Microbial production of fatty alcohols

Abstract

The present disclosure provides a modified bacterial cell and methods of use thereof to produce native and / or non-native unsaturated fatty acid derivative molecules. Provided is a biosynthetic route combining the activity of various enzymes, such as a fatty acyl-ACP desaturase and a fatty acyl reductase, to generate unsaturated fatty acids and derivatives thereof.

Description

[0001] MICROBIAL PRODUCTION OF FATTY ALCOHOLS BACKGROUND

[0002] Rising global demand for food combined with a growing concern about the environmental effects of overusing often toxic insecticides has magnified the need for more natural and sustainable means of crop protection such as insect pheromones, and precursors thereof, which are often fatty acids or derivatives thereof. The use of insect pheromones is a natural and non-toxic way of pest control and has gained attraction in agriculture in recent years. Insect pheromones can be used as lures in insect traps or sprayed on crops for insect mating disruption. However, so far synthetic pheromones are expensive to produce and are mostly synthesized from petroleum-derived feedstocks by sometimes complicated and / or low yielding chemical syntheses.

[0003] SUMMARY

[0004] In some aspects, the disclosure provides a recombinant bacterial cell comprising a acyl-[acyl-carrier-protein] desaturase (FAD, e.g., EC 1.14.19.11) and an alcohol-forming fatty acyl-CoA reductase (FAR, e.g., EC 1.2.1.84), wherein at least one of the FAD and the FAR is heterologous. In some embodiments, the recombinant bacterial cell produces at least one of a non-native fatty alcohol, a non-native fatty acetate, a non-native fatty acid, a non-native fatty ester, or a derivative of any of the foregoing. In aspects, the disclosure provides methods for making one or more of a non-native fatty alcohol, a non-native fatty acetate, a non-native fatty acid, a non-native fatty ester, or a derivative of any of the foregoing, by culturing a recombinant bacterial cell according to the disclosure, and recovering the non-native fatty alcohol, non-native fatty acetate, non-native fatty acid, non-native fatty ester, or a derivative thereof, from the culture.

[0005] In some embodiments, the disclosure provides methods of producing a non-native unsaturated fatty alcohol or derivative thereof comprising culturing recombinant bacterial cells disclosed herein and extracting the non-native unsaturated fatty alcohol or derivative thereof from the recombinant bacterial cells or the culture.

[0006] In some embodiments, the disclosure provides methods of producing a non-native unsaturated fatty acetate or derivative thereof comprising culturing recombinant bacterial cells disclosed herein and extracting the non-native unsaturated fatty acetate or derivative thereof from the recombinant bacterial cells or the culture. In some embodiments, the disclosure provides methods of producing a non-native unsaturated fatty acid or derivative thereof comprising culturing recombinant bacterial cells disclosed herein and extracting the non-native unsaturated fatty acid or derivative thereof from the recombinant bacterial cells or the culture.

[0007] In some embodiments, the disclosure provides methods of producing a non-native unsaturated fatty ester or derivative thereof comprising culturing recombinant bacterial cells disclosed herein and extracting the non-native unsaturated fatty ester or derivative thereof from the recombinant bacterial cells or the culture.

[0008] In some embodiments, the disclosure provides methods of producing a pheromone molecule or precursor thereof comprising culturing recombinant bacterial cells disclosed herein and extracting the pheromone molecule or precursor thereof from the recombinant bacterial cells or the culture.

[0009] In some embodiments, the disclosure provides a recombinant bacterial cell for producing at least one non-native unsaturated fatty alcohol or derivative thereof, wherein the non-native unsaturated fatty alcohol or derivative thereof selected from Z11-C16: 1-OH, Z9-C14: 1-OH, and Z13-C18:1-OH.

[0010] BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 (FIG. 1) shows a generic fatty acid-derived pheromone biosynthesis route via a acyl-[acyl-carrier-protein] desaturase (FAD, EC 1.14.19.11) and an alcohol-forming fatty acyl-CoA reductase (FAR) (EC 1.2.1.84), and optionally fatty alcohol acetyl transferase (FAA EC 2.3.1.84) with Z10-C12: 1-OH as an example. While most often described as acting on Acyl-CoA the FAR in this example directly converts Acyl-ACP to Fatty Alcohol.

[0012] Figure 2 (FIG. 2) shows a representative fatty acid-derived pheromone biosynthesis route for various Z11-C16 molecules.

[0013] Figure 3 (FIG. 3) is a pair of graphs showing fatty acid methyl ester (FAME) analysis of whole cell E. coli fatty acid from the cell pellets grown in LB with overexpressed heterologous fatty acyl-ACP desaturase. Controls were the same strain with a plasmid without heterologous desaturases. (Codon Optimization 1 = JKA0195, Codon Optimization 2 = JKA0196).

[0014] Figure 4 (FIG. 4) is a series of graphs showing the production of fatty alcohols (i.e., Z9-C16:1-OH, Z11-C16:1-OH, or C16:0-OH) by E. coli transformed with a plasmid encoding a FAD T32 variant (i.e., JKA0195, JKA0196, or a no FAD control) and a plasmid encoding an FAR variant (i.e., FAR019, FAR020, FARref, or no FAR control). Plasmid controls with either no FAD or no FAR sequence were used.

[0015] Figure 5 (FIG. 5) is a series of graphs showing production of fatty alcohols (FALC) by E. coli with and without varying combinations of FAD and FAR enzymes.

[0016] Figure 6 (FIG. 6) shows the fold change in production of fatty alcohol Z11-C16: 1-OH and total FALC by E. coli having FAD T32 (or no FAD as a control) and FAR022 or FARO 16.

[0017] Figure 7 (FIG. 7) shows the fermentation production of an E. coli strain with induced expression of FAD T32 and FAR022 resulting in production of Z9-C14:1-OH (-30.5 mg / L) and Z11-C16:1-OH (-85.9 mg / L). The data also demonstrates the distribution of acyl chain lengths from a fermentation run with minimal media.

[0018] Figure 8 (FIG. 8) shows the expression of a compatible FAD (FAD T36) with MaqFAR (in STR5726) increases the production of Z11-C16: 1-OH by about 28-fold relative to a control strain STR5733 (which expresses MaqFAR with another FAD not relevant for Z11-C16:1-OH).

[0019] Figure 9 (FIG. 9) shows the overexpression of FAD_T32, PetF and MaqFAR increases Z11-C16: 1-OH production in fermentation. The control has no FAD T32 or PetF present, while expressing MaqFAR.

[0020] Figure 10 (FIG. 10) Shows the attenuation of Fab A by replacing the WT FabAfSeq ID No: 42] to attenuated variant FabA2 [Seq ID No: 43], The control has WT Fab A. The FabA2 variant results in a decrease in Z9-C16:1-OH and increase in Z11-C16:1-OH. Addition of more FADT32 and PetF in the presence of FabA2 further decreases Z9-C16: 1-OH and further increases in Z11-C16:1-OH. In all cases MaqFAR is expressed.

[0021] DETAILED DESCRIPTION

[0022] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0023] Also incorporated by reference in their entirety are any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the world wide web at tigr.org and / or the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov.

[0024] Without being bound to any specific scientific theory, steps envisioned for the production of unsaturated fatty acid or its derivates include: 1) introducing a double bond to the position of interest, 2) decoupling the molecule of interest from native fatty acid synthesis, and 3) recovering the unsaturated fatty acid or its derivative. For the first step, desaturases are feasible candidate enzymes due to their diverse specificity for fatty acyl chain length and position of desaturation. For the second step, enzymes such as thioesterases or fatty acyl reductases are able to release either free fatty acids or their alcohol derivates and for the third step, once decoupled from the carrier protein the molecule can be exported out of the production host cells to facilitate recovery.

[0025] Three major classes of fatty acid desaturases exist depending on their main substrates including acyl-lipid, acyl-acyl carrier protein (acyl-ACP) and acyl-coenzyme A (acyl-CoA). See Cerone and Smith, IUBMB Life. 2022;74(ll): 1036-1051. These enzymes insert double bonds into fatty acids that are in a glycerolipid or other esterified form (e.g., bound to ACP or CoA). However, they differ significantly in several aspects. For example, acyl-lipid desaturases are membrane-bound enzymes associated with the endoplasmic reticulum, chloroplast membranes of plants, and plasmatic and thylakoid membranes of cyanobacteria. Acyl-lipid desaturases insert double bonds into fatty acids that are already esterified to glycerolipids in membranes. On the other hand, fatty acyl-CoA desaturases are membrane-bound enzymes associated with the endoplasmic reticulum in yeasts, fungi, and animal cells. They use fatty acids esterified to coenzyme A (CoA) as substrates and are ubiquitous among animals, fungi, and many bacteria. These enzymes can introduce double bonds at various positions (e.g., A9, A6, A5) depending on the specific enzyme. In contrast, acyl-ACP desaturases are soluble enzymes found primarily in the plastids of higher plants. They use fatty acids bound to acyl carrier protein (ACP) as substrates and typically catalyze the conversion of saturated fatty acids to monounsaturated acids, such as the conversion of stearic acid (Cl 8:0) to oleic acid (Cl 8: 1 ). Furthermore, the substrate specificity and regioselectivity of membrane-bound desaturases (fatty acyl-CoA and acyl-lipid) are largely determined by the interaction between the enzyme and the lipid head-group, with specific residues in the cytoplasmic domain and transmembrane helix playing crucial roles. Fatty acid biosynthesis in E. coli is distinct from that of yeasts or other eukaryotes. Bacteria, including E. coli, use a Type II fatty acid synthase (FAS) where each enzymatic step is independent and Acyl-ACP intermediates are accessible, whereas yeasts or other Eukaryotes use a type I FAS where saturated acyl-ACP molecules are elongated in a multifunctional enzyme and then releases as acyl -Co A which may be acted on by acyl-CoA desaturases (EC 1.14.19.1).

[0026] Against these background, this application provides the use of selected enzyme combinations (e.g., an acyl-ACP desaturase (FAD) and an alcohol-forming fatty acyl-CoA reductase (FAR), see Figures 1 and 2) with different specificity especially on fatty acyl chain length and positions to produce unsaturated fatty alcohol and derivatives thereof in bacteria.

[0027] Definitions

[0028] As used herein, “a”, “an”, and “the” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” discloses embodiments of exactly one element and embodiments including more than one element.

[0029] As used herein, term “about”, when used in reference to a value, refers to a value that is ±10% of the reference value, unless the context clearly indicates otherwise.

[0030] As used herein, the term “bioproduction” is intended to mean production of a compound (e.g., a fatty alcohol) by way of biological or enzymatic synthesis (as opposed to chemical synthesis). In some embodiments, bioproduction may be performed by a transgenic organism or microbe that has been engineered to express enzymes involved in the biological synthesis of a compound of interest (e.g., a fatty alcohol).

[0031] As used herein, the term “biosynthetic enzymes” refers to enzymes involved in the production of a particular compound or a class of compounds. In some embodiments, biosynthetic enzymes catalyze particular steps in a synthesis pathway that produces a particular compound or a class of compounds. In some embodiments, the term “biosynthetic enzymes” may also encompass enzymes that do not themselves catalyze synthetic reactions in a synthesis pathway, but that regulate the expression and / or activity of other enzymes that do so. In some embodiments, the biosynthetic enzymes are enzymes involved in the production of one or more fatty acids. In some embodiments, the biosynthetic enzymes are enzymes involved in the production of one or more fatty alcohols. In some embodiments, the biosynthetic enzymes include but are not limited to, for example, fatty acyl reductase (FAR) and fatty acyl-ACP desaturase (FAD). The term “endogenous” as used herein refers to a substance e.g. , a nucleic acid, protein, etc. that is produced from within a cell. Thus, an endogenous polynucleotide or polypeptide refers to a polynucleotide or polypeptide produced by 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 is a gene that was present in the cell when the cell was originally isolated from nature i.e., the gene is native to the cell. In other exemplary embodiments, an “endogenous” gene has been altered through recombinant techniques e.g., by altering the relationship of control and / or coding sequences. Thus, a heterologous gene may, in some exemplary embodiments, be endogenous to a host cell. Additionally, a variant (i.e., mutant) polypeptide may be produced from within the cell and would be considered endogenous polypeptide. In contrast, an “exogenous” polynucleotide or polypeptide, or other substance (e.g., fatty acid derivative, small molecule compound, etc.) refers to a polynucleotide or polypeptide or other substance that is not encoded or produced by the cell and which is therefore added to a cell, a cell culture, or assay from outside of the cell. A variant (i.e., mutant) polypeptide added to the cell, cell culture, or assay is one example of an exogenous polypeptide.

[0032] As used herein, the term “fatty acid” refers to an aliphatic carboxylic acid having the formula R-COOH wherein R is an aliphatic group having at least 4 carbons, typically between about 4 and about 28 carbon atoms (e.g., 4 to 18 carbon atoms, or 8 to 18 carbon atoms). The aliphatic R group can be saturated or unsaturated, branched or unbranched. Unsaturated “fatty acids” may be monounsaturated or polyunsaturated. A fatty acid can be produced within a cell through the process of fatty acid biosynthesis, through the reverse of fatty acid degradation or betaoxidation, 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 a cell 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. As used herein, the term “fatty acid derivative” refers to a product derived from a fatty acid. 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-ACP derivatives. Thus, a fatty acid derivative includes alkyl- thioesters and acylthioesters. 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 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, (CO- 1 (-hydroxy fatty acids, (CO-2 (-hydroxy fatty acids, (CO-3 (-hydroxy fatty acids, 10-hydroxy fatty acids, 1,3 fatty-diols, a,w- diols, a,w -3-hydroxy triols, co-hydroxy FAME, co-OH FAEE, etc.), and unsaturated fatty acid derivatives, including unsaturated compounds of each of the above mentioned fatty acid derivatives.

[0033] In a fatty acid, the "co" (omega) refers to the position of the first double bond, counting from the methyl end of the fatty acid chain; so, an "omega-3" fatty acid would have its first double bond on the third carbon from the methyl end, while an "omega-6" fatty acid would have its first double bond on the sixth carbon from the methyl end.

[0034] “Isomers” are different compounds that have the same molecular formula. “Stereoisomers” are isomers that differ only in the way the atoms are arranged in space. As used herein, the term “isomer” includes any and all geometric isomers and stereoisomers. For example, “isomers” include cis- and trans-i somers, E- and Z-isomers, R- and S-enantiomers, diastereomers, (d)-isomers, (1)-isomers, racemic mixtures thereof, and other mixtures thereof, as falling within the scope of this disclosure.

[0035] Stereoisomers of compounds (also known as optical isomers) include all chiral, diastereomeric, and racemic forms of a structure, unless the specific stereochemistry is expressly indicated. Thus, compounds can include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions. Both racemic and diastereomeric mixtures, as well as the individual optical isomers can be isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners. 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 where "Z" means the highest priority groups are on the same side and "E" means they are on opposite sides. Unless otherwise specified, structures depicting double bonds encompass both the “E” and “Z” isomers.

[0036] The optical activity of a compound can be analyzed via any suitable method, including but not limited to chiral chromatography and polarimetry, and the degree of predominance of one stereoisomer over the other isomer can be determined.

[0037] The term “genetically modified cell” or “modified cell” refers to a genetically modified cell (e.g., a prokaryotic cell) wherein the modification can be selected from e.g., increased expression of a gene, inhibited expression of a gene, knockout of a gene, introduction of new gene(s), introduction of mutant gene(s), or mutation / genetic alteration of gene(s), wherein the increased expression or inhibited expression of a gene can be achieved by using common techniques in the art, such as gene deletion, changed gene copy number, changed gene promoter (e.g., by using a strong or weak promoter), etc. A genetically modified cell may also include a cell that has been isolated. In some embodiments, a genetically modified cell is a transgenic cell capable of producing high levels of a compound or biomolecule of interest. In some embodiments, the genetically modified cell refers to a genetically modified microbe. An example of a microbe herein may be a microbial cell (e.g., bacteria).

[0038] As used herein, the term “heterologous” refers to a sequence (i.e., nucleic acid or polypeptide) that is not normally expressed by an organism (e.g., a “wild-type” organism) or that is expressed at a level or pattern of expression that is not normally found in nature. A “heterologous enzyme,” as used herein, refers to an enzyme that is encoded by a heterologous gene. However, it is also contemplated herein that a heterologous gene can encode an endogenous or homologous enzyme. As used herein, the term “heterologous gene” refers to a gene that occurs in a form not normally found in an organism (e.g., a microbe) in its natural, non-modified state. Thus, in some embodiments, a heterologous gene is a gene that is derived from a species that is different from the species of the host cell expressing the gene. In some embodiments, a heterologous gene is a modified version of a gene that is endogenous to the host cell (e.g., an endogenous gene subjected to manipulation and then introduced or transformed into the host cell). For example, in some embodiments, a heterologous gene has an endogenous coding sequence, but has modifications in the promoter sequence. Similarly, in other embodiments, a heterologous gene encodes the same amino acid sequence as an endogenous gene, but has modifications in codon usage and / or to noncoding regions (e.g., introns), and / or combinations thereof. In some embodiments, the heterologous gene is a gene that has been modified to overexpress a gene product of interest.

[0039] As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Methods for the calculation of a percent identity as between two provided sequences are known in the art. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences (or the complement of one or both sequences) for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). The nucleotides or amino acids at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, optionally taking into account the number of gaps, and the length of each gap, which may need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a computational algorithm, such as BLAST (basic local alignment search tool). For the purposes of this disclosure, a gene (e.g., a gene encoding a FAD) or a protein (e.g., a FAD) may comprise a nucleic acid sequence or an amino acid sequence that shares about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or up to about 100% sequence identity or homology with a reference or “parent” sequence.

[0040] As used herein with respect to a molecule such as a nucleic acid or polypeptide, or form thereof, “level” is used to refer to a measure indicative of an amount, concentration, ratio, or activity of the molecule, e.g., in a particular context such as a tissue, sample, organism, or a context representative thereof. An amount can be, for example, a mass or number of molecules. A concentration can be an amount relative to a context value, e.g., per a unit of mass or volume. A ratio can be a relationship between two values, such as an experimental value and a reference control value. Activity can be a measure of a function associated with a molecule, and can in various instances be measured relative to a context value, e.g., per a unit of mass or volume. Those of skill in the art will appreciate that the metric by which a level is expressed can vary depending, e.g., on the assay and purpose. Those of skill in the art will further appreciate that metrics such as amount, concentration, ratio, and activity are often interrelated and / or qualitatively or quantitatively informative of each other.

[0041] The term “recombinant” as used herein, refers to a genetically modified polynucleotide, polypeptide, cell, 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 fatty acid or derivative molecule according to the disclosure. A recombinant cell can be derived from a microorganism such as a bacterium. In exemplary embodiments, a “recombinant host cell” or “recombinant bacterial cell” is used to produce one or more non-native fatty acid derivatives including, but not limited to, non-native monounsaturated fatty acids, non-native monounsaturated fatty esters (e.g., waxes), fatty acid esters, fatty esters, fatty acid methyl esters (FAME), fatty acid ethyl esters (FAEE)), non-native monounsaturated fatty acyl acetate esters (FACE), non-native monounsaturated fatty alcohols (e.g., polyols), non-native monounsaturated fatty aldehydes, non-native monounsaturated fatty amines, non-native monounsaturated terminal olefins, non-native monounsaturated ketones, etc. Therefore, in some 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 non-native monounsaturated fatty acid derivative composition when cultured in the presence of a (simple) carbon source under conditions effective to express the polynucleotides.

[0042] Recombinant bacterial cells

[0043] In certain aspects, the disclosure provides a recombinant bacterial cell comprising one or more enzymes useful for producing fatty acids and / or derivatives thereof. In some embodiments, the recombinant bacterial cells comprise a fatty acyl-ACP desaturase (FAD). In some embodiments, the recombinant bacterial cells comprise a fatty acyl reductase (FAR). In some embodiments, the recombinant bacterial cells comprise a FAD and a FAR.

[0044] In some embodiments, the FAD is a heterologous FAD. In some embodiments, the FAR is a heterologous FAR. In some embodiments, the FAD and the FAR are heterologous.

[0045] In some embodiments, the recombinant bacterial cell comprises a transgene encoding FAD or a genetic modification capable of increasing the expression of FAD. In some embodiments, the recombinant bacterial cell comprises a transgene encoding FAR or a genetic modification capable of increasing the expression of FAR. In some embodiments, the recombinant bacterial cell provided herein expresses FAD. In some embodiments, the recombinant bacterial cell provided herein expresses FAR. In some embodiments, the recombinant bacterial cell provided herein expresses FAD and FAR

[0046] In some embodiments, at least one of FAD and FAR is encoded in the genome of the recombinant bacterial cell. In some embodiments, FAD is encoded in the genome of the recombinant bacterial cell. In some embodiments, FAR is encoded in the genome of the recombinant bacterial cell. In some embodiments, FAD and FAR are encoded in the genome of the recombinant bacterial cell.

[0047] In some embodiments, the recombinant bacterial cell comprises a first plasmid and a second plasmid. In some embodiments, the first plasmid encodes FAD. In some embodiments, the second plasmid encodes FAR.

[0048] In some embodiments, the FAD and / or FAR is cytosolic. In some embodiments, the FAD and / or FAR is membrane-associated. In some embodiments, the FAD and / or FAR is a partial protein with a N- and / or C-terminus truncation relative to the corresponding full-length protein.

[0049] In some embodiments, the recombinant bacterial cell further comprises a heterologous enzyme selected from a fatty acyl-ACP thioesterase and a fatty acyl-CoA ligase. In some embodiments, the recombinant bacterial cell further comprises a modification suppressing, attenuating, or knocking out the expression or activity of an endogenous dual 3 -hydroxy acyl-ACP dehydratase / isomerase. In some embodiments, the recombinant bacterial cell further comprises an heterologous dual 3-hydroxy-acyl-ACP dehydratase / isomerase (fabZ / A), wherein optionally the isomerase activity of the heterologous fabZ / A is capable of providing a fatty acid or fatty acid derivative molecule having one or more double bonds in a non-native position selected from co-3, co-5, co-6, co-8, co-9, co- 11, and col2.

[0050] In some embodiments, the recombinant bacterial cell described herein may or may not express endogenous FAD. In some embodiments, the recombinant bacterial cell described herein may or may not express endogenous FAR. In some embodiments, the recombinant bacterial cell comprises a transgene that encodes a wild-type FAD or a genetic modification capable of increasing the expression of a wild-type FAD. In some embodiments, the recombinant bacterial cell comprises a transgene that encodes a wild-type FAR or a genetic modification capable of increasing the expression of a wild-type FAR. In some embodiments, the recombinant bacterial cell comprises a first transgene that encodes a wild-type FAD or a first genetic modification capable of increasing the expression of a wild-type FAD and a second transgene that encodes a wild-type FAR or a second genetic modification capable of increasing the expression of a wild-type FAR. The engineered FAD may be a functionally active fragment of a wild-type FAD, a FAD fused with a tag (e.g., a peptide tag, such as a Myc tag, HaloTag, HA tag, Flag tag, His tag, biotin tag, or V5 tag) or a reporter protein (e.g., a GFP protein, a luciferase protein, etc.), and / or a mutated FAD with one or more amino acid deletions, insertions, and / or substitutions. In some embodiments, the mutated FAD maintains or increases its activity in converting saturated fatty acids into unsaturated fatty acids. The engineered FAR may be a functionally active fragment of a wild-type FAR, a FAR fused with a tag (e.g., a peptide tag, such as a Myc tag, HaloTag, HA tag, Flag tag, His tag, biotin tag, or V5 tag) or a reporter protein (e.g., a GFP protein, a luciferase protein, etc.), and / or a mutated FAR with one or more amino acid deletions, insertions, and / or substitutions. In some embodiments, the mutated FAR maintains or increases its activity in catalyzing the biosynthesis of fatty alcohols.

[0051] Exemplary amino acid sequences of FAD or FAR enzymes encompassed within the scope of compositions-of-matter and methods of the present disclosure are shown in Table 1.

[0052] Table 1. Fatty Acyl-ACP Desaturase, Fatty-Acyl Reductase and Other Sequences Name Amino Acid or Nucleotide Sequence SEQ (Alias) ID NO:

[0053] AAC49421.1, FAD T32 MASTSISKVNHIRKVGVTGVMAPQKIEIFKS myristyl-ACP desaturase MEEWGKHNILPLAKPVEKSWQPTDFLPDPS (Pelargonium x hortorum) SEGFMEEYNAFKERTRELPDEYFVVLAGDM ITEEALPTYQTLVNRPDEVADETGHSESPWA VWSRAWTAEENRHGDLLNKYLYLSGKLDM RQVEKTIQYLIALGQDIGTEKNPYHLF1YTSF

[0054] 1 QERATFISHANTAKLAQQHGDKQLAQICGTI AADEKRHETAYTRIVDKLFELDPDETMSCL AHMMKRKITMPAHLMRDGRDPHLFQHFSV VASRTGVYTVMDYINILEHFVEKWNIEKITA GLSDKGREAQDYVCKLGERLRKVEERAHQ RVVQADPIPFSWIFDRKV WP 007152038 MATQQQQNGASASGVLEQLRGKHVLITGTT A07_FAR_Marinobacter_algicola GFLGKVVLEKLIRTVPDIGGIHLLIRGNKRHP (FARref. mFAR, MaFAR) AARERFLNEIASSSVFERLRHDDNEAFETFLE ERVHCITGEVTESRFGLTPERFRALAGQVDA FINSAASVNFREELDKALKINTLCLENVAAL AELNSAMAVIQVSTCYVNGKNSGQITESVIK PAGESIPRSTDGYYEIEELVHLLQDKISDVKA RYSGKVLEKKLVDLGIREANNYGWSDTYTF TKWLGEQLLMKALSGRSLTIVRPSIIESALEE 2 PSPGWIEGVKVADAIILAYAREKVSLFPGKR SGIIDVIPVDLVANSIILSLAEALSGSGQRRIY QCCSGGSNPISLGKFIDYLMAEAKTNYAAY DQLFYRRPTKPFVAVNRKLFDVVVGGMRVP LS1AGKAMRLAGQNRELKVLKNLDTTRSEA TIFGFYTAPDYIFRNDSLMALASRMGELDRV LFPVDARQIDWQLYLCKIHLGGLNRYALKE RKLYSLRAADTRKKAA WP 036204264.1 MATQQQQNGASASGVLEQLRGKHVLITGTT A08_FAR_Marinobacter_salarius GFLGKVVLEKLIRTVPDIGGIHLLIRGNKRHP AARERFLNEIASSSVFERLRHDDNEAFESFLE ERVHCTTGEVTEPRFGLTQERFRALAGQVDA FINSAASVNFREELDKALKINTLCLENVAAL AELNSTMAVIQVSTCYVNGKNSGQITESVIK PAGESIPRSTDGYYETEELVHLLQDKISDVK ARYSGKVLEKKLVDLGIREANNYGWSDTYT FTKWLGEQLLMKALSGRSLTIVRPSIIESALE 3 EPSPGWIEGVKVADAIILAYAREKVSLFPGK RSGIIDVIPVDLVANSIILSLAEAISEPGHRRIY QCCSGGSNPISLGTFIDYLMAEAKSNYAAYD QLFYRRPTKPFVAVNRKLFDVVVGGMRVPL SIAGKALRLAGQNRELKVLKNLDTTRSLATI FGFYTAPDYIFRNDSLMALASRMGELDRVL FPVDARQIDWQLYLCKIHLGGLNRYALKER

[0055]

[0056] KLY SLRAAQTRKKA A WP_137437574.1 MEKQHLQTGTSASAVLEQLRGKHVLITGTT A09_FAR_Marinobacter_sp .PJ- 16 GFLGKVVLEKLIRAVPDIGGIHLLIRGNKRYP GARERFNNEIASSSVFERLRHEDNEAFENFL EDRVHCITGEVTEPRFGLTPDRFQALAGQVD AFINSAASVNFREELDKALKINTLCLENVAA LARLNSKMAVIQVSTCYVNGKNSGPVTESVI KPAGDAIPRSTGGYYEIDELVHLLQDKIADV RSRYSGKLLEKKLVDLGIREANIYGWSDTYT FTKWLGEQLLMKALSGRSLTIVRPSIIESALE 4 EPSPGWIEGVKVADAIILAYARGKVSLFPGK RSGIIDVIPVDLVANSIILSLAEAITEPAQRRIY QCCSGSSNPISLGKFIDHLMAEAKSNYADYE QLFYRQPTKPFIAVNRRLFDVVVGGMRLPLS IAGKALRLAGQNRELKVLKNLDTTRSLATIF GFYTAPDYVFRNDGLIALASRMGELDRVLF PVDARQIDWQLYLRKIHLGGLNRYALNERK LYSLRATQSRKKAA WP 135955356.1 MAKQQPKTGTSVSGVLEQLRGRDVLITGTT A 10_FAR_Marinobacter_orientalis GFLGKVVLEKLIRTVPDIGGIHLLIRGNKRYP DARERFLNEIASSSVFERLRQQDNEAFERFID ARVHCITGEVTESRFGLTPNRFQALAGQVD AFINSAASVNFREELDKALKINTLCLENIAAL ARLNSKMAVIQVSTCYVNGKNSGQVTESVI KPAGEAIPRSNGGYYEIDELVHLLQDKIADV RSRYSGKLLEKKLVDLGIREANIYGWSDTYT FTKWLGEQLLMKALSGRSLTIVRPSIIESALE 5 EPSPGWIEGVKVADAIILAYARGKVSLFPGK RSGIIDVIPVDLVANSIILSLAEAIAEPGQRRIY QCCSGSCNPVSLGEFIDHLMAEAKSSYADYS QLFYRKPTKPFIAVNRKLFDVVVGGMRLPLS FAGKALRLAGQNRELKVLKNLDTTRSLATIF GFYTAPDYIFHNDGLLALASRTAEVDRVLFP VDARQIDWQLYLRKIHLGGLNRYALSERRL YSLRAAQPRRKAA WP_012138772.1 MVQQLQTSELSSTVLEQLRGKHVLVTGTTG B07_FAR_Marinobacter_lipolyticus FLGKVVLEKLIRAVPDIGGIHLLIRGNKRHPN ARERFFHEIATSSVFERLRQEDNEAFEAFIEE RVHC1TGE VTKPRFGLTPERFTTLAN QADAFI NSAASVNFREELDKALTINTLCLNNVVELAR RNRKMAVIQVSTCYVNGKNSGQVTESVIKP AGESIPRSTAGYYEIEELVRLLEDKIADVRSR Y SGKVLEKKLVDLGIQEANRYGWSDTYTFT 6 KWLGEQLLMKALDQRALTIVRPSIIESALEE PAPGWIEGVKVADAIILAYAREKVTLFPGKR SGIIDVIPVDLVANAIILSLAEALAEAPQRRIY QCCSGSSNPISLGEFIDHLMAESKANYAAYD QLFYRQPSKPFIAVNRKLFDAVVGGMRVPL SLTSRVMRMLGQNRELKTLRNLDTSRSLATI

[0057]

[0058] FGFYTAPDYIFRNDSLQALASRMGERDQALF PVDARRIDWSLYLRKIHLAGLNQYALKERK LYSLRSAKARKQAA WP 150912695.1 MANKQTTSGESASGVLEQLSGKHVLITGTT B08_FAR_Marinobacter_halotolerans GFLGKVVLEKLIRAVPEIGGIHLLIRGNRNHS (FAR006) D ARSRFLNEIATS SVFDRLRHEDND AFESFM D SRIHCITGEVTEPRFGMDTAAFKKLAGNID AFVNSAASVNFREELDKALQINTLCLNNVA ALADLNPDMAVIQVSTCYVNGKNSGQVTES VIKPAGEA1PRSTNGYYE1EELVHLLEDKIAD VRARYSGKALEKKLVELGIREANQYGWSDT YTFTKWLGEQLLMKALAGRSLTILRPSIIESA 7 LEEPAPGWIEGVKVADAIILAYAREKVSLFP GKRSGIIDVIPVDLVANGIILSMAEALAVPGK RRIYQCCSGSSNPLSLGEFIDHVMAEARHHH DDYQQLFYRKPTKPFLAVNRRLFDMVVGGL RVPLSFADRALRLTGQNRELKMLKNLDTSR SLATIFGFYTAPDYIFRNDSLLALASRMGEL DRLLFPIDARQIDWSLYLRKIHIGGLNRYAL KERKVYSLRSAKTRKKAA WP_203301396.1 MATSQLNTGESSSKVLEQLRGKHVLITGTTG B09_FAR_Marinobacter_sedimmum FLGKVVLEKLIRTVPDIGGIHLLVRGNKRHP (FAR007) EARARFLDEIATS SVFERLRSDDNEAFEAFV EERVHCVTGEVTEPMFGLPAKQFQALAKGI D AVINS AASVNFREELDKALAINTRCLDNIA ELARQNSRLAVLQVSTCYVNGMNSGQVSEE VVKPAGEAVPRSVDGYYEIDELVRLLDDKI ADVRSRYSGKALEKKLVDLGIREANRYGWS DTYTFTKWLGEQLLMKALAGRSLTIVRPSII 8 ESAEEEPAPGWIEGVKVADA1ILAYAREKVT LFPGKRSGVIDVIPVDLVANSIILSLAEALSEP ADQRIYQCCSGSSNPISLGEFIDHLMVEAKA NYSDYDQLFYRRPSKPFVAVNRKLFDTVVG GVRIPLSFTSRMLKLLGHNRELKALRNLDTT RSLATIFGFYTAPDYVFQNDGLLALSSRMGG LDRVLFPVDARQIDWSVYLRKIHLAGLNRY ALKERKLYSLRSAKTRKKAA WP_138440763.1 MATSQLNTGESSSKVLEQLRGKHVLITGTTG B 1 O FAR Marinobacter alexandrii FLGKVVLEKLIRTVPDIGGIHLLVRGNKRHP (FAR008) EARARFLDEIATS SVFERLRSDDNEAFEAFV EERVHCVTGEVTEPMFGLPVYQFQALANGI DAVINS AASVNFREELDKALAINTRCLDNIA ELARQNRRLAVLQVSTCYVNGMNSGQVSE AVIKPAGEALPRSVDGHYEVDELVRLLDDKI

[0059] 9 ADVRSRYAGKALEKKLVDLGIREANRYGW SDTYTFTKWLGEQILMKALAGRSLTIVRPSII ESALEEPAPGWIEGVKVADAIILAYAREKVT LFPGKRSGVIDVIPVDLVANSIILSLAEALSEP AEQRIYQCCSGSSNPISLGEFIDHLMVEAKAS YADYDQLFYRRPTKPFIAVNRKLFDAVVGG

[0060]

[0061] VRIPLSFTSRILKVLGHNRELKVLRNLDTTRS LATIFGFYTAPDYIFQNDSLLALSSRMGGLD RVLFPIDARQIDWSVYLRKIHLAGLNRYALK ERKLYSLRSAKTRKKAA KPQ27819.1 MAMQQINQTES S SKVLGHLRGKRVLITGTT C07_FAR_Marinobacter_excellens GFLGKVVLEKLIRTVPDIGGIYLLIRGNKRHP (FAR009) D ARSRFLNEIATS SVFDRLREADLDSFESFID ERVHCITGEVTEPRFGMSEEACAKLADELD AVINSAASVNFREELDKALTINTLCLDNIAEL ALQNPQLAVLQVSTCYVNGMNSGQVTESV1 KPAGESIPRSSDGYYEIEELVRLLEDKIADVR ARYSGKTLEKKLVDLGIREANRYGWSDTYT FTKWLGEQLLMKALQGRSLTIVRPSIIESALE 10 EPAPGWIEGVKVADAIILAYAREKVTLFPGK RSGIIDVIPVDLVANAIILSLAEAIGEPGRRRI YQCCSGGSNPVSLGQFIDHLMAEAKANYAA YDHLFYRQPSKPFVAVNRRLFDLVVSGVRL PLSISDKVLKLLGNSRDLKMLKNLDTTQSLA TIFGFYTAPDYTFRNDDLMALSARMGGVDK VLFPVDARQIDWEVYLRKIHLAGLNRYALK ERKVYSLRATKSRQKAA WP_106672760.1 MAMQQVKQHESSSKVLGQLRGKRVLITGTT C08_FAR_Marinobacter_halophilus GFLGKVVLEKLIRTVPDIGGIYLLIRGNKRHP (FARO 10) DARGRFLNEIATSSVFDRLRETDPEGFETFIE HRVHCITGEVTEPRFGMTEDACARLASELD AVINSAASVNFREELDKALTINTLCLENIAGL AGLNSRLAVLQVSTCYVNGMNSGQVSESVI KPAGQ AIPRS SKGYYEIEEL VRLLQDKI AD V RARYSGKTLEKKLIDLGIQEANRYGWSDTY TFTKWLGEQLLMKALNGRSLT1VRPS11ESAL 11 EEPAPGWIEGVKVADAIILAYAREKVSLFPG KRSGIIDVIPVDLVANAIILSLAEALGEPGRR RIYQCCSGGSNPVSLGQFIDHLMAEAKANY AAYDHLFYRQPSKPFVAVNRALFDLVVSGV RLPLSVTDKVLKLLGHSRDLKVLKNLDTTQ SLATIFGFYTAPDYIFCNDELQALSARMGTV DKVLFPVDARQIDWEVYLRKIHLAGLNRYA LKERKVFSLRASRSRQKAA WP 036131572.1 MATQQTNPGESSSEVLDRLRGKHVLITGTTG C09_FAR_Marinobacter_nitratireducens FLGKVVLEKLIRAVPDIGGIHLLIRGNKRHPD (FAR011) ARGRFMNEIATSSVFERLRQEDNEAFESFVE DRVHCVTGEVTEPLFGLGADQFSKLAKGLD AVINSAASVNFREELDKALSINTRSLEGIAEL ARQNANLTVLQVSTCYVNGMNAGPVTESVI KPAGEDIPRSSEGYYEIDELVRLLDDKVADV 12 RSRYSGKVLEKKLVDLGIREANHYGWSDTY TFTKWLGEQLLMKALAGRALTIVRPSIIESA LEEPAPGWIEGVKVADAIILAYAREKVTLFP GKRSGVIDVIPVDLVANSIILGLAEAVSEPAQ RRIYQCCSGSSNPITLGEFIDHLMEEAKENYA

[0062]

[0063] EYDQLFYRKPSKPFIAVNRRLFDTVVGGVRI PLRITSRVLKMLGQSRELKALRNLDTSRSLA TIFGFYTAPDYIFRNDDLLALAARMGDVDK VLFPVDARQIDWAVYLRKIHMAGLNRYAL KERKLCSLRSAKARKKAA WP_072677872.1 MANQQVNQKESSSKVLGQLRGKRVLITGTT C 1 O FAR Marinobacter hydrocarbonocl GFLGKVVLEKLIRSVPDIGGIYLLIRGNKRHP asticus D ARSRFDNEI ATS S VFDRLRS VD AD WFE AFL (FARO 12) EERVHCITGEVTEPCFGMGEDAWQKLAGEL DAV1NSAASVNFREELDKALT1NTLCLEN1A GLARENAELAVLQVSTCYVNGMNSGPVTES VIKPAGESIPRSREGYYEIEELVQLLQDKIAD VRARYSGKTLEKKLVDLGIREANRYGWSDT YTFTKWLGEQLLMKALNRRSLTIVRPSIIESA 13 LEEPAPGWIEGVKVADAIILAYAREKVTLFP GKRSGVIDVIPVDLVANAIVLSLSEALAEPG RRRIYQCCSGSSNPVSLGQFIDHLMAESKAN YAAYDHLFYRQPSKPFIAVNRTLFDLVVSGV RLPLSVTDKVLKLFGNSRDLKMLKNLDTTQ SLATIFGFYTAPDYIFHNDELQALSARMGEV DKGLFPVDARQIDWEVYLRKIHLAGLNRYA LKERKVYSLRATKQRKKAA WP_072799080.1 MVRQQKGAGVSSSKVLEQLRGKHVLITGTT D07_FAR_Marinobacter_antarcticus GFLGKVVLEKLIRAVPDIGGVHLLIRGNARH (FARO 13) PDAHSRFLAEIATSSVFDRLRQENGQAFEVF LEERLHCVTGEATQPGFGLQPEAFRKLADSI DAVINSAASVNFREELDKALSINTLCLENIAE LARQNPSLAVIQVSTCYVNGMNSGQITESVI KPAGEGIPRSRAGYYEIEELVRLLKDSIADVR SRYSGKMLERKLVDLG1REANRYGWSDTYT LTKWLGEQLLMKGLEGRALTIVRPSIIESALE 14 EPVPGWIEGVKVADAIILAYAREKVTVFPGK RSGIIDVIPVDLVANSIILAVAEALGEPSRQW VYQCCSGSSNPVSLGEFIDHLMAEAKTNYA AYERLFYRQPVKPFFAVNRALFDALVGGAR IPLSMTRRMMRLLGQDRELKVLRNLDTTRS LATIFGFYTAPDYIFRNDDLLALASRMGEID KALFPVDARQIDWAVYLRKIHLAGLNRYAL KAKSKPRPRSSRARKQAA WP_094624693.1 MTKRQVSGGEAS SRVLEQLRGKKVLITGTT D08_FAR_Marinobacter_vinifirrnus GFLGKVVLEKLIRAVPDIGGIYLLIRGNKRHP (FARO 14) D ARSRFLNEIATS SVFDRLREADTEAFDAFL EDRIHCVTGEVTEPGFGLGEPACRKLAMEL DAVINSAASVNFREELDKALTINTLCLENIA QLARMNPALAVLQVSTCYVNGMNSGQVAE

[0064] 15 AVIKPVGEAIPQSPEGYYEIEDLVRLLHDKIE DVRSRYTGKALEKKLVELGIREANRYGWSD TYTFTKWLGEQLLMKALQGRSLTIVRPSIIES ALEEPAPGWIEGVKVADAIILAYAREKVTLF PGKRSGIIDVIPVDLVANAIIMGLAEALAEPG

[0065]

[0066] EQHIYQCCSGGSNPVTLGQFIDHLMAESKTN YAAYDHLFYRQPSKPFVAVNRGLFDLVVSG VRLPLSITDRVLKLLGNSRDLKWLRNLDTTQ SLATIFGFYTAPDYVFRNDRLQDLAERMGQ TDKALFPVDPKAIDWQHYLRKVHLAGLNR YALKERKVYSLKSSRQRKKAA WP 348701184.1 MATPHVSTES SSNVLETLRGKHVLITGTTGF D09_FAR_Oceanospirillales_bacterium LGKVVLEKLIRAVPDIGGIHLLIRGNKRHPD (FARO 15) ARSRFLNEVATS SVFERLRAEDNDGFEAFLE ERVHCVTGEVTEARFGLADAEFRALAGRLD AVINSAASVNFREELDKALAINTLCLHNIAD LAARNPDLAVIQVSTCYVNGMNSGQVTESV IKPAGEAIPRTAHGYYEISELVRLLQDKIAGV RSRYSGKTLEKKLVELGIREANRYGWSDTY TFTKWLGEQLLMKALAGRTLTIVRPSIIESAL 16 EEPVPGWIEGVKVADAIILAYAREKVTLFPG KRSGVIDVIPVDLVANAIVLALAEALSAPAG PRVYQCCSGSSNPISLGQFIDHLMAESKANY AAYDSLFYRQPTKPFVAVNRRLFDAIMGGV RLPLSLADRVFRLLGQSRELKMLRNLDTTRS LATIFGFYTAPDYIFRNDELLALSARMGAVD QALFPVDARRIDWSVYLRKIHLAGLNRYAL KERKLYSLRSAKARKKQAA TNE77515.1 MATQQTNPGESSSEVLDRLRGKHVLITGTTG D 1 O FAR Gammaproteobacteria bacteri FLGKVVLEKLIRAVPDIGGIHLLIRGNKRHPD um ARGRFMNEIATSSVFERLRQEDNEAFESFVE (FARO 16) DRVHCVTGEVTEPLFGLGADQFSKLAKGLD AVINSAASVNFREELDKALSINTRSLEGIAEL AKQNANLTVLQVSTCYVNGMNAGPVTESVI KPAGEDIPRSSEGYYE1DELVRLLDDKVADV RSRYSGKVLEKKLVDLGIREANHYGWSDTY TFTKWLGEQLLMKALAGRALTIVRPSIIESA 17 LEEPAPGWIEGVKVADAIILAYAREKVTLFP GKRSGVIDVIPVDLVANSIILGLAEAVSEPAQ RRIYQCCSGSSNPITLGEFIDHLMEEAKENYA EYDQLFYRKPSKPFIAVNRRLFDTVVGGVRI PLRITSRVLKMLGQSRELKALRNLDTSRSLA TIFGFYTAPDYIFRNDDLLALAARMGDVDK VLFPVDARQ1DWAVYLRK1HMAGLNRYAL KERKLYSLRSAKARKKAA WP_188437701.1 MAKQALGAWSSSRVLEQLRGKQILITGTTG E07_FAR_StreptosporangiumJomthonge FLGKVVLEKLIRAVPDIGGIHLLVRGNAQHR nse D ARSRFLAEIATS SVFDRLRGENPAAFDNFL (FARO 17) S SRLHCITGEVTQ AGFGLEPEAFRKLAGTID VWNSAASVNFREQLDTALSINTLCLENIAE LARQNPSLAVVQVSTCYVNGMNAGQVTES 18 VIKPAGEDIPRNPEGYYDIEELLRVLNDSIAD VRSRYTGKMLERKLVDLGIREANRYGWSDT YTFTKWLGEQLLMKALQGRALTIVRPSIVES ALEEPAPGWIEGVKVADAIILAYAREKVTLF

[0067]

[0068] PGKRSGIIDVVPVDLVANAIILAVAEALGEFP RVRIYQCC SGS SNPVSLGEFIDHLMAEAKAN YGAYENLFYRQPVKPFIAVNRTLFDALVGG ARIPLSVINSVTRLLRQDSDLRVLKNLDTTRS LATIFGFYTAPDYIFHNDDLLALAKRMGEAD KALFPVDARRIDWELYLRRIHLAGLNRYAL KARKPRRRKSTGVVTKAV MAA63608.1 MPQAVEPES SKILTSLRGKHVLITGTTGFLG E08_FAR_Alteromonadaceae_bacterium KWLEKLIRTVPDIGGIHLMIRGNRRHPKAH (FARO 18) DRF1NE1ACSSVFEREKLDDADAFDAF1ENKV HCVTGEVTAPQFGLTQARFEQLSGQIDAIINS AASVNFREELDKALEINALCLRNIVALSEAG GNLPVVQVSTCYVNGMNAGQAHEDVARPA KGGVTRHKDGYYDVDELIPVLQDKIADVKS RYSGAKLEKKLVELGICEANRYGWSDTYTF TKWMGEQLLNKGLKGAPLTILRPSIIESALEE 19 PAPGWIEGVKVADAIILAYAREKVAVFPGK RSGVIDVIPADLVANSIIMSLAELFVEPIRHRI YQCCSGSGNPITLGEFIDHLMTEAKENHASY DRLFYRRPRKPFIAVGRRLFDTVMGGVRLPL SVLDRAMKIAGSDRELKVLRNIDTTMSLATI FGFYTAPDYIFHNDKLLDLAERMGPEDKLLF PVDARQIDWPIYLRKIHMAGLNKYALSERK LYRLKTRKERQAA WP_243402318.1 MSEEHPNSASHSRVLNALSGKQVLITGTTGF E09_FAR_Tamilnaduibacter_salinus LGKVVLEKLIRSVPDIGGIHLLIRGNRHYPEA (FARO 19) RERFLEEIAASSVFERLRSEEPDRFEAFIDTRL HCVTGEITEPLFGLASHEFDALAAQLDAVIN SAASVNFREELDKALAINTDCLGNICELSRR GGNLPVIQVSTCYVNGMNAGDAKEALVNP ARSGMVQREDGTWDVDEVRHVLNDKIAEL RHCYSGNVLAEKLVDLGIREANRYGWSDTY TFTKWMGEQVLMQALHRAPLTIVRPAIIESA 20 LAEPSPGWIEGVKVADAVILAYAREKVSLFP GRRAGIIDVIPADLVGNSIILSLAEAVSSPAEQ RIYQCCSGATNPLRLGEMIDHVMTEARENH AAYDRLFHRQPTKPFIAVGRRLFDRVMGGV RLPLSLLDRVLKLAGSETELKALRNVRTTME LAT1FGFYTAPDYVFHNDRLLELADRMGSR DQTLFPVDARLIDWPTYLRKIHLAGLNRFAL SHRRLPRPTQRRKQNQRAA WP_159268210.1 MNTHKDVSQTLKLLKGKHILITGTTGFVGK E 1 O FAR Zhongshania aliphaticivorans VMLEKIMRTIPDIGGVYLLIRGSKKHINAESR (FAR020) FANEIATSSVFEFLKLNTPEYFNDFCHSKVH CISGEITEHQFGLPRVEFRQLASKVDAVVNC AASVNFREELDRALNINTISLRNIIEMSDLAG

[0069] 21 NVPVIQVSTCYVNGFNEGHIHETVTVPRHAD IPRHKEGYFEVGNLIEELEFKISQTKAKYSGK ALKRELIDLGIREANAYGWNDTYTFTKWLG EQLLLKSLRGYSLTLLRPSIIESTLREPSPGWI

[0070]

[0071] EGVKVADAILLAYAKEKVSFFPGKRSGVIDV IPADLVSNSILLSLAEQFKNPGNQHIYQCCSG SANPLTLGAFIDHLMAEAKENHGSYDKLFL RQPRKPFIAVNRKLFSAITLCLSVILSVISQTL VKLGMKRGLKARRNLDAAMALSTVFSFYT EPNYIFHNNKLLDLAKRMGSADQALFPVDS NAINWETYLRKVHMAGLNCYALKGRKQTN HLPSAVVINTTQEQAA WP 077528914.1 MESEQDNAAASESNVLGELQGKRVLITGVT F07_FAR_Halomonas_utahensis GFVGKVVLEKL1RTVPDIEGITLL1RGNRRHG (FAR021) TAGERFANEVATASVFDRLRVDDPERLDAF LDSRIQCVTGEITEPCFGMTRARFRELAAET DGVINVAASVDFREPLDRALAINALSLRNVT EFVQEAGDIPLVQVSTCYVNGYNRGHRHEE LVTPAGRGITRDPDGTCRVEDLIVTLQDRIA DLRSRYSGQKLQEELVNLGLREANRLGWN DTYTLTKWIGEQLLARNLRGGSVTILRPSIVE 22 STLEEPVPGWVEGVKVADAVIMAYARGNV MFFPGRRRGVIDVIPADLVANGIILSLTEQLQ SPGRQRIYQCC SGS SNPLRLGEFIDHVMTEA TENHQAYDHLFYQKPQLPFLAVNRRLFNTV SFGVGIPVNAVRRGLQWLGRDAELKTVEHF QTTMKLATTFGFYSCPDYTFHNDRLLEMAQ RMGDEDRRVFPVDARRINWRRYIRSVHLAG LNKYALKERKLYRMRQRRNRRRVA NP_OO 1036967.1 MSFINGTLDEHYQTVREFYDGKSVFITGATG F08_FAR_Bombyx_mori FLGKAYVEKLAYSCPGIVSIYILIRDKKGSNT EERMRKYLDQPIFSRIKYEHPEYFKKIIPISGD ITAPKLGLCDEERNILINEVSIVIHSAASVKLN DHLKFTLNTNVGGTMKVLELVKEMKNLAM FVYVSTAYSNTSQRILEEKLYPQSLNLNEIQK FAEEHYILGKDNDEMIKFIGNHPNTYAYTKA LAENLVAEEHGEIPTIIIRPSIITASAEEPVRGF 23 VDSWSGATAMAAFALKGWNNIMYSTGEEN IDLIPLDYVVNLTLVAIAKYKPTKEVTVYHV TTSDLNPISIRRIFIKLSEFASKNPTSNAAPFA ATTLLTKQKPLIKLVTFLMQTTPAFLADLW MKTQRKEAKFVKQHNLVVRSRDQLEFFTSQ SWLLRCERARVLSAALSDSDRAVFRCDPST1 DWDQYLPIYFEGINKHLFKNKL XP_028030838.1 MSHNGTLDEHYQTVSEFYDGKSVFITGATG F09_FAR_Bombyx mandarina FLGKAYVEKLAYSCPGIVSIYILIRNKKGSNT EERMRKYLDQPIFSRIKYEHPEYFKKIIPISGD ITAPKLGLCDEERNILINEVSIVIHSAASVKLN DHLKFTLNTNVGGTMKVLELVKEMKNLAM FVYVSTAYSNTSQRILEEKLYPQSLNLSEIQK 24 FAEEHYILGKDDDEMIKFIGNHPNTYAYTKA LAENLVAEEHGEIPTIIIRPSIITASAEEPVRGF VDSWSGATAMAAFTLKGWNYIMYSTGEEN IDLIPLDYVVNLTLVAIAKNKPTKEVTVYHV

[0072]

[0073] TTSDLNPISIRRIFIKLSEFASKNPTSNAAPFA ATTLLTKQKPLIKLVTFLMQTTPAFLADLW MKTQRKEAKFVKQHNLVVRSRDQLEFFTSQ SWLLRCERARVLSAGLSDSDRAVFRCDPSTI DWDQYLPIYFEGINKHLFKNKL AGD98718.1 MYQAIAECYEGQ SVFMTGGTGFLGKVLLEK F 1 O FAR Bicyclus anynana LLYSCPGIEKVYLLVREKQGATAQQRVQKL LEQPLFSRIKEEKPQAFEKIIPIVGDISEPQLGI KAEDEELLAEKVSIVYHVAATIKFNEPFEQA MN VN VAGTGRVLNLSKRMKN VKAF VY VST AFSNTDRKVIEEIIYPAPASLNEVKKLLEIGIT DAQVKELIRGRPNTYTFTKALAENLVADNH GNVPAVIIRPSIVSSSKREPMVGWIDNWFGA 25 S ALITTIMKGLNRVLLS SRDNSLDLIPVDYVS NLIIVAAAKCKCSKNVGVYNSSTSGENPLRI GRLAELIIADSNKHKFYDVPMPMVYFTRFK WVVLLITLFFQTLPAYIADIFLLIRGKKTRYV KIQSKVSFVRDTLEYFTSNTWSISSRQTTALS QSLSPSDRYLFPCDPTDIDWKDYIPTYCQGIR QFLCKT XP_030032183.1 MTQEFQNGSYQSVSDFYSGRSVFITGATGFL G07_FAR_Manduca_sexta GKAYVEKLAYECREIDKIYILIRHKKAVNLE QRIQNYLDQPFFTRLKEERPEVIQKIIPILGDI TAPKLGLKAEDEQTLIDKVSVVIHSAAVVKF NQALKDAINANVEGTRKVLSLAKLMKNVC TFVYISTAYSNCQEYVVEEVLYPPPADLEEA KKFAMKHYILGDDDKELIKNHPNTYTFSKA LAEQVVAENRGDLPTIIIRPSIVSASEREPVK 26 GWVDNLTAATGVITSLVKGWIHVIEASGDV VLD1IPLDYVVNETIVAASRSSLVPGLRVYHS CSSTTNPITLKNIYTHIKEFTSQNNNNASHHV SMFFIKNKALVSGLTCLMQISPAFMADMWL RCKGKQPKYLKLQSKVLQGRNMLEFFSTRS WQIHSEGACSLYSSLSPEDRKAFRCDTTHIE WREYIFHYLVGIERYLARKS XP_034826751.1 MCEYQAIADCYDGQSVFMTGGTGFLGKVL G08_FAR_Maniola_hyperantus LEKLLYSCSGIDK1YLLVREKQGATPQQRVQ KILEQPLFSRLKEEKPKALDKIVPIVGDISEPQ LGLKADDEELLVEKVSFVYHVAATIKFNEPF EQAMNVNVAGTGRVLNLTKRMKNIKAFLY V STAYSNTDRKVVEEVIYPAP ASLNEVKKLL EIGLSDKQVKELINGRPNTYTFTKALAEHLV ADNHGRIPAVIIRPSIVTSSEREPMVGWIDNW 27 FGASALFTSIMKGLNRVLLAEPDNRLDLIPV DYVSNLIIVAAARCKCAKKVDVYNCSTSGD NPITIGQFAELAIADSKKHKFYDVPMPMVFF TSYKWMVMMITLLTQTLPAYIADIFLLIRGK KARYVKLQSRVGHIRDTLEYFTLNTWSISAL KTRELSKSLSPSDRQLFPCDPTDIDWKTYVPI

[0074]

[0075] YCQGIRQFLCKR CAG4999510.1 MSRPDGIEDIYESIERHYAGKSVFITGGTGFL G09_FAR_Pamassius_apollo GKVFIEKLLYSCSEINKIFLLLRNKNNSDTSE RVKQLLELPIFERVRKEKPENFEKIVPLCGDV TLPNLGLSQEDEQMLIEEVSHVFHFAANVKF NENLKVAMKTNVEGTRRVVNLCQRIMNIEV FVYVSTAYSNTNEPVLEEIVYTAPVSIDEVY EILEQEVSEVSNEDKLKKLLCGRPNTYTFAK ALAEGLVSQEHGDFPTIIIRPSIVSASMHEPLT 28 GWVDNWFGATALIATIAMGLVRVVLTKST NTLDLIPVDYVSNLIVAAAAKCKRSKEVSVY NCCTSDANPFTVGLLGKLITEDSVKHNFHEII RPTLFCIQSEWLLNVLTLIMQTMPAFIADCW LRLTGRSPRFMKMQRKILQTRS ALQYFS SRS WQMKCQRTRDLF A SLS VSDRLEFPFDPTHIK WDMYIPIYCMGIRKFLIKAS AGD98718.1 MYQAIAECYEGQ SVFMTGGTGFLGKVLLEK H07_FAR_Bicyclus_anynana LLYSCPGIEKVYLLVREKQGATAQQRVQKL LEQPLFSRIKEEKPQAFEKIIPIVGDISEPQLGI KAEDEELLAEKVSIVYHVAATIKFNEPFEQA MNVNVAGTGRVLNLSKRMKNVKAFVYVST AFSNTDRKVIEEIIYPAPASLNEVKKLLEIGIT DAQVKELIRGRPNTYTFTKALAENLVADNH GNVPAVIIRPSIVSSSKREPMVGWIDNWFGA 29 S ALITTIMKGLNRVLLS SRDNSLDLIPVDYVS NLIIVAAAKCKCSKNVGVYNSSTSGENPLRI GRLAELIIADSNKHKFYDVPMPMVYFTRFK WVVLLITLFFQTLPAYIADIFLLIRGKKTRYV KIQSKVSFVRDTLEYFTSNTWSISSRQTTALS QSLSPSDRYLFPCDPTDIDWKDYIPTYCQGIR QFLCKT XP 047019706.1 MVVLTSKETKPSVAEFYAGKSVFITGGTGFL H08_FAR_Helicoverpa_zea GKVFIEKLLYSCPDIGNIYMLIREKKGLSVSE RIKQFLDDPLFTRLKEKRPADLEKIVLIPGDIT APDLGITSENEKMLIEKVSVIIHSAATVKFNE PLPTAWKINVEGTRMMLALSRRMKRIEVFIH ISTAYTNTNREVVDEILYPAPADINQVHQYV KDGISEEDTEKILNGRPNTYTFTKALTEHLV AEN QA Y VPT1I VRPS V VAA1KDEPIKGWLGN 30 WYGATGLTVFTAKGLNRVIYGHS S YIVDLIP VDY VANLVIAAGAKS SKSTELKVYNCCS S A CNPITIGKLMSMFAEDAIKQKSYAMPLPGW YIFTKYKWLVLLLTMLFQVIPAYITDLYRHLI GKNPRYIKLQSLVNQTRSSIDFFTSHSWVMK ADRVRELFASLSPADKYLFPCDPTDINWTHY IQDYCWGVRHFLEKKSTNK AFD04727.1 MVVLTSKETKPSVAEFYAGKSVFITGGTGFL H09_FAR_Helicoverpa_assulta GKIFIEKLLYSCPDIGNIYMLIREKKGLSVSER (FAR022) IKQFLDDPLFTRLKEKRPADLEKIVLIPGDIT 31

[0076] APDLGITSENEKMLIEKVSVIIHSAATVKFNE

[0077]

[0078] PLPTAWKINVEGTRMMLALSRRMKRIEVFIH ISTAYTNTNREVVDEILYPAPADIDQVHQYV KDGISEEETEKILNGRPN1YTFTKALTEHLV AENQAYVPTIIVRPSVVAAIKDEPIKGWLGN WYGATGLTVFTAKGLNRVIYGHS S YIVDLIP VDYVANLVIAAGAKS SKSTELKVYNCCS S A CNPITIGKLMSMFAEDAIKQKSYAMPLPGW YVFTKYKWLVLLLTILFQVIPAYITDLYRHLI GKNPRYIKLQSLVNQTRSSIDFFTSHSWVMK ADRVRELFASLSPADKYLFPCDPTDINWTHY IQDYCWGVRHFLEKKTTNK

[0079] Fatty acyl-ACP desaturase (a codon- ATGGCAAGCACCAGCATTAGCAAAGTTAA optimized coding sequence for CCATATTCGTAAAGTTGGTGTTACCGGTGT FAD T32) TATGGCACCGCAGAAAATTGAAATCTTCA AAAGCATGGAAGAGTGGGGCAAACATAA TATTCTGCCGCTGGCAAAACCGGTTGAAA AATCATGGCAGCCGACCGATTTTCTGCCG GATCCGAGTAGCGAAGGTTTTATGGAAGA ATATAACGCCTTTAAAGAACGCACCCGTG AACTGCCGGATGAATATTTTGTTGTTCTGG CAGGCGATATGATTACCGAAGAAGCACTG CCGACCTATCAGACCCTGGTTAATCGTCCG GATGAAGTTGCAGATGAAACCGGTCATAG CGAAAGCCCGTGGGCAGTTTGGAGCCGTG CCTGGACAGCCGAAGAAAATCGTCATGGT GATCTGCTGAACAAATATCTGTATCTGAG CGGTAAACTGGATATGCGTCAGGTTGAGA AAACCATTCAGTATCTGATTGCACTGGGTC AAGATATTGGCACAGAGAAAAATCCGTAT

[0080] 32 CACCTGTTTATCTACACCAGCTTTCAAGAA CGTGCAACCTTTATTAGCCATGCAAATACC GCAAAACTGGCACAGCAGCATGGTGATAA ACAGCTGGCACAGATTTGTGGCACCATTG CAGCCGATGAAAAACGCCATGAAACCGCA TATACCCGTATTGTGGATAAACTGTTTGAA CTGGATCCTGATGAAACCATGAGCTGTCT GGCACACATGATGAAACGTAAAATTACCA TGCCTGCACATCTGATGCGTGATGGTCGTG ATCCGCACCTGTTTCAGCATTTTAGCGTTG TTGCCAGCCGTACCGGTGTGTATACCGTTA TGGATTATATCAACATCCTGGAACACTTCG TGGAAAAATGGAACATTGAGAAAATTACC GCAGGCCTGAGCGATAAAGGTCGTGAAGC ACAGGATTATGTGTGTAAACTGGGTGAAC GTCTGCGTAAAGTGGAAGAACGCGCACAT CAGCGTGTTGTTCAGGCCGATCCGATTCCG TTTAGCTGGATTTTTGATCGTAAAGTGTAA WP_011785687.1 MAIQQVHHADTSSSKVLGQLRGKRVLITGT 33 fatty acyl-CoA reductase [Marinobacter TGFLGKVVLERLIRAVPDIGAIYLLIRGNKRH aquaeolei 1 PDARSRFLEEIATSSVFDRLREADSEGFDAFL

[0081]

[0082] MaqFAR EERIHCVTGEVTEAGFGIGQEDYRKLATELD AVINSAASVNFREELDKALAINTLCLRNIAG MVDLNPKLAVLQVSTCYVNGMNSGQVTES VIKPAGEAVPRSPDGFYEIEELVRLLQDKIED VQARYSGKVLERKLVDLGIREANRYGWSDT YTFTKWLGEQLLMKALNGRTLTILRPSIIESA LEEPAPGWIEGVKVADAIILAYAREKVTLFP GKRSGIIDVIPVDLVANSIILSLAEALGEPGRR RIYQCCSGGGNPISLGEFIDHLMAESKANYA AYDHLFYRQPSKPFLAVNRALFDLVISGVRL PLSLTDRVLKLLGNSRDLKMLRNLDTTQSL ATIFGFYTAPDYIFRNDELMALANRMGEVD KGLFPVDARLIDWELYLRKIHLAGLNRYAL KERKVYSLKTARQRKKAA AAC63059.1, FAD T33 MAMKLNALMTLQCPKRNMFTRIAPPQAGR 34 delta-4-palmitoyl-acyl carrier protein VRSKVSMASTLHASPLVFDKLKAGRPEVDE desaturase [Coriandrum sativum] LFNSLEGWARDNILVHLKSVENSWQPQDYL PDPTSDAFEDQVKEMRERAKDIPDEYFVVL VGDMITEEALPTYMSMLNRCDGIKDDTGAQ PTSWATWTRAWTAEENRHGDLLNKYLYLS GRVDMRMIEKTIQYLIGSGMDTKTENCPYM GFIYTSFQERATFISHANTAKLAQHYGDKNL AQVCGNIASDEKRHATAYTKIVEKLAEIDPD TTVIAFSDMMRKKIQMPAHAMYDGSDDML FKHFTAVAQQIGVYSAWDYCDIIDFLVDKW NVAKMTGLSGEGRKAQEYVCSLAAKIRRVE EKVQGKEKKAVLPVAFSWIFNRQIII AAA82160.1, FAD T34, TAD4 MALVFKSIGAHKTPPCTLNLASPALYHTRVT 35 dclta6-palmitoyl-acyl carrier protein MASTITHPPPLKDRKISSTRRVRTYPLAPEKA desaturase precursor [Thunbergia alata] EIFN SMHGW VEDTILPFLKP VEESWQPTDFE PDSTSDGFHEQVEELRKRTADLPDDYLVAL VGAMVTEEALPTYQTMLNTTDVIY DESGAS PVPWAVWTRAWTAEENRHGDIVNKYLYLS GRVDMKQIEKTIQYLIGSGMDPGADNNPYL AYIYTSYQERATAISFIGSLGRLARQKGEMK LAQICGTISADEKRHEAAYSKIVEKLFELDPE GTMLALAYMMKMKIVMPARLMHDGKDPD MFQHFSAVSQRLGIYTAKEYTDILEHMIAR WGVDKLTGLSGEGRRAQDYVCGLPMRFRK VEERAQAWAENISHVPFSWIFGRRV AAC49719.1, FAD T35 MQTLVFSTTNPSAWNTHGYTIKNPPKSTAVF 36 acyl-acyl carrier protein desaturase RRLPNVSAVASPPALRTLPPEKVQIFKSLES [Asclepias syriaca] WATQNVLPLLKPVEKSWQPTEFLPNPAQNF DDFTKEVHALRHRTSDLPDEFLVVLVGDMI TEEALPTYQSMINRLDGVKDETGACTSPWAI WTRAWTAEENRHGDLLKTYLYLSGRVDMT MIDKTIQYLLSSGMNTGTNRNPYFGFVYTSF QERATFVSHGNTARLAKERGDPVLAKICGSI ASDEKRHESAYSRIVEKLVEVDPSGAMLAIG EMMRMKITMPAHLMYDGQDPNLFAHFSAV

[0083]

[0084] AQRLGVYTAGDYADILEFLIQRWRLEKLEG LTDDARRAQEFVCGLAPRMRRLQEKADER AKKLRTTTGVKFSWIFNKELYL XP 002274652.1, FAD T36, FAD_Vv MA SMHRS VSREIKNTKKTS S SPRKVQ VTHS 37 stearoyl-[acyl-carrier-protein] 9- MPPHKIEIFKSMENWVEENVLIHLKPVEKC desaturase, chloroplastic [Vitis vinifera] WQPQDFLPHPASDGFHERVEELKERAKGIPD DYFVVLVGDMITEEALPTYQTLFNTTDGIRD ETGASPTSWATWTRAWTAEENRHGDLLNK YLYLSGRVDMKQIEKTIQYLIRAGMDFQTE NNPYLLF1YTSFQERATFISHGNTARLAKQH GDKSLAQICGIIASDEKRHETAYTKIVEKLFE IDPNGTVLAFADRMRKKITMPALLMYDGCD DDLFEHFSAVAQRLGVYTAKDYVDNLEFFV ERWNVEKLTGLS SEGRKAQDYVCGLAKRL RTLEERAQEKAKQAPTIPFSWIFDREVKL KAJ9701704.1, FAD T37 MNLNLNPLTFQPGKFPSLSLRPRSSNVSIAST 38 hypothetical protein PVL29 006888 ITSGSEEIKNTKKTSSSPRKVQVTHSMPPHKI [Vitis rotundifolia] KIFKSMENWVEKNVLIHLKPVEKCWQSQDF LPDPASDGFHERVKELQERAKGIPDDYFVVL VGHMITEEALPTYQTQINITDGIRDETGASPS SWATWTRAWTAEENRHGDLLNKYLYLSGR VDMKQIEKTIQYLIGAGMDAQNENSPYLGYI YTSFQERATFISHSNTARLAKEHGDKSLAQI LEKLFEIDPNGTVLAFADRMRKKITMPGLL MYDGCDDNLFEHFSAVAQRLGVYIAKDYV DNLEFFVERWNVEKLTGLSSEGREAQDYVC GLAQRLRKLEERAQERAKQAPTIPFSWILDR EVRL GKV29262.1, FAD T38 MALKFSSQSQKLPSSALPPMPSVRSPKFVMA 39 hypothetical protein SLEPl_g38200 STLHSASKEVDNLKKPFTPPREVHLQVTHSM [Rubroshorea leprosula] PPQKIEIFKSLEDWAENNILVHLKPVEKCWQ PQDFLPDPASDGFNEQVRELRERAKEIPDDY FVVLVGDMITEEALPTYQTMLNTLDGVKDE TGASPTSWAIWTRAWTAEENRHGDLLNKY LYLSGRVDMRQIEKTIQYLISSGMDPRTENS PYLGFIYTSFQERATFISHGNTARLAKEHGD LKLAQTCGSIAADEKRHETAYTKIVEKLFEID PDGTVLAFADMMRKKISMPAHLMYDGCDN NLFDHFSAVAQRLGVYTAKDYADILEFLVE RWKVKDLTGLSGEGRKAQDYVCGLPPRIRR LEERAQGRAKQAPRIPFSWIYDREVQL EOY04657.1, FAD T39 MHLISTKWALYLLSLSFAKCVFSLLWSKVK 40 Plant stearoyl-acyl-carrier-protein LLKQKPKENSQRGLKHQISREKNQNQANAE desaturase family protein [Theobroma QMALKLNPITSQSQKLPYFALPPMASLRSPK cacao] FFMASTLRSGSKEVENVKKPFMPPREVHVQ VTHSMPPQKIEIFKSLENWAEQNILVHLKPV EKCWQPQDFLPDPASDGFDEQVKELRERAK EIPDDYFVVLVGDMITEEALPTYQTMLNTLD GVLDETGASLTSWAIWTRAWTAEENRHGD

[0085]

[0086] LLNKYLYLSGRVDMRQIEKTIQYLIGSGMDP RTENSPYLGFIYTSFQERATFISHGNTARLAK EHGDFKLAQICGTIASDERRHETAYTKIVEK LFEIDPDGTVLAFADMMRKKISMPAHLMYD GRDDNLFDHFSAVAQRLGVYTAKDYADILE FLVERWKVKELTGLSADGRKAQDYVCGLPP RIRRLEERAQGRAKQAPSIPFSWIFDREVKL NP 197127.1, FAD T40 MSMALLLTSPAMKQKPAVITSPRRGSSPSRR 41 Plant stearoyl-acyl-carrier-protein LRVSCVTTNPARKKNETCNHFRPIKEVNNQL desaturase family protein | Arabidopsis THT1PQEKLE1FKSMENWAEQKLLPYLKPVE thaliana] D SWQPQDFLPAPENDDEFYDRVKEIRERTKE IPDDYFVVLVGDMITEEALPTYQTTLNTLDG VKDETGGSLSPWAVWIRAWTAEENRHGDL LNKYLYLTGRVDMRHVEKTIQYLIGSGMDS KFENNPYNGFIYTSFQERATFISHGNTARLAT TYGDVTLAKICGTIAADEKRHETAYTKIVEK LFEIDPDGSVQALASMMKKRITMPAHLMHD GRDNDLFDHYAAVAQRIGVYTAADY AGILE FLLRRWKVESLGLGLSGEGRRAQEYLCTLP QRIKRLEERANDRVKLVSKPSVSFSWVFGR DVKL

[0087] FabA [Escherichia coli] MVDKRESYTKEDLLASGRGELFGAKGPQLP 42 APNMLMMDRVVKMTETGGNFDKGYVEAE LDINPDLWFFGCHFIGDPVMPGCLGLDAMW QLVGFYLGWLGGEGKGRALGVGEVKFTGQ VLPTAKKVTYRIHFKRIVNRRLIMGLADGEV LVDGRLIYTASDLKVGLFQDTSAF

[0088] FabA2 (attenuated variant) [Escherichia MVDKRES YTKEDLLA SGRGELFGAKGPQLP 43 coli] APNMLMMDRVVKMTETGGNFDKGYVEAE LDINPDLWFFGCHFIGDPVMPGCLGLDAMW QLVGFYLGWLGGEDKGRALGVGEVKFTGQ VLPTAKKVTYRIHFKRIVNRRLIMGLADGEV

[0089]

[0090] LVDGRLIYTASDLKVGLFQDTSAF

[0091] In some embodiments, the recombinant bacterial cells are engineered to produce fatty acids and / or derivatives thereof. In some embodiments, the recombinant bacterial cells are engineered to produce monounsaturated fatty acids. In some embodiments, the recombinant bacterial cells are engineered to produce non-native fatty acids and / or derivatives thereof. In some embodiments, the recombinant bacterial cells are engineered to produce non-native unsaturated fatty acids and / or derivatives thereof. In some embodiments, the recombinant bacterial cells are engineered to produce at least one of a non-native fatty alcohol, a non-native fatty acetate, a non-native fatty acid, a non-native fatty ester, or a derivative of any of the foregoing.

[0092] In some embodiments, the non-native fatty alcohol, the non-native fatty acetate, the non-native fatty acid, the non-native fatty ester, and / or the derivative thereof comprises one or more double bonds at a non-native position. In some embodiments, the non-native position is selected from the group consisting of co-3, cn-5, co-6, co-8, co-9, co- 11, and col2.

[0093] In some embodiments, the recombinant bacterial cell produces at least one of a non-native fatty alcohol, a non-native fatty acetate, a non-native fatty acid, a non-native fatty ester, or a derivative of any of the foregoing at a level higher than a control cell (e.g., a parent strain) not containing the heterologous FAD or FAR.

[0094] In some embodiments, the non-native fatty alcohol, the non-native fatty acetate, the non-native fatty acid, the non-native fatty ester, or a derivative of any of the foregoing is a pheromone (e.g., an insect pheromone) or a precursor thereof. In some embodiments, the recombinant bacterial cells are engineered to produce at least one of a non-native unsaturated fatty alcohol, a non-native unsaturated fatty acetate, a non-native unsaturated fatty acid, a non-native unsaturated fatty ester, or a derivative of any of the foregoing. In some embodiments, the non-native unsaturated fatty alcohol, the non-native unsaturated fatty acetate, the non-native unsaturated fatty acid, the non-native unsaturated fatty ester, or a derivative of any of the foregoing is a pheromone or a precursor thereof.

[0095] In some embodiments, the disclosure provides a recombinant bacterial cell for producing at least one non-native fatty alcohol or derivative thereof. In some embodiments, the disclosure provides a recombinant bacterial cell for producing at least one non-native unsaturated fatty alcohol or derivative thereof. In some embodiments, the non-native unsaturated fatty alcohol or derivative thereof is enriched in Z isomers. In some embodiments, the non-native unsaturated fatty alcohol or derivative thereof is an co-5 unsaturated fatty alcohol or derivative thereof. In some embodiments, the non-native fatty alcohol or derivative thereof is selected from zll-C16:l-OH, z9-C14:l-OH, and zl3-C18:l-OH. In some embodiments, the co-5 unsaturated fatty alcohol or derivative thereof is selected from z 11 -C 16 : 1 -OH, z9-C 14 : 1 -OH, and z 13 -C 18 : 1 -OH.

[0096] In some embodiments, the recombinant bacterial cell is engineered to produce at least one of a non-native fatty alcohol, a non-native fatty acetate, a non-native fatty acid, a non-native fatty ester, or a derivative thereof at a level higher than a control cell (e.g., a parent strain) not containing the heterologous FAD or FAR. In some embodiments, the recombinant bacterial cell is engineered to produce at least one of a non-native unsaturated fatty alcohol, a non-native unsaturated fatty acetate, a non-native unsaturated fatty acid, a non-native unsaturated fatty ester, or a derivative thereof at a level higher than a control cell not containing the heterologous FAD or FAR. In some embodiments, the recombinant bacterial cell is engineered to produce at least one of a non-native fatty alcohol, a non-native fatty acetate, a non-native fatty acid, a non-native fatty ester, or a derivative thereof at a yield of from about 1 g / L to about 300 g / L (e g., from about 10 g / L to about 200 g / L, from about 20 g / L to about 200 g / L, from about 50 g / L to about 150 g / L, from about 75 g / L to about 100 g / L, from about 100 g / L to about 150 g / L, from about 150 g / L to about 200 g / L, from about 200 g / L to about 300 g / L, or from about 250 g / L to about 300 g / L), from about 300 g / L to about 500 g / L, from about 350 g / L to about 500 g / L, from about 400 g / L to about 500 g / L, from about 450 g / L to about 500 g / L, from about 10 g / L to about 400 g / L, from about 10 g / L to about 300 g / L, from about 10 g / L to about 200 g / L, from about 10 g / L to about 100 g / L, from about 10 g / L to about 90 g / L, from about 10 g / L to about 80 g / L, from about 10 g / L to about 70 g / L, from about 10 g / L to about 60 g / L, from about 10 g / L to about 50 g / L, from about 10 g / L to about 40 g / L, from about 10 g / L to about 30 g / L, from about 10 g / L to about 20 g / L, from about 5 g / L to about 10 g / L, from about 10 g / L to about 450 g / L, from about 20 g / L to about 400 g / L, from about 30 g / L to about 350 g / L, from about 40 g / L to about 300 g / L, from about 50 g / L to about 250 g / L, from about 60 g / L to about 200 g / L, from about 70 g / L to about 150 g / L, or from about 80 g / L to about 100 g / L- In some embodiments, the recombinant bacterial cell is engineered to produce a non-native fatty alcohol at a yield of from about 1 g / L to about 300 g / L (e.g., from about 10 g / L to about 200 g / L, from about 20 g / L to about 200 g / L, from about 50 g / L to about 150 g / L, from about 75 g / L to about 100 g / L, from about 100 g / L to about 150 g / L, from about 150 g / L to about 200 g / L, from about 200 g / L to about 300 g / L, or from about 250 g / L to about 300 g / L), from about 300 g / L to about 500 g / L, from about 350 g / L to about 500 g / L, from about 400 g / L to about 500 g / L, from about 450 g / L to about 500 g / L, from about 10 g / L to about 400 g / L, from about 10 g / L to about 300 g / L, from about 10 g / L to about 200 g / L, from about 10 g / L to about 100 g / L, from about 10 g / L to about 90 g / L, from about 10 g / L to about 80 g / L, from about 10 g / L to about 70 g / L, from about 10 g / L to about 60 g / L, from about 10 g / L to about 50 g / L, from about 10 g / L to about 40 g / L, from about 10 g / L to about 30 g / L, from about 10 g / L to about 20 g / L, from about 5 g / L to about 10 g / L, from about 10 g / L to about 450 g / L, from about 20 g / L to about 400 g / L, from about 30 g / L to about 350 g / L, from about 40 g / L to about 300 g / L, from about 50 g / L to about 250 g / L, from about 60 g / L to about 200 g / L, from about 70 g / L to about 150 g / L, or from about 80 g / L to about 100 g / L. In some embodiments, the recombinant bacterial cell is engineered to produce a non-native fatty acetate at a yield of from about 1 g / L to about 300 g / L (e.g., from about 10 g / L to about 200 g / L, from about 20 g / L to about 200 g / L, from about 50 g / L to about 150 g / L, from about 75 g / L to about 100 g / L, from about 100 g / L to about 150 g / L, from about 150 g / L to about 200 g / L, from about 200 g / L to about 300 g / L, or from about 250 g / L to about 300 g / L), from about 300 g / L to about 500 g / L, from about 350 g / L to about 500 g / L, from about 400 g / L to about 500 g / L, from about 450 g / L to about 500 g / L, from about 10 g / L to about 400 g / L, from about 10 g / L to about 300 g / L, from about 10 g / L to about 200 g / L, from about 10 g / L to about 100 g / L, from about 10 g / L to about 90 g / L, from about 10 g / L to about 80 g / L, from about 10 g / L to about 70 g / L, from about 10 g / L to about 60 g / L, from about 10 g / L to about 50 g / L, from about 10 g / L to about 40 g / L, from about 10 g / L to about 30 g / L, from about 10 g / L to about 20 g / L, from about 5 g / L to about 10 g / L, from about 10 g / L to about 450 g / L, from about 20 g / L to about 400 g / L, from about 30 g / L to about 350 g / L, from about 40 g / L to about 300 g / L, from about 50 g / L to about 250 g / L, from about 60 g / L to about 200 g / L, from about 70 g / L to about 150 g / L, or from about 80 g / L to about 100 g / L- In some embodiments, the recombinant bacterial cell is engineered to produce a non-native fatty acid at a yield of from about 1 g / L to about 300 g / L (e.g., from about 10 g / L to about 200 g / L, from about 20 g / L to about 200 g / L, from about 50 g / L to about 150 g / L, from about 75 g / L to about 100 g / L, from about 100 g / L to about 150 g / L, from about 150 g / L to about 200 g / L, from about 200 g / L to about 300 g / L, or from about 250 g / L to about 300 g / L), from about 300 g / L to about 500 g / L, from about 350 g / L to about 500 g / L, from about 400 g / L to about 500 g / L, from about 450 g / L to about 500 g / L, from about 10 g / L to about 400 g / L, from about 10 g / L to about 300 g / L, from about 10 g / L to about 200 g / L, from about 10 g / L to about 100 g / L, from about 10 g / L to about 90 g / L, from about 10 g / L to about 80 g / L, from about 10 g / L to about 70 g / L, from about 10 g / L to about 60 g / L, from about 10 g / L to about 50 g / L, from about 10 g / L to about 40 g / L, from about 10 g / L to about 30 g / L, from about 10 g / L to about 20 g / L, from about 5 g / L to about 10 g / L, from about 10 g / L to about 450 g / L, from about 20 g / L to about 400 g / L, from about 30 g / L to about 350 g / L, from about 40 g / L to about 300 g / L, from about 50 g / L to about 250 g / L, from about 60 g / L to about 200 g / L, from about 70 g / L to about 150 g / L, or from about 80 g / L to about 100 g / L.

[0097] In some embodiments, the recombinant bacterial cell is engineered to produce a non-native fatty ester at a yield of from about 1 g / L to about 300 g / L (e.g., from about 10 g / L to about 200 g / L, from about 20 g / L to about 200 g / L, from about 50 g / L to about 150 g / L, from about 75 g / L to about 100 g / L, from about 100 g / L to about 150 g / L, from about 150 g / L to about 200 g / L, from about 200 g / L to about 300 g / L, or from about 250 g / L to about 300 g / L), from about 300 g / L to about 500 g / L, from about 350 g / L to about 500 g / L, from about 400 g / L to about 500 g / L, from about 450 g / L to about 500 g / L, from about 10 g / L to about 400 g / L, from about 10 g / L to about 300 g / L, from about 10 g / L to about 200 g / L, from about 10 g / L to about 100 g / L, from about 10 g / L to about 90 g / L, from about 10 g / L to about 80 g / L, from about 10 g / L to about 70 g / L, from about 10 g / L to about 60 g / L, from about 10 g / L to about 50 g / L, from about 10 g / L to about 40 g / L, from about 10 g / L to about 30 g / L, from about 10 g / L to about 20 g / L, from about 5 g / L to about 10 g / L, from about 10 g / L to about 450 g / L, from about 20 g / L to about 400 g / L, from about 30 g / L to about 350 g / L, from about 40 g / L to about 300 g / L, from about 50 g / L to about 250 g / L, from about 60 g / L to about 200 g / L, from about 70 g / L to about 150 g / L, or from about 80 g / L to about 100 g / L.

[0098] In some embodiments, the species of the recombinant bacterial cell is Escherichia coli, Salmonella spp., Vibrio natriegens, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Xanthomonas axonopodis, Pseudomonas syringae, Xyella fastidiosa, Marinobacter aquaeolei, Yersinia pestis. or Vibrio cholerae. In some embodiments, the species of the recombinant bacterial cell is Escherichia coli .

[0099] In some embodiments, the recombinant bacterial cell comprises a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% identical to any one of SEQ ID NOs: 2 to 31 and 33.

[0100] In some embodiments, the recombinant bacterial cell comprises a FAR comprising an amino acid sequence at least 95% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 2 to 31 and 33.

[0101] In some embodiments, the recombinant bacterial cell comprises a FAR comprising an amino acid sequence selected from any one of SEQ ID NOs: 2 to 31 and 33.

[0102] In some embodiments, the recombinant bacterial cell comprises a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% identical to any one of SEQ ID NOs: 1 and 34 to 41, or is encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% identical to SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAD comprising an amino acid sequence at least 95% identical or at least 97% identical, or at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 1 and 34 to 41, or is encoded by a nucleotide sequence at least 95% identical to SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAD comprising the amino acid sequence of any one of SEQ ID NO: 1 and 34-41, or is encoded by SEQ ID NO: 32.

[0103] In some embodiments, the recombinant bacterial cell comprises a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% identical to any one of SEQ ID NOs: 2 to 31 and 33, and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% identical to any one of SEQ ID NOs: 1 and 34 to 41.

[0104] In some embodiments, the recombinant bacterial cell comprises a FAR comprising an amino acid sequence at least 95% identical or at least 97% identical, or at least 98% identical, or at least 99% identical to any one of SEQ ID NOs: 2 to 31 and 33, and a FAD comprising an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 1 and 34 to 41.

[0105] In some embodiments, the recombinant bacterial cell comprises a FAR comprising an amino acid sequence selected from any one of SEQ ID NOs: 2 to 31 and 33, and a FAD comprising the amino acid sequence of any one of SEQ ID NO: 1 and 34-41.

[0106] In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 2 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 3 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 4 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 5 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 6 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 7 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 8 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 9 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 10 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 11 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 12 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 13 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 14 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 15 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 16 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 17 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 18 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 19 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 20 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 21 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 22 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 23 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 24 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 25 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 26 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 27 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 28 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 29 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 30 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 31 or 33 and a FAD comprising the amino acid of any one of SEQ ID NO: 1 and 34-41.

[0107] In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 2 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 3 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 4 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 5 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 6 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 7 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 8 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 9 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 10 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 11 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 12 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 13 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 14 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 15 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 16 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 17 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 18 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 19 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 20 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 21 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 22 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 23 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 24 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 25 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 26 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 27 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 28 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 29 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 30 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the recombinant bacterial cell comprises a FAR comprising the amino acid of SEQ ID NO: 31 or 33 and a FAD encoded by SEQ ID NO: 32.

[0108] In some embodiments, the disclosure provides a recombinant bacterial cell comprising a codon-optimized nucleotide sequence encoding a FAD. In some embodiments, the disclosure provides a recombinant bacterial cell comprising a codon-optimized nucleotide sequence encoding a FAR. In some embodiments, the disclosure provides a recombinant bacterial cell comprising a codon-optimized nucleotide sequence encoding a FAD and a codon-optimized nucleotide sequence encoding a FAR. In some embodiments, the codon-optimized nucleotide sequence encoding the FAD is present in the genome of the recombinant bacterial cell. In some embodiments, the codon-optimized nucleotide sequence encoding the FAR is present in the genome of the recombinant bacterial cell.

[0109] In some embodiments, the codon-optimized nucleotide sequence encoding the FAD is present in the genome of the recombinant bacterial cell and the codon-optimized nucleotide sequence encoding the FAR is present in the genome of the recombinant bacterial cell.

[0110] In some embodiments, the codon-optimized nucleotide sequence encoding the FAD is encoded by a first plasmid present in the recombinant bacterial cell. In some embodiments, the codon-optimized nucleotide sequence encoding the FAR is encoded by a second plasmid present in the recombinant bacterial cell.

[0111] In some embodiments, the codon-optimized nucleotide sequence encoding the FAD is encoded by a first plasmid present in the recombinant bacterial cell and the codon-optimized nucleotide sequence encoding the FAR is encoded by a second plasmid present in the recombinant bacterial cell.

[0112] In some embodiments, the recombinant bacterial cell comprises a non-native fatty alcohol, a non-native fatty acetate, a non-native fatty acid, a non-native fatty ester, or a derivative of any of the foregoing that is a pheromone or a precursor thereof.

[0113] In some aspects, the disclosure provides a recombinant bacterial cell for producing at least one non-native unsaturated fatty alcohol or derivative thereof, wherein the non-native unsaturated fatty alcohol or derivative thereof selected from the group consisting of zl 1-C16: 1-OH, z9-C 14: 1-OH, and Z13-C18: 1-OH. Enzymes for fatty acyl production

[0114] In some embodiments, the present disclosure provides a recombinant bacterial cell comprising one or more enzymes to produce fatty acids and / or derivatives thereof. In some embodiments, the disclosure provides a recombinant cell comprising one or more heterologous enzymes including a fatty acyl-ACP desaturase (FAD), a fatty acyl reductase (FAR), a heterologous acyl-ACP thioesterase, a heterologous ferredoxin, a heterologous 3-hydroxyacyl-ACP-dehydratase (FabZ), a heterologous carboxylic acid reductase, a heterologous alcohol dehydrogenase, a heterologous phosphopantetheinyl transferase, a heterologous alcohol acetyl-CoA transferase, a heterologous co-hydroxylase, a heterologous alcohol oxidase / dehydrogenase, a heterologous fatty acid metabolism regulator protein (fadR), a heterologous aldehyde hydrogenase, and a heterologous P-ketoacyl-ACP synthase. In some aspects, one or more heterologous enzymes used in a recombinant bacterial cell provided here are engineered variants with tailored or improved activities and / or selectivity.

[0115] Fatty acyl-ACP desaturase (FAD)

[0116] In some embodiments, the recombinant bacterial cell comprises a fatty acyl-ACP desaturase (FAD). The terms “fatty acyl-ACP desaturase,” “FAD,” and “acyl-ACP desaturase” are used interchangeably herein. FADs are enzymes belonging to the oxidoreductase family of enzymes. FADs catalyze dehydrogenation reactions by converting a single bond between two carbon atoms (C-C) to a double bond (C=C) on the acyl chain of a fatty acid molecule in a stereospecific manner. The term “ACP” is used herein to refer to the acyl carrier proteins, known in the art to be cofactors of fatty acid biosynthesis machinery.

[0117] In some embodiments, the FAD is heterologous. In some embodiments, the recombinant bacterial cell comprises a nucleic acid molecule encoding a FAD enzyme disclosed herein. In some embodiments, the FAD, or a polynucleotide encoding the FAD, is produced by the recombinant bacterial cell. In some embodiments, the FAD belongs to the classification EC 1.14.19.2. In some embodiments, the FAD is a variant of myristyl-ACP desaturase. In some embodiments, the FAD is myristyl-ACP desaturase. In some embodiments, the FAD is cytosolic. In some embodiments, the FAD is membrane-associated. In some embodiments, the FAD is a partial protein with aN- and / or C -terminus truncation relative to the corresponding full-length protein. In some embodiments, the FAD comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41 (see Table 1). In some embodiments, the FAD comprises an amino acid sequence at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41 (see Table 1).

[0118] In some embodiments, the FAD is encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32 (see Table 1). In some embodiments, the FAD comprises an amino acid sequence at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32 (see Table 1).

[0119] Fatty acyl reductase (FAR)

[0120] In some embodiments, the recombinant bacterial cell comprises a fatty acyl reductase (FAR). The terms “fatty acyl reductase” and “FAR” are used interchangeably herein. FARs are alcohol-forming enzymes that catalyze the reduction of thioester bonds found in acyl-acyl carrier proteins (acyl-ACPs) and acyl-coenzyme A’s (acyl-CoAs). Some FARs perform one reduction step to produce aldehydes, while others can perform two sequential reduction steps to produce alcohols. In some aspect, a FAR is a fatty acyl-CoA reductase. In another aspect, a FAR is a fatty acyl-ACP reductase.

[0121] In some embodiments, the FAR is heterologous. In some embodiments, the recombinant bacterial cell comprises a nucleic acid molecule encoding a FAR enzyme disclosed herein. In some embodiments, the FAR, or a polynucleotide encoding the FAR, is produced by the recombinant bacterial cell. In some embodiments, the FAR is a FAR listed in Table 1. In some embodiments, the FAR is cytosolic. In some embodiments, the FAR is membrane-associated. In some embodiments, the FAR is a partial protein with a N- and / or C-terminus truncation relative to the corresponding full-length protein.

[0122] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 2 (see Table 1). In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 3 (see Table 1).

[0123] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 4 (see Table 1).

[0124] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 5 (see Table 1).

[0125] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 6 (see Table 1).

[0126] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 7 (see Table 1).

[0127] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 8 (see Table 1).

[0128] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 9 (see Table 1).

[0129] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 10 (see Table 1).

[0130] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 11 (see Table 1).

[0131] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 12 (see Table 1).

[0132] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 13 (see Table 1).

[0133] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 14 (see Table 1).

[0134] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 15 (see Table 1).

[0135] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 16 (see Table 1).

[0136] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 17 (see Table 1). In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 18 (see Table 1).

[0137] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 19 (see Table 1).

[0138] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 20 (see Table 1).

[0139] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 21 (see Table 1).

[0140] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 22 (see Table 1).

[0141] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 23 (see Table 1).

[0142] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 24 (see Table 1).

[0143] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 25 (see Table 1).

[0144] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 26 (see Table 1).

[0145] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 27 (see Table 1).

[0146] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 28 (see Table 1).

[0147] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 29 (see Table 1).

[0148] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: ID NO: 30 (see Table 1).

[0149] In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 31 or 33 (see Table 1).

[0150] FAD and FAR in recombinant bacterial cells

[0151] In some embodiments, the recombinant bacterial cell comprises a FAD disclosed herein and a FAR disclosed herein. In some embodiments, the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 2 to 31 and 33, and the FAD comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41.

[0152] In some embodiments, the FAR comprises an amino acid sequence at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 2 to 31 and 33, and the FAD comprises an amino acid sequence at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41.

[0153] In some embodiments, the FAR comprises an amino acid sequence selected from any one of SEQ ID NOs: 2 to 31 and 33, and the FAD comprises an amino acid sequence selected from SEQ ID NO: 1.

[0154] Acyl-ACP thioesterase

[0155] In some embodiments, the recombinant bacterial cell comprises an acyl-ACP thioesterase. The term “acyl-ACP thioesterase,” as used herein, refers to an enzyme that catalyzes the hydrolysis of thioester bonds to terminate fatty acyl extension. In some embodiments, the acyl-ACP thioesterase is native to the recombinant bacterial cell. In some embodiments, the acyl-ACP thioesterase is heterologous to the recombinant bacterial cell. In some embodiments, the acyl-ACP thioesterase is endogenous, wherein the enzyme, or a polynucleotide encoding the enzyme (e.g., RNA), is produced by the cell. In some embodiments, the acyl-ACP thioesterases disclosed herein may belong to EC 3.1.2.14. In some embodiments, the particular acyl-ACP thioesterase determines the chain length of the fatty acid molecule or derivative thereof. In some embodiments, the acyl-ACP thioesterase is a plant FatA-type thioesterase (which is specific for acyl-ACPs with C16 and C18 chain lengths), a plant FatB-type thioesterase (which is specific for acyl-ACPs with a C14 chain length), or a bacterial acyl-ACP thioesterase (for example, the thioesterase from C. thermocellum that hydrolyzes acyl-ACPs of various chain lengths, including z9-tetradecanol). Carboxylic acid reductase In some embodiments, the recombinant bacterial cell comprises a carboxylic acid reductase. As used herein, the term “carboxylic acid reductase” refers to an enzyme that converts a fatty acid to its corresponding aldehyde. In some embodiments, the carboxylic acid reductase is native to the recombinant bacterial cell. In some embodiments, the carboxylic acid reductase is heterologous to the recombinant bacterial cell. In some embodiments, the heterologous carboxylic acid reductase is endogenous, wherein the enzyme, or a polynucleotide encoding the enzyme (e.g., RNA), is produced by the cell. In some embodiments, the carboxylic acid reductase disclosed herein may belong to EC 1.2.1.30.

[0156] Ferrodoxin

[0157] In some embodiments, the recombinant bacterial cell comprises a ferrodoxin. As used herein, the term “ferredoxin” refers to an iron-sulfur protein that mediates the transfer of electrons in metabolic reactions. In some embodiments, the ferredoxin is native to the recombinant bacterial cell. In some embodiments, the ferredoxin is heterologous to the recombinant bacterial cell. In some embodiments, the ferredoxin is endogenous, wherein the enzyme, or a polynucleotide encoding the enzyme (e.g., RNA), is produced by the cell. In some embodiments ferredoxin refers to PetF, ferredoxin reductase (PetH), and flavodoxin / ferredoxin-NADP+reductase (Fpr). In some embodiments, the ferredoxin described herein may belong to EC 1.18.1.2.

[0158] Alcohol dehydrogenase

[0159] In some embodiments, the recombinant bacterial cell comprises an alcohol dehydrogenase. 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, the alcohol dehydrogenase is native to the recombinant bacterial cell. In some embodiments, the alcohol dehydrogenase is heterologous to the recombinant bacterial cell. 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. In some embodiments, the alcohol dehydrogenase is endogenous, wherein the enzyme, or a polynucleotide encoding the enzyme (e.g., RNA), is produced by the cell.

[0160] a>-hydroxylase

[0161] In some embodiments, the recombinant bacterial cell comprises a co-hydroxylase. As used herein, the term “co-hydroxylase” refers to an enzyme that hydrolyzes a fatty acid derivative in the co-position. In some embodiments, the co-hydroxylase is native to the recombinant bacterial cell. In some embodiments, the co-hydroxylase is heterologous to the recombinant bacterial cell. In some embodiments, the co-hydroxylase is endogenous, wherein the enzyme, or a polynucleotide encoding the enzyme (e.g., RNA) is produced by the cell. In some embodiments, the co-hydroxylase may belong to EC 1.14.15.3 or 1.14.14.80.

[0162] Alcohol acetyl-CoA transferase

[0163] In some embodiments, the recombinant bacterial cell comprises an alcohol acetyl-CoA transferase. As used herein, the term “alcohol acetyl-CoA transferase” refers to an enzyme that converts an alcohol to an acetyl ester. In some embodiments, the alcohol acetyl-CoA transferase is native to the recombinant bacterial cell. In some embodiments, the alcohol acetyl-CoA transferase is heterologous to the recombinant bacterial cell. In some embodiments, the alcohol acetyl-CoA transferase is endogenous, wherein the enzyme, or a polynucleotide encoding the enzyme (e.g., RNA), is produced by the cell. In some embodiments, the alcohol acetyl-CoA transferase belongs to EC 2.3.1.84.

[0164] Phosphopantetheinyl transferase

[0165] In some embodiments, the recombinant bacterial cell comprises a phosphopantetheinyl transferase. As used herein, the term “phosphopantetheinyl transferase” refers to an enzyme that transfers the 4'-phosphopantetheine moiety from CoA to the acyl carrier protein. In some embodiments, the phosphopantetheinyl transferase is native to the recombinant bacterial cell. In some embodiments, the phosphopantetheinyl transferase is heterologous to the recombinant bacterial cell. For example, the entD gene codes for a phosphopantetheinyl transferase.

[0166] Overexpression of native E. coli entD, a phosphopantetheinyl transferase, enables the activation of carboxylic acid reductase CarB from apo-CarB to holo-CarB, thereby allowing conversion of free fatty acids into fatty aldehydes, which can then be converted to fatty alcohols by a fatty aldehyde reductase. See, for example, U.S. Pat. No. 9,340,801, which is incorporated herein by reference in ts entirety. In some embodiments, the heterologous phosphopantetheinyl transferase is endogenous, wherein the enzyme, or a polynucleotide encoding the enzyme (e.g., RNA), is produced by the cell. In some embodiments, the phosphopantetheinyl transferase may belong to EC 2.7.8.7.

[0167] Aldehyde dehydrogenase

[0168] In some embodiments, the recombinant bacterial cell comprises an aldehyde dehydrogenase. As used herein, the term “aldehyde dehydrogenase” refers to enzymes that converts aldehydes to carboxylic acids. In some embodiments, the aldehyde dehydrogenase is native to the recombinant bacterial cell. In some embodiments, the aldehyde dehydrogenase is heterologous to the recombinant bacterial cell. In some embodiments, the aldehyde dehydrogenase is endogenous, wherein the enzyme, or a polynucleotide encoding the enzyme (e.g., RNA) is produced by the cell. In some embodiments, the aldehyde dehydrogenase may be described by the number EC 1.2.1.3. O-ketoacyl-A CP-synthase

[0169] In some embodiments, the recombinant bacterial cell comprises a O-ketoacyl-ACP-synthase. As used herein, the term “O-ketoacyl-ACP-synthase,” which includes “FabB” or “FabF,” refers to enzymes that catalyzes the condensation reaction to elongate the fatty acid chain. In some embodiments, the P-ketoacyl-ACP synthase is native to the recombinant bacterial cell. In some embodiments, the P-ketoacyl-ACP synthase is heterologous to the recombinant bacterial cell. In some embodiments, the heterologous -ketoacyl-ACP-synthase is endogenous, wherein the enzyme or a polynucleotide encoding the enzyme (e.g., RNA) is produced by the cell. In some embodiments, the P-ketoacyl-ACP-synthase may be described by the number EC 2.3.1.41.

[0170] Methods of producing fatty acids

[0171] In some aspects, the disclosure provides methods of producing fatty acid molecules. In some embodiments, the disclosure provides methods of producing fatty acids and derivatives thereof. In some embodiments, the disclosure provides methods of producing non-native fatty acids and derivatives thereof. In some embodiments, the disclosure provides methods of producing non-native unsaturated fatty acids and derivatives thereof.

[0172] In some embodiments, the methods disclosed herein produce fatty acids (e.g., unsaturated fatty alcohols) and derivatives thereof that are isomers. In some embodiments, the fatty acids and derivatives thereof produced by the methods disclosed herein are cis-, trans-, E-, Z-isomers, R-, S-enantiomers, diastereomers, (d)-isomers, (l)-isomers, or racemic mixtures thereof. In some embodiments, the fatty acids or derivatives thereof are produced in mixtures that are racemic. In some embodiments, the fatty acids or derivatives thereof produced using the methods disclosed herein are enriched for one isomer. In some embodiments, the fatty acids or derivatives thereof produced using the methods disclosed herein are enriched for a Z-isomer. In some embodiments, the fatty acids or derivatives thereof produced using the methods disclosed herein are enriched for an E-isomer. In some embodiments, an enriched composition comprises at least from about 51% to about 100%) (e.g., about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%) of a specific isomer (e.g., Z-isomer).

[0173] In some embodiments, the disclosure provides a method of producing a non-native unsaturated fatty alcohol or derivative thereof comprising culturing a plurality of recombinant bacterial cells herein disclosed and extracting the non-native unsaturated fatty alcohol or derivative thereof from the plurality of recombinant bacterial cells. In some embodiments, the non-native unsaturated fatty alcohol or derivative thereof is enriched in Z isomers. In some embodiments, the non-native unsaturated fatty alcohol or derivative thereof is an co-5 unsaturated fatty alcohol or derivative thereof. In some embodiments, the non-native unsaturated fatty alcohol or derivative thereof i s sei ected from z 11 -C 16 : 1 -OH, z9-C 14 : 1 -OH, and z 13 -C 18 : 1 -OH.

[0174] In embodiments, the method comprises chemically converting the non-native unsaturated fatty alcohol or derivative thereof from a Z-isomer form to an E-isomer form to achieve a desired Z / E-isomer ratio. In embodiments, the Z / E-isomer ratio is from about 5:95 to about 95:5. In embodiments, the Z / E-isomer ratio is at or around about 0:100, or about 5:95, or about 10:90, or about 15:85, or about 20:80, or about 25:75, or about 30:70, or about 35:65, or about 40:60, or about 45:55, or about 50:50, or about 55:45, or about 60:40, or about 65:35, or about 70:30, or about 75:25, or about 80:20, or about 85:15, or about 90:10, or about 95:5, or about 100:0. In embodiments, the Z / E-isomer ratio is within a range selected from 0:100 to 10:90, 10:90 to 20:80, 20:80 to 30:70, 30:70 to 40:60, 40:60 to 50:50, 50:50 to 60:40, 60:40 to 70:30, 70:30 to 80:20, 80:20 to 90: 10, and 90: 10 to 100:0. In embodiments, the Z / E-isomer ratio is within a range selected from 0: 100 to 20:80, 20:80 to 40:60, 40:60 to 60:40, 60:40 to 80:20, and 80:20 to 100:0. In embodiments, the Z / E-isomer ratio is within a range selected from 0: 100 to 20:80, 20:80 to 40:60, 40:60 to 60:40, 60:40 to 80:20, and 80:20 to 100:0. In embodiments, the Z / E-isomer ratio is within a range selected from 0:100 to 30:70, 30:70 to 60:40, and 60:40 to 100:0.

[0175] In some embodiments, the disclosure provides a method of producing a non-native unsaturated fatty acetate or derivative thereof comprising culturing a plurality of recombinant bacterial cells herein disclosed and extracting the non-native unsaturated fatty acetate or derivative thereof from the plurality of recombinant bacterial cells. In some embodiments, the non-native unsaturated fatty acetate or derivative thereof is selected from zl 1-hexadecen-l-yl acetate, z9-tetradecen-l-yl acetate, and zl3-octadecen-l-yl acetate.

[0176] In some embodiments, the disclosure provides a method of producing a non-native unsaturated fatty acid or derivative thereof comprising culturing a plurality of recombinant bacterial cells herein disclosed and extracting the non-native unsaturated fatty acid or derivative thereof from the plurality of recombinant bacterial cells. In some embodiments, the non-native unsaturated fatty acid or derivative thereof is selected from Zl 1 -hexadecenoic acid, Z9-tetradecenoic acid, and Z 13-octadecenoic acid.

[0177] In some embodiments, the disclosure provides a method of producing a non-native unsaturated fatty ester or derivative thereof comprising culturing a plurality of recombinant bacterial cells herein disclosed and extracting the non-native unsaturated fatty ester or derivative thereof from the plurality of recombinant bacterial cells. In some embodiments, the non-native unsaturated fatty ester or derivative thereof is selected from Zl 1 -hexadecenol, Zl 1 -hexadecenyl acetate, Zl 1-hexadecenal, Z9-tetradecenal, Z9-tetradecenyl acetate, Z9-tetradecenol, Z13-octadecenal, Z13-octadecen-l-yl acetate.

[0178] In some embodiments, the disclosure provides a method of producing a pheromone molecule or precursor thereof comprising culturing a plurality of recombinant bacterial cells herein disclosed and extracting the pheromone molecule or precursor thereof from the plurality of recombinant bacterial cells. In some embodiments, the pheromone molecule or precursor thereof is selected from Zl 1 -hexadecenol, Zll-hexadecenyl acetate, Zl 1-hexadecenal, Z9-tetradecenal, Z9-tetradecenyl acetate, Z9-tetradecenol, Z13-octadecenal, Z13-octadecen-l-yl acetate.

[0179] In some embodiments, the methods disclosed herein comprise producing a fatty acid molecule as herein disclosed by culturing a recombinant bacterial cell of the present disclosure. In some embodiments, the methods comprise culturing the recombinant bacterial cell under conditions in which the fatty acid molecule is produced and extracting the fatty acid molecule. The fatty acid molecule produced using these methods may be, for example, a non-native unsaturated fatty alcohol disclosed herein, a non-native unsaturated fatty acetate disclosed herein, a non-native unsaturated fatty acid disclosed herein, or a non-native unsaturated fatty ester disclosed herein. In some embodiments, the fatty acid molecule produced using these methods may be, for example, a pheromone. In some embodiments, the method comprises expressing in a recombinant bacterial cell at least one of a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 2 to 31 and 33, and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41.

[0180] In some embodiments, the method comprises expressing in a recombinant bacterial cell at least one of a FAR comprising an amino acid sequence selected from any one of SEQ ID NOs: 2 to 31 and 33, and a FAD comprising an amino acid sequence identical to any one of SEQ ID NOs: 1 and 34 to 41.

[0181] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 2 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 2 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0182] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 3 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 3 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0183] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 4 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 4 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0184] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 5 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 5 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0185] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 6 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 6 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0186] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 7 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 7 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0187] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 8 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 8 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0188] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 9 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 9 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0189] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 10 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 10 and a FAD comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 11 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 11 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0190] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 12 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 12 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0191] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 13 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 13 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0192] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 14 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 14 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0193] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 15 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 16 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0194] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 17 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 17 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0195] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 18 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 18 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0196] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 19 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 19 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0197] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 20 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 20 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0198] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 21 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 21 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0199] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 22 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 22 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0200] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 23 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 23 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0201] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 24 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 24 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0202] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 25 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 25 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0203] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 26 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 26 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0204] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 27 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 27 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0205] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 28 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 28 and a FAD comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 29 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 29 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0206] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 30 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 30 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0207] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 31 or 33 and a FAD comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 1 and 34 to 41. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 31 or 33 and a FAD comprising the amino acid sequence of SEQ ID NO: 1.

[0208] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 2 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 2 and a FAD encoded by SEQ ID NO: 32.

[0209] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 3 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 3 and a FAD encoded by SEQ ID NO: 32.

[0210] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 4 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 4 and a FAD encoded by SEQ ID NO: 32.

[0211] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 5 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 5 and a FAD encoded by SEQ ID NO: 32.

[0212] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 6 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 6 and a FAD encoded by SEQ ID NO: 32.

[0213] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 7 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 7 and a FAD encoded by SEQ ID NO: 32.

[0214] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 8 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 8 and a FAD encoded by SEQ ID NO: 32.

[0215] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 9 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 9 and a FAD encoded by SEQ ID NO: 32.

[0216] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 10 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 10 and a FAD encoded by SEQ ID NO: 32.

[0217] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 11 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 11 and a FAD encoded by SEQ ID NO: 32.

[0218] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 12 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 12 and a FAD encoded by SEQ ID NO: 32.

[0219] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 13 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 13 and a FAD encoded by SEQ ID NO: 32.

[0220] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 14 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 14 and a FAD encoded by SEQ ID NO: 32.

[0221] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 15 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 15 and a FAD encoded by SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 16 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 16 and a FAD encoded by SEQ ID NO: 32.

[0222] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 17 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 17 and a FAD encoded by SEQ ID NO: 32.

[0223] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 18 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 18 and a FAD encoded by SEQ ID NO: 32.

[0224] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 19 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 19 and a FAD encoded by SEQ ID NO: 32.

[0225] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 20 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 20 and a FAD encoded by SEQ ID NO: 32.

[0226] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 21 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 21 and a FAD encoded by SEQ ID NO: 32.

[0227] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 22 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 22 and a FAD encoded by SEQ ID NO: 32.

[0228] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 23 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 23 and a FAD encoded by SEQ ID NO: 32.

[0229] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 24 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 24 and a FAD encoded by SEQ ID NO: 32.

[0230] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 25 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 25 and a FAD encoded by SEQ ID NO: 32.

[0231] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 26 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 26 and a FAD encoded by SEQ ID NO: 32.

[0232] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 27 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 27 and a FAD encoded by SEQ ID NO: 32.

[0233] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 28 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 28 and a FAD encoded by SEQ ID NO: 32.

[0234] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 29 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 29 and a FAD encoded by SEQ ID NO: 32.

[0235] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 30 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 30 and a FAD encoded by SEQ ID NO: 32.

[0236] In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 31 or 33 and a FAD encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32. In some embodiments, the method comprises expressing in a recombinant bacterial cell a FAR comprising the amino acid sequence of SEQ ID NO: 31 or 33 and a FAD encoded by SEQ ID NO: 32.

[0237] Conditions under which the fatty acid molecule is produced will be known to those of ordinary skill in the art. In some embodiments, the recombinant bacterial cells can be grown under conditions that are selected, for example based upon cell culture parameters associated with optimized growth of specific bacterial cells used in an embodiment of the disclosure. In some embodiments, the recombinant bacterial cells are grown or cultured at a temperature of from 25-40° C (e.g., 32-37° C), for a period of time of at least 4 hours, or at least 8 hours, or at least 12 hours, or at least 24 hours, or at least 48 hours, or at least 72 hours. In embodiments, the period of time is from about 12 hours to about 72 hours, or from about 24 hours to about 48 hours. In some embodiments, the recombinant bacterial cells are grown at a pH from about 6.5 to about 8.5 (e.g., from about 6.5 to about 7.5). In some embodiments, the pH of the cell culture media can be about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, or about 8.1.

[0238] In some embodiments, the fatty acid molecule or derivative thereof is purified prior to its use. In some embodiments, the fatty acid molecule 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, or at least about 99% free) from other components with which they are associated.

[0239] In some embodiments, the fatty acid molecules or derivatives thereof are insoluble or highly insoluble in water. In such cases, the fatty acid molecules or derivatives thereof are in a separate phase from the environment in which the recombinant bacterial cells reside (e.g., cell culture media). In some embodiments, the fatty acid molecules or derivatives thereof are solid at room temperature. In another embodiment, the fatty acid molecules or derivatives thereof (e.g., fatty alcohols) are liquid.

[0240] In some embodiments, the fatty acid molecules are extracted using standard methods known in the art as described in U.S. Patent Publication No: 2024 / 0229086A1, herein incorporated by reference in its entirety. In some embodiments, fatty acid molecules are extracted by separating the recombinant bacterial cells from the cell culture medium using centrifugation and withdrawal of fatty acid-containing supernatant after centrifugation. Purification and quantification of fatty acids produced using the methods herein disclosed is conducted using standard methods and equipment known in the art, e.g., analysis of fatty acids using high-performance liquid chromatography (HPLC) and identification of fatty acids extracted using mass spectrometry (e.g., gas chromatographic mass spectrometry (GC-MS)).

[0241] Vectors

[0242] In some embodiments, the disclosure provides vectors comprising a nucleotide sequence that encodes one or more heterologous enzymes. In some embodiments, the disclosure provides a vector comprising at least one nucleotide sequence encoding one or more of the enzymes disclosed herein. In some embodiments, the disclosure provides a vector comprising a nucleotide sequence encoding a FAD. In some embodiments, the disclosure provides a vector comprising a nucleotide sequence encoding a FAR. In some embodiments, the disclosure provides a vector comprising a nucleotide sequence encoding a FAD and a FAR. In some embodiments, the disclosure provides a first vector comprising a first nucleotide sequence encoding a FAD. In some embodiments, the disclosure provides a second vector comprising a second nucleotide sequence encoding a FAR. In some embodiments, the disclosure provides a first vector comprising a first nucleotide sequence encoding a FAD and a second vector comprising a second nucleotide sequence encoding a FAR.

[0243] In some embodiments, the disclosure provides vectors comprising a nucleotide sequence encoding one or more heterologous enzymes, including one or more of a heterologous acyl-ACP desaturase, a heterologous acyl-ACP thioesterase, a heterologous ferredoxin, a heterologous 3-hydroxyacyl-ACP-dehydratase (FabZ), a heterologous carboxylic acid reductase, a heterologous alcohol dehydrogenase, a heterologous phosphopantetheinyl transferase, a heterologous alcohol acetyl-CoA transferase, a heterologous (o-hydroxylase, a heterologous alcohol oxidase / dehydrogenase, a heterologous fatty acid metabolism regulator protein (fadR), a heterologous aldehyde hydrogenase, and a heterologous -ketoacyl-ACP synthase.

[0244] In some embodiments, the vector comprises a nucleotide sequence encoding the one or more enzymes disclosed herein and one or more regulatory elements. In some embodiments, the one or more regulatory elements are operably linked to nucleotide sequence(s) encoding the one or more enzymes disclosed herein (e.g., FAD and / or FAR). In some embodiments, the vector comprises a nucleotide sequence encoding more than one of the enzymes recited above, the vector may comprise a single heterologous regulatory element that directs expression of multiple enzymes or multiple heterologous regulatory elements that independently direct expression of each of the enzymes encoded by the vector.

[0245] In some embodiments, a vector described herein comprises a promoter operably linked to the nucleotide sequence encoding the one or more enzyme(s) disclosed herein. In certain embodiments, the promoter is a developmentally-regulated promoter, an organelle-specific promoter, a tissue-specific promoter, an inducible promoter, a constitutive promoter, or a cellspecific promoter.

[0246] In some embodiments, a vector described herein comprises at least one sequence which may be (a) an expression control sequence (or regulatory element) operatively coupled to the nucleotide sequence encoding the one or more enzyme(s) disclosed herein; (b) a selection marker operatively coupled to the nucleotide sequence encoding the one or more enzyme(s) disclosed herein; (c) a marker sequence operatively coupled to the nucleotide sequence encoding the one or more enzyme(s) disclosed herein; (d) a purification moiety operatively coupled to the nucleotide sequence encoding the one or more enzyme(s) disclosed herein; (e) a secretion sequence operatively coupled to the nucleotide sequence encoding the one or more enzyme(s) disclosed herein; and (f) a targeting sequence operatively coupled to the nucleotide sequence encoding the one or more enzyme(s) disclosed herein.

[0247] In some embodiments, the vectors disclosed herein comprise a nucleotide sequence described herein in a form suitable for expression of the nucleotide sequence in a host cell. It will be appreciated by those skilled in the art that the design of the vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. The vectors described herein can be introduced into host cells to produce polypeptides, including fusion polypeptides, encoded by the nucleotide sequences as described herein.

[0248] Expression of genes encoding polypeptides in prokaryotes, for example, Escherichia coli, is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. In some embodiments, fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino- or carboxy-terminus of the recombinant polypeptide. In some embodiments, such fusion vectors serve one or more of the following three purposes: (1) to increase expression of the recombinant polypeptide; (2) to increase the solubility of the recombinant polypeptide; and (3) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. In some embodiments, a proteolytic cleavage site is introduced to a fusion expression vector at the junction of the fusion moiety and the recombinant polypeptide. This enables separation of the recombinant polypeptide from the fusion moiety after purification of the fusion polypeptide. Examples of such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, and enterokinase. Exemplary fusion expression vectors include pGEX (Pharmacia Biotech, Inc., Piscataway, NJ; Smith et al., Gene, 67: 31-40 (1988)), pMAL (New England Biolabs, Beverly, MA), and pRITS (Pharmacia Biotech, Inc., Piscataway, N.J.), which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant polypeptide.

[0249] Suitable expression systems for both prokaryotic and eukaryotic cells are well known in the art; see, e.g., Sambrook et al., “Molecular Cloning: A Laboratory Manual,” second edition, Cold Spring Harbor Laboratory (1989). Examples of inducible, non-fusion E. coli expression vectors include pTrc (Amann et al., Gene, 69: 301-315 (1988)) and PET lid (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA, pp. 60-89 (1990)). In certain embodiments, a nucleotide sequence of the disclosure is operably linked to a promoter derived from bacteriophage T5. Examples of vectors for expression in yeast include pYepSecl (Baldari et al., EMBO J, 6: 229-234 (1987)), pMFa (Kurjan et al., Cell, 30: 933-943 (1982)), pJRY88 (Schultz et al., Gene, 54: 113-123 (1987)), pYES2 (Invitrogen Corp., San Diego, CA), and picZ (Invitrogen Corp., San Diego, CA). Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include, for example, the pAc series (Smith et al., Mol. CellBiol., 3: 2156-2165 (1983)) and the pVL series (Lucklow et al., Virology, 170: 31-39 (1989)). Examples of mammalian expression vectors include pCDM8 (Seed, Nature, 329: 840 (1987)) and pMT2PC (Kaufman et al., EMBO J., 6: 187-195 (1987)).

[0250] In some embodiments, the vectors disclosed herein can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-preci pitati on, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in, for example, Sambrook et al. (supra).

[0251] For stable transformation of bacterial cells, it is known that, depending upon the expression vector and transformation technique used, only a small fraction of cells will take-up and replicate the expression vector. In order to identify and select these transformants, a gene that encodes a selectable marker (e.g., resistance to an antibiotic) can be introduced into the recombinant bacterial cells along with the gene of interest. In some embodiments, selectable markers include those that confer resistance to drugs such as, but not limited to, ampicillin, kanamycin, chloramphenicol, or tetracycline. Nucleic acids encoding a selectable marker can be introduced into a recombinant bacterial cell on the same vector as that encoding an enzyme disclosed herein or can be introduced on a separate vector. Cells stably transformed with the introduced nucleic acid can be identified by growth in the presence of an appropriate selection drug.

[0252] Extraction, derivatization, and analytical methods

[0253] In exemplary embodiments, non-native unsaturated fatty alcohol derivatives are identified by assaying for the production of non-native unsaturated fatty alcohol derivatives (e.g., an ro3- monounsaturted fatty alcohol, an co5 -monounsaturted fatty alcohol, co9-monounsaturted fatty alcohol, oil 1 -monounsaturted fatty alcohol, etc.) by a recombinant microbial host strain. In exemplary embodiments, small aliquots of media samples were used to assay the amount of fatty alcohols produced after the cells were removed by centrifugation. For example, 200 uL of the culture broth is mixed with 300 uL of butylacetate then vigorously mixed to extract fatty molecules to the organic phase. After phase separation by centrifugation, organic layers were collected and used for further sample preparation or direct analysis. In exemplary embodiments, Gas-Chromatography with Flame-Ionization Detection (GC-FID) is used to assay the non-native monounsaturated fatty acid derivative. GC-FID is known in the art (see e.g., Adlard, E. R.; Handley, Alan J. (2001). Gas chromatographic techniques and applications. London: Sheffield Academic). However, any appropriate method for quantitation and analysis may be used e.g., mass spectrometry (MS), Gas Chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography (TLC), etc. In exemplary embodiments, fatty alcohols were derivatized with N,O-Bis(trimethylsilyl)trifluoroacetamide with trimethylchlorosilane (BSTFA, Supelco 15238). For example, 60 uL of the butyl acetate extract was mixed with 30 uL BSTFA and placed in an oven at 50 degC for 60 min to produce trimethylsilane (TMS) derivatives of fatty alcohol species for analysis of different fattyl alcohol products.

[0254] The position of the double bond can be confirmed e.g., either by using authentic standards or by GC / MS of their dimethyl disulfide (DMDS) adducts (see e.g., Nichols et al. 1986, J. Microbiol. Methods 5: 49-55).

[0255] EXAMPLES

[0256] Example 1: Testing FAD T32

[0257] FAD T32 (SEQ ID NO: 1) was cloned in an E. coli plasmid under a strong T7 promoter with N-terminal chloroplast transit peptide removed. Two different codon optimized nucleotide sequences were used to construct CoOptl or CoOpt2 plasmids. Two separate E. coli strains were used to test the activity of FAD T32: K-12 MG1655 (K strain) or BL21.DE3 (B strain). In this example, K-strain had a genome integrated T7 RNA polymerase under the leaky inducible promoter. 50 mL of LB broth with carbenicillin (100 ug / mL) in a 250 mL baffled flask was inoculated with an aliquot of pre-grown overnight culture. E. coli cultures were maintained at 30 °C with shaking at 250 rpm, then IPTG was added to a final concentration of 1 mM for overnight incubation at 20 °C. Cell pellets after overnight incubation were used for whole cell fatty acid methyl ester (FAME) assay for fatty acid species profiling. Both Z9-C14:1-FAME which was the primary product of the heterologous desaturase FAD_T32 and Z11-C16: 1-FAME which was the secondary product of the same enzyme due to C2 elongation with native E. coli fatty acid biosynthesis were detected (FIG.

[0258] 3).

[0259] Example 2: Screening of fatty acyl reductases to produce C14-C16 fatty alcohols in E. coli Different fatty acyl reductases (see Table 2) were cloned in an E. coli plasmid under a strong T7 promoter or a leaky inducible LacUV5 promoter. A wild type strain with a genome integrated T7 RNA polymerase under the leaky inducible promoter were used to test the activity of fatty acyl reductases by measuring fatty alcohols produced during cultivation. 285 pL of minimal media (see Table 3 below for ingredients of minimal media) with added glucose in a 96 well plate with 100 pg / mL of carbenicillin was inoculated with 1 pL of overnight cultures in LB with carbenicillin. Cultures were grown overnight, then IPTG was added to the final concentration of 1 mM. IPTG-induced cultures were further incubated in a shaking incubator for 30 hrs at 30 °C. Cultures were extracted with butyl acetate then fatty alcohols extracted to the organic layer were analyzed by GC.

[0260] Table 2. Fatty alcohol produced with overexpressed fatty acyl reductases

[0261] clone CIO: C12: C12: C12: C14: C16: C16: C18: C18: Total

[0262] O-OH O-OH 2-OH 0- O-OH 1-OH O-OH 1-OH O-OH FA

[0263] FFA

[0264] control 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 B07_FAR_Mari 0.00 1.19 0.00 0.00 97.30 105.1 207.1 0.00 4.99 415.7 nobacter lipolyti 4 1 3

[0265] cus

[0266] B08_FAR_Mari 0.00 0.00 0.00 0.00 11.65 21.18 126.7 64.76 5.03 229.4 nobacter halotol 9 1

[0267] erans

[0268] BlO FAR Mari 0.00 0.00 0.00 0.00 7.49 9.94 88.65 50.97 5.34 162.3 nobacter alexan 9

[0269] drii

[0270] C07_FAR_Mari 0.00 0.00 0.00 0.00 54.01 60.00 208.2 0.00 5.21 327.4 nobacter excelle 3 5

[0271] ns

[0272] C09_FAR_Mari 0.00 1.03 0.00 0.00 57.95 48.39 123.5 0.00 4.73 235.6 nobacter nitratir 7 8

[0273]

[0274] educens clone CIO: C12: C12: C12: C14: C16: C16: C18: C18: Total O-OH O-OH 2-OH 0- O-OH 1-OH O-OH 1-OH O-OH FA FFA

[0275] D07_FAR_Mari 0.00 0.00 0.00 0.00 0.00 4.10 4.48 0.00 0.00 8.58 nobacter antarcti

[0276] cus

[0277] DIO FAR Gam 0.00 0.00 0.00 0.00 46.09 50.11 174.6 0.00 5.21 276.0 maproteobacteria 4 bacterium

[0278] E07_FAR_Strept 0.00 0.00 0.00 0.00 0.00 4.10 4.25 0.00 0.00 8.35 osporangiumjo

[0279] mthongense

[0280] ElO FAR Zhon 0.00 0.00 0.00 0.00 6.67 6.46 56.78 24.47 4.68 99.07 gsliania aliphati

[0281] civorans

[0282] F07_FAR_Halo 0.00 0.00 0.00 0.00 3.72 7.25 59.72 48.16 5.42 124.2 monas utaliensis 8 F09 FAR Bomb 0.00 0.00 0.00 0.00 0.00 0.00 4.26 0.00 0.00 4.26 yx mandarina

[0283] FlO FAR Bicyc 0.00 0.00 0.00 0.00 12.23 4.22 13.13 0.00 0.00 29.58 lus anynana

[0284] FAR006 0.00 0.00 0.00 0.00 9.52 15.45 56.65 16.65 0.00 98.26 FAR007 0.00 0.00 0.00 0.00 59.52 81.12 160.7 0.00 0.00 301.3

[0285] 2 6 FAR008 0.00 0.00 0.00 0.00 13.74 17.90 74.97 39.59 0.00 146.1

[0286] 9 FAR009 0.00 0.00 0.00 0.00 64.78 48.16 108.9 45.50 0.00 267.4

[0287] 9 4 FARO 10 0.00 0.00 0.00 0.00 17.58 7.07 32.77 14.86 0.00 72.28 FARO 11 0.00 2.50 0.00 0.00 128.6 132.1 139.7 0.00 0.00 403.0

[0288] 4 5 4 3 FARO 12 0.00 2.51 0.00 0.00 102.3 109.4 111.8 58.73 0.00 384.8

[0289] 0 9 3 5 FARO 13 0.00 0.00 0.00 0.00 0.00 4.11 4.26 0.00 0.00 8.37 FARO 14 0.00 1.34 0.00 0.00 101.4 104.6 133.7 56.62 4.90 402.7

[0290] 4 1 9 1 FARO 15 0.00 1.59 0.00 0.00 75.31 57.49 100.4 42.00 0.00 276.8

[0291] 3 3 FARO 16 0.00 5.33 0.00 0.00 143.9 94.97 114.9 58.52 4.88 422.5

[0292] 1 7 9 FARO 17 0.00 0.00 0.00 0.00 58.41 63.25 132.8 60.76 4.61 319.8

[0293] 3 7 FARO 18 0.00 0.00 0.00 0.00 3.17 4.68 19.42 10.66 0.00 37.92 FARO 19 0.00 0.00 0.00 0.00 86.78 166.5 154.0 0.00 4.66 412.0

[0294] 3 8 5 FAR020 0.00 0.00 0.00 0.00 50.77 45.39 125.3 57.51 0.00 278.9

[0295] 1 9 FAR021 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 FARref 0.00 0.00 0.00 0.00 82.38 92.51 134.9 0.00 0.00 309.8

[0296] 4 3 G07_FAR_Man 0.00 0.00 0.00 0.00 0.00 0.00 4.11 0.00 0.00 4.11

[0297]

[0298] duca sexta clone CIO: C12: C12: C12: C14: C16: C16: C18: C18: Total O-OH O-OH 2-OH 0- O-OH 1-OH O-OH 1-OH O-OH FA FFA

[0299] G08_FAR_Mani 0.00 0.00 0.00 0.00 17.95 5.43 30.18 10.66 0.00 64.23 ola hyperantus

[0300] H09 FAR Helic 0.00 7.14 0.00 0.00 21.63 5.02 0.00 0.00 0.00 33.79

[0301]

[0302] overpa assulta

[0303] Table 3. Minimal Media

[0304] Component Concentration Ammonium sulfate 10 g / L

[0305] MOPS buffer stock, 1 M 200 mL / L

[0306] Ferrous sulfate, heptahydrate 3 mg / L

[0307] Sodium chloride 700 mg / L

[0308] Potassium phosphate monobasic 5 g / L

[0309] Biotin 0.5 mg / L

[0310] Thiamine 1 mg / L

[0311] lOOOx Trace Metals 1 mL / L

[0312] Sterile IM MgSO4 solution 1 mM

[0313] Sterile 500 g / L glucose solution 40mL / L

[0314]

[0315] Table 4. Minimal Media (MIMM with low nitrogen for microtiter plate based culture)

[0316] Component (for 1 L) Amount unit 50% Glucose 80 mL lOx Salts No Nitrogen (MIMM) 100 mL 200g / L Ammonium Chloride 3.75 mL MOPS buffer, stock IM, pH 7.4 200 mL Citric Acid (IM) 10.4 mL MIMM Metals (lOOx) 10 mL MIMM Vitamins (lOOx) 10 mL

[0317] lOx Salts No Nitrogen (MIMM) (for 1 L)

[0318] Sodium chloride 7 g

[0319]

[0320] Potassium phosphate monobasic 50 _s _ lOOx MIMM Metals (for 1 L)

[0321] Ferrous sulfate, heptahydrate 1000 mg Manganese sulfate, monohydrate 300 mg

[0322] Teknova trace metals (T 1001 ) 100 mL Magnesium sulfate IM (T3003, Technova) 100 mL

[0323] lOOx MIMM Vitamins (for 250 mL)

[0324] Biotin 125 mg

[0325]

[0326] Thiamine 25 mg

[0327] Example 3: Production of non-native unsaturated fatty alcohols by co-expression of heterologous fatty acyl-ACP desaturase and fatty acyl reductase

[0328] Two FAD T32 clones (JKA0195 and JKA0196, see FIG. 4) with different codon optimized sequences were cloned under a strong T7 promoter in a plasmid with pBR322 origin of replication that confers the resistance to carbenicillin. Three fatty acyl reductases (FARref (SEQ ID NO: 2), FAR019 (SEQ ID NO: 20), and FAR020 (SEQ ID NO: 21), see FIG. 4) were individually cloned under the strong T7 promoter in a plasmid with pl5A origin of replication that confers the resistance to tetracycline. Plasmid controls with either no fatty acyl-ACP desaturase or no fatty acyl reductases were used as controls. K-12 MG1655 strain with an integrated T7 RNA polymerase under a leaky inducible promoter was used to transform various combinations of plasmids encoding a desaturase or a reductase control plasmids. Cultures were grown as described in Example 2 with the following changes. 100 ug / mL of carbenicillin and 15 ug / mL of tetracycline were used to maintain the culture. Cultures were incubated at 20 °C after IPTG was added to induce the production of the heterologous enzymes.

[0329] Example 4: Level of native and non-native fatty alcohols produced from E. coli strains were affected by the culture condition

[0330] 300 pL minimal media cultivation in a 96-well plate used while testing two different temperatures after IPTG was added to the media to induce the heterologous enzyme production. All other conditions were the same as described in Examples 2 and 3. Three fatty alcohol (FALC) sample injections were used to generate data with a single end timepoint. FIG. 4 shows that the present of a FAD (Z9-C14-ACP desaturase JKA0195 or JKA0196 noted in the figure) is necessary to produce Z11-C 16:1 -OH in this assay regardless of FAR enzyme.

[0331] Example 5: Production of fatty alcohols with FAD T32 and FAR022

[0332] Fatty alcohols Z9-C14:1-OH and Zll-C16:l-0H were produced in a small scale fermentation with an E. coli base strain with induced expression of FAD T32 and FAR022 (FIG. 7). The base strain with integrated constitutively expressed FAR022 and FAD_T32 is shown in FIG. 7. Constitutively expressed FAD T32 resulted in production of Z9-C14:1-OH (-30.5 mg / L) andZll-C16:l-OH (-85.9 mg / L) (FIG. 7). Distribution of production in a fermentation run with minimal media is shown in FIG. 7.

[0333] Example 6: Example of FAD and FAR combination capable of providing Z11-C16:1-OH enrichment.

[0334] Production of Z9-C16: 1-OH and Z11-C16: 1-OH were measured using E. coli strains expressing FAD T40 (CTRL FAD in STR5733, a control FAD enzyme) or FAD T36 (SEQ ID NO: 37, FAD vv Bball, STR5726) under an inducible promoter. Both strains also expressed MaqFAR (SEQ ID NO: 33) under another inducible promoter. Microtiter cultures were made in a low nitrogen and high glucose media to compare fatty alcohol productions between strains. STR5726 with FAD T36 showed a higher Z11-C16: 1-OH titer at 263 mg / L (compared to ~9.4mg / L detected in STR5733 with FAD T40 (FIG. 8)). This demonstrates successful implementation of a FAD in combination with a FAR to significantly increase the amount of Z11-C16: 1-OH by about 28-fold. This strain also shows higher Z9-C 16: 1-OH production (going from 673 mg / L to 898 mg / L)

[0335] Example 7: FAD T32 increases Z11-C16:1-OH production in fermentation.

[0336] An E. coli strain co-expressing FAD T32, PetF, and MaqFAR was subject to fermentation runs and exhibited increased level of Z 11 -C 16: 1 -OH relative to a control strain that expressed MaqFAR (upon IPTG induction) without FAD T32 (FIG. 9). This suggests that in the presence of FAR enzymes (e.g., MaqFAR), E. coli can make Z11-C16:1-OH without a FAD. However, an appropriate FAD can increase the Z 11 -C 16 : 1 -OH titer.

[0337] Example 8: FabA2 decreases Z9-C16:1-OH production and further increases Z11-C16:1-OH production in fermentation.

[0338] An E. coli strain co-expressing FAD T32, PetF, and MaqFAR was subject to fermentation runs and compared to a strain where the WT FabA [Seq ID No: 42] was replaced with FabA2 [Seq ID No: 43], and additionally to a strain where WT FabA was replaced with FabA2 and an additional copy of FADT32 and PetF. In all cases the originally co-expressed FAD T32, PetF, and MaqFAR were present. The FabA2 variant results in a decrease in Z9-C16:1-OH and increase in Z11 -Cl 6:1 -OH (FIG. 10). Addition of more FADT32 and PetF in the presence of FabA2 further decreases Z9-C16:1-OH and further increases inZll-C16:l-OH (FIG 11).

[0339] EQUIVALENTS

[0340] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS1. A recombinant bacterial cell comprising a fatty acyl-ACP desaturase (FAD) and a fatty acyl reductase (FAR), wherein at least one of the FAD and the FAR is heterologous.

2. The recombinant bacterial cell of claim 1, wherein the recombinant bacterial cell produces at least one of a non-native fatty alcohol, a non-native fatty acetate, a non-native fatty acid, a nonnative fatty ester, or a derivative of any of the foregoing.

3. The recombinant bacterial cell of claim 2, wherein the non-native fatty alcohol, the non-native fatty acetate, the non-native fatty acid, the non-native fatty ester, and / or the derivative thereof comprises one or more double bonds at a non-native position.

4. The recombinant bacterial cell of claim 3, wherein the non-native position is selected from the group consisting of co-3, cn-4, co-5, co-6, co-8, cn-9, co-10, co-11, and wl2.

5. The recombinant bacterial cell of claim 1, wherein the recombinant bacterial cell produces at least one of a non-native fatty alcohol, a non-native fatty acetate, a non-native fatty acid, a non-native fatty ester, or a derivative of any of the foregoing at a level higher than a control cell not containing the heterologous FAD or FAR.

6. The recombinant bacterial cell of claim 1, wherein at least one of the FAD and FAR is encoded in the genome of the recombinant bacterial cell.

7. The recombinant bacterial cell of claim 1, wherein the recombinant bacterial cell comprises a first plasmid and a second plasmid, wherein the first plasmid encodes the FAD and the second plasmid encodes the FAR.

8. The recombinant bacterial cell of any one of claims 1-7, wherein the FAD and / or FAR is cytosolic.

9. The recombinant bacterial cell of any one of claims 1-7, wherein the FAD and / or FAR is membrane-associated.

10. The recombinant bacterial cell of any one of claims 1-9, wherein the FAD and / or FAR is a partial protein with aN- and / or C-terminus truncation relative to the corresponding full-length protein.

11. The recombinant bacterial cell of any one of claims 1-10, wherein the recombinant bacterial cell further comprises a heterologous ferredoxin and / or a heterologous ferredoxin reductase.

12. The recombinant bacterial cell of any one of claims 1-10, wherein the recombinant bacterial cell further overexpresses a ferredoxin and / or a ferredoxin reductase.

13. The recombinant bacterial cell of claim 12, wherein the ferredoxin and / or the ferredoxin reductase are endogenous to the bacterial cell.

14. The recombinant bacterial cell of any one of claims 1-13, wherein the recombinant bacterial cell further comprises a modification suppressing, attenuating, or knocking out the expression or activity of an endogenous dual 3-hydroxy acyl-ACP dehydratase / isomerase.

15. The recombinant bacterial cell of any one of claims 1-14, wherein the recombinant bacterial cell further comprises a heterologous dual 3-hydroxy-acyl-ACP dehydratase / isomerase (fabZ / A), wherein optionally the isomerase activity of the heterologous fabZ / A is capable of providing a fatty acid or fatty acid derivative molecule having one or more double bonds in a non-native position selected from the group consisting of co-3, co-4, ®-5, co-6, co-8, co-9, co-10, co-11, and col2.

16. The recombinant bacterial cell of any one of claims 1-15, wherein the recombinant bacterial cell is of a bacterial species selected from Escherichia coh, Salmonella spp., Vibrio natriegens, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Xanthomonas axonopodis, Pseudomonas syringae, Xyellafastidiosa, an Marinobacter aquae olei.

17. The recombinant bacterial cell of any one of claims 1-16, wherein the recombinant bacterial cell is an Escherichia coli cell.

18. The recombinant bacterial cell of any one of claims 1-17, wherein the FAR comprises an Enzyme Commission (EC) number of 1.2.1.80 (long-chain acyl-[acyl-carrier-protein] reductase) or 1.2.1.84 (alcohol-forming fatty acyl-CoA reductase).

19. The recombinant bacterial cell of any one of claims 1 -Error! Reference source not found., wherein the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% identical to any one of SEQ ID NOs: 2 to 31 and 33.

20. The recombinant bacterial cell of claim 19, wherein the FAR comprises an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 2 to 31 and 33.

21. The recombinant bacterial cell of claim 19, wherein the FAR comprises an amino acid sequence selected from any one of SEQ ID NOs: 2 to 31 and 33.

22. The recombinant bacterial cell of any one of claims 1-21, wherein the FAD comprises an Enzyme Commission (EC) number of 1.14.19.11 (acyl-[acyl-carrier-protein] 4-desaturase), or 1.14.19.2 (stearoyl-[acyl-carrier-protein] 9-desaturase), or 1.14.19.26 (acyl-[acyl-carrier-protein] 6-desaturase), or 1.4.19 (myristoyl-[acyl-carrier-protein] 9-desaturase).

23. The recombinant bacterial cell of any one of claims 1-22, wherein the FAD comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% identical to any one of SEQ ID NOs: 1 and 34 to 41, or is encoded by a nucleotide sequence at least 60%, at least 70%, at least 80% or at least 90% identical to SEQ ID NO: 32.

24. The recombinant bacterial cell of claim 23, wherein the FAD comprises an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 1 and 34 to 41, or is encoded by a nucleotide sequence at least 95% identical to SEQ ID NO: 32.

25. The recombinant bacterial cell of claim 23, wherein the FAD comprises the amino acid sequence of SEQ ID NO: 1, or is encoded by SEQ ID NO: 32.

26. The recombinant bacterial cell of any one of claims 1-25, wherein:i) the FAR comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% identical to any one of SEQ ID NOs: 2 to 31 and 33, andii) the FAD comprises an amino acid sequence at least 60%, at least 70%, at least 80% or at least 90% identical to any one of SEQ ID NOs: 1 and 34 to 41.

27. The recombinant bacterial cell of claim 26, wherein:i) the FAR comprises an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 2 to 31 and 33, andii) the FAD comprises an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 1 and 34 to 41.

28. The recombinant bacterial cell of claim 26, wherein:i) the FAR comprises an amino acid sequence selected from any one of SEQ ID NOs: 2 to 31 and 33, andii) the FAD comprises the amino acid sequence of any one of SEQ ID NO: 1 and 34-41.

29. The recombinant bacterial cell of claim 26, wherein the FAR comprises a sequence at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identical to the amino acid of SEQ ID NO: 31 or 33; and wherein the FAD comprises a sequence at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identical to the amino acid of SEQ ID NO: 1 or 37.

30. The recombinant bacterial cell of claim 26, wherein the FAR comprises a sequence at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identical to the amino acid of SEQ ID NO: 31; and wherein the FAD comprises a sequence at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identical to the amino acid of SEQ ID NO: 1.

31. The recombinant bacterial cell of claim 26, wherein the FAR comprises a sequence at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identical to the amino acid of SEQ ID NO: 31 ; and wherein the FAD comprises a sequence at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identical to the amino acid of SEQ ID NO: 37.

32. The recombinant bacterial cell of claim 26, wherein the FAR comprises a sequence at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identical to the amino acid of SEQ ID NO: 33; and wherein the FAD comprises a sequence at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identical to the amino acid of SEQ ID NO: 1.

33. The recombinant bacterial cell of claim 26, wherein the FAR comprises a sequence at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identical to the amino acid of SEQ ID NO: 33; and wherein the FAD comprises a sequence at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identical to the amino acid of SEQ ID NO: 37.

34. The recombinant bacterial cell of any one of claims 1 to 33, comprising:i) a codon-optimized nucleotide sequence encoding the FAD, and / orii) a codon-optimized nucleotide sequence encoding the FAR.

35. The recombinant bacterial cell of any one of claims 2-34, wherein the non-native fatty alcohol, the non-native fatty acetate, the non-native fatty acid, the non-native fatty ester, or a derivative of any of the foregoing is a pheromone or a precursor thereof.

36. A method of producing a non-native unsaturated fatty alcohol or derivative thereof comprising culturing a recombinant bacterial cell of any one of claims 1-35, and extracting the non-native unsaturated fatty alcohol or derivative thereof from the recombinant bacterial cells or the culture.

37. The method of claim 36, wherein the non-native unsaturated fatty alcohol or derivative thereof is enriched in Z isomers.

38. The method of claim 36 or 37, further comprising: chemically converting the non-native unsaturated fatty alcohol or derivative thereof from a Z-isomer form to an E-isomer form to achieve a desired Z / E-isomer ratio.

39. The method of claim 38, wherein the Z / E-isomer ratio is from about 5:95 to about 95:5.

40. The method of any one of claims 36 to 39, wherein the non-native unsaturated fatty alcohol or derivative thereof is an OJ-5 unsaturated fatty alcohol or derivative thereof.

41. The method of claim 40, wherein the co-5 unsaturated fatty alcohol or derivative thereof is selected from z 11 -C 16 : 1 -OH, z9-C 14 : 1 -OH, and z 13 -C 18 : 1 -OH.

42. A method of producing a non-native unsaturated fatty acetate or derivative thereof comprising culturing recombinant bacterial cell of any one of claims 1 to 35, and extracting the non-native unsaturated fatty acetate or derivative thereof from the recombinant bacterial cells or the culture.

43. The method of claim 42, wherein the non-native unsaturated fatty acetate or derivative thereof is selected from zl 1-hexadecen-l-yl acetate, z9-tetradecen-l-yl acetate, and zl3-octadecen-1-yl acetate.

44. A method of producing a non-native unsaturated fatty acid or derivative thereof comprising culturing recombinant bacterial cells of any one of claims 1 to 35, and extracting the non-native unsaturated fatty acid or derivative thereof from the recombinant bacterial cells or the culture.

45. The method of claim 44, wherein the non-native unsaturated fatty acid or derivative thereof is selected from Zl 1 -hexadecenoic acid, Z9-tetradecenoic acid, and Z13-octadecenoic acid.

46. A method of producing a non-native unsaturated fatty ester or derivative thereof comprising culturing recombinant bacterial cells of any one of claims 1-35, and extracting the non-native unsaturated fatty ester or derivative thereof from the recombinant bacterial cells or the culture.

47. The method of claim 46, wherein the non-native unsaturated fatty ester or derivative thereof is selected from Zl 1 -hexadecenol, Zl 1 -hexadecenyl acetate, Zl 1 -hexadecenal, Z9-tetradecenal, Z9-tetradecenyl acetate, Z9-tetradecenol, Z13-octadecenal, and Z13-octadecen-l-yl acetate.

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