C18 chain length.

SUMMARY

[0008] This disclosure provides a novel biochemical pathway (for example, see FIG. 2) that combines the acyl-ACP dependent fatty acid biosynthetic pathway with two steps from the acyl- CoA dependent pathway. This novel pathway circumvents the disadvantages described above to efficiently produce various trans-2 fatty acids and derivatives thereof.

[0009] Disclosed herein is a recombinant microbe comprising: (1) either a combination of a 3-hydroxy acyl-ACP thioesterase (that uses 3-hydroxy acyl-ACP as a substrate) and an acyl-CoA synthetase, or a 3-hydroxy acyl ACP:CoA transacylase; and (2) a heterologous R-3-hydroxy-acyl- CoA dehydratase or a heterologous R-specific enoyl-CoA hydratase; and, optionally, (3) one or more fatty acid derivative enzymes, such as, for example, an ester synthase, an acyl-CoA reductase, or an acyl-CoA thioesterase. The recombinant microbe may further comprise a P-keto- acyl-ACP synthase I, an enoyl- ACP reductase, and/or a FadR. In some embodiments, the activity of acyl-CoA dehydrogenase (FadE) is attenuated (or deleted).

[0010] The recombinant microbe may be a recombinant bacteria (e.g., y-proteobacteria or cyanobacteria), a recombinant yeast, or a recombinant algae. The recombinant microbe may produce one or more trans-2 unsaturated fatty acids or derivatives thereof, including one or more of trans-2-hexadecenoic acid, trans-2-hexadecenoic acid ethyl ester, trans-2-hexadecenoic acid methyl ester, trans-2-tetradecenoic acid, trans-2-tetradecenoic acid ethyl ester, trans-2- tetradecenoic acid methyl ester, trans-2-dodecenoic acid, trans-2-dodecenoic ethyl ester, trans-2- dodecenoic acid methyl ester, trans-2-decenoic acid, trans-2-decenoic acid ethyl ester, trans-2- decenoic acid methyl ester, trans-2-octenoic acid, trans-2-octenoic acid ethyl ester, and trans-2- octenoic acid methyl ester.

[0011] Also described herein are modified biosynthetic pathways and methods for producing one or more trans-2 unsaturated fatty acids or derivatives thereof, said method comprising culturing, on a carbon source, a recombinant microbe containing the enzymes/pathways described above.

[0012] Also disclosed herein are fatty ester compositions comprising trans-2 fatty acid methyl esters, fatty acid methyl esters (FAMEs), and/or 3-hydroxy-FAMEs; or comprising trans-2 fatty acid ethyl esters, fatty acid ethyl esters (FAEEs), and/or 3-hydroxy-FAEEs, wherein the predominant chain length of the fatty esters in the composition is C8, CIO, or C12.

[0013] Also disclosed herein is the use of the recombinant microbe comprising the enzymes and/or pathways described above to produce trans-2 unsaturated fatty acids or derivatives thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1A-1B depict alternative biochemical pathways for the production of trans-2 unsaturated fatty acids. FIG. 1A depicts an acyl- ACP dependent fatty acid biosynthetic pathway. FIG. IB depicts an acyl-ACP independent fatty acid biosynthetic pathway.

[0015] FIG. 2 depicts an example of a novel, efficient (modified) biochemical pathway for the production of trans-2 unsaturated fatty acid derivatives.

[0016] FIG. 3 depicts an example of a biochemical pathway for the production of trans-2 unsaturated fatty acid derivatives with an additional double bond. DETAILED DESCRIPTION

I. Definitions

[0017] The following definitions refer to the various terms used above and throughout the disclosure.

[0018] As used herein, singular articles such as “a” and “an” and “the” and similar referents in the context of describing the elements are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0019] As used herein, the term "or" is used to mean "and/or" unless explicitly indicated to refer to alternatives only or if the alternatives are mutually exclusive.

[0020] As used herein, “about” is understood by persons of ordinary skill in the art and may vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which the term “about” is used, “about” will mean up to plus or minus 10% of the particular term. As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence, “about 10%” means “about 10%” and also means “10%. ”

[0021] As will be understood by one skilled in the art, for any and all purposes, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Furthermore, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 atoms refers to groups having 1, 2, or 3 atoms. Similarly, a group having 1-5 atoms refers to groups having 1, 2, 3, 4, or 5 atoms, and so forth.

[0022] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. In particular, this disclosure utilizes routine techniques in the field of recombinant genetics, organic chemistry, fermentation, and biochemistry.

[0023] The term “fatty acid” as used herein, refers to an aliphatic carboxylic acid having the formula RCOOH wherein R is an aliphatic group having at least 4 carbons, typically between about 4 and about 28 carbon atoms. The aliphatic R group can be saturated or unsaturated and branched or unbranched. Branched aliphatic R groups may include branches comprising lower alkyl branches, such as a C1-C4 alkyl, preferably in a co-1 or co-2 position. In some embodiments, the branched aliphatic R group may be methyl in the co-1 or co-2 position. Unsaturated fatty acids may be monounsaturated or polyunsaturated. A “trans-2 fatty acid” refers to a fatty acid with a trans double bond in the 2 position, where the carboxyl group carbon is assigned position number 1. A “3 -hydroxy fatty acid” refers to a fatty acid with a hydroxy (OH) group in the 3 position, where the carboxyl group carbon is assigned position number 1. A “3-hydroxy” or “3-OH” fatty acid or fatty acid derivative can also be referred to as a “beta-hydroxy,” “beta-OH”, or “0- hydroxy” or “0-OH” fatty acid or fatty acid derivative.

[0024] The term “omega” or “co” as used herein, with respect to positioning within the carbon chain, refers to the last carbon in the chain, farthest from the carboxyl group, in a fatty acid or fatty acid derivative, or farthest from the thioester group, for example, in a fatty acyl-CoA or fatty acyl-ACP molecule. When a number is appended to the term “omega” or “co,” that number denotes the position with respect to the omega carbon. For example, a substituent at the omega- 1 (co-1) position is attached to the penultimate carbon. For example, a C12 fatty acid, with a hydroxy group at the co position can be referred to as 12-hydroxy dodecanoic acid; a C12 fatty acid with a hydroxy group at the co-1 position can be referred to as 11-hydroxy dodecanoic acid; a C12 fatty acid with a hydroxy group at the co-2 position can be referred to as 10-hydroxy dodecanoic acid, and so forth. The omega (co) numbering of the double bond position in a compound does not indicate the geometric isomerism of the compound; thus, as used herein, co7-hexadecenoic acid can have a cis or a trans double bond, or the term may refer to a mixture of cis and trans isomers thereof.

[0025] The position of a double bond within a carbon chain in any of the fatty acids or derivatives thereof provided herein also can be described by the upper-case Greek letter “A”, or “delta”, followed by a number, which refers to the position of the double bond with respect to the carboxyl group (in a fatty acid or derivative thereof), or with respect to the thioester group (in a fatty acyl-CoA or fatty acyl-ACP), where the carbon of the carboxyl or thioester group is designated as position number 1. For example, A9-hexadecenoic acid refers to a C 16 fatty acid containing a double bond between carbon numbers 9 and 10, where the carboxyl carbon is at position number 1. Similarly, A7-hexadecenoic acid has a double bond between carbon numbers 7 and 8, with the carboxyl carbon having position number 1. A7-hexadecenoic acid and A9- hexadecenoic acid can also be referred to as co9-hexadecenoic acid and co7-hexadecenoic acid, respectively. The delta (A) numbering of the double bond position in a compound does not indicate the geometric isomerism of the compound; thus, as used herein, A9-hexadecenoic acid can refer to Z9-hexadecenoic acid (or cis-9- or (9Z) -hexadecenoic acid), or to E9 -hexadecenoic acid (or trans-9- or (9E)-hexadecenoic acid), or to a mixture thereof.

[0026] The fatty acids and derivatives thereof provided herein can be described in terms of their geometric isomerism. Geometric isomers can be represented by the symbol which denotes a bond that can be a single, double, or triple bond as described herein. Provided herein are various geometric isomers and mixtures thereof resulting from the arrangement of substituents around a carbon-carbon double bond. Substituents around a carbon-carbon double bond are designated as being in the "Z" or "E" configuration wherein the terms "Z" and "E" are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting double bonds encompass both the "E" and "Z" isomers.

[0027] Substituents around a carbon-carbon double bond alternatively can be referred to as "cis" or "trans," where "cis" represents substituents on the same side of the double bond and "trans" represents substituents on opposite sides of the double bond. The term "cis" represents substituents on the same side of the plane of the ring, and the term "trans" represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated "cis/trans."

[0028] The fatty acid or fatty acids, as used herein, can be produced within a cell through the process of fatty acid biosynthesis, through the reverse of fatty acid degradation or beta (P)- oxidation, or they can be fed to a cell. As is well known in the art, fatty acid biosynthesis is generally a malonyl-CoA dependent synthesis of acyl-ACPs or acyl CoAs, while the reverse of beta-oxidation is acetyl-CoA dependent and results in the synthesis of acyl-CoAs. Fatty acids fed to cells are converted to acyl-CoAs and can be converted to acyl-ACPs. Fatty acids can be synthesized in a cell by natural fatty acid biosynthetic pathways or can be synthesized from heterologous fatty acid biosynthetic pathways that comprise a combination of fatty acid biosynthetic and/or degradation enzymes that result in the synthesis of acyl-CoAs and/or Acyl- ACPs.

[0029] The term “fatty acid derivative” as used herein, refers to a product derived from a fatty acid, or from a fatty acyl thioester, such as a fatty acyl-ACP or a fatty acyl-CoA. Thus, a fatty acid derivative is a compound that includes a fatty acid as defined above with a modification. In general, fatty acid derivatives include malonyl-CoA derived compounds including acyl-ACP or acyl-CoA derivatives. Thus, a fatty acid derivative includes alkyl-thioesters and acyl-thioesters. Further, a fatty acid derivative includes a molecule/compound that is derived from a metabolic pathway that includes a fatty acid derivative enzyme. Exemplary fatty acid derivatives include fatty acids, fatty acid esters (e.g., waxes), fatty acid methyl esters (FAME), fatty acid ethyl esters (FAEE), fatty alcohol acetate esters (FACE), fatty amines, fatty amides, fatty aldehydes, fatty alcohols, hydrocarbons (e.g., alkanes, alkenes, etc.), ketones, terminal olefins, internal olefins, 3- hydroxy fatty acid derivatives, bifunctional fatty acid derivatives (e.g., co-hydroxy fatty acids, (cohydroxy fatty acids, (co-hydroxy fatty acids, (co-hydroxy fatty acids, 10-hydroxy fatty acids, 1,3 fatty-diols, a,co-diols, a, co-3 -hydroxy triols, co-hydroxy FAME, co-OH FAEE, etc.), and unsaturated fatty acid derivatives, including unsaturated versions of each of the above mentioned fatty acid derivatives. The fatty acid derivatives can be saturated or unsaturated, and/or can be branched or unbranched. Unsaturated fatty acid derivatives can be monounsaturated or polyunsaturated. The fatty acid derivative typically contains between about 4 and about 28 carbon atoms, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 carbon atoms. A fatty acid alkyl ester can be a methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or other alkyl ester. A “trans-2 fatty acid derivative” refers to fatty acid derivatives having a trans double bond at the 2 position.

[0030] The expression “fatty acid composition” as used herein, refers to a composition of trans-2 unsaturated fatty acids or derivatives thereof, for example a fatty acid composition produced by recombinant microbes described herein, such as a recombinant proteobacterium comprising a 3-hydroxy acyl-ACP thioesterase; an acyl-CoA synthetase; one of a heterologous R- 3-hydroxy-acyl-CoA dehydratase or heterologous /^-specific enoyl-CoA hydratase; and one of an ester synthase, acyl-CoA reductase, or acyl-CoA thioesterase. A fatty acid derivative composition may comprise a single fatty acid derivative species or may comprise a mixture of fatty acid derivative species. In some exemplary embodiments, the mixture of fatty acid derivatives includes more than one type of fatty acid derivative product (e.g., fatty acids, fatty acid esters, fatty alcohols, fatty alcohol acetates, fatty aldehydes, fatty amine, bifunctional fatty acid derivatives, and non-native monounsaturated fatty acid derivatives, etc.). In other exemplary embodiments, the mixture of fatty acid derivatives includes a mixture of trans-2 unsaturated fatty acid esters (or another fatty acid derivative) with different chain lengths, saturation and/or branching characteristics. In other exemplary embodiments, the mixture of fatty acid derivatives comprises predominantly one type of fatty acid derivative e.g., trans-2 fatty acid methyl ester or a trans-2 fatty acid ethyl ester. In still other exemplary embodiments, a fatty acid derivative composition comprises a mixture of more than one type of fatty acid derivative product e.g., fatty acid derivatives with different chain lengths, saturation and/or branching characteristics. In still other exemplary embodiments, a “fatty acid derivative composition” comprises a mixture of fatty esters and 3-hydroxy esters. In still other exemplary embodiments, a fatty acid derivative composition comprises a mixture of fatty alcohols and fatty aldehydes, for example a mixture of monounsaturated fatty alcohols or fatty aldehydes. In other exemplary embodiments, the mixture of fatty acid derivatives includes a mixture of trans-2 fatty acid derivatives with different chain lengths, saturation and/or functional group characteristics.

[0031] As used herein, the expression “fatty acid derivative biosynthetic/biosynthesis pathway” refers to a biochemical pathway that produces fatty acid derivatives. The enzymes that comprise a “fatty acid derivative biosynthetic/biosynthesis pathway” are thus referred to herein as “fatty acid derivative biosynthetic/biosynthesis polypeptides” or equivalently “fatty acid derivative enzymes.” As discussed supra, and elsewhere herein, the term “fatty acid derivative” includes a molecule or compound derived from a biochemical pathway that includes a fatty acid derivative enzyme. Thus, a thioesterase enzyme (e.g. , an enzyme having thioesterase activity, such as EC 3.2.1.14) is a “fatty acid derivative biosynthetic/biosynthesis polypeptide” or equivalently, a “fatty acid derivative enzyme.” Thus, the term "fatty acid derivative enzymes" or equivalently "fatty acid derivative biosynthetic/biosynthesis polypeptides" refers, collectively and individually, to enzymes that may be expressed or overexpressed (e.g., in a host cell, microbe, or microorganism) to produce fatty acid derivatives, such as, e.g., a fatty acid methyl ester (FAME) or a fatty acid ethyl ester (FAEE). Additional non-limiting examples of "fatty acid derivative enzymes" or equivalently "fatty acid derivative biosynthetic/biosynthesis polypeptides" include, e.g., fatty acid synthases, lactonizing enzymes, thioesterases, acyl-CoA synthetases, acyl-CoA reductases, acyl-ACP reductases, alcohol dehydrogenases, alcohol oxidases, aldehyde dehydrogenases, alcohol O-acyltransferases, fatty alcohol-forming acyl-CoA reductases, fatty acid decarboxylases, fatty aldehyde decarbonylases and/or oxidative deformylases, carboxylic acid reductases, fatty alcohol O-acetyl transferases, hydroxylating enzymes (including, for example omega-hydroxylases, oxygenases, or monooxygenases), hydratases, desaturases, ester synthases, transaminases (aminotransferases), etc. "Fatty acid derivative enzymes" or equivalently "fatty acid derivative biosynthetic/biosynthesis polypeptides" convert substrates into fatty acid derivatives. The substrate for a fatty acid derivative enzyme can be an intermediate of a fatty acid derivative biosynthetic/biosynthesis pathway. For example, a fatty acyl-ACP can be a substrate for a thioesterase, which converts the acyl-ACP to a free fatty acid, and the free fatty acid (as an intermediate), in turn, can be a substrate for a carboxylic acid reductase, which converts the fatty acid to a fatty aldehyde. Further, the fatty aldehyde can act as an intermediate, and can be a substrate for an alcohol dehydrogenase, which converts the fatty aldehyde intermediate into a fatty alcohol product. The expression “fatty acid composition” or “fatty acid derivative composition” as used herein, refers to a composition of fatty acids and/or fatty acid derivatives thereof, that contain one or more fatty acids and/or fatty acid derivatives.

[0032] Sequence Accession numbers throughout this description were obtained from databases provided by the NCBI (National Center for Biotechnology Information) maintained by the National Institutes of Health, U.S.A, (which are identified herein as “NCBI Accession Numbers” or alternatively as “GenBank Accession Numbers” or alternatively a simply “Accession Numbers”), and from the UniProt Knowledgebase (UniProtKB) and Swiss-Prot databases provided by the Swiss Institute of Bioinformatics (which are identified herein as “UniProtKB Accession Numbers”). [0033] The term “enzyme classification (EC) number” refers to a number that denotes a specific polypeptide sequence or enzyme. EC numbers classify enzymes according to the reaction they catalyze. EC numbers are established by the nomenclature committee of the international union of biochemistry and molecular biology (IUBMB), a description of which is available on the IUBMB enzyme nomenclature website on the world wide web.

[0034] As used herein, the terms “isolated” and “purified,” with respect to products (such as monounsaturated fatty acids and derivatives disclosed herein), refers to products that are separated from cellular components, cell culture media, or chemical or synthetic precursors. The monounsaturated fatty acids and derivatives disclosed herein produced by the methods disclosed herein can be relatively immiscible in the fermentation broth, as well as in the cytoplasm. Therefore, in exemplary embodiments, the trans-2 fatty acids and derivatives disclosed herein collect in an organic phase extracellularly and are thereby “isolated”.

[0035] As used herein, the terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues that is typically 12 or more amino acids in length. Polypeptides less than 12 amino acids in length are referred to herein as “peptides.” The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The term “recombinant polypeptide” refers to a polypeptide that is produced by recombinant techniques, wherein generally DNA or RNA encoding the expressed protein is inserted into a suitable expression vector that is in turn used to transform a host cell to produce the polypeptide. In some exemplary embodiments, DNA or RNA encoding an expressed peptide, polypeptide, or protein is inserted into the host chromosome via homologous recombination or other means well known in the art and is so used to transform a host cell to produce the peptide or polypeptide. Similarly, the terms “recombinant polynucleotide” or “recombinant nucleic acid” or “recombinant DNA” are produced by recombinant techniques that are known to those of skill in the art (see e.g., methods described in Sambrook et al. (Sambrook et al., Molecular Cloning— A Laboratory Manual, Cold Spring Harbor Press 4^th Edition (Cold Spring Harbor, N.Y. 2012) and/or Current Protocols in Molecular Biology (Volumes 1-3, John Wiley & Sons, Inc. (1994-1998) and Supplements 1-115 (1987- 2016).).

[0036] When referring to two nucleotide or polypeptide sequences, the “percentage of sequence identity” between the two sequences is determined by comparing the two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The “percentage of sequence identity” is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0037] Thus, the expression “percent identity,” or equivalently “percent sequence identity,” “homology, or “homologous” in the context of two or more nucleic acid sequences or peptides or polypeptides, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (e.g., about 50% identity, preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured e.g., using a BLAST or BLAST 2.0 sequence comparison algorithm with default parameters (see e.g., Altschul etal. (1990) J. Mol. Biol. 215(3):403-410) and/or the NCBI web site at ncbi.nlm.nih.gov/BLAST/) or by manual alignment and visual inspection. Percent sequence identity between two nucleic acid or amino acid sequences also can be determined using e.g., the Needleman and Wunsch algorithm that has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 (Needleman and Wunsch (1970) J. Mol. Biol. 48:444-453). The percent sequence identity between two nucleotide sequences also can be determined using the GAP program in the GCG software package, using a NWSgapdna. CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. One of ordinary skill in the art can perform initial sequence identity calculations and adjust the algorithm parameters accordingly. A set of parameters that may be used if a practitioner is uncertain about which parameters should be applied to determine if a molecule is within a sequence identity limitation of the claims, are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. Additional methods of sequence alignment are known in the biotechnology arts (see, e.g., Rosenberg (2005) BMC Bioinformatics 6:278; Altschul et al. (2005) FEBS J. 272(20):5101-5109).

[0038] Two or more nucleic acid or amino acid sequences are said to be “substantially identical,” when they are aligned and analyzed as discussed above and are found to share about 50% identity, preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region. Two nucleic acid sequences or polypeptide sequences are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences are the same when aligned for maximum correspondence as described above. This definition also refers to, or may be applied to, the compliment of a test sequence. Identity is typically calculated over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length, or over the entire length of a given sequence.

[0039] The term “endogenous” as used herein refers to a substance e.g. , a nucleic acid, protein, enzyme, etc. that is produced from within a cell and/or that is naturally occurring or naturally found inside a cell. Similarly, an endogenous pathway (such as a fatty acid biosynthesis pathway or a fatty acid derivative pathway) is one that is naturally occurring or naturally found inside a cell.. Thus, an endogenous nucleic acid sequence, gene, polynucleotide, or polypeptide refers to a nucleic acid sequence, gene, polynucleotide, or polypeptide produced by and found inside the cell. In some exemplary embodiments an endogenous polypeptide or polynucleotide is encoded by the genome of the parental cell (or host cell). In other exemplary embodiments, an endogenous polypeptide or polynucleotide is encoded by an autonomously replicating plasmid carried by the parental cell (or host cell). In some exemplary embodiments, an endogenous gene or nucleic acid sequence is a gene or nucleic acid sequence that was present in the cell when the cell was originally isolated from nature, i.e., the gene is native to the cell.

[0040] In contrast, an “exogenous” nucleic acid sequence, gene, polynucleotide, or polypeptide (e.g., an enzyme), or other substance (e.g., fatty acid derivative, small molecule compound, etc.), as used herein, refers to a nucleic acid sequence, gene, polynucleotide, or polypeptide or other substance that is not encoded by or produced by the cell, and which is therefore added to a cell, a cell culture, or assay, from outside of the cell. A nucleic acid sequence encoding a variant (i.e., mutant) polypeptide, when added to the cell, is one example of an exogenous nucleic acid sequence. Similarly, a nucleic acid sequence encoding a fatty acid biosynthesis enzyme or fatty acid derivative enzyme, when introduced into a cell (e.g., in a vector, such as a plasmid), is considered an exogenous nucleic acid sequence. The exogenous nucleic acid sequence can encode a polypeptide or an enzyme that is also otherwise endogenous or native to the cell. Such an encoded polypeptide or enzyme can be considered “exogenously expressed.” For example, to achieve overexpression of an endogenous gene, additional copies of the gene can be introduced into the cell (e.g., in a vector, such as a plasmid); such additional copies of the endogenous gene can be considered as “exogenous” (e.g., exogenous gene(s) or an exogenous nucleic acid sequence(s)), because the additional copies are introduced into the cell from outside the cell. An “exogenous gene” or “exogenous nucleic acid sequence” also refers to a native (or endogenous) gene or nucleic acid sequence that is deregulated (e.g., upregulated or attenuated) or otherwise altered or modified, for example, by operably linking it to a regulatory element, such as a heterologous, or non-native, or non-naturally occurring, regulatory element (e.g., a promoter, enhancer, 5’-UTR, ribosome binding site, etc.); such a deregulated or altered gene or nucleic acid sequence can be on a chromosome or can be on a plasmid. An exogenous nucleic acid sequence or exogenous gene can also be used to express or overexpress a heterologous polypeptide or enzyme in a cell. Thus, an exogenous nucleic acid sequence or an exogenous gene can encode a polypeptide (e.g., an enzyme) that is native to the cell, that is otherwise endogenous to the cell, or that is heterologous to the cell.

[0041] The term “heterologous” as used herein refers to a polypeptide or polynucleotide which is in a non-native state. Thus, a polynucleotide or a polypeptide is “heterologous” to a cell when the polynucleotide and/or the polypeptide and the cell are not found in the same relationship to each other in nature. Therefore, a polynucleotide or polypeptide sequence is “heterologous” to an organism or a second sequence if it originates from a different organism, different cell type, or different species, or, if from the same species, it is modified from its original form. Thus, in an exemplary embodiment, a polynucleotide or polypeptide is “heterologous” when it is not naturally present in a given organism. For example, a polynucleotide sequence that is native to cyanobacteria can be introduced into a host cell of E. coli (a proteobacterium) by recombinant methods, and the polynucleotide from cyanobacteria is then heterologous to the E. coli cell (i.e., the now recombinant E.coli cell).

[0042] Similarly, a polynucleotide or polypeptide is heterologous when it is modified from its native form or from its relationship with other polynucleotide sequences or is present in a recombinant host cell in a non-native state. Thus, in an exemplary embodiment, a heterologous polynucleotide or polypeptide comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, a promoter operably linked to a nucleotide coding sequence derived from a species different from that from which the promoter was derived. Alternatively, in another example, if a promoter is operably linked to a nucleotide coding sequence derived from a species that is the same as that from which the promoter was derived, then the operably-linked promoter and coding sequence are “heterologous” if the coding sequence is not naturally associated with the promoter (e.g. a constitutive promoter operably linked to a developmentally regulated coding sequence that is derived from the same species as the promoter). In other exemplary embodiments, a heterologous polynucleotide or polypeptide is modified relative to the wild-type sequence naturally present in the corresponding wild-type host cell, e.g., an intentional modification e.g., an intentional mutation in the sequence of a polynucleotide or polypeptide or a modification in the level of expression of the polynucleotide or polypeptide. Typically, a heterologous nucleic acid or polynucleotide is recombinantly produced. A heterologous polynucleotide, polypeptide, or enzyme, for example, is typically exogenous to the cell, or exogenously expressed (or overexpressed) in the cell, i.e., is introduced into or added to the cell from outside the cell.

[0043] As used herein the term “native” refers to the form of a nucleic acid, protein, polypeptide or a fragment thereof that is isolated from nature, or to a nucleic acid, protein, polypeptide or a fragment thereof that is in its natural state without intentionally introduced mutations in the structural sequence and/or without any engineered changes in expression such as e.g., changing a developmentally regulated gene to a constitutively expressed gene. As used herein, “native” also refers to “wildtype” or “wild-type,” in which the nucleic acid, protein, polypeptide, or a fragment thereof is present in both sequence, quantity, and relative quantity as typically found in the organism as naturally found. Wild-type organisms may serve as a control and/or reference for determination of cellular functions, such as to identity and/or quantity fatty acid(s) and derivatives thereof produced. A native gene, nucleic acid sequence, polypeptide, or enzyme, for example, is typically endogenous to a cell, i.e., found in or produced by the cell. An exogenous nucleic acid sequence or an exogenous gene can encode a native polypeptide or enzyme, for example, where additional copies of a native gene or nucleic acid sequence are added to the cell from outside the cell, or where a native gene or nucleic acid sequence is deregulated or altered, e.g., by operably coupling it to a regulatory element that is not native or endogenous to the cell.

[0044] The term “non-native” is used herein to refer to nucleic acid sequences, amino acid sequences, polypeptide sequences, enzymes, fatty acids and derivatives thereof, and/or small molecules that do not occur naturally in the host. Heterologous genes and polypeptides are considered “non-native.” A nucleic acid sequence or amino acid sequence that has been removed from a host cell, subjected to laboratory manipulation, and introduced or reintroduced into a host cell, is also considered “non-native.” Synthetic or partially synthetic genes introduced into a host cell are “non-native.” Non-native genes further include genes that are endogenous and/or native to the host microorganism but that are operably linked to one or more heterologous regulatory sequences that have been recombined into the host genome. A naturally occurring gene under the control of a heterologous regulatory sequence is considered “non-native.” In some embodiments, an organism comprising a non-native gene may be utilized as a control and/or reference for an organism having additional and/or different variations from wildtype organisms. [0045] The term “gene” as used herein, refers to nucleic acid sequences e.g., DNA sequences, which encode either an RNA product or a protein product, as well as operably linked nucleic acid sequences that affect expression of the RNA or protein product e.g., expression control sequences such as e.g., promoters, enhancers, ribosome binding sites, translational control sequences, etc.). The term “gene product” refers to either the RNA (e.g., tRNA, mRNA) and/or protein expressed from a particular gene.

[0046] The term “expression” or “expressed” as used herein in reference to a gene, refers to the production of one or more transcriptional and/or translational product(s) of a gene. In exemplary embodiments, the level of expression of a DNA molecule in a cell is determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell. The term “expressed genes” refers to genes that are transcribed into messenger RNA (mRNA) and then translated into protein, as well as genes that are transcribed into other types of RNA, such as e.g., transfer RNA (tRNA), ribosomal RNA (rRNA), and regulatory RNA, which are not translated into protein.

[0047] The level of expression of a nucleic acid molecule in a cell or cell free system is influenced by “expression control sequences” or equivalently “regulatory sequences” or “regulatory elements.” Expression control sequences, regulatory sequences, or regulatory elements are known in the art and include, for example, promoters, enhancers, polyadenylation signals, transcription terminators, nucleotide sequences that affect RNA stability, internal ribosome entry sites (IRES), and the like, that provide for the expression of the polynucleotide sequence in a host cell. In exemplary embodiments, “expression control sequences” interact specifically with cellular proteins involved in transcription (see e.g., Maniatis et al., Science, 236: 1237-1245 (1987); Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990)). In exemplary methods, an expression control sequence, regulatory sequence, or regulatory element is operably linked to a polynucleotide sequence. By “operably linked” is meant that a polynucleotide sequence and an expression control sequence(s) or regulatory element(s) are functionally connected so as to permit expression of the polynucleotide sequence when the appropriate molecules (e.g., transcriptional activator proteins) contact the expression control sequence(s). In exemplary embodiments, operably linked promoters are located upstream of the selected polynucleotide sequence in terms of the direction of transcription and translation. In some exemplary embodiments, operably linked enhancers can be located upstream, within, or downstream of the selected polynucleotide.

[0048] As used herein, the phrase “expression of said nucleotide sequence is modified relative to the wild-type nucleotide sequence,” refers to a change e.g., an increase or decrease in the level of expression of a native nucleotide sequence or a change e.g., an increase or decrease in the level of the expression of a heterologous or non-native polypeptide-encoding nucleotide sequence as compared to a control nucleotide sequence e.g., wild-type control. In some exemplary embodiments, the phrase “the expression of said nucleotide sequence is modified relative to the wild-type nucleotide sequence,” refers to a change in the pattern of expression of a nucleotide sequence as compared to a control pattern of expression e.g., constitutive expression as compared to developmentally timed expression.

[0049] A “control” sample e.g., a control nucleotide sequence, a control polypeptide sequence, a control cell, etc., or value) refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample. For example, in an exemplary embodiment, a test sample comprises a trans-2 fatty acid derivative composition made by a recombinant microbe that comprises a 3-hydroxy acyl-ACP thioesterase; an acyl-CoA synthetase; one of a heterologous R-3-hydroxy-acyl-CoA dehydratase or heterologous R-specific enoyl-CoA hydratase; and one of an ester synthase, acyl-CoA reductase, or acyl-CoA thioesterase, as disclosed herein, while the control sample comprises a trans-2 free fatty acid or derivative thereof composition made by the corresponding or designated microbe that does not comprise a the combination of enzymes described herein. Additionally, a control cell or microorganism may be referred to as a corresponding wild-type or host cell. One of skill will recognize that controls can be designed for assessment of any number of parameters. Furthermore, one of skill in the art will understand which controls are valuable in a given situation and will be able to analyze data based on comparisons to control values.

[0050] The term “overexpressed” or “up-regulated” as used herein, refers to a gene whose expression is elevated in comparison to a control level of expression. In exemplary embodiments, overexpression of a gene is caused by an elevated rate of transcription as compared to the native transcription rate for that gene. In other exemplary embodiments, overexpression is caused by an elevated rate of translation of the gene compared to the native translation rate for that gene. Methods of testing for overexpression are well known in the art, for example transcribed RNA levels can be assessed using rtPCR and protein levels can be assessed using SDS page gel analysis. [0051] In other embodiments, the polypeptide, polynucleotide, or hydrocarbon having an altered level of expression is “attenuated” or has a “decreased level of expression” or is “down- regulated.” As used herein, these terms mean to express or cause to be expressed a polynucleotide, polypeptide, or hydrocarbon in a cell at a lesser concentration than is normally expressed in a corresponding control cell (e.g., wild-type cell) under the same conditions. In other words, the term “attenuate” means to weaken, reduce, or diminish. For example, a polypeptide can be attenuated by modifying the polypeptide to reduce its activity (e.g., by modifying a nucleotide sequence that encodes the polypeptide).

[0052] A polynucleotide or polypeptide can be attenuated using any method known in the art. For example, in some exemplary embodiments, the expression of a gene or polypeptide encoded by the gene is attenuated by mutating the regulatory polynucleotide sequences which control expression of the gene. In other exemplary embodiments, the expression of a gene or polypeptide encoded by the gene is attenuated by overexpressing a repressor protein, or by providing an exogenous regulatory element that activates a repressor protein. In still other exemplary embodiments, DNA- or RNA-based gene silencing methods are used to attenuate the expression of a gene or polynucleotide. In some embodiments, the expression of a gene or polypeptide is completely attenuated, e.g., by deleting all or a portion of the polynucleotide sequence of a gene. [0053] The degree of overexpression or attenuation can be 1.5-fold or more, e.g., 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, or 15-fold or more. Alternatively, or in addition, the degree of overexpression or attenuation can be 500-fold or less, e.g., 100-fold or less, 50-fold or less, 25-fold or less, or 20-fold or less. Thus, the degree of overexpression or attenuation can be bounded by any two of the above endpoints. For example, the degree of overexpression or attenuation can be 1.5-500-fold, 2-50-fold, 10-25-fold, or 15-20-fold.

[0054] As used herein, “modified activity” or an “altered level of activity” of a protein/polypeptide in a recombinant host cell refers to a difference in one or more characteristics in the activity the protein/polypeptide as compared to the characteristics of an appropriate control protein e.g., the corresponding parent protein or corresponding wild-type protein. Thus, in exemplary embodiments, a difference in activity of a protein having “modified activity” as compared to a corresponding control protein is determined by measuring the activity of the modified protein in a recombinant host cell and comparing that to a measure of the same activity of a corresponding control protein in an otherwise isogenic host cell. Modified activities can be the result of, for example, changes in the structure of the protein e.g., changes to the primary structure, such as e.g., changes to the protein’s nucleotide coding sequence that result in changes in substrate specificity, changes in observed kinetic parameters, changes in solubility, etc.); changes in protein stability (e.g., increased or decreased degradation of the protein) etc.

[0055] The term “recombinant” as used herein, refers to a genetically modified polynucleotide, polypeptide, cell, tissue, or organism. When used with reference to a cell, the term “recombinant” indicates that the cell has been modified by the introduction of a heterologous nucleic acid or protein or has been modified by alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified and that the derived cell comprises the modification. Thus, for example, “recombinant cells” or equivalently “recombinant host cells” may be modified to express genes that are not found within the native (non-recombinant) form of the cell or may be modified to abnormally express native genes e.g., native genes may be overexpressed, underexpressed or not expressed at all. In exemplary embodiments, a “recombinant cell” or “recombinant host cell” is engineered to express a heterologous enzyme pathway capable of producing a bifunctional fatty acid derivative molecule. A recombinant cell can be derived from a microorganism or microbe such as a bacterium (including proteobacterium and cyanobacterium), archaea, a virus, algae, or a fungus. In addition, a recombinant cell can be derived from a plant or an animal cell. In exemplary embodiments, a “recombinant host cell” or “recombinant cell” is used to produce one or more non-native monounsaturated fatty acid derivatives including, but not limited to, trans-2 fatty acids, non-native monounsaturated fatty esters e.g., waxes), trans-2 fatty acid esters, trans-2 fatty esters, trans-2 fatty acid methyl esters (FAME), trans-2 fatty acid ethyl esters (FAEE)), trans-2 fatty acyl acetate esters (FACE), trans-2 fatty alcohols (e.g., polyols), trans-2 fatty aldehydes, trans-2 fatty amines, trans-2 fatty amides, trans-2-co-hydroxy fatty esters, trans-2-co-carboxy fatty esters, trans-2-a, co-fatty diacids, transact, co-fatty diesters, trans-2-a, co-fatty diols, trans-2 terminal olefins, trans-2 ketones, etc. Therefore, in some exemplary embodiments a “recombinant host cell” is a “production host” or equivalently, a “production host cell”. In some exemplary embodiments, the recombinant cell includes one or more polynucleotides, each polynucleotide encoding a polypeptide having fatty acid biosynthetic enzyme activity, wherein the recombinant cell produces a trans-2 fatty acid derivative composition when cultured in the presence of a (simple) carbon source under conditions effective to express the polynucleotides.

[0056] When used with reference to a polynucleotide, the term “recombinant” indicates that the polynucleotide has been modified by comparison to the native or naturally occurring form of the polynucleotide or has been modified by comparison to a naturally occurring variant of the polynucleotide. In an exemplary embodiment, a recombinant polynucleotide (or a copy or complement of a recombinant polynucleotide) is one that has been manipulated by the hand of man to be different from its naturally occurring form. Thus, in an exemplary embodiment, a recombinant polynucleotide is a mutant form of a native gene or a mutant form of a naturally occurring variant of a native gene wherein the mutation is made by intentional human manipulation e.g., made by saturation mutagenesis using mutagenic oligonucleotides, through the use of UV radiation, mutagenic chemicals, chemical synthesis etc. Such a recombinant polynucleotide might comprise one or more point mutations, deletions and/or insertions relative to the native or naturally occurring variant form of the gene. Similarly, a polynucleotide comprising a promoter operably linked to a second polynucleotide (e.g., a coding sequence) is a “recombinant” polynucleotide. Thus, a recombinant polynucleotide comprises polynucleotide combinations that are not found in nature. A recombinant protein (discussed supra) is typically one that is expressed from a recombinant polynucleotide, and recombinant cells, tissues, and organisms are those that comprise recombinant sequences (polynucleotide and/or polypeptide).

[0057] The term “vector,” as used herein, refers to a polynucleotide sequence that contains a gene of interest (e.g. , it encodes one or more proteins or enzymes described herein) and a promoter operably linked to the fatty acid biosynthetic polynucleotide sequence of interest. Once a polynucleotide sequence(s) encoding a fatty acid biosynthetic pathway polypeptide has been prepared and isolated, various methods may be used to construct expression cassettes, vectors, and other DNA constructs. The skilled artisan is well aware of the genetic elements that must be present on an expression construct/vector in order to successfully transform, select, and propagate the expression construct in host cells. Techniques for manipulation of nucleic acids such as subcloning nucleic acid sequences into expression vectors, labeling probes, DNA hybridization are well known in the art.

[0058] As used herein, the term “microbe” or “microorganism” refers generally to a microscopic organism. Microbes can be prokaryotic or eukaryotic. Exemplary prokaryotic microbes include e.g., bacteria (including y-proteobacteria), archaea, cyanobacteria, etc. An exemplary proteobacterium is Escherichia coli. Exemplary eukaryotic microorganisms include e.g., yeast, protozoa, algae, etc. In exemplary embodiments, a “recombinant microbe” is a microbe that has been genetically altered and thereby expresses or encompasses a heterologous nucleic acid sequence and/or a heterologous peptide, polypeptide, or protein.

[0059] A microbe as used herein, can grow on a carbon source e.g., a simple carbon source. The recombinant microbe may be a gamma proteobacterium (also known as a y-proteobacterium), a cyanobacterium, a yeast or an algae. In some embodiments, the recombinant proteobacterium may be Escherichia coli, Salmonella spp., Vibrio natriegens, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Xanthomonas axonopodis, Pseudomonas syringae, Xyella fastidiosa, Marinobacter aquaeolei, Yersinia pestis, Bacillus spp., Lactobacillus spp., Zymomonas spp., Streptomyces spp., or Vibrio cholerae. In some embodiments, the recombinant cyanobacterium may be Synechococcus elongatus PCC7942 or Synechocystis sp. PCC6803. In some embodiments, the recombinant yeast may be Saccharomyces cerevisiae, Scheffersomyces stipitis, Schizosaccharomyces pombe, Kluyveromyces marxianus, K. lactis, Pichia pastoris, Hansenula polymorpha, or Yarrowia lipolytica. In some embodiments, the recombinant algae may be Botryococcus braunii, Nannochloropsis gaditina, Chlamydomonas reinhardtii, Chlorella vulgaris, Spirulina platensis, Ostreococcus tauri, Phaeodactylum tricornutum, Symbiodinium sp., algal phytoplanktons, Saccharina japonica, Chlorococcum spp., and Spiro gyra spp.

[0060] As used herein, the term “culture” typically refers to a liquid media comprising viable cells. In one embodiment, a culture comprises cells reproducing in a predetermined culture media under controlled conditions, for example, a culture of recombinant host cells grown in liquid media comprising a selected carbon source and nitrogen.

[0061] “Culturing” or “cultivation” refers to growing a population of recombinant host cells (e.g., recombinant microbes) under suitable conditions in a liquid or on a solid medium. In particular embodiments, culturing refers to the fermentative bioconversion of a substrate to an end-product. Culturing media are well-known and individual components of such culture media are available from commercial sources, e.g., under the Difco™ and BBL™ trademarks. In one non-limiting example, the aqueous nutrient medium is a “rich medium” comprising complex sources of nitrogen, salts, and carbon, such as YP medium, comprising 10 g/L of peptone and 10 g/L yeast extract of such a medium.

[0062] A “production host” or equivalently a “production host cell” is a cell used to produce products. As disclosed herein, a production host is typically modified to express or overexpress selected genes, or to have attenuated expression of selected genes. Thus, a production host or a “production host cell” is a recombinant host or equivalently a recombinant host cell. Non-limiting examples of production hosts include e.g., recombinant microbes as disclosed above.

[0063] As used herein, the terms “purify,” “purified,” or “purification” mean the removal or isolation of a molecule from its environment by, for example, isolation or separation. “Substantially purified” molecules are at least about 60% free (e.g., at least about 65% free, at least about 70% free, at least about 75% free, at least about 80% free, at least about 85% free, at least about 90% free, at least about 95% free, at least about 96% free, at least about 97% free, at least about 98% 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. [0064] As used herein, the term “carbon source” refers to a substrate or compound suitable to be used as a source of carbon for prokaryotic or simple eukaryotic cell growth. Carbon sources can be in various forms, including, but not limited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, and gases (e.g., CO and CO2). Exemplary carbon sources include, but are not limited to, monosaccharides, such as glucose, fructose, mannose, galactose, xylose, and arabinose; oligosaccharides, such as fructo-oligosaccharide and galactooligosaccharide; polysaccharides such as starch, cellulose, pectin, and xylan; disaccharides, such as sucrose, maltose, cellobiose, and turanose; cellulosic material and variants such as hemicelluloses, methyl cellulose and sodium carboxymethyl cellulose; 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 biomass. In other embodiments, the carbon source is glucose. In other embodiments the carbon source is sucrose. In other embodiments the carbon source is glycerol. In other embodiments, the carbon source is a simple carbon source such as e.g., glucose. In other embodiments, the carbon source is a renewable carbon source. In other embodiment, the carbon source is natural gas. In other embodiments the carbon source comprises one or more components of natural gas, such as methane, ethane, or propane. In other embodiments, the carbon source is flu gas or synthesis gas. In still other embodiments, the carbon source comprises one or more components of flu or synthesis gas such as carbon monoxide, carbon dioxide, hydrogen, etc. As used herein, the term “carbon source” or “simple carbon source” specifically excludes oleochemicals such as e.g., saturated or unsaturated fatty acids.

II. Enzymes

[0065] As used herein, the term “3-hydroxy acyl-ACP thioesterase” refers to an enzyme that uses 3-hydroxy-acyl-ACP as a substrate and hydrolyzes it to the corresponding 3-hydroxy fatty acid. The 3-hydroxy fatty acid is then converted to 3-hydroxy-acyl-CoA by an acyl-CoA synthetase (further described below). Thus, the combination of 3-hydroxy acyl-ACP thioesterase and an acyl-CoA synthetase represents a two-step pathway for converting 3-hydroxy acyl-ACP to 3-hydroxy-acyl-CoA. The 3-hydroxy acyl-ACP thioesterase may be native to the recombinant microbe (i.e., from or derived from the same species), or may be heterologous (i.e., from or derived from a different species). In some embodiments, the 3-hydroxy acyl-ACP thioesterase may be endogenous, wherein the enzyme, or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA) is produced by the cell. In another embodiment, the 3-hydroxy acyl-ACP thioesterase is heterologous, wherein a polynucleotide encoding the enzyme is exogenous, and is not produced by the cell, but instead is added to the cell from outside the cell. In some embodiments, the acyl- ACP thioesterases described herein may belong to EC 3.1.2.14 or EC 3.1.2.21, and can also be referred to as an acyl-ACP hydrolase. Examples of 3-hydroxy-acyl-ACP thioesterases with source microbes are shown in Table 1 below.

[0066] As used herein, the term “3-hydroxy acyl-ACP:CoA transacylase” or “3-hydroxy acyl- ACP:CoA acyltransferse” refers to an enzyme that transfers the 3-hydroxy acyl moiety from an ACP thioester to a CoA thioester. A 3-hydroxy acyl-ACP:CoA transacylase may also exhibit 3- hydroxy acyl-ACP thioesterase activity. Thus, the 3-hydroxy acyl-ACP:CoA transacylase represents a one-step pathway for converting 3-hydroxy acyl-ACP to 3-hydroxy-acyl-CoA. It may be native to the recombinant microbe or may be heterologous. In some embodiments, the 3- hydroxy acyl-ACP:CoA transacylase may be endogenous, wherein the enzyme, or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA) is produced by the cell. In another embodiment, the 3-hydroxy acyl-ACP:CoA transacylase may be heterologous, wherein a polynucleotide encoding the enzyme is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell. In some embodiments, the 3-hydroxy acyl-ACP:CoA transacylase described herein may belong to EC 2.4.1.-. Examples of 3-hydroxy acyl-ACP:CoA transacylases with source microbes are shown in Table 1 below. [0067] In certain embodiments, the 3-hydroxy-acyl-ACP thioesterase is any one of those listed in Table 1 below, or comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1- 11 or 40, or is a homolog of any of the enzymes listed in Table 1 or a homolog of any one of SEQ ID NOs: 1-11 or 40, having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%F, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, sequence identity thereto. [0068] Table 1: Examples of 3-hydroxy-acyl-ACP thioesterases (3OH-TE) and 3-hydroxy acyl-ACP:CoA transacylases (3OH-TA)

[0069] As used herein, the term “acyl-CoA synthetase” (alternatively “acyl-CoA synthase” or “acyl-CoA ligase”) refers to enzymes that can convert or reactivate free 3-hydroxy fatty acids (e.g., prepared by 3-hydroxy acyl-ACP thioesterase described above) to the corresponding 3- hydroxy-acyl-CoAs. The acyl-CoA synthetase may be native to the recombinant microbe or may be heterologous. In some embodiments, the acyl-CoA synthetase may be endogenous, wherein the enzyme or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA) is produced by the cell. In another embodiment, the acyl-CoA synthetase may be heterologous, wherein a polynucleotide encoding the enzyme is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell. Acyl-CoA synthetase may be described by the number EC 6.2.1.3, and can also be referred to as a long-chain-fatty acid CoA ligase, a fatty acid CoA ligase, a fatty acyl-CoA synthase, a fatty acyl-CoA synthetase, or an acyl-CoA ligase. Examples of acyl- CoA synthetases with source microbes are shown in Table 2 below.

[0070] In certain embodiments, the acyl-CoA synthetase is any one of those listed in Table 2 below, or comprises the amino acid sequence set forth in any one of SEQ ID NOs: 12-19, or is a homolog of any of the enzymes listed in Table 2 or a homolog of any one of SEQ ID NOs: 12-19, having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, sequence identity thereto.

[0071] Table 2: Examples of acyl-CoA synthetases

[0072] As used herein, the term “R-3-hydroxy-acyl-CoA dehydratase” refers to enzymes that convert 3-hydroxy-acyl-CoA to trans-2-enoyl-CoA. The R-3-hydroxy-acyl-CoA dehydratase may be native to the recombinant microbe or may be heterologous. In some embodiments, the R-3- hydroxy-acyl-CoA dehydratase may be endogenous, wherein the enzyme or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA) is produced by the cell. In another embodiment, the R-3-hydroxy-acyl-CoA dehydratase may be heterologous, wherein a polynucleotide encoding the enzyme is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell. R-3-hydroxy-acyl-CoA dehydratase may be described by the number EC 4.2.1.134 or EC 4.2.1.55. [0073] As used herein, the term “R-specific enoyl-CoA hydratase” refers to enzymes that also convert 3-hydroxy-acyl-CoA to trans-2-enoyl-CoA. The R-specific enoyl-CoA hydratase may be native to the recombinant microbe or may be heterologous. In some embodiments, the R- specific enoyl-CoA hydratase may be endogenous, wherein the enzyme or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA) is produced by the cell. In another embodiment, the R- specific enoyl-CoA hydratase may be heterologous, wherein a polynucleotide encoding the enzyme is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell. R-specific enoyl-CoA hydratase may be described by the number EC 4.2.1.119 or EC 4.2.1.17. The R-3-hydroxy-acyl-CoA dehydratase and R-specific enoyl-CoA hydratase are interchangeable in the biochemical synthetic pathways for producing trans-2 unsaturated fatty acid derivatives.

Examples of 3-hydroxy-acyl-CoA dehydratases or R-specific enoyl-CoA hydratases are shown in Table 3 below.

[0074] In certain embodiments, 3-hydroxy-acyl-CoA dehydratase or the R-specific enoyl- CoA hydratase is any one of those listed in Table 3 below, or comprises the amino acid sequence set forth in any one of SEQ ID NOs: 20-32, or is a homolog of any of the enzymes listed in Table

3 or a homolog of any one of SEQ ID NOs: 20-32, having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, sequence identity thereto.

[0075] Table 3: Examples of 3-hydroxy-acyl-CoA dehydratases or R-specific enoyl-CoA hydratases

[0076] As used herein, the term “trans-2-enoyl-CoA reductase” refers to an enzyme that reduces or converts trans-2-enoyl-CoA to the corresponding, fully reduced acyl-CoA. When NADH or NADPH is a cofactor of the reaction, the reduction is irreversible. The trans-2-enoyl- CoA reductase may be native to the recombinant cell or microbe (i.e., from or derived from the same species), or it may be heterologous (i.e., from or derived from a different species). The trans- 2-enoyl-CoA reductase may be endogenous, wherein the enzyme, or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA) is produced by the cell. Alternatively, the trans-2-enoyl- CoA reductase may be heterologous, wherein a polynucleotide, nucleic acid sequence, or gene encoding the enzyme is exogenous, and is not produced by the cell, but instead is added to the cell from outside the cell. In some embodiments, the trans-2-enoyl-CoA reductase described herein may belong to EC 1.3.1.44. In some embodiments, the trans-2-enoy-CoA reductase can be referred to as TER and/or FabV.

[0077] As used here, the term “ester synthase” refers to an enzyme that esterifies or converts trans-2-enoyl-CoA to the corresponding trans-2 fatty ester. The ester synthase may be native to the recombinant microbe or may be heterologous. In some embodiments, the ester synthase may be endogenous, wherein the enzyme or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA) is produced by the cell. In another embodiment, the ester synthase may be heterologous, wherein a polynucleotide encoding the enzyme is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell. The ester synthase may be described by the number EC 2.3.1.20.

[0078] As used herein, the term “acyl-CoA reductase” refers to an enzyme that reduces trans- 2-enoyl-CoA to the corresponding trans-2 fatty aldehyde. The acyl-CoA reductase may be native to the recombinant microbe or may be heterologous. In some embodiments, the heterologous acyl- CoA reductase may be endogenous, wherein the enzyme or a polynucleotide encoding the enzyme (e.g., RNA) is produced by the cell. In another embodiment, the heterologous acyl-CoA reductase may be exogenous, wherein the enzyme or a polynucleotide encoding the enzyme is not produced by the cell, but instead is added to the cell from outside the cell. Acyl-CoA reductase may be described by the number EC 1.2.1.50 or EC 1.2.1.84.

[0079] As used here, the term “acyl-CoA thioesterase” refers to an enzyme that converts acyl- CoA or trans-2-enoyl-CoA to the corresponding fatty acid or trans-2 fatty acid, respectively. The acyl-CoA thioesterase may be native to the recombinant microbe or may be heterologous. In some embodiments, acyl-CoA thioesterase may be endogenous, wherein the enzyme or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA) is produced by the cell. In another embodiment, the acyl-CoA thioesterase may be heterologous, wherein a polynucleotide encoding the enzyme is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell. The acyl-CoA thioesterase may be described by the number EC 3.1.2.2 or EC 3.1.2.20, and can also be referred to as a fatty-acyl-CoA hydrolase, a long-chain fatty-acyl-CoA hydrolase, or an acyl-CoA hydrolase.

[0080] As used here, the term “acyl-ACP thioesterase” refers to an enzyme that converts acyl- ACP or 3 -hydroxy acyl-ACP to the corresponding fatty acid or 3 -hydroxy fatty acid, respectively. The acyl-ACP thioesterase may be native to the recombinant, cell, microorganism, or microbe, or may be heterologous. The acyl-ACP thioesterase may be endogenous, wherein the enzyme or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA) is produced by the cell. Alternatively, the acyl-ACP thioesterase may be heterologous, wherein a polynucleotide, nucleic acid sequence, or gene encoding the enzyme is not produced by the cell, but instead is added to the cell from outside the cell. The acyl-ACP thioesterase may be described by the number EC 3.1.2.14 or EC 3.1.2.21, and can also be referred to as an acyl-ACP hydrolase.

[0081] As used herein, the term “P-ketoacyl-ACP-synthase,” which includes P-ketoacyl-ACP synthase I, e.g., “FabB” and/or P-ketoacyl-ACP synthase II, e.g., “FabF,” refers to enzymes that catalyze the condensation reactions to elongate the fatty acid chain. The P-ketoacyl-ACP synthase may be native to the recombinant cell or microbe or may be heterologous. The P-ketoacyl-ACP- synthase may be endogenous, wherein the enzyme or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA) is produced by the cell. Alternatively, the P-ketoacyl-ACP-synthase may be heterologous, wherein a polynucleotide, nucleic acid, or gene encoding the enzyme is not produced by the cell, but instead is added to the cell from outside the cell. P-ketoacyl-ACP- synthase may be described by the number EC 2.3.1.41 (P-ketoacyl-ACP-synthase I; e.g., FabB), or EC 2.3.1.179 (P-ketoacyl-ACP-synthase II; e.g., FabF).

[0082] As used herein, the term “enoyl-ACP reductase,” which includes “FabI” refers to enzymes which can convert trans-2-enoyl-ACP to the corresponding acyl-ACP. The enoyl-ACP reductase may be native to the recombinant microbe or may be heterologous. In some embodiments, the enoyl-ACP reductase may be endogenous, wherein the enzyme or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA) is produced by the cell. In another embodiment, the enoyl-ACP reductase may be heterologous, wherein a polynucleotide encoding the enzyme is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell. Enoyl-ACP reductase may be described by the number EC 1.3.1.10, EC 1.3.1.38, EC 1.3.1.39, or EC 1.3.1.104.

[0083] As used herein, the term “alcohol dehydrogenase” refers to an enzyme that catalyzes the interconversion between aliphatic alcohols (e.g., aliphatic medium-chain alcohols) and their corresponding aldehydes. In some embodiments, and under some conditions, the alcohol dehydrogenase converts an alcohol into an aldehyde. In some embodiments and under some conditions, the alcohol dehydrogenase converts an aldehyde into an alcohol. The alcohol dehydrogenase may be native to the recombinant cell or microbe or may be heterologous. The alcohol dehydrogenase may be endogenous, wherein the enzyme, or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA), is produced by the cell. Alternatively, the alcohol dehydrogenase may be heterologous, wherein a polynucleotide, nucleic acid sequence, or gene, encoding the enzyme is not produced by the cell, but instead is added to the cell from outside the cell. The alcohol dehydrogenase may belong to EC 1.1.1.1 or EC 1.1.1.2, or EC 1.1.1.-, and can also be referred to as an aldehyde reductase.

[0084] As used herein, the term “alcohol-O-acetyl-transferase” (also known as “alcohol-O- acetyltransferase”) refers to an enzyme that catalyzes the interconversion between acetyl-CoA and an alcohol, and CoA and an acetyl ester. The alcohol-O-acetyl-transferase may be native to the recombinant cell or microbe or may be heterologous. The alcohol-O-acetyl-transferase may be endogenous, wherein the enzyme, or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA), is produced by the cell. Alternatively, the alcohol-O-acetyl-transferase may be heterologous, wherein a polynucleotide, nucleic acid sequence, or gene, encoding the enzyme is not produced by the cell, but instead is added to the cell from outside the cell. The alcohol-O- acetyl-transferase may belong to EC 2.3.1.84.

[0085] As used herein, the term “carboxylic acid reductase” refers to an enzyme that converts a fatty acid to its corresponding fatty aldehyde. The carboxylic acid reductase may be native to the recombinant cell or microbe or may be heterologous. The carboxylic acid reductase may be endogenous, wherein the enzyme, or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA), is produced by the cell. Alternatively, the carboxylic acid reductase may be heterologous, wherein a polynucleotide, nucleic acid sequence, or gene, encoding the enzyme is not produced by the cell, but instead is added to the cell from outside the cell. The carboxylic acid reductase described herein may belong to EC 1.2.1.30, and can also be referred to as a carboxylate reductase. [0086] As used herein, the term “acyl-CoA reductase” refers to an enzyme that converts a fatty acyl-CoA to its corresponding fatty aldehyde. The acyl-CoA reductase may be native to the recombinant cell or microbe or may be heterologous. In some embodiments, the acyl-CoA reductase may be endogenous, wherein the enzyme or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA) is produced by the cell. In another embodiment, the acyl-CoA reductase may be heterologous, wherein a polynucleotide, nucleic acid sequence, or gene, encoding the enzyme is not produced by the cell, but instead is added to the cell from outside the cell. Acyl-CoA reductase may be described by the number EC 1.2.1.50.

[0087] As used herein, the term “fatty alcohol forming acyl-CoA reductase” refers to an enzyme or polypeptide that catalyzes the reduction of fatty acyl-CoAs to fatty aldehydes, and that catalyzes the subsequent reduction of the fatty aldehydes to fatty alcohols. The fatty alcohol forming acyl-CoA reductase may be native to the recombinant cell or microbe, i.e., from or derived from the same species as the recombinant cell or microbe, or may be heterologous, i.e., from or derived from an organism or species that is different from the recombinant cell or microbe. The native or heterologous fatty alcohol forming acyl-CoA reductase can be expressed, or can be overexpressed, in the recombinant cell or microbe. In some embodiments, the heterologous native fatty alcohol forming acyl-CoA reductase (FAR) may be endogenous, wherein the enzyme or a polynucleotide encoding the enzyme (e.g., RNA, mRNA, or DNA) is produced by the cell. In another embodiment, the fatty alcohol forming acyl-CoA reductase is heterologous (to the recombinant cell or microbe), and the polynucleotide, nucleic acid sequence, or gene, encoding the enzyme, is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell. Fatty alcohol forming acyl-CoA reductase may be described by EC 1.2.1.84 and can be alternatively referred to as alcohol-forming fatty acyl-CoA reductase. In some embodiments, the fatty alcohol forming acyl-CoA reductase is native to the cell and is overexpressed. In other embodiments, the fatty alcohol forming acyl-CoA reductase is heterologous to the cell and is expressed in the cell.

[0088] As used herein, the term “aldehyde dehydrogenase” refers to enzymes that convert aldehydes to carboxylic acids. The aldehyde dehydrogenase may be native to the recombinant cell or microbe, i.e., from or derived from the same species as the recombinant cell or microbe, or may be heterologous, i.e., from or derived from an organism or species that is different from the recombinant cell or microbe. The native or heterologous aldehyde dehydrogenase can be expressed, or can be overexpressed, in the recombinant cell or microbe.

[0089] In some embodiments, the native aldehyde dehydrogenase may be endogenous, wherein the enzyme, or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA) is produced by the cell. For example, the recombinant cell or microbe can comprise an endogenous nucleic acid sequence or endogenous gene encoding the native aldehyde dehydrogenase. [0090] In other embodiments, the native aldehyde dehydrogenase can be encoded by an exogenous nucleic acid sequence or an exogenous gene, such that the encoding nucleic acid sequence or gene is added to the cell from outside the cell, for example, where overexpression of the native enzyme is desired. In another embodiment, the aldehyde dehydrogenase is heterologous (to the recombinant cell or microbe), and the polynucleotide, nucleic acid sequence, or gene, encoding the enzyme, is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell. Aldehyde dehydrogenases may be described by EC 1.2.1.3. In some embodiments, the aldehyde dehydrogenase is native to the cell and is overexpressed. In other embodiments, the aldehyde dehydrogenase is heterologous to the cell and is expressed in the cell. [0091] As used herein, the term “co-hydroxylase” or “omega-hydroxylase” refers to an enzyme or polypeptide that hydroxylates a fatty acid or fatty acid derivative in the co-position (omegaposition), i.e., adds a hydroxy (-OH) group to the co-position of the fatty acid or derivative thereof. The omega- (co)-position indicates the reduced end of a fatty acid derivative, or the position of the last carbon along the fatty acid derivative chain (farthest from the carboxyl group, for example). The co-hydroxylase may be native to the recombinant cell or microbe i.e., from or derived from the same species as the recombinant cell or microbe, or may be heterologous, i.e., from or derived from an organism or species that is different from the recombinant cell or microbe. The native or heterologous co-hydroxylase synthase can be expressed, or can be overexpressed, in the recombinant cell or microbe. In some embodiments, the native co-hydroxylase may be endogenous, wherein the enzyme, or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA) is produced by the cell. For example, the recombinant cell or microbe can comprise an endogenous nucleic acid sequence or endogenous gene encoding the native co-hydroxylase. In another embodiment, the co-hydroxylase is heterologous (to the recombinant cell or microbe), and the polynucleotide, nucleic acid sequence, or gene, encoding the enzyme, is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell. In some embodiments, the co-hydroxylase may belong to EC 1.14.15.3 or 1.14.14.80, and can alternatively be referred to as a monooxygenase, an alkane 1 -monooxygenase, an alkane 1 -hydroxylase, a fatty acid omega- hydroxylase, or a long chain fatty acid omega-monooxygenase.

[0092] As used herein, “FadR” refers to a transcriptional regulator of fatty acid degradation. FadR inhibits and/or represses transcription of genes required for fatty acid transport and P- oxidation.

[0093] In any of the embodiments described herein, any one or more of the fatty acid biosynthesis enzymes and/or fatty acid derivative enzymes described herein, can be native or heterologous to the recombinant cell or microbe (or microorganism). For example, a native enzyme or polypeptide is from or derived from the same species as the recombinant cell or microbe. A heterologous enzyme or polypeptide is from or derived from an organism or species that is different from the recombinant cell or microbe. Any of the native or heterologous enzymes or polypeptides described herein can be expressed, or can be overexpressed, in the recombinant cell or microbe.

[0094] The native enzyme or polypeptide, or the encoding polynucleotide sequence or gene, can be endogenous, i.e., found in and produced within the cell. For example, the recombinant cell or microbe or microorganism can comprise an endogenous nucleic acid sequence or endogenous gene encoding the native enzyme or polypeptide. In other embodiments, the native enzyme or polypeptide can be encoded by an exogenous nucleic acid sequence or an exogenous gene, such that the encoding nucleic acid sequence or gene is added to the cell from outside the cell, for example, where overexpression of the native enzyme or polypeptide is desired. Overexpression of a native enzyme or polypeptide, such as any described herein, can also be achieved by other methods known in the art, such as, for example, by placing the encoding nucleic acid sequence or gene under control of a different (e.g., a more active, or constitutively active, or stronger) promoter, or by modifying the native or endogenous promoter, or by modifying other associated regulatory elements. In such a case, the encoding nucleic acid sequence with the modified or altered regulatory element(s) is considered an exogenous nucleic acid sequence.

[0095] The gene or nucleic acid sequence encoding a native enzyme or polypeptide can be a non-native variant, for example, where the gene or nucleic acid sequence is operably linked to a non-native regulatory element; in such a case, the non-native gene or nucleic acid sequence typically is referred to herein as an exogenous gene or nucleic acid sequence, even though it can encode a native polypeptide or enzyme.

[0096] In other embodiments, any of the enzymes or polypeptides described herein can be a heterologous enzyme or polypeptide, and the polynucleotide, nucleic acid sequence, or gene, encoding the enzyme, is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell.

[0097] A native, endogenous, or heterologous enzyme or polypeptide can be expressed or overexpressed in the recombinant cell or microbe or microorganism. For example, in some embodiments, an enzyme or polypeptide is native and is expressed in the recombinant cell or microbe by an endogenous nucleic acid sequence or gene. In other embodiments, the polypeptide or enzyme is native to the cell and is overexpressed, for example, where the recombinant cell or microbe contains an exogenous nucleic acid sequence encoding the native enzyme or polypeptide. In other embodiments, the enzyme or polypeptide is heterologous to the recombinant cell or microbe, and can be expressed or overexpressed in the recombinant cell or microbe by an exogenous nucleic acid sequence.

III. Recombinant microbes comprising novel biochemical pathways

[0098] As discussed herein, a novel (or modified) biosynthetic pathway engineered in a microbe to produce trans-2 unsaturated fatty acids and derivatives thereof is provided. An example of this the novel biochemical synthetic pathway is depicted in FIG. 2. In FIG. 2, the novel pathway harnesses the high efficiency of acyl-ACP dependent fatty acid biosynthesis. Once the desired chain length of the target trans-2-enoyl fatty acid derivative product is reached, a thioesterase (e.g., having EC 3.1.2.14, or EC 3.1.2.21) is employed that hydrolyses (R)-3-hydroxy- acyl-ACP from the acyl-ACP dependent fatty acid biosynthesis to the corresponding 3-hydroxy fatty acid. Free 3-hydroxy fatty acid is then reactivated to the corresponding R-3-hydroxy-acyl- CoA using an acyl-CoA synthetase (also known as an acyl-CoA ligase, EC 6.2.1.3). Alternatively, a 3-hydroxy-acyl-ACP:CoA transacylase (EC 2.4.1.-) can be employed to directly convert (R)-3- hydroxy-acyl-ACP to the corresponding (R)-3-hydroxy-acyl-CoA. Then, an R-3-hydroxy-acyl- CoA dehydratase (EC 4.2.1.134, EC 4.2.1.55) or an R-specific enoyl-CoA hydratase (EC 4.2.1.119, EC 4.2.1.17) (these enzymatic activities are interchangeable) is employed to convert 3- hydroxy-acyl-CoA to trans-2-enoyl-CoA. At this point, trans-2-enoyl-CoA enters the biochemical pathway toward the target product, e.g., trans-2 fatty acids, trans-2 fatty esters, trans- 2 fatty aldehydes, trans-2 fatty alcohols, trans-2 fatty alcohol acetates, etc.

[0099] For example, trans-2-enoyl-CoA can be esterified to the corresponding trans-2 fatty ester when an ester synthase (EC 2.3.1.20) is employed, or it can be reduced to the corresponding trans-2 fatty aldehyde when an acyl-CoA reductase (EC 1.2.1.50, EC 1.2.1.84) is employed, or it can be reduced to a trans-2 fatty alcohol when an acyl-CoA reductase and an alcohol dehydrogenase (EC 1.1.1.1) are employed or when a fatty alcohol forming acyl-CoA reductase is used, or it can be converted to an trans-2-fatty alcohol acetate when, in addition, an alcohol- acetyl- CoA transferase (EC 2.3.1.84) is employed. Free trans-2-fatty acids can be produced by chemical hydrolysis of the trans-2-fatty esters or by employing an acyl-CoA specific thioesterase (see FIG. 2).

[00100] In some embodiments, the trans-2-enoyl-thioesters (trans-2-enoyl-CoA and/or trans- 2-enoyl-ACP) can be hydrolyze to the corresponding trans-2-fatty acids by a thioesterase with the appropriate activity. The trans-2-fatty acid can then be converted to a variety of trans-2-fatty acid derivatives by the appropriate fatty acid derivative enzymes. For example, the trans-2-fatty acid can be converted to a trans-2-fatty aldehyde by a carboxylic acid reductase (CAR); to a trans-2- fatty alcohol by a CAR and an alcohol dehydrogenase (ADH); to a trans-2-fatty amine by a CAR and a transaminase; to a trans-2-a,co-diol by a CAR, ADH, and an omega-hydroxylase/oxygenase; to a trans-2-co-hydroxy fatty acid by an omega-hydroxylase; to a trans-2-a, co-diacid by an omega- hydroxylase/oxygenase, an alcohol dehydrogenase/oxidase, and an aldehyde dehydrogenase/oxidase, etc.

[00101] In this biochemical pathway, R-3-hydroxy-acyl-CoA dehydratase and R-specific enoyl-CoA hydratase are interchangeable as they are carrying out the same reversible enzymatic reaction, i.e. the reversible interconversion of a 3-hydrox- acyl-CoA and a 2-trans-enoyl-CoA. Under certain physiological conditions such enzymes may favor the dehydration of a 3-hydroxy- acyl-CoA to a 2-trans-enoyl-CoA (i.e., a R-3-hydroxy-acyl-CoA dehydratase), or under certain physiological conditions such an enzyme may favor the hydration of a 2-trans-enoyl-CoA to a 3- hydroxy-acyl-CoA to (i.e., a trans-2-enoyl-CoA hydratase).

[00102] The efficiency of the novel pathway to produce trans-2 fatty acids and derivatives thereof may be increased in a number of ways. For example, when a thioesterase is employed that has high hydrolytic activity, or specificity, or selectivity, towards 3-hydroxy-acyl-ACPs, and low or no hydrolytic activity, or specificity, or selectivity, towards acyl-ACPs and acyl-thioesters with CoenzymeA (CoA) (i.e., acyl-CoA, 3-hydroxy-acyl-CoA or trans-2-enoyl-CoA), the production of trans-2 fatty acids and derivatives thereof may be increased. If a thioesterase has undesirable hydrolytic activity towards acyl-ACP or acyl-CoA thioesters, it can be engineered or evolved such that it possesses only low or no hydrolytic activity towards those thioesters. Once the thioesterase has low or no such hydrolytic activity, the efficiency of the novel pathway to produce trans-2 fatty acids or derivatives thereof is increased.

[00103] Additionally or alternatively, the efficiency of the novel pathway to produce trans-2 fatty acids or derivatives thereof may also be increased when an ester synthase or an acyl-CoA- reductase is employed that has high activity toward trans-2-enoyl CoA and low or no activity towards 3-hydroxy-acyl-CoA or acyl-thioesters with acyl carrier protein (ACP) (i.e. acyl-ACP or 3-hydroxy-acyl-ACP). If an ester synthase or acyl-CoA reductase has undesirable activity toward acyl-ACP or 3-hydroxy-acyl-ACP thioesters, it can be engineered or evolved such that it possesses only low or no activity toward those thioesters. Once the ester synthase or acyl-CoA reductase has low or no such activity, the efficiency of the novel (or modified) pathway to produce trans-2 fatty acids or derivatives thereof is increased.

[00104] Additionally or alternatively, the efficiency of the novel pathway to produce trans-2 fatty acids or derivatives thereof may also be increased when an ester synthase is engineered or evolved to more efficiently convert trans-2-enoyl-CoA and methanol, ethanol, propanol, isopropanol, butanol, isobutanol, or allyl alcohol to the corresponding trans-2 fatty acid methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or allyl ester.

[00105] Additionally or alternatively, the efficiency of the novel pathway to produce trans-2 fatty acids or derivatives thereof may also be increased when an acyl-CoA-reductase is engineered or evolved to more efficiently convert trans-2-enoyl-CoA to trans-2 fatty aldehydes.

[00106] Additionally or alternatively, the efficiency of the novel pathway to produce trans-2 fatty acids or derivatives thereof may also be increased when an alcohol-acetyl-CoA transferase is engineered or evolved to more efficiently convert trans-2 fatty alcohols to trans-2 fatty alcohol acetates.

[00107] Additionally or alternatively, if the producing microbial host encodes a trans-2-enoyl- CoA reductase (enoyl-CoA reductase) or a polypeptide with trans-2-enoyl-CoA reductase (enoyl- CoA reductase) activity, the efficiency of the novel pathway to produce trans-2 fatty acids or derivatives thereof may also be increased by attenuating or eliminating the trans-2-enoyl-CoA reductase (enoyl-CoA reductase) activity. In contrast to the biochemical pathways depicted in FIG. 1, the novel pathway may not require trans-2-enoyl-CoA reductase (enoyl-CoA reductase) for balanced and “high flux” fatty acid biosynthetic activity, therefore, attenuating trans-2-enoyl- CoA reductase (enoyl-CoA reductase) activity may not decrease the overall fatty acid derivative titer or increase 3-hydroxy fatty acid derivative side product formation.

[00108] Additionally or alternatively, if the producing microbial host contains a trans-2-enoyl- ACP reductase (EC 1.3.1.10, EC 1.3.1.10, EC 1.3.1.38, EC 1.3.1.39, EC 1.3.1.104) that also possesses trans-2-enoyl-CoA reductase activity, e.g., FabI of E. coli (see, e.g., Bergeler et al. 1994, J. Biol. Chem. 269: 5493-5496; Vick et al., AEM 2015, vol. 81:1406-1416), the gene encoding such a trans-2-enoyl-ACP reductase may be replaced (e.g., attenuated and replaced or deleted/knocked out and replaced) with a trans-2-enoyl-ACP reductase gene that encodes an enzyme possessing lower or no trans-2-enoyl-CoA reductase activity, e.g. FabL or FabI from Bacillus subtilus (see, e.g., Heath et al. 2000, J. Biol. Chem. 275: 40128-40133; Vick et al. , AEM 2015, vol. 81:1406-1416). In addition, Fabl-type or FabL-type trans-2-enoyl-ACP reductases from other microbes, FabK-type trans-2-enoyl-ACP reductases (see, e.g., Marrakchi et al. 2003, Biochem. J. 370:1055-1062) or FabV-type trans-2-enoyl-ACP reductases (see, e.g., Massengo- Tiasse & Cronan 2008, J. Biol. Chem. 283: 1308-1316) may be employed for the same purpose, if they do not possess significant trans-2-enoyl-CoA reductase activity. In addition, a trans-2- enoyl-ACP reductase with undesirable trans-2-enoyl-CoA reductase activity may be engineered or evolved such that it possesses only low or no trans-2-enoyl-CoA reductase activity, while maintaining high trans-2-enoyl-ACP reductase activity. Once the producing microbial host encodes a trans-2-enoyl-ACP reductase with low or no trans-2-enoyl-CoA reductase activity, the efficiency of the novel pathway to produce trans-2 fatty acids or derivatives thereof may be increased.

[00109] Additionally or alternatively, the efficiency of the novel pathway for producing trans- 2 fatty acids or derivatives thereof may also be increased when the (R)-3-hydroxy acyl-ACP dehydratase (EC 4.2.1.59) activity of the producing microbial host is attenuated.

[00110] Additionally or alternatively, the efficiency of the novel pathway for producing trans- 2 fatty acids or derivatives thereof may also be increased when the expression of the acyl carrier protein (ACP) of the producing microbial host is attenuated.

[00111] An alternative way to produce trans-2 fatty acids or derivatives thereof is also provided herein. For example, 3-hydroxy fatty acids can be fed/added to the producing microbial host exogenously. In this case expression of a thioesterase is not necessary. The exogenously added 3-hydroxy fatty acid is activated to the corresponding 3-hydroxy acyl-CoA by acyl-CoA synthetase (also known as acyl-CoA ligase). Then R-3 -hydroxy acyl-CoA dehydratase or an R- specific enoyl-CoA hydratase (these enzymatic activities are interchangeable) is employed to convert 3-hydroxy acyl-CoA to trans-2-enoyl-CoA. At this point the trans-2-enoyl-CoA enters the biochemical pathway toward the target product, e.g., trans-2-fatty acids, trans-2-fatty esters, trans-2-fatty alcohols or trans-2-fatty alcohol acetates (see FIG. 2).

[00112] The method also allows for producing trans-2 fatty acids or derivatives thereof in any biochemical pathways that include 3-hydroxy fatty acid, 3-hydroxy-acyl-ACP, and/or 3-hydroxy- acyl-CoA intermediates. Additional examples are biochemical pathways towards trans-2 fatty amines, trans-2 co-hydroxy fatty esters, trans-2 co-carboxy fatty esters, trans-2 a/co-fatty diesters, etc. For example, when a biochemical pathway described in FIG. 2 is combined with an cohydroxylase (or co-oxygenase), e.g., a cypl53A family P450 enzyme or an alkB type enzyme (EC 1.14.15.3), then an co-hydroxylated trans-2 fatty acid, such as 10-hydroxy-trans-2-decenoic acid, or an co-hydroxylated trans-2 fatty acid alkyl ester, such as 10-hydroxy-trans-2-decenoic acid methyl ester, etc., can be produced.

[00113] The method also allows for producing a trans-2 fatty ester composition that comprise trans-2 fatty acids esterified with various alcohols, e.g. methanol for producing trans-2- fatty acid methyl ester, ethanol for producing trans-2- fatty acid ethyl ester, propanol for producing trans-2- fatty acid propyl ester, isopropanol for producing trans-2- fatty acid isopropyl ester, butanol for producing trans-2- fatty acid butyl ester, isobutanol for producing trans-2- fatty acid isobutyl ester, allyl alcohol for producing trans-2- fatty acid allyl alcohol ester, etc. The alcohol is added to the culture medium during fermentation or is made endogenously by the producing recombinant microbial host.

[00114] The method also allows for producing trans-2 fatty acids and derivatives thereof of various chain lengths, e.g., even- or odd-chain C6 to C18. For example, if the fatty acid derivative pathway is an ester synthase and methanol is added, then (2e)-hexenoic acid methyl ester, (2e)- heptenoic acid methyl ester, (2e)-octenoic acid methyl ester, (2e)-nonenoic acid methyl ester, (2e)- decenoic acid methyl ester, (2e)-dodecenoic acid methyl ester, (2e)-tridecenoic acid methyl ester, (2e) -tetradecenoic acid methyl ester, (2e) -pentadecenoic acid methyl ester, (2e)-hexadecenoic acid methyl ester, (2e)-heptadecenoic acid methyl ester, (2e)-octadecenoic acid methyl ester can be produced. Altenratively, if the fatty acid derivative pathway is an ester synthase and ethanol is added then (2e)-hexenoic acid ethyl ester, (2e)-heptenoic acid ethyl ester, (2e)-octenoic acid ethyl ester, (2e)-nonenoic acid ethyl ester, (2e)-decenoic acid ethyl ester, (2e)-dodecenoic acid ethyl ester, (2e)-tridecenoic acid ethyl ester, (2e) -tetradecenoic acid ethyl ester, (2e) -pentadecenoic acid ethyl ester, (2e) -hexadecenoic acid ethyl ester, (2e) -heptadecenoic acid ethyl ester, (2e)- octadecenoic acid ethyl ester can be produced. A method of producing odd-chain fatty acids or derivatives thereof is described in U.S. patent application 2012/0070868, which is incorporated herein by reference in its entirety.

[00115] The method also allows for producing trans-2 fatty acids or derivatives thereof with iso- or anteiso-branched acyl-chains. A method of producing branched-chain fatty acids and derivatives thereof is described in U.S. patent application 2011/0244532, which is incorporated herein by reference in its entirety.

[00116] The method also allows for producing trans-2 fatty acid derivatives with a second double bond, which is incorporated during acyl-ACP dependent fatty acid biosynthesis in for example the “co7” position, i.e., seven carbons from the reducing end of the acyl chain. For example, if the thioesterase hydrolyses (5Z)-3-hydroxy dodecenoyl-ACP, then depending on the fatty acid derivative pathway employed (2e,5z)-dodecadienyl alkyl ester, (2e,5z)-dodecadien-l- al, (2e,5z)-dodecadien-l-ol, (2e,5z)-dodecadien-l-ol acetate, etc., can be produced by the recombinant microbial host (see FIG. 3).

[00117] Thus, a recombinant microbe comprising: (1) either a combination of a 3-hydroxy acyl-ACP thioesterase that uses 3-hydroxy acyl-ACP as a substrate, and an acyl-CoA synthetase to convert a 3-hydroxy acyl-ACP to 3-hydroxy acyl-CoA via a 3-hydroxy fatty acid intermediate, or a 3-hydroxy acyl-ACP:CoA transacylase, which directly converts the 3-hydroxy acyl-ACP to 3-hydroxy acyl-CoA; (2) a heterologous 7?-3-hydroxy-acyl-CoA dehydratase or heterologous R- specific enoyl-CoA hydratase; and, optionally, (3) an additional fatty acid derivative enzyme, such as, for example, an ester synthase, an acyl-CoA reductase, or an acyl-CoA thioesterase, is provided herein, wherein the recombinant microbe optionally does not comprise an enoyl-CoA reductase, or wherein the recombinant microbe optionally comprises an attenuated acyl-CoA reductase activity. Additionally, the recombinant microbe may further comprise one or more enzymes such as P-keto-acyl-ACP synthase (I, II, or III), a (heterologous) enoyl-ACP reductase, and a FadR. The recombinant microbe may comprise a trans-2-enoyl-CoA reductase (enoyl-CoA reductase) activity that is attenuated compared to a wildtype microbe. As described elsewhere herein, if the recombinant microbe comprises a trans-2-enoyl-ACP reductase (enoyl-ACP reductase) that also has trans-2-enoyl-CoA reductase (enoyl-CoA reductase) activity, the trans-2-enoyl-ACP reductase (enoyl-ACP reductase) can be attenuated, or can be attenuated or deleted and replaced with another enzyme comprising trans-2-enoyl-ACP reductase (enoyl-ACP reductase) activity but comprising little to no trans-2-enoyl-CoA reductase (enoyl-CoA reductase) activity. Additionally or alternatively, the trans-2-enoyl-ACP reductase (enoyl-ACP reductase) comprising trans-2- enoyl-CoA reductase (enoyl-CoA reductase) activity can be engineered or modified or mutated, such that it has less or no trans-2-enoyl-CoA reductase (enoyl-CoA reductase) activity.

[00118] The recombinant microbe described herein may be a bacterium, cyanobacterium, yeast, or algae.

[00119] The recombinant microbe may be a recombinant proteobacterium, such as a recombinant y-proteobacterium. In particular, the y-proteobacterium may be Escherichia coli, Salmonella spp., Vibrio natriegens, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Xanthomonas axonopodis, Pseudomonas syringae, Xyella fastidiosa, Marinobacter aquaeolei, Yersinia pestis, Bacillus spp., Lactobacillus spp., Zymomonas spp., Streptomyces spp., or Vibrio cholerae. Specifically, the y-proteobacterium may be Escherichia coli.

[00120] Additionally or alternatively, the recombinant microbe may be a cyanobacterium such as Synechococcus elongatus PCC7942. or Synechocystis sp. PCC6803.

[00121] Additionally or alternatively, the recombinant microbe may be a yeast such as Saccharomyces cerevisiae, Scheffersomyces stipitis, Schizosaccharomyces pombe, Kluyveromyces marxianus, K. lactis, Pichia pastoris, Hansenula polymorpha, and Yarrowia lipolytica or an algae such as Botryococcus braunii, Nannochloropsis gaditina, Chlamydomonas reinhardtii, Chlorella vulgaris., Spirulina platensis, Ostreococcus tauri, Phaeodactylum tricornutum, Symbiodinium sp., algal phytoplanktons, Saccharina japonica, Chlorococum spp., and Spiro gyra spp. [00122] Additionally or alternatively, the acyl-ACP thioesterase or 3-hydroxy acyl-ACP:CoA transacylase may be PhaG, FatBl, FatB2, FatB3, an acyl-ACP thioesterase from Lactobacillus plantarum, or an acyl-ACP thioesterase from Anaerococcus tetradius. Alternatively, the acyl- ACP thioesterase or 3-hydroxy acyl-ACP:CoA transacylase may be any one disclosed in Table 1, or a homolog thereof with the same activity.

[00123] The acyl-CoA synthetase may be FadD3, FadD-I, FadD-II, FadD, or IcfB. Alternatively, the heterologous acyl-CoA synthetase may be any one disclosed in Table 2, or a homolog thereof with the same activity.

[00124] The heterologous R-3-hydroxy acyl-CoA dehydratase or heterologous /^-specific enoyl-CoA hydratase may be PhaJl, PhaJ3, PhaJ4, or MaoC. Alternatively, the heterologous R- 3-hydroxy acyl-CoA dehydratase or heterologous R-specific enoyl-CoA hydratase may be any one disclosed in Table 3, or a homolog thereof with the same activity.

[00125] Additionally or alternatively, the recombinant microbe may be a recombinant proteobacterium and the 3-hydroxy acyl-ACP thioesterase is FatBl, the acyl-CoA synthetase is FadD, and the heterologous R-3-hydroxy acyl-CoA dehydratase or heterologous R-specific enoyl- CoA hydratase may be PhaJ 1 from P. putida, PhaJ4 from P. putida, PhaJ3 from P. aeruginosa, or PhaJ4 from P. aeruginosa. In particular, the recombinant proteobacterium may further comprise a native or heterologous FadR and a native or heterologous FabB, and FadE expression can optionally be attenuated (or deleted) compared to a wildtype or reference proteobacterium.

[00126] Additionally or alternatively, the recombinant microbe may be a recombinant proteobacterium and the 3-hydroxy acyl-ACP thioesterase is FatB2, the acyl-CoA synthetase is FadD3, and the heterologous R-specific enoyl-CoA hydratase may be PhaJ4. The recombinant proteobacterium may further comprise a native or heterologous FadR, and FadE expression may be attenuated (or deleted) compared to a wildtype or reference proteobacterium.

[00127] Additionally or alternatively, the recombinant microbe is a recombinant proteobacterium and the 3-hydroxy acyl-ACP thioesterase is PhaG, the acyl-CoA synthetase is FadD3, and the heterologous R-specific enoyl-CoA hydratase is PhaJ4. The recombinant proteobacterium may further comprise a native or heterologous FadR, and FadE expression is optionally attenuated (or deleted) compared to a wildtype or reference proteobacterium.

[00128] Additionally or alternatively, the recombinant microbe produces a trans-2 unsaturated fatty acid or a derivative thereof. The recombinant microbe may produce one or more of a trans- 2 fatty acid, a trans-2 fatty acid ester, a trans-2 fatty acid methyl ester, a trans-2 fatty acid ethyl ester, a trans-2-unsaturated fatty aldehyde, a trans-2-unsaturated fatty alcohol, a trans-2- unsaturated fatty alcohol acetate, a trans-2-unsaturated fatty amine, a trans-2-diester, a trans-2- diacid, a trans-2-diol, a trans-2 unsaturated co-hydroxy fatty ester, or a trans-2 unsaturated cohydroxy fatty acid. The trans-2 unsaturated fatty acid or derivative thereof may be one or more of trans-2-hexadecenoic acid, trans-2-hexadecenoic acid ethyl ester, trans-2-hexadecenoic acid methyl ester, trans-2-tetradecenoic acid, trans-2-tetradecenoic acid ethyl ester, trans-2- tetradecenoic acid methyl ester, trans-2-dodecenoic acid, trans-2-dodecenoic ethyl ester, trans-2- dodecenoic acid methyl ester, trans-2-decenoic acid, trans-2-decenoic acid ethyl ester, trans-2- decenoic acid methyl ester, trans-2-octenoic acid, trans-2-octenoic acid ethyl ester, and trans-2- octenoic acid methyl ester.

[00129] In some exemplary embodiments, the host cell (e.g., a recombinant microbe; or a recombinant bacterium, proteobacterium, cyanobacterium, yeast, or algae) may further comprise genetic manipulations and alterations to enhance or otherwise fine tune the production of the target fatty acids or derivatives thereof. The optional genetic manipulations can be used interchangeably from one host cell to another, depending on what other heterologous enzymes and what native enzymatic pathways are present in the host cell. Some optional genetic manipulations include one or more of the following modifications described below.

[00130] The gene encoding acyl-CoA dehydrogenase (e.g., FadE) can optionally be attenuated or deleted in the recombinant cells, microbes, or microorganisms provided herein. FadE (Acyl- CoA dehydrogenase) catalyzes the first step in fatty acid utilization/degradation (P-oxidation cycle), which is the oxidation of acyl-CoA to 2-enoyl-CoA (see e.g., Campbell, J.W. and Cronan, J.E. Jr (2002) J. Bacteriol. 184(13):3759-3764; and Lennen, R.M. and Pfleger, B.F (2012) Trends Biotechnol. 30(12):659-667). Since FadE initiates the P-oxidation cycle, when E. coli lacks FadE, it cannot grow on fatty acids as a carbon source (see e.g., Campbell, J.W. and Cronan supra). The same effect can be achieved by attenuating or deleting other enzymes from the P-oxidation cycle, e.g., FadA, which is a 3-ketoacyl-CoA thiolase, or FadB, which is a dual 3 -hydroxy acyl-CoA- dehydrogenase/dehydratase.

[00131] However, when a microbe such as E. coli is grown on a carbon source other than fatty acids, e.g., when it is grown on sugar, acetate, etc., FadE attenuation is optional, because under such conditions, FadE expression is repressed by FadR. Therefore, when cells are grown on a simple carbon source, such as, e.g., glucose, the FadE gene product is already attenuated. Accordingly, when grown on a carbon source other than fatty acids, a FadE mutation/deletion or attenuation is optional.

[00132] In some embodiments, the fatty acid biosynthetic pathway in the production host uses the precursors acetyl-CoA and malonyl-CoA. E. coli or other host organisms engineered to overproduce these components can serve as the starting point for subsequent genetic engineering steps to provide the specific output product (such as, fatty acids, fatty esters, hydrocarbons, fatty alcohols, etc.). Several different modifications can be made, either in combination or individually, to the host cell or strain, to obtain increased acetyl-CoA, malonyl-CoA, fatty acid, and/or fatty acid derivative production. See, for example, U.S. Patent Application Publication 2010/0199548, which is incorporated herein by reference in its entirety. For example, to increase malonyl-CoA production, one or more of the acetyl-CoA carboxylase subunits, including AccA, AccB, AccC, and/or AccD, can be expressed or overexpressed in the recombinant cell or microbe.

[00133] Other exemplary modifications of a host cell include, e.g., overexpression of nonnative and/or native and/or variants of genes involved in the synthesis of acyl-ACP. In general, increasing acyl-ACP synthesis increases the amount of acyl-ACP, which is the substrate of thioesterases, ester synthases, and acyl-ACP reductases. Exemplary enzymes that increase acyl- ACP production include, e.g., enzymes that make up the “fatty acid synthase” (FAS). As is known in the art (see e.g., U.S. 2010/0199548) FAS enzymes are a group of enzymes that catalyze the initiation and elongation of acyl chains. The acyl carrier protein (ACP) along with the enzymes in the FAS pathway control the length, degree of saturation, and branching of the fatty acids produced. FAS pathway enzymes include, for example, AccABCD, FabD, FabH, FabG, FabA, FabB, FabZ, FabF, FabI, FabK, FabU, FabM, FabQ, FabV, FabX, FabR, and FadR, and homologs thereof and corresponding enzymes with the same activities that are derived from other organisms or species. Depending upon the desired product, one or more of these genes can be attenuated, deleted, downregulated, expressed, upregulated, or over-expressed, or otherwise modified or deregulated. The functions or exemplary uses for FAS genes (e.g., accA, accB, accC, accD, fabA, fabB, fabD, fabF, fabG, fabH, fabl, fabR, fabV, fabZ, fabK, fabU, fabM, fabX) are provided in Table A below. Table A also provides the functions or exemplary uses genes encoding other enzymes, including, for example, certain fatty acid derivative genes (e.g., acyl-CoA synthetases, thioesterases, ester synthases, alcohol dehydrogenases, acyl-CoA reductases, etc.). Any one or more of the FAS or other enzymes described herein in can be expressed or overexpressed in the recombinant cells or microbes provided herein, including heterologously expressed or overexpressed. Additionally or alternatively, the expression or activity of any one or more of the genes can be altered, deregulated, or modified, for example, by attenuation, downregulation, or deletion of one or more genes and their encoded products.

[00134] In some embodiments, the recombinant cells, microbes, or microorganisms provided herein contain pathways that use a renewable feedstock, such as glucose, to produce fatty acids and derivatives thereof. Glucose is converted to an acyl-ACP by the native organism. Polynucleotides that code for polypeptides with fatty acid degradation enzyme activity can be optionally attenuated depending on the desired product. Non-limiting examples of such polypeptides are acyl-CoA synthetase (FadD) and acyl-CoA dehydrogenase (FadE). For example, FadR is a key regulatory factor involved in fatty acid degradation and fatty acid biosynthetic pathways (Cronan et al., Mol. Microbiol., 29(4): 937-943 (1998)). The E. coli enzyme FadD and the fatty acid transport protein FadL are components of a fatty acid uptake system. FadL mediates transport of fatty acids into the bacterial cell, and FadD mediates formation of acyl-CoA esters. When no other carbon source is available, exogenous fatty acids are taken up by bacteria and converted to acyl-CoA esters, which can bind to the transcription factor FadR and depress the expression of the fad genes that encode proteins responsible for fatty acid transport (FadL), activation (FadD), and P-oxidation (FadA, FadB, and FadE,). When alternative sources of carbon are available, bacteria synthesize fatty acids as acyl-ACPs, which are used for phospholipid synthesis, but are not substrates for P-oxidation. Thus, acyl-CoA and acyl-ACP are both independent sources of fatty acids that can result in different end-products (Caviglia et al., J. Biol. Chem., 279(12): 1163-1169 (2004)).

[00135] In some embodiments, a host strain may overexpress one or more of the FAS genes (e.g., any one or more of those described above and/or known in the art). Exemplary FAS genes that may be overexpressed include, e.g., FadR from Escherichia coli (see, e.g., GenBank Accession No. NP_415705.1), FabB from Escherichia coli (see, e.g., UniProtKB Accession No. P0A953), or FabZ from Escherichia coli (see, e.g., UniProtKB Accession No. P0A6Q6) or FabZ Acinetobacter baylyi (see, e.g., UniProtKB Accession No. Q6FCG4), as well as homologs thereof and corresponding enzymes, with the same activities, that are derived from other organisms or species. In another embodiment, the host strain encompasses optional overexpression of one or more genes, including, for example, fadR, fabA, fabD, fabG, fabH, fabV, and/or fabF. Examples of such genes axc fadR from Escherichia coli, fabA from Salmonella typhimurium (NP_460041), fabD from Salmonella typhimurium (NP_460164), fabG from Salmonella typhimurium (NP_460165), fabH from Salmonella typhimurium (NP_460163), fabV from Vibrio cholera (YP_001217283), and tabF from Clostridium acetobutylicum (NP_350156). In some exemplary embodiments, the overexpression of one or more of these genes, which code for enzymes and regulators in fatty acid biosynthesis, serves to further increase the titer of fatty acids and fatty acid derivative compounds under particular culture conditions.

[00136] Also provided herein are cell cultures comprising any of the recombinant cells, microbes, or microorganism, described herein.

IV. Compositions [00137] Provided herein are fatty acid compositions (or fatty acid derivative compositions) comprising fatty acids and/or derivatives thereof, including, but not limited to, for example, fatty esters, fatty alcohols, fatty alcohol acetates, fatty aldehydes, fatty amines, co-hydroxy fatty acids, co-carboxy fatty acids, co-hydroxy fatty esters, co-carboxy fatty esters, a, co-fatty diacids, a, co-fatty diols, and a, co-fatty diesters, that are produced by the recombinant cells or microbes (or cell cultures comprising them), or the methods or pathways described herein. The compositions can comprise trans-2-fatty acids or derivatives thereof, including trans-2-unsaturated versions of fatty esters, fatty alcohols, fatty alcohol acetates, fatty aldehydes, fatty amines, fatty amides, co-hydroxy fatty acids, co-carboxy fatty acids, co-hydroxy fatty esters, co-carboxy fatty esters, a, co-fatty diacids, a, co-fatty diols, and a, co-fatty diesters, for example. The compositions can further comprise 3- hydroxy (3-OH) fatty acids or derivatives thereof, including 3-OH fatty esters, 1,3-fatty diols, 1,3- fatty dialcohol acetates, 3-OH fatty aldehydes, 3-OH fatty amines, 3-OH fatty amides, co-hydroxy fatty acids with a 3-OH group, co-carboxy fatty acids with a 3-OH group, co-hydroxy fatty esters with a 3-OH group, co-carboxy fatty esters with a 3-OH group, a, co-fatty diacids with a 3-OH group, a, co-fatty diols with a 3-OH group, or a, co-fatty diesters with a 3-OH group, or a combination thereof.

[00138] Further contemplated herein are fatty ester compositions comprising trans-2 fatty acid alkyl esters, 3-hydroxy fatty acid alkyl esters, such as a trans-2 fatty acid methyl ester, a trans-2 fatty acid ethyl ester, a trans-2-unsaturated fatty aldehyde, a trans-2-unsaturated fatty alcohol, a trans-2-unsaturated fatty alcohol acetate or a trans-2 unsaturated co-hydroxy fatty acid.

[00139] Additionally or alternatively, the fatty ester composition may comprise trans-2 fatty acid methyl ester, fatty acid methyl ester (FAME) and 3-hydroxy-FAME; or may comprise trans- 2 fatty acid ethyl ester, fatty acid ethyl ester (FAEE) and 3-hydroxy-FAEE, wherein the predominant chain length of the fatty esters in the composition is C8, CIO or C12.

[00140] The fatty ester composition may be prepared by culturing the recombinant microbe described herein in the presence of a carbon source to produce a culture and adding an alcohol such as, methanol or ethanol, to the culture. In particular, the fatty ester composition may comprise a trans-2 fatty acid methyl ester, such as trans-2-hexadecenoic acid methyl ester, trans-2- tetradecenoic acid methyl ester, trans-2-dodecenoic acid methyl ester, trans-2-decenoic acid methyl ester, or trans-2-octenoic methyl ester. Additionally or alternatively, the fatty ester composition may comprise a trans-2 fatty acid ethyl ester, such as trans-2-hexadecenoic acid ethyl ester, trans-2-tetradecenoic acid ethyl ester, trans-2-dodecenoic acid ethyl ester, trans-2-decenoic acid ethyl ester, and trans-2-octenoic acid ethyl ester. [00141] For example, the compositions provided herein can contain at least about 0.01%-99%, or 10-99%, or 20-99%, or 30-99%, or 40-99%, or 50-99%, or 10-90%, or 20-90%, or 30-90%, or 40-90%, or 50-90%, or 10-80%, or 20-80%, or 30-80%, or 40-80%, or 50-80%, or 60-80%, or 70- 80%, or more, by weight of the composition, of one or more trans-2-fatty acids or derivatives thereof. For example, the compositions provided herein can contain at least about 0.01% 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more, by weight of the composition, of one or more trans-2-fatty acids or derivatives thereof. The composition can further comprise about 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, or less, by weight of the composition, of one or more 3-hydroxy fatty acids or derivatives thereof; and/or can comprise about 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, or less, by weight of the composition, of a fatty acid or fatty acid derivative (that is not a trans-2-fatty acid or derivative thereof, and is not a 3-hydroxy fatty acid or derivative thereof).

[00142] For example, provided herein is a fatty acid derivative composition, comprising at least about 90 wt%, or more, of a trans-2-fatty acid or derivative thereof; and 10 wt% or less of other fatty acid species or a fatty acid or derivative thereof that is not a trans-2-fatty acid or derivative thereof, including, for example, fatty acids, 3-hydroxy fatty acids, and derivatives thereof. In another embodiment, provided herein is a composition comprising at least about 80 wt%, or more, of a trans-2-fatty acid or derivative thereof; and 20 wt% or less of other fatty acid species, or a fatty acid or derivative thereof that is not a trans-2-fatty acid or derivative thereof, including, for example, fatty acids, 3-hydroxy fatty acids, and derivatives thereof. Also provided herein is a composition comprising at least about 99 wt% of a trans-2-fatty acid or derivative thereof; and 1 wt% or less of other fatty acid species, or a fatty acid or derivative thereof that is not a trans-2- fatty acid or derivative thereof, including, for example, fatty acids, 3-hydroxy fatty acids, and derivatives thereof. In another embodiment, provided herein is a composition comprising at least about 70 wt%, or more, of a trans-2-fatty acid or derivative thereof; and 30 wt% or less of other fatty acid species, or a fatty acid or derivative thereof that is not a trans-2-fatty acid or derivative thereof, including, for example, fatty acids, 3-hydroxy fatty acids, and derivatives thereof. In another embodiment, provided herein is a composition comprising at least about 75 wt%, or more, of a trans-2-fatty acid or derivative thereof; and 25 wt% or less of other fatty acid species, or a fatty acid or derivative thereof that is not a trans-2-fatty acid or derivative thereof, including, for example, fatty acids, 3 -hydroxy fatty acids, and derivatives thereof. In another embodiment, provided herein is a composition comprising at least about 85 wt%, or more, of a trans-2-fatty acid or derivative thereof; and 15 wt% or less of other fatty acid species, or a fatty acid or derivative thereof that is not a trans-2-fatty acid or derivative thereof, including, for example, fatty acids, 3-hydroxy fatty acids, and derivatives thereof. In another embodiment, provided herein is a composition comprising at least about 95 wt%, or more, of a trans-2-fatty acid or derivative thereof; and 5 wt% or less of other fatty acid species, or a fatty acid or derivative thereof that is not a trans-2-fatty acid or derivative thereof, including, for example, fatty acids, 3-hydroxy fatty acids, and derivatives thereof. In another embodiment, provided herein is a composition comprising at least about 60 wt%, or more, of a trans-2-fatty acid or derivative thereof; and 40 wt% or less of other fatty acid species, or a fatty acid or derivative thereof that is not a trans-2- fatty acid or derivative thereof, including, for example, fatty acids, 3-hydroxy fatty acids, and derivatives thereof. In another embodiment, provided herein is a composition comprising at least about 65 wt%, or more, of a trans-2-fatty acid or derivative thereof; and 35 wt% or less of other fatty acid species, or a fatty acid or derivative thereof that is not a trans-2-fatty acid or derivative thereof, including, for example, fatty acids, 3-hydroxy fatty acids, and derivatives thereof. In another embodiment, provided herein is a composition comprising at least about 55 wt%, or more, of a trans-2-fatty acid or derivative thereof; and 45 wt% or less of other fatty acid species, or a fatty acid or derivative thereof that is not a trans-2-fatty acid or derivative thereof, including, for example, fatty acids, 3-hydroxy fatty acids, and derivatives thereof. In another embodiment, provided herein is a composition comprising at least about 50 wt%, or more, of a trans-2-fatty acid or derivative thereof; and 50 wt% or less of other fatty acid species, or a fatty acid or derivative thereof that is not a trans-2-fatty acid or derivative thereof, including, for example, fatty acids, 3-hydroxy fatty acids, and derivatives thereof.

[00143] In some embodiments, the compositions produced by the modified pathways, methods, and recombinant microbes provided herein, comprise more trans-2-fatty acids or derivatives thereof than a composition produced by a corresponding pathway or microbe that does not comprise (a) either (i) a combination of a 3-hydroxy-acyl-ACP thioesterase, that uses 3- hydroxyacyl-ACP as a substrate, and an acyl-CoA synthetase; or (ii) a 3-hydroxy acyl-ACP:CoA transacylase; and (b) a heterologous R-3-hydroxy-acyl-CoA dehydratase or a heterologous R- specific enoyl-CoA hydratase; and optionally, wherein the biosynthetic pathway or recombinant microbe does not comprise an enoyl-CoA reductase, or comprises attenuated enoyl-CoA reductase activity. For example, the compositions produced by the modified pathways, methods, and recombinant microbes provided herein, can comprise at least about 1.1- fold, 1.2- fold, 1.3- fold,

1.4- fold, 1.5- fold, 1.6- fold, 1.7- fold, 1.8- fold, 1.9- fold, 2.0- fold, 2.1- fold, 2.2- fold, 2.3- fold,

2.4- fold, 2.5- fold, 2.6- fold, 2.7- fold, 2.8- fold, 2.9- fold, 3.0- fold, 3.5- fold, 4.0- fold, 4.5- fold, 5.0-fold, 5.5- fold, 6.0- fold, 6.5- fold, 7.0- fold, 7.5- fold, 8.0- fold, 8.5- fold, 9.0- fold, 9.5- fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65- fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, or 5000-fold, or more, trans-2-fatty acids or derivatives thereof, than a composition produced by a corresponding pathway or microbe that does not comprise (a) either (i) a combination of a 3- hydroxy-acyl-ACP thioesterase, that uses 3 -hydroxy acyl- ACP as a substrate, and an acyl-CoA synthetase; or (ii) a 3-hydroxy acyl-ACP:CoA transacylase; and (b) a heterologous R-3-hydroxy- acyl-CoA dehydratase or a heterologous R-specific enoyl-CoA hydratase; and optionally, wherein the biosynthetic pathway or recombinant microbe does not comprise an enoyl-CoA reductase, or comprises attenuated enoyl-CoA reductase activity.

[00144] In some embodiments, the compositions produced by the modified pathways, methods, and recombinant microbes provided herein, can comprise at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 1500%, 2000%, 2500%, 3000%, 3500%, 4000%, 4500%, or 5000%, or more, trans-2-fatty acids or derivatives thereof, than a composition produced by a corresponding pathway or microbe that does not comprise (a) either (i) a combination of a 3-hydroxy-acyl-ACP thioesterase, that uses 3 -hydroxy acyl- ACP as a substrate, and an acyl-CoA synthetase; or (ii) a 3- hydroxy acyl-ACP:CoA transacylase; and (b) a heterologous R-3-hydroxy-acyl-CoA dehydratase or a heterologous R-specific enoyl-CoA hydratase; and optionally, wherein the biosynthetic pathway or recombinant microbe does not comprise an enoyl-CoA reductase or comprises an attenuated enoyl-CoA reductase, or attenuated enoyl-CoA reductase activity.

[00145] The trans-2-fatty acids and derivatives thereof, and/or the compositions comprising the trans-2-fatty acids and derivatives thereof, produced by the recombinant microbes provided herein, or by the modified pathways provided herein, or by the methods provided herein, can also be isolated and purified, and can be used in compositions to make products, such as fragrances, flavors, pheromones, fuels, nutritional supplements, dietary supplements, pharmaceuticals, and/or nutraceuticals, and/or precursors thereof.

V. Methods of producing a trans-2 unsaturated fatty adds or a derivatives thereof

[00146] Methods of producing a trans-2 unsaturated fatty acid or a derivative thereof are described herein. The method comprises culturing a recombinant microbe described above in the presence of a carbon source to produce a culture. The method may further comprise isolating the trans-2 unsaturated fatty acid or derivative thereof from the culture. Alcohol may be added to the culture to produce a trans-2 unsaturated fatty acid alkyl ester. The trans-2 unsaturated fatty acid alkyl ester may be trans-2 fatty acid methyl ester or trans-2 fatty acid ethyl ester, or both. The trans-2 fatty acid alkyl ester may be trans-2-hexadecenoic acid ethyl ester, trans-2-hexadecenoic acid methyl ester, trans-2-tetradecenoic acid ethyl ester, trans-2-tetradecenoic acid methyl ester, trans-2-dodecenoic acid ethyl ester, trans-2-dodecenoic acid methyl ester, trans-2-decenoic acid ethyl ester, trans-2-decenoic acid methyl ester, trans-2-octenoic acid ethyl ester, trans-2-octenoic methyl ester, or a combination thereof.

[00147] Additionally or alternatively, a trans-2 unsaturated fatty acid or derivative thereof, prepared by the method described above is provided. The trans-2 unsaturated fatty acid or derivative thereof may be purified. The trans-2 unsaturated fatty acid or derivative thereof may be purified by any known conventional method. For example, the trans-2 unsaturated fatty acid or derivative thereof may be purified by a two-step centrifugation and water-washing; decanting centrifugation and solvent extraction from a biomass; and/or a whole broth extraction with a water immiscible solvent.

VI. Uses

[00148] The recombinant microbes described herein may be used for a variety of purposes. In a particular embodiment, the recombinant microbes may be used to produce a trans-2 unsaturated fatty acid or derivative thereof, or to produce a composition (e.g., a fatty acid derivative composition), comprising a trans-2 unsaturated fatty acid or derivative thereof.

[00149] The trans-2 unsaturated fatty acid or derivative thereof prepared by the cultured and/or fermented recombinant microbe can be used in a composition. The trans-2 unsaturated fatty acid or derivative thereof may be a fermentation product of the recombinant microbe. Alternatively, the composition may comprise one or more (e.g., two, three, four, five, or more) particular species of trans-2 unsaturated fatty acid or derivative thereof. The composition may be a fragrance, pheromone, nutraceutical, nutritional, dietary, or pharmaceutical composition or product, or a precursor thereof.

[00150] Additionally or alternatively, the trans-2 unsaturated fatty acid or derivative thereof may be prepared at a time and/or location that is different than when the composition is prepared. For example, the trans-2 unsaturated fatty acid or derivative thereof may be produced by the recombinant microbe in one location (e.g., a first facility, city, state, or country), transported to another location (e.g., a second facility, city, state, or country) and incorporated into the composition comprising the trans-2 unsaturated fatty acid or derivative thereof.

[00151] Additionally or alternatively, the trans-2 unsaturated fatty acid or derivative thereof may be purified, for example, prior to its use in a composition. The trans-2 unsaturated fatty acid or derivative may be purified to a purity of at least about 60% free (e.g., at least about 65% free, at least about 70% free, at least about 75% free, at least about 80% free, at least about 85% free, at least about 90% free, at least about 95% free, at least about 96% free, at least about 97% free, at least about 98% free, at least about 99% free) from other components with which they are associated.

[00152] Additionally or alternatively, the trans-2 unsaturated fatty acid or derivative thereof may be insoluble or highly insoluble in water. In such cases, the trans-2 unsaturated fatty acid or derivative thereof may be in a separate phase from the environment in which the recombinant proteobacteria reside (e.g., fermentation broth). The trans-2 unsaturated fatty acid or derivative thereof may be solid at room temperature. Alternatively, the trans-2 unsaturated fatty acid or derivative thereof (e.g., alcohol derivatives) may be a liquid.

[00153] Additional purification steps may be required depending on the final product applications and specifications. These steps may include saponification, bleaching, and eventually distillation if high purity of a single chain length is required. All these are standard unit operations used regularly in the industry.

[00154] Additionally or alternatively, purification of the trans-2 unsaturated fatty acid or derivatives thereof may involve isolating and recovering trans-2 unsaturated fatty acids. Purification of trans-2 unsaturated fatty acids differs from the separation of alcohols in that the fatty acids mixed with the biomass are both solids.

[00155] Two different approaches can be applied:

[00156] One approach includes recovery of the solid phase of biomass plus product via decanting centrifugation, followed by solvent extraction of the product from the biomass with an appropriate solvent (i.e., methanol or ethanol). The fatty acids dissolve in the solvent and the biomass is removed either by centrifugation or filtration. The recovery of the fatty acids is then completed by evaporating the solvent. Depending on the application the product can be further used as a solution in the solvent or as a solid. Other purification steps including distillation could be applied to meet final specifications. [00157] Another approach includes recovery of the product via whole broth extraction with a water immiscible solvent. In this approach, the fermentation broth is contacted in either batch or continuous schemes with an appropriate solvent (i.e., butyl acetate, medium chain alcohols or esters) to allow for the complete dissolution of the product in the solvent. The light organic solvent phase can be separated from the water phase in a similar way as those described for the recovery of the long chain alcohols. Once a clear solvent phase has been obtained, the final product is again recovered by solvent evaporation.

[00158] Additionally or alternatively, the trans-2 unsaturated fatty acid or derivative thereof may be prepared by the recombinant microbe, or a composition comprising the trans-2 unsaturated fatty acid or derivative thereof may be prepared by the recombinant microbe which is incorporated into a product. This product is made by combining, mixing, or otherwise using the trans-2 unsaturated fatty acid or derivative thereof produced by the recombinant microbe in combination with other or more additional components to prepare the product. The product may comprise one or more than one (e.g., two, three, four, five, or more) trans-2 unsaturated fatty acids or derivatives thereof prepared by the recombinant microbe. In particular, the product may be a pheromone or precursor thereof, a fragrance or precursor thereof, a pharmaceutical agent or precursor thereof, or a nutritional supplement or precursor thereof.

VII. Modified Biosynthetic Pathways

[00159] Provided herein are modified biosynthetic pathways for the production of trans-2-fatty acids or derivatives thereof, or for the production of compositions comprising trans-2-fatty acids or derivatives thereof. For example, in certain embodiments, provided is a modified biosynthetic pathway, comprising: (a) one or more polypeptides for converting a 3-hydroxy-acyl-ACP to a corresponding 3-hydroxy-acyl-CoA, wherein the one or more polypeptides correspond to: (i) a 3- hydroxy acyl-ACP:CoA transacylase; or (ii) a 3-hydroxy-acyl-ACP thioesterase, and an acyl-CoA synthetase; and (b) a heterologous R-3-hydroxy-acyl-CoA dehydratase or a heterologous R- specific enoyl-CoA hydratase. In some embodiments, the modified biosynthetic pathway additionally (optionally) comprises attenuated enoyl-CoA reductase activity. In some embodiments, the modified biosynthetic pathway, comprises (a) a 3-hydroxy acyl-ACP:CoA transacylase; and (b) a heterologous R-3-hydroxy-acyl-CoA dehydratase or a heterologous R- specific enoyl-CoA hydratase, and optionally comprises attenuated enoyl-CoA reductase activity. In some embodiments, the modified biosynthetic pathway comprises (a) a 3-hydroxy-acyl-ACP thioesterase, and an acyl-CoA synthetase; and (b) a heterologous R-3-hydroxy-acyl-CoA dehydratase or a heterologous R-specific enoyl-CoA hydratase; wherein the modified biosynthetic pathway optionally comprises attenuated enoyl-CoA reductase activity. In certain embodiments, any of the modified biosynthetic pathways can further comprise one or more of: (i) one or more enzymes or polypeptides corresponding to an ester synthase, a P-keto-acyl-ACP synthase I, a P- keto-acyl-ACP synthase II, an alcohol dehydrogenase, an alcohol-O-acetyl-transferase, a fatty- alcohol-forming acyl-CoA reductase, an acyl-CoA reductase, an acyl-CoA thioesterase, an enoyl- ACP reductase, a carboxylic acid reductase, a desaturase, an omega-hydroxylase, a transaminase (or aminotransferase), an amine dehydrogenase, a CoA-ligase/transferase, an aldehyde decarbonylase, an aldehyde oxidative deformylase, a decarboxylase, one or more subunits of an acetyl-CoA carboxylase (AccABCD), an OleA, an OleBCD, an OleABCD, an OleACD, or an aldehyde dehydrogenase; (ii) a FadR that is optionally overexpressed; (iii) an attenuation or deletion of acyl-CoA dehydrogenase activity; (iv) an attenuation of trans-2-enoyl-CoA reductase activity; (v) an attenuation of an endogenous trans-2-enoyl-ACP reductase that also has trans-2- enoyl-CoA reductase activity; and/or (vi) a heterologous trans-2-enoyl-ACP reductase with lower or no trans-2-enoyl-CoA reductase activity compared to the endogenous trans-2-enoyl-ACP reductase. In some embodiments, the modified biosynthetic pathways produce a composition comprising an increased amount of trans-2-fatty acids or derivatives thereof, compared to a biosynthetic pathway that does not comprise (a) and (b). In yet a further embodiment, the composition can further comprise a reduced amount of 3-hydroxy fatty acids or derivatives thereof, compared to a biosynthetic pathway that does not comprise (a) and (b). In some embodiments, the modified biosynthetic pathways comprising a trans-2-fatty acid; a trans-2-fatty ester; a trans-2-fatty alcohol; a trans-2-fatty alcohol acetate ester; a trans-2-fatty aldehyde; a trans- 2-fatty amine; a trans-2-fatty amide; a trans-2-fatty 1,3-diol; a trans-2-fatty diol; a trans-2-fatty triol; a trans-2-fatty tetrol; a trans-2-co-hydroxy fatty acid; a trans-2-co-carboxy fatty acid; a trans- 2-co-hydroxy fatty ester, a trans-2-co-carboxy fatty ester, a trans-2-a, co-fatty diacid, a trans-2-a, cofatty diester, or a trans-2-a, co-fatty diol, or a combination thereof. In some embodiments, the modified biosynthetic pathway produces a composition, such as a fatty acid derivative composition, comprising at least about 90 weight (wt)%, or more, of a trans-2-fatty acid or derivative thereof; and about 10 wt%, or less, of a fatty acid or derivative thereof that is not a trans-2-fatty acid or derivative thereof.

EXAMPLES

[00160] The following examples are provided to further illustrate the invention disclosed herein but should not be construed as in any way limiting its scope.

[00161] Example 1: Small scale fermentation

[00162] 40 pL LB culture (from an LB culture growing in a 96 well plate) was used to inoculate

360 pL LB media containing 0.5% of the desired alcohol (either methanol or ethanol), which was then incubated with shaking for approximately 4 hours at 32°C. 80 pL of the LB seed containg 0.5% alcohol was used to inoculate 320 pL N-lim media containing 2% of the desired alcohol. (Table 4). Antibioitics, such as spectinomycin or kanamycin, were added as needed at a final concentraiton of 100 pg/mL for maintenance of plasmids. After growing at 32°C for 2 hours, the cultures were induced with isopropyl P-d-1 -thiogalactopyranoside (IPTG) (final concentration 1 mM). The cultures were then incubated at 32°C with shaking for 20 hours (unless otherwise noted), after which they were extracted with the extraction protocol detailed below.

[00163] Table 4 N-lim Media Formulation

* Aminolevulinic acid is only added for fatty acid derivatives, not for production of free fatty acids.

[00164] Free fatty acid species extraction and analytical Protocol:

[00165] To each well to be extracted, 400 pL of butyl acetate containing 500 mg/L 1- undecanoic acid methyl ester as an internal standard was added. The 96 well plates were then heat- sealed and shaken for 30 minutes at 2000 rpm. After shaking, the plates were centrifuged for 10 minutes at 4500 rpm at room temperature to separate the aqueous and organic layers. 50 pL of the organic layer was transferred to a 96 well plate and derivatized with 50 pL of TMS/BSTFA. The plate was subsequently heat sealed and stored at -20°C until evaluated by either GC-FID or GC- MS.

[00166] Fatty acid analytics: The GC-MS parameters used to generate chromatograms and mass spectra for compounds identification were as follows:

[00167] Table 5

[00168] The mass spectrometry parameters are shown in Table 6.

[00169] Table 6

[00170] The GC-FID parameters used to quantify each compound are shown in Table 7:

[00171] Table 7

[00172] The protocols detailed above represents standard conditions, which may be modified as necessary. [00173] Example 2: Production of trans-2 fatty add derivatives by recombinant strains expressing the acyl-ACP thioesterase FatBl from Umbellularia and various heterologous enoyl-CoA hydratases

[00174] This example describes that recombinant microbes, exemplified herein by E. coli strains, expressing a heterologous acyl-ACP thioesterase, such as FatB l from Umbellularia California, an acyl-CoA synthetase, and an ester synthase, when further expressing heterologous enoyl CoA hydratases, produced a fatty acid ester composition comprising significant amounts of trans-2 fatty acid methyl esters when methanol was added, and produced a fatty acid ester composition comprising significant amounts of trans-2 fatty acid ethyl esters when ethanol was added.

[00175] E. coli strain stEP.979 was engineered to have attenuated acyl-CoA dehydrogenase (FadE; SEQ ID NO: 33), and to overexpress acyl-CoA synthetase (FadD) (SEQ ID NO: 18). Strain stEP.979 also comprised a plasmid, derived from a pCL1920 vector, which contains an SC101 replicon, a spectinomycin resistance marker, and nucleic acids encoding: i) an acyl-ACP thioesterase from U. californica (Uniprot_Q41635) (SEQ ID NO: 6); ii) an ester synthase from Eimnobacter (Uniprot_A6GSQ9) (SEQ ID NO: 34); iii) a P-ketoacyl-acyl-ACP synthase I (FabB from E. coli) (SEQ ID NO: 35); and iv) a transcriptional regulator (FadR from E. coli) (SEQ ID NO: 36), all under control of an IPTG-inducible Ptrc promoter.

[00176] Enoyl-CoA hydratase genes encoding PhaJ4 from P. putida (“PhaJ4_P.put”) (SEQ ID NO: 21), PhaJ3 from P. aeruginosa (“PhaJ3_P.aer”) (SEQ ID NO: 22), or PhaJ4 from P. aeruginosa (“PhaJ4_P,aer”) (SEQ ID NO: 23), each were separately cloned into a pACYC- derivative vector (comprising a pl5A replicon and a kanamycin resistance marker). The enoyl- CoA hydratase genes were placed under the control of an IPTG-inducible Ptrc promoter. The plasmids comprising phaJ4 from P. putida, phaJ3 from P. aeruginosa, and phaJ4 from P. aeruginosa were transformed into E. coli strain stEP.979, to generate strains sAS.412, sAS.413, and sAS.414, respectively. Strain sAS.410 contains an empty pACYC-derivative vector (i.e., does not express an enoyl-CoA hydratase) and serves as a control strain.

[00177] Each of strains sAS.412, sAS.413, and sAS.414, and control strain sAS.410 were subjected to small scale fermentation as described in Example 1. The culture was supplemented with either methanol or ethanol and product analysis was performed as described above. All strains produced fattty acid methyl esters (FAME) and 3 -hydroxy fatty acid methyl esters (3OH-FAME) when methanol was added, or produced fatty acid ethyl esters (FAEE) and 3-hydroxy fatty acid ethyl esters (3OH-FAEE) when ethanol was added. All strains also produced small amounts of free fatty acids (FFAs) and 3-hydroxy fatty acids (3OH-FFA). The predominant chain length of the fatty acid derivatives was C12. The most abundant product was dodecanoic acid methyl ester or dodecanoic acid ethyl ester when the recombinant E. coli was cultured in methanol or ethanol, respectively.

[00178] When strains sAS.412, sAS.413, and sAS.414 were cultured in methanol, the strains produced trans-2 dodecenoic acid methyl ester with a retention time of 8.571 minutes on the chromatography column. The retention time and ion fragmentation pattern by mass spectrometry were consistent with trans-2 dodecenoic acid methyl ester of a trans-2 dodecenoic acid standard. The control strain sAS.410, which does not express heterologous enoyl-CoA hydratase, did not produce trans-2 dodecenoic acid methyl ester.

[00179] When sAS.412, sAS.413, and sAS.414 were cultured in ethanol, the strains also produced trans-2-dodecenoic acid ethyl ester, with a retention time of 9.162 minutes on the chromatography (GC) column. The retention time and ion fragmentation pattern by mass spectrometry were consistent with trans-2-dodecenoic acid ethyl ester synthesized by ethyl esterification of trans-2-dodecenoic acid. Strains sAS.412, sAS.413, and sAS.414 produced siginificantly more trans-2-dodecenoic acid ethyl ester than the control strain sAS.410, which did not express a heterologous enoyl-CoA hydratase.

[00180] The amounts of FAME/FAEE, 3OH-FAME/3OH-FAEE and trans-2 fatty acid methyl or ethyl esters, produced by all four strains is shown in Table 8. As can be seen, all three strains coexpressing a thioesterase, an acyl-CoA synthetase, and ester synthase and three different enoyl- CoA hydratases produced significant amounts of trans-2 fatty acid methyl and ethyl esters, particularly trans-2-dodecenoic acid ethyl ester and trans-2-dodecenoic acid methyl ester, and smaller amounts of trans-2-decenoic acid ethyl ester.

[00181] Table 8: Titers of FAME/FAEE, 3-hydroxy FAME/FAEE and 2-trans FAME/FAEE produced by recombinant strains with and without enoyl-CoA hydratase expression.

[00182] These results indicate that the expression of an enoyl-CoA hydratase produces a composition with an increased amount of trans-2-fatty acid derivatives, in comparison to a control strain that does not express an enoyl-CoA hydratase. The results also show that the expression of an enoyl-CoA hydratase results in the reduction of the amount of 3-hydroxy fatty acid derivatives.

[00183] Example 3: Production of trans-2 fatty add derivatives by recombinant strains expressing thioesterase FatB2 from Cuphea and a heterologous enoyl-CoA hydratase

[00184] This example describes that recombinant E. coli strains, expressing a heterologous acyl-ACP thioesterase, such as FatB2 from Cuphea, an acyl-CoA synthetase, and an ester synthase, when further expressing a heterologous enoyl-CoA hydratase, produced significant amounts of trans-2 fatty acid methyl esters when methanol was added, and produced significant amounts of trans-2 fatty acid ethyl esters when ethanol was added.

[00185] E. coli strain sAS.559 was prepared as a control strain that lacks an enoyl-CoA hydratase. It was otherwise isogenic to strain sAS.560 described below.

[00186] E. coli strain sAS.560 comprised a chromosomal copy of an attenuated acyl-CoA dehydrogenase (FadE), and a chromosomal copy of an overexpressed transcriptional regulator, FadR. The chromosome further comprised nucleic acids encoding a heterologous acyl-CoA synthetase FadD3 from P. putida (Uniprot_Q88PT5) (SEQ ID NO: 12) and a heterologous enoyl- CoA hydratase, PhaJ4 from P. putida (“PhaJ4_P.put”) (SEQ ID NO: 21), placed under control of an IPTG inducible Pte promoter. The strain comprised two plasmids. The first plasmid was a pCL- derivative vector (comprising a SC101 replicon and spectinomycin resistance marker) containing a heterologous acyl-ACP thioesterase (FatB2) from Cuphea hookeriana (UniProtKB Accession No.Q39514) (SEQ ID NO: 8) under control of an IPTG-inducible Ptrc promotor. The second plasmid was a pACYC-derivative vector (comprising a pl5A replicon and a kanamycin resistance marker) containing an ester synthase from Limnobacter (Uniprot_A6GSQ9) (SEQ ID NO: 34), placed under control of an IPTG-inducible Ptrc promoter.

[00187] E. coli strain sRG.843 comprised a chromosomal copy of an attenuated acyl-CoA dehydrogenase (FadE), and a chromosomal copy of an overexpressed transcriptional regulator, FadR. The strain comprised two plasmids. The first plasmid was a pCL-derivative vector (comprising a SC 101 replicon and spectinomycin resistance marker) containing, in an operon, nucleic acid sequences encoding a heterologous acyl-ACP thioesterase (FatB2) from C. hookeriana (Uniprot_Q39514) and an acyl-CoA synthetase (FadD3) from P. putida (Uniprot_Q88PT5) under control of an IPTG-inducible Ptrc promotor, and an enoyl-CoA hydratase PhaJ4, from P. putida (Uniprot_Q88PT5) (“PhaJ4_P.put”). The second plasmid was a pACYC-derivative vector (comprising a pl5A replicon and a kanamycin resistance marker) containing an ester synthase from Limnobacter (Uniprot_A6GSQ9) under control of an IPTG- inducible Ptrc promoter.

[00188] Each of strains sAS.559 (control), sAS.560, and sRG.843 were subjected to small scale fermentation as described in Example 1. The culture was supplemented with either 2% methanol or 2% ethanol and product analysis was performed as described above. All strains produced FAME and 3OH-FAME when methanol was added, or FAEE and 3OH-FAEE when ethanol was added. All strains also produced trace amounts of FFAs and 3OH-FFA. The predominant chain length of the fatty acid derivatives was C8. The most abundant product was octanoic acid methyl or ethyl ester when the recombinant E. coli cultured in methanol or ethanol, respectively. [00189] When sAS.560 and sRG.843 were cultured in methanol, the strains produced trans-2 octenoic acid methyl ester with a retention time of 4.529 minutes on the chromatography column. The retention time and ion fragmentation pattern by mass spectrometry were consistent with a trans-2 octenoic acid methyl ester sample prepared from the authentic standard of trans-2 octenoic acid. The control strain sAS.559, which did not express a heterologous enoyl-CoA hydratase, did not produce trans-2 octenoic acid methyl ester.

[00190] When sAS.560 and sRG.843 were cultured in ethanol, the strains produced trans-2 octenoic acid ethyl ester and trans-2 decenoic acid ethyl ester with retention times of 6.376 minutes and 7.517 minutes, respectively, on the (GC) chromatography column. The retention times and ion fragmentation patterns by mass spectrometry were consistent with samples of trans- 2 octenoic acid ethyl ester and trans-2 decenoic acid ethyl ester that were prepared from authentic standards of the corresponding free acids. Strains sAS.560 and sRG.843 produced siginificantly more trans-2 octenoic acid ethyl ester and trans-2 decenoic acid ethyl ester than the control strain sAS.560, which did not express a heterologous enoyl-CoA hydratase.

[00191] The amounts of FAME/FAEE, 3OH-FAME/FAEE and trans-2 fatty acid methyl or ethyl esters of all strains is shown in Table 9. As can be seen, the two strains coexpressing a thioesterase, an acyl-CoA synthetase, and ester synthase and three different enoyl-CoA hydratases produced significant amounts of trans-2 fatty acid methyl and ethyl esters, in particular trans-2- octenoic acid ethyl ester, trans-2-decenoic acid ethyl ester, and trans-2-octenoic acid methyl ester. [00192] Table 9: FAME/FAEE, 3 -hydroxy FAME/FAEE and 2-trans fatty ester titer and composition of recombinant E. coli strains expressing thioesterase FatB2 from C. hookeria with and without enoyl-CoA hydratase expression.

[00193] These results indicate that the expression of an enoyl-CoA hydratase produces a composition with an increased amount of trans-2-fatty acid derivatives, in comparison to a control strain that does not express an enoyl-CoA hydratase. The results also show that the expression of an enoyl-CoA hydratase results in the reduction of the amount of 3 -hydroxy fatty acid derivatives. [00194] Example 4: Production of trans-2 fatty add derivatives by recombinant strains expressing thioesterase/transacylase PhaG from P. putida and a heterologous enoyl-CoA hydratase

[00195] This example describes that recombinant microbes (exempliefied herein by E. coli), expressing the heterologous acyl-ACP thioesterase/acyl-ACP:CoA transacylase PhaG from Pseudomonas, an ester synthase and, optionally, an acyl-CoA synthetase, and further expressing a heterologous enoyl-CoA hydratase, produced significant amounts of trans-2 fatty acid ethyl esters when ethanol was added.

[00196] E. coli strain sSX.041 was a control strain that lacked a chromosomal copy of an enoyl- CoA hydratase. It was otherwise isogenic to strain sSX.039 described below.

[00197] E. coli strain sSX.039 comprised a chromosomal copy of an attenuated acyl-CoA dehydrogenase (FadE), and a chromosomal copy of an overexpressed transcriptional regulator, FadR. The chromosome further comprised nucleic acids/genes encoding a heterologous acyl-CoA synthetase (FadD3 from P. putida (Uniprot_Q88PT5)), and a heterologous enoyl-CoA hydratase, PhaJ4 from P. putida (“PhaJ4_P.put”), placed under the control of an IPTG inducible Pte promoter. The strain comprised two plasmids. The first plasmid was a pCE-derivative vector (comprising a SC 101 replicon and spectinomycin resistance marker) containing a nucleic acid sequence/gene encoding a heterologous acyl-ACP thioesterase/acyl-ACP:CoA transacylase PhaG from P. putida (UniProt_O85207) (SEQ ID NO: 1), controlled by the IPTG-inducible Ptrc promoter. The second plasmid was a pACYC-derivative vector (comprising a pl5A replicon and kanamycin resistance marker) containing a nucleic acid sequence/gene encoding a heterologous ester synthase from Limnobacter (UniProt_A6GSQ9), controlled by the ITPG-inducible Ptrc promoter.

[00198] Each of strains sSX.041 (control without an enoyl-CoA hydratase) and sSX.039 were subjected to small scale fermentation as described in Example 1. The culture was supplemented with 2% ethanol and product analysis was performed as described above. All strains produced FAEE and 3OH-FAEE when ethanol was added. All strains also produced trace amounts of FFAs and 3OH-FFA. The predominant chain length of the fatty acid derivatives was CIO. The most abundant product was decanoic acid ethyl ester.

[00199] When sSX.039 was cultured in ethanol, it produced significantly more trans-2 octenoic acid ethyl ester, trans-2 decenoic acid ethyl ester, and trans-2 dodecenoic acid ethyl ester than the control strain sSX.041 which lacked a chromosomal copy of an enoyl-CoA hydratase. The amounts of FAEE, 3OH-FAEE and trans-2 fatty acid ethyl esters produced by both strains is shown in Table 10. [00200] Table 10: FAEE, 3-hydroxy FAEE and trans-2-fatty acid ethyl ester titers produced by recombinant strains expressing acyl-ACP thioesterase/acyl-ACP:CoA transacylase PhaG, with and without enoyl-CoA hydratase expression.

[00201] These results also demonstrate that the expression of an enoyl-CoA hydratase produces a composition with an increased amount of trans-2-fatty acid derivatives, in comparison to a composition produced by a control strain that does not express an enoyl-CoA hydratase. The results also show that the expression of an enoyl-CoA hydratase results in the reduction of the amount of 3-hydroxy fatty acid derivatives in the composition.

[00202] Examples 2-4 demonstrate that the expression of an enoyl-CoA hydratase results in the production of trans-2-enoyl-CoAs, which can then give rise to trans-2-fatty acids (for example, by the action of a thioesterase that can hydrolyze trans-2-enoyl-CoAs to trans-2-fatty acids), or to trans-2-fatty acid derivatives, such as, for example, trans-2-fatty esters, by the action of an ester synthase on the trans-2-enoyl-CoAs. Other trans-2-fatty acid derivatives can be produced by the expression of the appropriate fatty acid derivative enzymes. For example, expression of an acyl- CoA reductase can convert the trans-2-enoyl-CoAs to the corresponding trans-2-fatty aldehydes, and the expression of an acyl-CoA reductase and a transaminase (aminotransferase) or an amine dehydrogenase can convert the trans-2-enoyl-CoAs to the corresponding trans-2-fatty amines. Alternatively, expression of an acyl-CoA reductase and an alcohol dehydrogenase, or expression of a fatty alcohol-forming acyl-CoA reductase, can convert the trans-2-enoyl-CoAs to the corresponding trans-2-fatty alcohols. Further expression of an alcohol-O-acetyltransferase, for example, can convert the trans-2-fatty alcohols to trans-2-fatty alcohol acetate esters.

[00203] As described elsewhere herein, other fatty acid derivative enzymes or pathways can be used to prepare other trans-2-fatty acid derivatives. For example, trans-2-fatty acids can be converted to trans-2-fatty aldehydes by a carboxylic acid reductase (CAR), or to trans-2-fatty amines by a CAR and a transaminase, or to trans-2-fatty alcohols by a CAR and an alcohol dehydrogenase (ADH), or to trans-2-omega-hydroxy fatty acids by an omega-hydroxylase, and so forth, as described elsewhere herein and as known in the art. [00204] Example 5: Alternative ways to producing trans-2 fatty acid derivatives

[00205] Alternatively, trans-2 fatty acid derivatives can be obtained by employing exclusively an acyl-ACP dependent fatty acid biosynthetic pathway (z.e., the trans-2-fatty acid derivatives can be produced directly from trans-2 enoyl-ACP), or by exclusively using an acyl-CoA dependent (z.e., an acyl-ACP independent) fatty acid biosynthetic pathway (e.g., a reversal of P-oxidation). These pathways are shown in FIG 1.

[00206] For example, in an acyl-ACP dependent fatty acid biosynthetic pathway (see, FIG. 1A), the trans-2-enoyl-ACP intermediate is converted to the corresponding trans-2-fatty acid by the action of a thioesterase, particularly one that has a higher specificity and/or selectivity for trans-2-enoyl-ACP and/or that can compete with enoyl-ACP reductase, to reduce or prevent the conversion of the trans-2-enoyl-ACPs to the corresponding acyl-ACPs. The trans-2-fatty acids can then be converted to a variety of fatty acid derivatives by the appropriate fatty acid derivative enzyme(s). For example, the trans-2-fatty acids can be converted to: i) trans-2-fatty aldehydes by a carboxylic acid reductase (CAR); ii) trans-2-fatty alcohols by a CAR and alcohol dehydrogenase; iii) a trans-2-fatty amine by a CAR and a transaminase; or iv) a trans-2-co-hydroxy fatty acid by an omega-hydroxylase.

[00207] These are exemplary pathways towards trans-2-fatty acid derivatives from trans-2- fatty-acyl-ACP; other trans-2-fatty acid derivatives can be prepared by other fatty acid derivative enzyme pathways, as known in the art and/or described herein.

[00208] The same pathways can be used when the fatty acid biosynthetic pathways is exclusively acyl-CoA dependent, however, in this case, the trans-2-enoyl-CoA is converted to a trans-2-fatty acid by a thioesterase that has a higher specificity and/or selectivity for trans-2-enoyl- CoA and/or that can compete with enoyl-CoA reductase, to reduce or prevent the conversion of the trans-2-enoyl-CoAs to the corresponding acyl-CoAs. The trans-2-fatty acids and trans-2- enoyl — CoAs can be converted into various fatty acid derivatives by the appropriate fatty acid derivative enzyme(s), as known in the art and/or described herein. [00209] Example 6: Production of trans-2 fatty alcohols by recombinant strains expressing an acyl-CoA reductase and a heterologous enoyl-CoA hydratase

[00210] This example illlustarates that recombinant microbes (e.g., E. coli) expressing a heterologous acyl-ACP thioesterase/acyl-ACP:CoA transacylase PhaG from Pseudomonas, a heterologous acyl-CoA reductase, and optionally, an acyl-CoA synthetase, and further expressing a heterologous enoyl-CoA hydratase, produce significant amounts of trans-2 fatty alcohols.

[00211] An E. coli MG1655 derivative strain is engineered to encode a deregulated (i.e., overexpressed) acyl-CoA synthetase (FadD), such as by replacing the native promoter with a synthetic or heterologous promoter, or an expressed or overexpressed heterologous acyl-CoA synthetase, such as FadD3 from P. putida (UniProt_Q88PT5).

[00212] A heterologous acyl-CoA reductase from Acinteobacter baylyi (UniProt_Q6F7B8) (SEQ ID NO: 38) or a heterologous acyl-CoA reductase from Marinobacter (GenBank ABM19582) (SEQ ID NO: 39), and an alcohol dehydrogenase from Acinetobacter (UniProt_Q6F6R9) (SEQ ID NO: 37) are cloned into pACYC-derivative vector (comprising a pl5A replicon and a kanamycin resistance marker) and placed under the control of an IPTG- inducible Ptrc promoter.

[00213] A second plasmid is prepared that comprises: a first operon comprising the enoyl-CoA hydratase gene phaJ from P. putida under the control of an IPTG-inducible PT5 promoter; and a second operon comprising a gene encoding a heterologous acyl-ACP/ acyl-ACP:CoA transacylase thioesterase PhaG from P. putida (UniProt_O85207), placed under the control of an IPTG-inducible Ptrc promoter. The second plasmid is a pCL1920-derivative vector, which contains an SC 101 replicon and a spectinomycin resistance marker.

[00214] Both plasmids are transformed into the E. coli MG1655 derivative strain and the strain is subjected to small scale fermentation as described in Example 1. The strain produces fatty alcohols, such as, for example, octanol and decanol; 1,3-diols, such as 1,3 -octanediol and 1,3- decanediol; and it produces 2-trans unsaturated fatty alcohols, such as 2-trans-octenol and 2-trans- decenol.

[00215] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[00216] Particular embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those particular embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[00217] Sequence Table

Claims

CLAIMS What is claimed is

1. A recombinant microbe, comprising:

(a) one or more polypeptides for converting a 3-hydroxy-acyl-ACP to a corresponding 3- hydroxy-acyl-CoA, wherein the one or more polypeptides correspond to:

(i) a 3-hydroxy acyl-ACP:CoA transacylase; or

(ii) a 3-hydroxy-acyl-ACP thioesterase, and an acyl-CoA synthetase; and

(b) a heterologous R-3-hydroxy-acyl-CoA dehydratase or a heterologous R-specific enoyl- CoA hydratase; wherein the recombinant microbe optionally comprises attenuated enoyl-CoA reductase activity; and wherein the recombinant microbe produces trans-2-unsaturated fatty acids or derivatives thereof, or produces a fatty acid derivative composition comprising trans-2-unsaturated fatty acids or derivatives thereof.

2. The recombinant microbe of claim 1, wherein: the 3-hydroxy acyl-ACP:CoA transacylase comprises activity to convert a 3-hydroxy- acyl-ACP to a 3-hydroxy-acyl-CoA; the 3-hydroxy-acyl-ACP thioesterase comprises activity to convert a 3-hydroxy-acyl-ACP to a corresponding 3-hydroxy fatty acid; the acyl-CoA synthetase comprises activity to convert the 3-hydroxy fatty acid to a corresponding 3-hydroxy-acyl-CoA; and the heterologous R-3-hydroxy-acyl-CoA dehydratase or the heterologous R-specific enoyl-CoA hydratase comprises activity to convert a 3-hydroxy-acyl-CoA to a corresponding trans-2-enoyl-CoA.

3. The recombinant microbe of claim 1 or claim 2, wherein the 3-hydroxy-acyl-ACP thioesterase or the 3-hydroxy-acyl-ACP:CoA transacylase is PhaG, FatBl, FatB2, FatB3, an acyl- ACP thioesterase from Lactobacillus plantarum, or an acyl- ACP thioesterase from Anaerococcus tetradius.

4. The recombinant microbe of any one of claims 1-3, wherein the 3-hydroxy-acyl-ACP thioesterase or the 3-hydroxy-acyl-ACP:CoA transacylase comprises an amino acid sequence set forth in any one of SEQ ID NOs:l-l l or 40, or is a homolog thereof, or comprises an amino acid sequence having at least 30% sequence identity to any one of SEQ ID NOs:l-l l or 40.

5. The recombinant microbe of any one of claims 1-4, wherein the acyl-CoA synthetase is FadD3 (PP_0763), FadD-I, FadD-II, FadD, or IcfB.

6. The recombinant microbe of any one of claims 1-5, wherein the acyl-CoA synthetase comprises an amino acid sequence set forth in any one of SEQ ID NOs:12-19, or is a homolog thereof, or comprises an amino acid sequence having at least 30% sequence identity to any one of SEQ ID NOs:12-19.

7. The recombinant microbe of any one of claims 1-6, wherein the heterologous R-3- hydroxy-acyl-CoA dehydratase or the heterologous R-specific enoyl-CoA hydratase is PhaJl, PhaJ3, PhaJ4, MaoC, MaoC9, or an MaoC family dehydratase.

8. The recombinant microbe of any one of claims 1-7, wherein the heterologous R-3- hydroxy-acyl-CoA dehydratase or the heterologous R-specific enoyl-CoA hydratase comprises an amino acid sequence set forth in any one of SEQ ID NOs:20-32, or is a homolog thereof, or comprises an amino acid sequence having at least 30% sequence identity to any one of SEQ ID NOs:20-32.

9. The recombinant microbe of any one of claims 1-8, further comprising an ester synthase; an acyl-CoA reductase; or an acyl-CoA thioesterase.

10. The recombinant microbe of any one of claims 1-9, further comprising one or more enzymes or polypeptides corresponding to an ester synthase, a P-keto-acyl-ACP synthase I, a P- keto-acyl-ACP synthase II, an alcohol dehydrogenase, an alcohol-O-acetyl-transferase, a fatty- alcohol-forming acyl-CoA reductase, an acyl-CoA reductase, an acyl-CoA thioesterase, an enoyl- ACP reductase, a carboxylic acid reductase, a desaturase, an omega-hydroxylase, a transaminase (or aminotransferase), an amine dehydrogenase, a CoA-ligase/transferase, an aldehyde decarbonylase, an aldehyde oxidative deformylase, a decarboxylase, one or more subunits of an acetyl-CoA carboxylase (AccABCD), an OleA, an OleBCD, an OleABCD, an OleACD, or an aldehyde dehydrogenase.

11. The recombinant microbe of any one of claims 1-10, further expressing or overexpressing

FadR.

12. The recombinant microbe of any one of claims 1-11, wherein acyl-CoA dehydrogenase expression and/or activity is attenuated or deleted compared to a corresponding wild-type microbe.

13. The recombinant microbe of claim 12, wherein the acyl-CoA dehydrogenase is FadE.

14. The recombinant microbe of any one of claims 1-13, wherein enoyl-CoA reductase or trans-2-enoyl-CoA reductase activity is attenuated compared to a corresponding wild-type microbe.

15. The recombinant microbe of any one of claims 1-14, wherein: the expression and/or activity of an endogenous trans-2-enoyl-ACP reductase that also has trans-2-enoyl-CoA reductase activity is attenuated compared to a corresponding wild-type microbe; and the recombinant microbe is engineered to further express a heterologous trans-2-enoyl- ACP reductase with lower or no trans-2-enoyl-CoA reductase activity compared to the endogenous trans-2-enoyl-ACP reductase.

16. The recombinant microbe of any one of claims 1-15, wherein the recombinant microbe produces a trans-2 unsaturated fatty acid or a derivative thereof, or produces a composition comprising a trans-2 unsaturated fatty acid or a derivative thereof, wherein: the trans-2 unsaturated fatty acid or a derivative thereof is a trans-2-fatty acid; a trans-2- fatty ester; a trans-2-fatty alcohol; a trans-2-fatty alcohol acetate ester; a trans-2-fatty aldehyde; a trans-2-fatty amine; a trans-2-fatty amide; a trans-2-fatty 1,3-diol; a trans-2-fatty diol; a trans-2- fatty triol; a trans-2-fatty tetrol; a trans-2-co-hydroxy fatty acid; a trans-2-co-carboxy fatty acid; a trans-2-co-hydroxy fatty ester, a trans-2-co-carboxy fatty ester, a trans-2-a, co-fatty diacid, a trans- 2-a, co-fatty diester, or a trans-2-a, co-fatty diol, or a combination thereof.

17. The recombinant microbe of any one of claims 1-15, wherein the recombinant microbe produces a fatty acid derivative composition comprising:

(i) one or more of a trans-2-fatty acid or derivative thereof; (ii) one or more of a 3-hydroxy fatty acid or derivative thereof; and

(iii) one of more of a fatty acid or derivative thereof.

18. The recombinant microbe of claim 17, wherein:

(i) the one or more trans-2-fatty acids or derivatives thereof comprise a trans-2-fatty acid; a trans-2-fatty ester; a trans-2-fatty alcohol; a trans-2-fatty alcohol acetate ester; a trans-2-fatty aldehyde; a trans-2-fatty amine; a trans-2-fatty amide; a trans-2-fatty 1,3-diol; a trans-2-fatty diol; a trans-2-fatty triol; a trans-2-fatty tetrol; a trans-2-co-hydroxy fatty acid; a trans-2-co-carboxy fatty acid; a trans-2-co-hydroxy fatty ester, a trans-2-co-carboxy fatty ester, a trans-2-a, co-fatty diacid, a trans-2-a, co-fatty diester, or a trans-2-a, co-fatty diol, or a combination thereof;

(ii) the one or more 3-hydroxy fatty acids or derivatives thereof comprise a 3-hydroxy fatty acid; a 3-hydroxy fatty acid ester; a fatty 1,3-diol; a fatty alcohol 1,3-diacetate; a 3-hydroxy fatty aldehyde; a 3-hydroxy fatty amine; a 3-hydroxy fatty amide; a fatty diol with a 3-hydroxy group; a fatty triol with a 3-hydroxy group; a fatty tetrol with a 3-hydroxy group; an co-hydroxy fatty acid with a 3-hydroxy group, an co-carboxy fatty acid with a 3-hydroxy group, an co-hydroxy fatty ester with a 3-hydroxy group, an co-carboxy fatty ester with a 3-hydroxy group, an a, co-fatty diacid with a 3-hydroxy group, an a, co-fatty diester with a 3-hydroxy group, or an a, co-fatty diol with a 3- hydroxy group, or a combination thereof; and

(iii) the one or more fatty acids or derivatives thereof comprise a fatty acid, a fatty ester, a fatty alcohol, a fatty alcohol acetate ester, a fatty aldehyde, a fatty amine, a fatty amide, a fatty diol, a fatty triol, a fatty tetrol, an co-hydroxy fatty acid, an co-carboxy fatty acid, an co-hydroxy fatty ester, an co-carboxy fatty ester, an a, co-fatty diacid, an a, co-fatty diester, or an a, co-fatty diol, or a combination thereof.

19. The recombinant microbe of any one of claims 1-18, wherein the trans-2 unsaturated fatty acid or derivative thereof is a trans-2-unsaturated fatty acid, a trans-2 fatty acid methyl ester, a trans-2 fatty acid ethyl ester, a trans-2-unsaturated fatty aldehyde, a trans-2-unsaturated fatty alcohol, a trans-2-unsaturated fatty alcohol acetate or a trans-2 unsaturated co-hydroxy fatty acid.

20. The recombinant microbe of claim 19, wherein the trans-2 unsaturated fatty acid or derivative thereof is one or more of trans-2-hexadecenoic acid, trans-2-hexadecenoic acid ethyl ester, trans-2-hexadecenoic acid methyl ester, trans-2-tetradecenoic acid, trans-2-tetradecenoic acid ethyl ester, trans-2-tetradecenoic acid methyl ester, trans-2-dodecenoic acid, trans-2- dodecenoic acid ethyl ester, trans-2-dodecenoic acid methyl ester, trans-2-decenoic acid, trans-2- decenoic acid ethyl ester, trans-2-decenoic acid methyl ester, trans-2-octenoic acid, trans-2- octenoic acid ethyl ester, and trans-2-octenoic acid methyl ester.

21. The recombinant microbe of any one of claims 1-20, wherein the recombinant microbe produces an increased amount of trans-2-fatty acids or derivatives thereof, compared to a corresponding microbe that does not comprise (a) a 3-hydroxy-acyl-ACP thioesterase, and an acyl-CoA synthetase, or a 3-hydroxy acyl-ACP:CoA transacylase; and (b) a heterologous R-3- hydroxy-acyl-CoA dehydratase or a heterologous R-specific enoyl-CoA hydratase.

22. The recombinant microbe of any one of claims 1-20, wherein the recombinant microbe produces an increased amount of a trans-2-fatty acid or derivatives thereof, compared to a corresponding microbe that does not comprise a heterologous R-3-hydroxy-acyl-CoA dehydratase or a heterologous R-specific enoyl-CoA hydratase.

23. The recombinant microbe of any one of claims 1-22, wherein the recombinant microbe produces a reduced amount of a 3-hydroxy fatty acid or derivative thereof, compared to a corresponding microbe that does not comprise a heterologous R-3-hydroxy-acyl-CoA dehydratase or a heterologous R-specific enoyl-CoA hydratase.

24. The recombinant microbe of any one of claims 1-22, wherein the recombinant microbe produces a reduced amount of a 3-hydroxy fatty acid or derivative thereof, compared to a corresponding microbe that does not comprise (a) a 3-hydroxy-acyl-ACP thioesterase and an acyl- CoA synthetase, or a 3-hydroxy acyl-ACP:CoA transacylase; and (b) a heterologous R-3-hydroxy- acyl-CoA dehydratase or a heterologous R-specific enoyl-CoA hydratase.

25. The recombinant microbe of any one of claims 1-24, wherein the recombinant microbe is a bacterium, a cyanobacterium, a yeast, or an algae.

26. The recombinant microbe of claim 25, wherein the recombinant microbe is a recombinant y-proteobacterium.

27. The recombinant microbe of claim 26, wherein the recombinant y-proteobacterium is Escherichia coli, Salmonella spp., Vibrio natriegens, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Xanthomonas axonopodis, Pseudomonas syringae, Xyella fastidiosa, Marinobacter aquaeolei, Yersinia pestis, Bacillus spp., Lactobacillus spp., Zymomonas spp., Streptomyces spp., or Vibrio cholerae.

28. The recombinant microbe of claim 26, wherein the y-proteobacterium is Escherichia coli.

29. The recombinant microbe of claim 25, wherein the recombinant microbe is a cyanobacterium selected from Synechococcus elongatus PCC7942 and Synechocystis sp. PCC6803.

30. The recombinant microbe of claim 25, wherein: the recombinant microbe is a yeast selected from Saccharomyces cerevisiae, Scheffersomyces stipitis, Schizosaccharomyces pombe, Kluyveromyces marxianus, K. lactis, Pichia pastoris, Hansenula polymorpha, and Yarrowia lipolytica', or the recombinant microbe is an algae selected from Botryococcus braunii, Nannochloropsis gaditina, Chlamydomonas reinhardtii, Chlorella vulgaris, Spirulina platensis, Ostreococcus tauri, Phaeodactylum tricornutum, Symbiodinium sp., algal phytoplanktons, Saccharina japonica, Chlorococum spp., and Spiro gyra spp.

31. The recombinant microbe of any one of claims 1-30, comprising an acyl-ACP dependent fatty acid biosynthetic pathway for the production of fatty acids and derivatives thereof.

32. A cell culture, comprising the recombinant microbe of any one of claims 1-31.

33. A method for producing a trans-2 unsaturated fatty acid or a derivative thereof, the method comprising culturing the recombinant microbe of any one of claims 1-31, or the cell culture of claim 32, in the presence of a carbon source.

34. The method of claim 33, further comprising isolating and/or purifying the trans-2 unsaturated fatty acid or derivative thereof.

35. A method for preparing a fatty acid derivative composition, the method comprising culturing the recombinant microbe of any one of claims 1-31, or the cell culture of claim 32, in the presence of a carbon source, wherein: the fatty acid derivative composition comprises a fatty acid or derivative thereof; and the fatty acid derivative composition comprises an increased amount of a trans-2- unsaturated fatty acid or derivative thereof, compared to a fatty acid derivative composition prepared by culturing a corresponding microbe that does not express (a) a 3-hydroxy-acyl-ACP thioesterase and an acyl-CoA synthetase, or a 3 -hydroxy acyl-ACP:CoA transacylase; or (b) a heterologous / -3-hydroxy acyl-CoA dehydratase or a heterologous //-specific enoyl-CoA hydratase; or a combination of (a) and (b).

36. The method of claim 35, wherein the fatty acid derivative composition further comprises a reduced amount of 3 -hydroxy fatty acids or derivatives thereof, compared to a fatty acid derivative composition prepared by culturing a corresponding microbe that does not express (a) a 3-hydroxy-acyl-ACP thioesterase and an acyl-CoA synthetase, or a 3-hydroxy acyl-ACP:CoA transacylase; or (b) a heterologous 7?-3-hydroxy acyl-CoA dehydratase or a heterologous R- specific enoyl-CoA hydratase; or a combination of (a) and (b).

37. The method of claim 35 or claim 36, further comprising isolating and/or purifying the fatty acid derivative composition.

38. The method of any one of claims 33-37, wherein the trans-2-unsaturated fatty acid or derivative thereof is a trans-2-fatty acid; a trans-2-fatty ester; a trans-2-fatty alcohol; a trans-2- fatty alcohol acetate ester; a trans-2-fatty aldehyde; a trans-2-fatty amine; a trans-2-fatty amide; a trans-2-fatty 1,3-diol; a trans-2-fatty diol; a trans-2-fatty triol; a trans-2-fatty tetrol; a trans-2-co- hydroxy fatty acid; a trans-2-co-carboxy fatty acid; a trans-2-co-hydroxy fatty ester, a trans-2-co- carboxy fatty ester, a trans-2-a, co-fatty diacid, a trans-2-a, co-fatty diester, or a trans-2-a, co-fatty diol, or a combination thereof.

39. The method of any one of claims 35-38, wherein the fatty acid or derivative thereof is a fatty acid, a fatty ester, a fatty alcohol, a fatty alcohol acetate ester, a fatty aldehyde, a fatty amine, a fatty amide, a fatty diol, a fatty triol, a fatty tetrol, an co-hydroxy fatty acid, an co-carboxy fatty acid, an co-hydroxy fatty ester, an co-carboxy fatty ester, an a, co-fatty diacid, an a, co-fatty diester, or an a, co-fatty diol, or a combination thereof.

40. The method of any one of claims 36-39, wherein the 3-hydroxy fatty acid or derivative thereof is a 3-hydroxy fatty acid; a 3-hydroxy fatty acid ester; a fatty 1,3-diol; a fatty alcohol 1,3- diacetate; a 3-hydroxy fatty aldehyde; a 3-hydroxy fatty amine; a 3-hydroxy fatty amide; a fatty diol with a 3 -hydroxy group; a fatty triol with a 3 -hydroxy group; a fatty tetrol with a 3 -hydroxy group; an co-hydroxy fatty acid with a 3-hydroxy group, an co-carboxy fatty acid with a 3-hydroxy group, an co-hydroxy fatty ester with a 3-hydroxy group, an co-carboxy fatty ester with a 3-hydroxy group, an a, co-fatty diacid with a 3-hydroxy group, an a, co-fatty diester with a 3-hydroxy group, or an a, co-fatty diol with a 3-hydroxy group, or a combination thereof.

41. The method of any one of claims 33-40, further comprising adding an alcohol to the culture or medium to produce a trans-2 unsaturated fatty acid alkyl ester.

42. The method of claim 41, wherein the trans-2 unsaturated fatty acid alkyl ester is trans-2 fatty acid methyl ester or a trans-2 fatty acid ethyl ester.

43. The method of claim 42, wherein the trans-2 fatty acid alkyl ester is one or more of trans- 2-hexadecenoic acid ethyl ester, trans-2-hexadecenoic acid methyl ester, trans-2-tetradecenoic acid ethyl ester, trans-2-tetradecenoic acid methyl ester, trans-2-dodecenoic acid ethyl ester, trans- 2-dodecenoic acid methyl ester, trans-2-decenoic acid ethyl ester, trans-2-decenoic acid methyl ester, trans-2-octenoic acid ethyl ester, and trans-2-octenoic acid methyl ester.

44. A fatty acid derivative, or a fatty acid derivative composition, prepared by the method of any one of claims 33-43.

45. The fatty acid derivative or the fatty acid derivative composition of claim 44, wherein the fatty acid derivative or fatty acid derivative composition is purified.

46. The fatty acid derivative or the fatty acid derivative composition of claim 44 or claim 45, wherein the fatty acid derivative or the fatty acid derivative composition is purified by a two-step centrifugation and water- washing; decanting centrifugation and solvent extraction from a biomass; or whole broth extraction with a water immiscible solvent; or a combination thereof.

47. A composition, comprising fatty acids or derivatives thereof, wherein: the composition comprises at least about 50-90%, or more, by weight of the composition, of one or more trans-2-fatty acids or derivatives thereof.

48. The composition of claim 47, comprising a trans-2-fatty acid; a trans-2-fatty ester; a trans-

2-fatty alcohol; a trans-2-fatty alcohol acetate ester; a trans-2-fatty aldehyde; a trans-2-fatty amine; a trans-2-fatty amide; a trans-2-fatty 1,3-diol; a trans-2-fatty diol; a trans-2-fatty triol; a trans-2- fatty tetrol; a trans-2-co-hydroxy fatty acid; a trans-2-co-carboxy fatty acid; a trans-2-co-hydroxy fatty ester, a trans-2-co-carboxy fatty ester, a trans-2-a, co-fatty diacid, a trans-2-a, co-fatty diester, or a trans-2-a, co-fatty diol, or a combination thereof.

49. The composition of claim 47 or claim 48, further comprising a fatty acid or derivative thereof that is a fatty acid, a fatty ester, a fatty alcohol, a fatty alcohol acetate ester, a fatty aldehyde, a fatty amine, a fatty amide, a fatty diol, a fatty triol, a fatty tetrol, an co-hydroxy fatty acid, an co-carboxy fatty acid, an co-hydroxy fatty ester, an co-carboxy fatty ester, an a, co-fatty diacid, an a, co-fatty diester, or an a, co-fatty diol, or a combination thereof.

50. The composition of any one of claims 47-49, further comprising a 3-hydroxy fatty acid or derivative thereof that is a 3-hydroxy fatty acid; a 3-hydroxy fatty acid ester; a fatty 1,3-diol; a fatty alcohol 1,3 -diacetate; a 3-hydroxy fatty aldehyde; a 3-hydroxy fatty amine; a 3-hydroxy fatty amide; a fatty diol with a 3-hydroxy group; a fatty triol with a 3-hydroxy group; a fatty tetrol with a 3-hydroxy group; an co-hydroxy fatty acid with a 3-hydroxy group, an co-carboxy fatty acid with a 3-hydroxy group, an co-hydroxy fatty ester with a 3-hydroxy group, an co-carboxy fatty ester with a 3-hydroxy group, an a, co-fatty diacid with a 3-hydroxy group, an a, co-fatty diester with a

3-hydroxy group, or an a, co-fatty diol with a 3-hydroxy group, or a combination thereof.

51. The composition of claim 49, wherein the composition comprises about 10% or less, by weight of the composition, of a fatty acid or fatty acid derivative that is not a trans-2-fatty acid or derivative thereof.

52. The composition of claim 50, wherein the composition comprises about 5% or less, by weight of the composition, of a 3-hydroxy fatty acid or derivative thereof.

53. A fatty ester composition, comprising: one or more trans-2 fatty acid methyl esters, one or more fatty acid methyl esters (FAMEs), and one or more 3-hydroxy-FAMEs; or one or more trans-2 fatty acid ethyl esters, one or more fatty acid ethyl esters (FAEEs), and one or more 3-hydroxy-FAEEs; wherein the predominant chain length of the fatty esters in the composition is C8, CIO, or

C12.

54. The composition of claim 23, wherein the composition is prepared by culturing the recombinant microbe of any one of claims 1-31 in the presence of a carbon source to produce a culture, and adding either methanol or ethanol to the culture.

55. The composition of claim 53 or claim 54, wherein: the trans-2 fatty acid methyl ester is trans-2-dodecenoic acid methyl ester, trans-2-decenoic acid methyl ester, or trans-2-octenoic acid methyl ester; and the trans-2 fatty acid ethyl ester is trans-2-dodecenoic acid ethyl ester, trans-2-decenoic acid ethyl ester, or trans-2-octenoic acid ethyl ester.

56. A trans-2 unsaturated fatty acid or derivative thereof prepared by the method of any one of claims 33-43.

57. The trans-2 unsaturated fatty acid or derivative thereof of claim 65, wherein the trans-2 unsaturated fatty acid or derivative thereof is purified.

58. The trans-2 unsaturated fatty acid or derivative thereof of any claim 56 or claim 57, wherein the trans-2 unsaturated fatty acid or derivative thereof is purified by a two-step centrifugation and water- washing; decanting centrifugation and solvent extraction from a biomass; whole broth extraction with a water immiscible solvent; or a combination thereof.

59. A modified biosynthetic pathway, comprising:

(i) a 3-hydroxy acyl-ACP:CoA transacylase; or

(ii) a 3-hydroxy-acyl-ACP thioesterase, and an acyl-CoA synthetase; and

(b) a heterologous R-3-hydroxy-acyl-CoA dehydratase or a heterologous R-specific enoyl- CoA hydratase; wherein the modified biosynthetic pathway optionally comprises attenuated enoyl-CoA reductase activity; and wherein the modified biosynthetic pathway produces trans-2-unsaturated fatty acids or derivatives thereof, or produces a fatty acid derivative composition comprising trans-2- unsaturated fatty acids or derivatives thereof.

60. A modified biosynthetic pathway, comprising:

(a) a 3-hydroxy acyl-ACP:CoA transacylase; and

61. A modified biosynthetic pathway, comprising:

(a) a 3-hydroxy-acyl-ACP thioesterase, and an acyl-CoA synthetase; and

62. The modified biosynthetic pathway of any one of claims 59-61, further comprising one or more of:

(i) one or more enzymes or polypeptides corresponding to an ester synthase, a P-keto-acyl- ACP synthase I, a P-keto-acyl-ACP synthase II, an alcohol dehydrogenase, an alcohol-O-acetyl- transferase, a fatty-alcohol-forming acyl-CoA reductase, an acyl-CoA reductase, an acyl-CoA thioesterase, an enoyl-ACP reductase, a carboxylic acid reductase, a desaturase, an omega- hydroxylase, a transaminase (or aminotransferase), an amine dehydrogenase, a CoA- ligase/transferase, an aldehyde decarbonylase, an aldehyde oxidative deformylase, a decarboxylase, one or more subunits of an acetyl-CoA carboxylase (AccABCD), an OleA, an OleBCD, an OleABCD, an OleACD, or an aldehyde dehydrogenase; (ii) a FadR that is optionally overexpressed;

(iii) an attenuation or deletion of acyl-CoA dehydrogenase activity;

(iv) an attenuation of trans-2-enoyl-CoA reductase activity;

(v) an attenuation of an endogenous trans-2-enoyl-ACP reductase that also has trans-2- enoyl-CoA reductase activity; and/or

(vi) a heterologous trans-2-enoyl-ACP reductase with lower or no trans-2-enoyl-CoA reductase activity compared to the endogenous trans-2-enoyl-ACP reductase.

63. The modified biosynthetic pathway of any one of claims 59-62, wherein the modified biosynthetic pathway further produces one or more fatty acids or derivatives thereof, or one or more 3 -hydroxy-fatty acids or derivatives thereof, or a combination thereof.

64. The modified biosynthetic pathway of any one of claims 59-63, wherein the modified biosynthetic pathways produces a composition comprising an increased amount of trans-2-fatty acids or derivatives thereof, compared to a biosynthetic pathway that does not comprise (a) and (b).

65. The modified biosynthetic pathway of claim 64, wherein the composition further comprises a reduced amount of 3-hydroxy fatty acids or derivatives thereof, compared to a biosynthetic pathway that does not comprise (a) and (b).

66. The modified biosynthetic pathway of any one of claims 59-65, wherein the composition comprises a trans-2-fatty acid; a trans-2-fatty ester; a trans-2-fatty alcohol; a trans-2-fatty alcohol acetate ester; a trans-2-fatty aldehyde; a trans-2-fatty amine; a trans-2-fatty amide; a trans-2-fatty 1,3-diol; a trans-2-fatty diol; a trans-2-fatty triol; a trans-2-fatty tetrol; a trans-2-co-hydroxy fatty acid; a trans-2-co-carboxy fatty acid; a trans-2-co-hydroxy fatty ester, a trans-2-co-carboxy fatty ester, a trans-2-a, co-fatty diacid, a trans-2-a, co-fatty diester, or a trans-2-a, co-fatty diol, or a combination thereof.

67. The modified biosynthetic pathway of any one of claims 59-66, wherein the modified biosynthetic pathway produces a fatty acid derivative composition comprising: at least about 90 weight (wt)%, or more, of a trans-2-fatty acid or derivative thereof; and about 10 wt%, or less, of a fatty acid or derivative thereof that is not a trans-2-fatty acid or derivative thereof.

68. Use of the recombinant microbe of any one of claims 1-31, or the cell culture of claim 32, or the method of any one of claims 33-43, or the modified biosynthetic pathway of any one of claims 59-67, for the production of trans-2 unsaturated fatty acids or derivatives thereof, or for the production of a composition comprising trans-2 unsaturated fatty acids or derivatives thereof.

69. Use of the recombinant microbe of any one of claims 1-31, the cell culture of claim 32, the method of any one of claims 33-43, or the modified biosynthetic pathway of any one of claims 59-67, for the preparation of a fragrance, flavor, pheromone, nutritional supplement, dietary supplement, pharmaceutical, or nutraceutical, ingredient or product, or a precursor thereof.

70. Use of the fatty acid derivative, the fatty acid derivative composition, the composition, the trans-2 unsaturated fatty acid or derivative thereof, or the fatty ester composition of any one of claims 44-58, for the preparation of a fragrance, flavor, pheromone, nutritional supplement, dietary supplement, pharmaceutical, or nutraceutical, ingredient or product, or a precursor thereof.

71. A fragrance, flavor, pheromone, nutritional supplement, dietary supplement, pharmaceutical, or nutraceutical, ingredient or product, or a precursor thereof, prepared by the recombinant microbe of any one of claims 1-31, the cell culture of claim 32, the method of any one of claims 33-43, or the modified biosynthetic pathway of any one of claims 59-67.

72. A fragrance, flavor, pheromone, fuel, nutritional supplement, dietary supplement, pharmaceutical, or nutraceutical, ingredient or product, or a precursor thereof, comprising the fatty acid derivative, the fatty acid derivative composition, the composition, the trans-2 unsaturated fatty acid or derivative thereof, or the fatty ester composition of any one of claims 44-58.