WO2023178193A1

WO2023178193A1 - Acyl-acp thioesterase variants and uses thereof

Info

Publication number: WO2023178193A1
Application number: PCT/US2023/064461
Authority: WO
Inventors: Andreas W. Schirmer; Emanuela Emilova POPOVA; Leland Ken WONG; Kevin Holden
Original assignee: Genomatica, Inc.
Priority date: 2022-03-16
Filing date: 2023-03-15
Publication date: 2023-09-21

Abstract

Variant acyl-ACP thioesterases and recombinant cells, microorganisms, or microbes, including proteobacteria and cyanobacteria, comprising the variant acyl-ACP thioesterases, are provided herein. The recombinant microbes produce monounsaturated free fatty acids and/or derivatives thereof, including palmitoleic acid and palmitoleic acid ethyl ester. Methods of producing monounsaturated free fatty acids or derivatives thereof are also provided, in addition to cell cultures and fatty acid derivative compositions produced by the recombinant microbes. The recombinant microbes may be used to produce fragrances, flavors, pheromones, pharmaceutical agents, nutraceuticals, nutritional or dietary supplements, or precursors thereof.

Description

ACYL-ACP THIOESTERASE VARIANTS AND USES THEREOF

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0001] This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted concurrently herewith as the sequence listing xml file entitled “ST26_SL_10_Mar_2023.xml”, file size 89 KiloBytes (KB), created on 10 March 2023. The aforementioned sequence listing is hereby incorporated by reference in its entirety.

FIELD

[0002] The disclosure relates to the field of specialty chemicals and methods for their production or preparation. The disclosure provides variant acyl-ACP thioesterases, recombinant cells or microbes or microorganisms that are engineered to express the variant acyl-ACP thioesterases, and methods of producing saturated and monounsaturated fatty acids and derivatives thereof, particularly monounsaturated fatty acids and derivatives thereof, such as medium-chain to long-chain monounsaturated fatty acids and derivatives thereof, using the described engineered or recombinant cells, microbes, or microorganisms (or cell cultures containing the recombinant cells, microbes, or microorganism). Compositions comprising monounsaturated fatty acids and derivatives thereof, such as long-chain monounsaturated fatty acids and derivatives thereof, for example, palmitoleic acid and/or derivatives thereof (e.g., palmitoleic acid ethyl ester), are also provided herein. Also provided are uses of the recombinant cells, microbes, microorganisms, or cell cultures for the preparation of the saturated and monounsaturated fatty acids and derivatives thereof and/or the compositions comprising the monounsaturated fatty acids and derivatives thereof, as well as uses for the saturated and monounsaturated fatty acids and derivatives thereof and/or the compositions, for example, in the preparation of nutritional, dietary, nutraceutical, pharmaceutical, pheromone, fuels (e.g., biofuels, biodiesel), flavor, and/or fragrance ingredients, products, and/or compositions, as well as precursors thereof.

BACKGROUND

[0003] Monounsaturated fatty acids and derivatives thereof are attractive and useful as the basis for many different products. For example, monounsaturated fatty acids and derivatives thereof are a component of good nutrition (see e.g., Nettleton J.A (2016) Ann Nutr Metab. 68:249-257) and they serve as the basis for production of numerous useful molecules and products, such as, e.g., nutritional or dietary supplements or ingredients, nutraceuticals, pheromones, flavors, and fragrances (see, e.g., International Patent Application Publication No. WO 2016/157719). Additionally, monounsaturated fatty acids are also ideal components for biodiesel since monounsaturated fatty acids improve fluidity at low temperatures and contribute to oxidative stability of the biodiesel product (see e.g., Yujin Cao et al. (2014) Biotechnol Biofuels , 7: 59).

[0004] Medium-chain to long-chain (e.g., C14-C20) fatty acids and derivatives thereof, including, but not limited to, fatty acids, fatty aldehydes, fatty alcohols, fatty alcohol acetate esters (FACE), fatty acid esters (including, but not limited to, for example, methyl, ethyl, and acetate esters), and bifunctional fatty acid derivatives (including, but not limited to, for example, omega-hydroxy (co-hydroxy) fatty acids, omega-hydroxy fatty acid esters, alpha, omega(a,co)- diols, a, co-diacids, and a,co-diesters), particularly monounsaturated medium-chain to long-chain fatty acids and derivatives thereof, are important components of nutritional or dietary supplements, nutraceuticals, pheromones, flavors, and fragrances. Such monounsaturated medium-chain to long-chain fatty acids and derivatives thereof include those with a double bond in the omega-3 (co-3), omega-5 (co-5), omega-7 (co-7), omega-9 (co-9), or omega-11 (co-11) positions.

[0005] Long-chain fatty acids, such as fatty acids with sixteen or eighteen carbon chain lengths, e.g., hexadecanoic acid (palmitic acid) (C16:0), A7-hexadecenoic acid (C16:l), A9- hexadecenoic acid (palmitoleic acid) (C16:l), octadecanoic acid (stearic acid) (C18:0), All- octadecenoic acid (vaccenic acid) (C18:l), or A9-octadecenoic acid (oleic acid) (C18:l), are important oleochemicals (chemical compounds derived from natural fats and oils). For example, palmitoleic acid, which is naturally found in a variety of animal fats, plant/vegetable oils, and marine oils, has several beneficial effects on health, including for example, beneficial effects on insulin sensitivity, cholesterol metabolism, inflammation, and cardiovascular health. However, naturally occurring oils do not comprise equal amounts of the various long-chain fatty acids. Whereas natural oils, such as, for example, palm, sunflower, canola, or rapeseed oil, have a high content of palmitic acid, stearic acid, and/or oleic acid, natural oils generally do not comprise a high content of palmitoleic acid or palmitoleic acid derivatives. For example, dietary sources of palmitoleic acid include salmon, cod liver oil, macadamia oil, and sea buckthorn oil, which only contain about 6%, 7%, 17%, and 32-42% or g/lOOg of total fatty acids, respectively, of palmitoleic acid (see, e.g., Frigolet et al. (2016) Adv. Nutr. 8( 1): 173S- 18 IS). The natural or dietary sources of palmitoleic acid are expensive and can have a limited shelf life, and typically contain high amounts, e.g., up to 80 wt%, of other components, including, for example, saturated fatty acids, which can be unhealthy; monounsaturated fatty acids, such as oleic acid; and a variety of polyunsaturated fatty acids (PUFAs). These additional components dilute and/or counteract the beneficial effects of palmitoleic acid and are difficult to separate from the palmitoleic acid. For example, macadamia oil and sea buckthorn oil contain roughly 60-65 weight (wt)% and 20-30 wt% of the monounsaturated A9-C18:l oleic acid, respectively. Thus, oils and compositions comprising a high content of long-chain fatty acids or derivatives thereof, particularly monounsaturated versions, such as palmitoleic acid and its derivatives, are needed.

[0006] Other monounsaturated fatty acids and derivatives thereof of interest include, for example, A7 -hexadecenoic acid ((Z7)-hexadecenoic acid) and its derivative, 16-hydroxy-7(Z)- hexadecenoic acid, which are precursors of natural ambrettolide, a musk/fragrance ingredient naturally found in ambrette seeds; and A9-hexadecenoic acid ((Z9)-hexadecenoic acid) and its derivative, 16-hydroxy-9(Z)-hexadecenoic acid, which are precursors of cis-isoambrettolide, a musk/fragrance ingredient. Additional monounsaturated fatty acid derivatives of interest include, for example, Z9-hexadecenol, Zl l -hexadecenol, Zl l-hexadecenal, Zl l-hexadecenyl acetate, Z9-tetradecenol, Z9-tetradecenal, Z9 -tetradecenyl acetate, Zl l -octadecenol, Zl l-octadecenal, Zl l -octadecenyl acetate, Z13-octadecenol, Z13-octadecenal, and Z13-octadecenyl acetate, which are fatty alcohol, fatty aldehyde, and fatty acid acetate derivatives of Z9-hexadecenoic acid (palmitoleic acid), Zl l-hexadecenoic acid, Z9-tetradecenoic acid, Zl l-octadecenoic acid, and Z13-octadecenoic acid, and are insect pheromones or precursors thereof that are important for the protection of crops. In some embodiments, the monounsaturated fatty acids and derivatives thereof include, for example, Z7-tetradecenoic acid, Z9-tetradecenoic acid, Z7- hexadecenoic acid, Z9-hexadecenoic acid (palmitoleic acid), Zl l-hexadecenoic acid, Z13- hexadecenoic acid, Z9-octadecenoic acid, Zl l-octadecenoic acid, Z13-octadecenoic acid, Z15- octadecenoic acid, Z7 -hexadecenoic acid ester, Z9-hexadecenoic acid ester (palmitoleic acid ester), Zl l-hexadecenoic acid ester, Z13 -hexadecenoic acid ester, Z9-octadecenoic acid ester, Zl l-octadecenoic acid ester, Z13-octadecenoic acid ester, Z15-octadecenoic acid ester, 16- hydroxy-7(Z)-hexadecenoic acid, 16-hydroxy-9(Z)-hexadecenoic acid, 16-hydroxy-7(Z)- hexadecenoic acid ester, 16-hydroxy-9(Z)-hexadecenoic acid ester, Z9-hexadecenol, Zl l- hexadecenol, Zl l-hexadecenal, Zl l-hexadecenyl acetate, Z9-tetradecenol, Z9-tetradecenal, Z9- tetradecenyl acetate, Zl l-octadecenol, Zl l-octadecenal, Zl l -octadecenyl acetate, Z13- octadecenol, Z13-octadecenal, or Z13-octadecenyl acetate, or a combination thereof; in particular embodiments, the esters are methyl esters or ethyl esters.

[0007] Most sources of monounsaturated fatty acids and derivatives thereof for nutrition and nutraceuticals, biodiesel, and for use as pheromone, flavor and/or fragrance molecules or ingredients, or precursors thereof, depend on plant or animal origins, and thus, can be limited in both quantity and quality. As discussed above, the typical sources of monounsaturated fatty acids and derivatives thereof are also expensive and contain large amounts of other components that are difficult to separate. In recent years, technology for the production of fatty acids and fatty acid derivatives has been successfully developed. See, for example, U.S. Patent Nos 9,951,322; 9,890,401; 9,879,239; 9,873,865; 9,758,769; 9,683,247; 9,683,219; 9,670,512; 9,598,706; 9,587,231; and 9,481,899 (each of which is incorporated herein by reference in its entirety). It would be greatly beneficial to be able to use such technology for the industrial scale production of monounsaturated fatty acids and derivatives thereof. In particular, it would be greatly beneficial to use recombinant microbes (or cells or microorganisms) to prepare such monounsaturated fatty acids and derivatives thereof.

[0008] Recombinant microbes (e.g., bacteria, such as proteobacteria and cyanobacteria; yeast; and algae) possess many advantages for the industrial production of fatty acid derivatives (see e.g., Front Microbiol. 2014; 5: 172). However, there are drawbacks when it comes to the production of monounsaturated fatty acids and derivatives thereof. Production of some co-7 (omega-7) monounsaturated fatty acids, such as palmitoleic acid and derivatives thereof, is difficult because microbes generally do not naturally produce such compounds at high yields. Thus, wild-type microbes are limited in the breadth of monounsaturated fatty acids and derivatives thereof that they can produce.

[0009] Consequently, new methods are needed for the production of medium to long-chain fatty acids, including monounsaturated fatty acids, such as palmitoleic acid, and derivatives thereof.

SUMMARY

[0010] Provided herein are variant acyl-ACP thioesterases (also referred to herein as variant acyl-ACP thioesterase polypeptides, variant thioesterases, thioesterase variants, or acyl-ACP thioesterase variants), comprising one or more amino acid modifications or mutations relative to the corresponding wild-type, unmodified, or reference acyl-ACP thioesterase. The variant acyl- ACP thioesterases can also be referred to as mutant, modified, or non-naturally occurring acyl- ACP thioesterases. The variant acyl-ACP thioesterases can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more, amino acid modifications or mutations, relative to a wild-type, unmodified, or reference thioesterase. In particular, provided herein are variant acyl-ACP thioesterases comprising one or more amino acid modifications or mutations (e.g, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more), relative to the full-length wild-type FatA acyl-ACP thioesterase from Arabidopsis lhaliana. whose amino acid sequence is set forth in SEQ ID NO:2, and/or relative to the mature wild-type FatA acyl-ACP thioesterase from Arabidopsis thaliana, whose amino acid sequence is set forth in SEQ ID NO:3, wherein the amino acid modifications or mutations can comprise amino acid substitutions (or replacements), amino acid deletions, and/or amino acid additions or insertions. In some embodiments, the variant acyl-ACP thioesterase comprises amino acid deletions and/or substitutions. For example, the variant acyl-ACP thioesterase can comprise a deletion in all or a portion of the plastid transit peptide (or transit peptide, or leader sequence), corresponding to amino acid residues 1-38, 2-38, 1-51, 2-51, 1-66, 2-66, 1-67, 2-67, 1-68, or 2- 68, of SEQ ID NO:2. In certain embodiments, the variant acyl-ACP thioesterase comprises a deletion of the plastid transit peptide corresponding to amino acid residues 2-51 of SEQ ID NO:2, and is thus a variant of the mature wild-type thioesterase polypeptide set forth in SEQ ID NO:3. In certain embodiments, the variant acyl-ACP thioesterase has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the wild-type, unmodified, or reference thioesterase sequence set forth in SEQ ID NO:2 or SEQ ID NO:3.

[0011] Also provided herein is a variant acyl-ACP thioesterase having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:22. In various embodiments, the variant acyl-ACP thioesterase comprises one or more amino acid substitutions (i.e., at least one amino acid substitution) relative to the sequence set forth in SEQ ID NO:3. In other embodiments, the variant acyl-ACP thioesterase comprises one or more amino acid substitutions (i.e., at least one amino acid substitution) relative to the sequence set forth in SEQ ID NO:2.

[0012] For example, provided herein is a variant acyl-ACP thioesterase, comprising one or more amino acid mutations (or substitutions) at one or more positions corresponding to positions 20, 40, 47, 50, 58, 82, 83, 147, 186, 292, 299, 303, or 305, or a combination thereof, with reference to SEQ ID NO:3, or at one or more positions corresponding to positions 70, 90, 97, 100, 108, 132, 133, 197, 236, 342, 349, 353, or 355, or a combination thereof, with reference to SEQ ID NO:2. For example, provided herein is a variant acyl-ACP thioesterase, comprising one or more amino acid mutations or substitutions, or comprising at least one amino acid mutation or substitution, corresponding to D20S, V40M, S47E, T50R, N58G, T82D, T83C, T83K, V147A, S186L, L292G, I299T, I299V, T3O3Q, or L305R, or a combination thereof, with reference to SEQ ID NOG, or comprising one or more amino acid mutations or substitutions, or comprising at least one amino acid mutation or substitution, corresponding to D70S, V90M, S97E, T100R, N108G, T132D, T133C, T133K, V197A, S236L, L342G, I349T, I349V, T353Q, or L355R, or a combination thereof, with reference to SEQ ID NO:2.

[0013] The variant acyl-ACP thioesterase can be a variant of a full-length or mature unmodified or wild-type or reference acyl-ACP thioesterase, such as the unmodified or wildtype or reference acyl-ACP thioesterase set forth in SEQ ID NO:2 or SEQ ID NOG. Such variant acyl-ACP thioesterase can comprise an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOG or SEQ ID NOG, and/or can comprise one or more amino acid mutations (such as substitutions) at one or more positions corresponding to positions 20, 40, 47, 50, 58, 82, 83, 147, 186, 292, 299, 303, and/or 305, with reference to SEQ ID NOG, or at one or more positions corresponding to positions 70, 90, 97, 100, 108, 132, 133, 197, 236, 342, 349, 353, and/or 355, with reference to SEQ ID NOG.

[0014] In some embodiments, the variant acyl-ACP thioesterase can include up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or more, amino acid modifications (or amino acid substitutions) compared to a reference, unmodified, or wild-type thioesterase or polypeptide sequence, such as, for example, the sequence set forth in SEQ ID NOG, or SEQ ID NOG, or SEQ ID NOG2. In some embodiments, the variant thioesterase includes up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications or amino acid substitutions. In some embodiments, the variant thioesterase includes up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, in addition to a deletion in all or a portion of the plastid-transit peptide (or transit peptide or leader sequence). In some embodiments, the variant thioesterase includes up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, in addition to a deletion of residues 1-38, 2-38, 1-51, 2-51, 1-66, 2-66, 1-67, 2-67, 1-68, or 2-68 of SEQ ID NOG, which correspond to all or a portion of the plastid-transit peptide.

[0015] In some embodiments, the variant acyl-ACP thioesterase comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21. In certain embodiments, the variant acyl-ACP thioesterase comprises the sequence set forth in any one of SEQ ID NOs:4-21. For example, the variant acyl-ACP thioesterase comprises the sequence set forth in SEQ ID NOG, or SEQ ID NOG, or SEQ ID NOG, or SEQ ID NOG, or SEQ ID NOG, or SEQ ID NOG, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, or SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO:18, or SEQ ID NO:19, or SEQ ID NO:20, or SEQ ID NO:21.

[0016] In some embodiments, the variant acyl-ACP thioesterase, when expressed in a cell, such as a recombinant cell or microbe, results in the production of a saturated and/or monounsaturated medium-chain to long-chain fatty acid or derivative thereof or a composition comprising a saturated and/or monounsaturated medium-chain to long-chain fatty acid or derivative thereof. In certain embodiments, the variant acyl-ACP thioesterase, when expressed in a cell, such as a recombinant cell or microbe, results in the production of a monounsaturated long-chain fatty acid or derivative thereof, such as a C 16:1 and/or Cl 8:1 fatty acid or derivative thereof. In some embodiments, the variant acyl-ACP thioesterase can exhibit one or more improved properties selected from increased thioesterase activity, increased specificity for a substrate, and/or increased selectivity for a substrate, in comparison to a corresponding wildtype, unmodified, or reference acyl-ACP thioesterase polypeptide. In some embodiments, the variant acyl-ACP thioesterase exhibits one or more improved properties selected from increased thioesterase activity, increased specificity for a substrate, and/or increased selectivity for a substrate, compared to SEQ ID NO:3. In other embodiments, the variant acyl-ACP thioesterase exhibits one or more improved properties selected from increased thioesterase activity, increased specificity for a substrate, and/or increased selectivity for a substrate, compared to SEQ ID NO:2. In particular embodiments, the increased thioesterase activity results in an increased amount, titer, yield, and/or productivity of a saturated or monounsaturated medium-chain to long-chain fatty acid or derivative thereof, and the increased specificity and/or selectivity is towards a saturated or monounsaturated medium-chain to long-chain acyl-ACP substrate. In some embodiments, the saturated or monounsaturated medium-chain to long-chain fatty acid or derivative thereof, or the saturated or monounsaturated medium-chain to long-chain acyl-ACP substrate is a C14-C20 fatty acid or derivative thereof, or a C14-C20 acyl-ACP substrate. In particular embodiments, the increased thioesterase activity results in an increased amount, titer, yield, and/or productivity of a monounsaturated long-chain fatty acid or derivative thereof, and the increased specificity and/or selectivity is towards a monounsaturated long-chain acyl-ACP substrate. In specific embodiments, the monounsaturated long-chain fatty acid, fatty acid derivative, or acyl-ACP, is a C16:l and/or a C18:l fatty acid, fatty acid derivative, or acyl-ACP. In some embodiments, the increased specificity and/or selectivity of the variant acyl-ACP thioesterase results in an increased amount, titer, yield, and/or productivity of a monounsaturated long-chain fatty acid or derivative thereof, such as a C 16:1 and/or Cl 8:1 fatty acid or derivative thereof, and/or the increased specificity and/or selectivity of the variant acyl-ACP thioesterase results in a fatty acid or fatty acid derivative composition with an increased percentage of monounsaturated long-chain (e.g., C16:l and/or C18:l) fatty acids or derivatives thereof, compared to the unmodified, wild-type, or reference thioesterase.

[0017] In some embodiments, the medium-chain to long-chain saturated or monounsaturated fatty acid or derivative thereof is a C14, C15, C16, C17, C18, C19, or C20 saturated or monounsaturated fatty acid or derivative thereof. In some embodiments, the fatty acid derivative is a saturated or monounsaturated free fatty acid, fatty alcohol, fatty diol (e.g., 1,3-fatty diol or alpha, omega(a,co)-diol), fatty aldehyde, fatty amine, fatty amide, fatty acid ester, fatty acid acetate ester, fatty alcohol acetate ester, hydroxy-fatty acid (including omega-hydroxylated and subterminally -hydroxylated versions), hydroxy-fatty acid ester (including omega-hydroxylated and subterminally-hydroxylated versions), alpha, omega(a,co)-fatty acid diester, a, co-diacid, co- carboxy fatty ester, alpha, omega (a,co)-free fatty acid, a derivative with a free fatty acid on one end and a fatty acid ester on the other end, a derivative with a free fatty acid on one end and an amine on the other end, and/or a derivative with a fatty acid ester on one end and an amine on the other end. The fatty acid ester derivative can be, for example, a fatty acid methyl ester (FAME), fatty acid ethyl ester (FAEE), or fatty acid propyl, isopropyl, butyl, or isobutyl ester, or can be a fatty acid acetate ester or a fatty alcohol acetate ester (FACE). In certain embodiments, the monounsaturated fatty acid or derivative thereof comprises a double bond at position 7 in the carbon chain between C7 and Cs from the reduced end of the fatty acid or derivative thereof (i.e., an omega-7 or co-7 double bond). In some embodiments, the monounsaturated fatty acid or derivative thereof comprises a double bond at the omega-3 (co-3), omega-5 (co-5), omega-7 (co- 7), omega-9 (co-9), or omega-11 (co-11) position. In some embodiments, the fatty acid or derivative thereof is a straight chain or a branched chain fatty acid or derivative thereof. In some embodiments, the fatty acid or derivative thereof is a Z7-tetradecenoic acid, Z9-tetradecenoic acid, Z7 -hexadecenoic acid, Z9-hexadecenoic acid (palmitoleic acid), Zl l-hexadecenoic acid, Z13-hexadecenoic acid, Z9-octadecenoic acid, Zl l -octadecenoic acid, Z13-octadecenoic acid, Z15-octadecenoic acid, Z7-hexadecenoic acid ester, Z9-hexadecenoic acid ester (palmitoleic acid ester), Zl l-hexadecenoic acid ester, Z13-hexadecenoic acid ester, Z9-octadecenoic acid ester, Zl l-octadecenoic acid ester, Z13-octadecenoic acid ester, Z15-octadecenoic acid ester, 16-hydroxy-7(Z)-hexadecenoic acid, 16-hydroxy-9(Z)-hexadecenoic acid, 16-hydroxy-7(Z)- hexadecenoic acid ester, 16-hydroxy-9(Z)-hexadecenoic acid ester, Z9-hexadecenol, Zl l- hexadecenol, Zl l-hexadecenal, Zl l -hexadecenyl acetate, Z9-tetradecenol, Z9-tetradecenal, Z9- tetradecenyl acetate, Zl l-octadecenol, Zl l-octadecenal, Zl l -octadecenyl acetate, Z13- octadecenol, Z13-octadecenal, and/or Z13-octadecenyl acetate. In some embodiments, the fatty acid or derivative thereof is palmitoleic acid, or palmitoleic acid ethyl ester, or a combination thereof.

[0018] Also provided herein are nucleic acid sequences or nucleic acid molecules encoding the variant acyl-ACP thioesterases provided herein, and vectors, such as plasmids (e.g., a bacterial plasmid), comprising the nucleic acid sequences encoding the variant acyl-ACP thioesterases. In embodiments, the nucleic acid molecules or nucleic acid sequences encoding the variant acyl-ACP thioesterases are exogenous nucleic acid sequences. In some embodiments, the nucleic acid molecule or nucleic acid sequence comprises a sequence of nucleotides set forth in any one of SEQ ID NOs: 32-49. The nucleic acid sequences set forth in SEQ ID NOs:32-49 encode the amino acid sequences corresponding to SEQ ID NOs:4-21. In some embodiments, the variant acyl-ACP thioesterases provided herein are encoded by the nucleic acid sequences set forth in SEQ ID NOs:32-49, and/or by degenerates of the sequences of SEQ ID NOs:32-49, whereby a degenerate nucleic acid sequence can perform the same function or yield the same output, i.e., encode the same polypeptide (e.g., a variant acyl-ACP thioesterase provided herein), as the original or reference nucleic acid sequence it is a degenerate of. In some embodiments, the nucleic acid sequence is operably linked to one or more heterologous regulatory elements. Also provided herein is an isolated cell or cell culture, or recombinant cells (also referred to herein as recombinant host cells), such as recombinant microorganisms or recombinant microbes, comprising the nucleic acid sequences or vectors.

[0019] Also provided herein is an isolated cell or cell culture, or recombinant cells (also referred to herein as recombinant host cells), such as recombinant microorganisms or recombinant microbes, comprising the variant acyl-ACP thioesterases (or variant acyl-ACP thioesterase polypeptides) described herein. For example, provided herein is an isolated cell or cell culture, or recombinant cells, recombinant microorganisms, or recombinant microbes, comprising a variant acyl-ACP thioesterase having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:22 as a heterologous or heterologously expressed acyl-ACP thioesterase. The heterologous or heterologously expressed acyl-ACP thioesterase may comprise one or more amino acid substitutions relative to SEQ ID NO:3, or relative to SEQ ID NO:2. Also provided herein are cell cultures comprising the recombinant (host) cell(s), microorganism(s), or microbe(s) that contain or express the variant acyl-ACP thioesterases provided herein or that contain the nucleic acid sequences encoding the variant acyl-ACP thioesterases provided herein.

[0020] In some embodiments, the isolated cell(s), or the recombinant cell(s), microorganism(s), or microbe(s), comprise(s) a variant acyl-ACP thioesterase polypeptide comprising one or more amino acid modifications or mutations (i.e., at least one amino acid mutation) relative to the full-length wild-type FatA acyl-ACP thioesterase from Arabidopsis thaliana, set forth in SEQ ID NO:2, and/or relative to the mature wild-type FatA acyl-ACP thioesterase from Arabidopsis thaliana, set forth in SEQ ID NO:3, wherein the amino acid modifications or mutations can comprise amino acid substitutions (or replacements), amino acid deletions, and/or amino acid additions or insertions. In certain embodiments, the recombinant cell(s), microorganism(s), or microbe(s) comprise(s) a variant acyl-ACP thioesterase having one or more amino acid modifications, such as amino acid substitutions, relative to SEQ ID NO:3, or comprise(s) a variant acyl-ACP thioesterase with at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:3.

[0021] Also provided herein is a recombinant cell, microbe, or microorganism, comprising the variant acyl-ACP thioesterase of any one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21, or a recombinant cell, microbe, or microorganism, comprising a variant acyl-ACP thioesterase with at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. In certain embodiments, the recombinant cell, microbe, or microorganism comprises a variant acyl-ACP thioesterase with at least one amino acid mutation (such as a substitution, insertion, deletion, etc.), such as for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, amino acid mutations (or substitutions), relative to the corresponding unmodified or wild-type thioesterase, or relative to the sequence set forth in SEQ ID NO:2, or SEQ ID NO:3. In certain embodiments, the variant acyl-ACP thioesterase comprises at least one amino acid mutation (e.g., substitution, insertion, deletion, etc.) at a position corresponding to position 20, 40, 47, 50, 58, 82, 83, 147, 186, 292, 299, 303, or 305, or a combination thereof, with reference to SEQ ID NO:3, or at a position corresponding to position 70, 90, 97, 100, 108, 132, 133, 197, 236, 342, 349, 353, or 355, or a combination thereof, with reference to SEQ ID NO:2. For example, provided herein is a recombinant cell, microbe, or microorganism, comprising a variant acyl-ACP thioesterase having one or more amino acid mutations or substitutions, or comprising at least one amino acid mutation or substitution, corresponding to D20S, V40M, S47E, T50R, N58G, T82D, T83C, T83K, V147A, S186L, L292G, I299T, I299V, T3O3Q, or L305R, or a combination thereof, with reference to SEQ ID NOG, or comprising one or more amino acid mutations or substitutions, or comprising at least one amino acid mutation or substitution, corresponding to D70S, V90M, S97E, T100R, N108G, T132D, T133C, T133K, V197A, S236L, L342G, I349T, I349V, T353Q, or L355R, or a combination thereof, with reference to SEQ ID NO:2.

[0022] In various embodiments, the recombinant cell, microorganism, or microbe further comprises one or more additional enzymes or polypeptides, such as one or more additional fatty acid biosynthesis or fatty acid derivative enzymes or polypeptides. In some embodiments, the recombinant cell, microorganism, or microbe further comprises a heterologous fatty acid biosynthesis enzyme and/or the fatty acid derivative enzyme. The recombinant cell, microorganism, or microbe can further comprise one or more of a heterologous P-ketoacyl-ACP synthase (e.g., FabB and/or FabF), a heterologous acyl-CoA synthetase, a heterologous acyl- CoA reductase, a heterologous fatty alcohol forming acyl-CoA reductase, a heterologous ester synthase, a heterologous omega-hydroxylase (or monooxygenase or oxygenase), a heterologous carboxylic acid reductase, a heterologous desaturase, a heterologous aldehyde dehydrogenase, and/or a heterologous alcohol dehydrogenase. In some embodiments, the recombinant cell, microorganism, or microbe, comprising a variant acyl-ACP thioesterase provided herein, further comprises one or more additional fatty acid biosynthesis or fatty acid derivative enzymes or polypeptides, including, but not limited to, for example, a P-ketoacyl-ACP synthase (I, II, or III), an acyl-CoA synthetase, an acyl-CoA reductase, a fatty alcohol forming acyl-CoA reductase, an ester synthase, an omega-hydroxylase (or monooxygenase or oxygenase), a carboxylic acid reductase, a desaturase, a transaminase (or aminotransferase), an amine dehydrogenase, a CoA- ligase/transferase, an alcohol-O-acetyl transferase, an aldehyde decarbonylase, an aldehyde oxidative deformylase, a decarboxylase, one or more subunits (e.g., AccA, AccB, AccC, and/or AccD) of an acetyl-CoA carboxylase (AccABCD), an OleA, an OleBCD, an OleABCD, an OleACD, an aldehyde dehydrogenase, or an alcohol dehydrogenase, or any combination thereof. The one or more additional fatty acid biosynthesis or fatty acid derivative enzymes or polypeptides can be heterologous, i.e., from a different species than the recombinant cell, microorganism, or microbe, or can be native, i.e., from the same species as the recombinant cell, microorganism, or microbe. The one or more heterologous fatty acid biosynthesis or fatty acid derivative enzymes or polypeptides can be expressed or can be overexpressed, or a combination thereof, in the recombinant cell, microbe, or microorganism. The heterologous enzyme or polypeptide is encoded by an exogenous nucleic acid sequence or gene, that can be expressed or overexpressed. The native enzyme or polypeptide can be encoded by an endogenous or exogenous gene or nucleic acid sequence, and can be expressed or overexpressed. Where a native enzyme or polypeptide is overexpressed, it is typically encoded by an exogenous gene or nucleic acid sequence. The native fatty acid biosynthesis or fatty acid derivative enzyme or polypeptide can be overexpressed, for example, by engineering the cell, microorganism or microbe to contain or express multiple copies of the encoding gene or nucleic acid, or by other techniques known in the art, such as by placing the encoding gene or nucleic acid under the control of a constitutive, inducible, or strong promoter, or by operably linking the encoding gene or nucleic acid sequence to another non-native regulatory element (e.g., 5’-UTR, ribosome binding site (RBS), or start codon, or a combination thereof). A variant of a native gene, for example, a variant with a non-native regulatory element or elements, can be considered as an exogenous gene, particularly where the whole gene, including the coding sequence and the regulatory elements, are introduced into the cell from outside the cell. The native fatty acid biosynthesis or fatty acid derivative enzyme or polypeptide can be exogenously expressed (or overexpressed), i.e., the nucleic acid sequence encoding the enzyme or polypeptide is introduced into the cell from the outside.

[0023] The recombinant (host) cell, microorganism, or microbe can be a recombinant bacterium (e.g., a y-proteobacterium, an a-proteobacterium, or a cyanobacterium), a recombinant yeast, or a recombinant algae. The recombinant cell, microorganism or microbe, or a cell culture comprising the recombinant cell, microorganism, or microbe, can produce one or more saturated or monounsaturated C14-C20 (e.g., C16, C16:l, C18, and/or C18:l) fatty acids or derivatives thereof, or a composition containing one or more saturated or monounsaturated fatty acids or derivatives thereof. In some embodiments, the monounsaturated fatty acid or derivative thereof comprises a double bond at position 7 in the carbon chain between C7 and Cs from the reduced end of the fatty acid or derivative thereof (i.e., an omega-7 or co-7 double bond). In some embodiments, the monounsaturated fatty acid or derivative thereof comprises a double bond at the omega-3 (co-3), omega-5 (co-5), omega-7 (co-7), omega-9 (co-9), or omega- 11 (co-11) position. In particular embodiments, the recombinant cell, microorganism or microbe, or a cell culture comprising the recombinant cell, microorganism, or microbe, produces one or more monounsaturated C14-C20, such as C16:l and/or C18:l, fatty acids or derivatives thereof, or produces a composition comprising one or more monounsaturated C14-C20, such as C16:l and/or Cl 8:1, fatty acids or derivatives thereof. In particular embodiments, the recombinant cell, microorganism, or microbe, produces a composition comprising a monounsaturated C16:l fatty acid or derivative thereof.

[0024] For example, the recombinant cell, microorganism or microbe, or a cell culture comprising the recombinant cell, microorganism, or microbe, can produce one or more monounsaturated free fatty acids, or a composition containing one or more monounsaturated free fatty acids, such as, but not limited to, for example, Z7-tetradecenoic acid, Z9-tetradecenoic acid, Z13-hexadecenoic acid, Zl l -hexadecenoic acid, Z9-hexadecenoic acid (palmitoleic acid), Z7-hexadecenoic acid, Z15-octadecenoic acid, Z13-octadecenoic acid, Zl l-octadecenoic acid, Z9-octadecenoic acid, Z7-octadecenoic acid, Z7-hexadecenoic acid ester, Z9-hexadecenoic acid ester (palmitoleic acid ester), Zl l -hexadecenoic acid ester, Z13-hexadecenoic acid ester, Z9- octadecenoic acid ester, Zl l-octadecenoic acid ester, Z13-octadecenoic acid ester, Z15- octadecenoic acid ester, 16-hydroxy-7(Z)-hexadecenoic acid, 16-hydroxy-9(Z)-hexadecenoic acid, 16-hydroxy-7(Z)-hexadecenoic acid ester, 16-hydroxy-9(Z)-hexadecenoic acid ester, Z9- hexadecenol, Zl l -hexadecenol, Zl l-hexadecenal, Zl l -hexadecenyl acetate, Z9-tetradecenol, Z9-tetradecenal, Z9-tetradecenyl acetate, Zl l-octadecenol, Zl l-octadecenal, Zl l-octadecenyl acetate, Z13-octadecenol, Z13-octadecenal, Z13-octadecenyl acetate and/or derivatives thereof. In some embodiments, the recombinant cell, microorganism or microbe, or a cell culture comprising the recombinant cell, microorganism, or microbe, can produce palmitoleic acid (Z9- hexadecenoic acid), or palmitoleic acid ethyl ester (Z9-hexadecenoic acid ethyl ester), or a combination thereof. Alternatively, or additionally, the recombinant cell, microorganism or microbe, or a cell culture comprising the recombinant cell, microorganism, or microbe, can produce one or more saturated fatty acids or derivatives thereof, such as one or more C14, C16, and/or C18 saturated fatty acids or derivatives thereof, including, for example, tetradecanoic acid, hexadecenoic acid, and/or octadecanoic acid, and/or derivatives thereof. Fatty acid derivatives include, for example, saturated and/or monounsaturated fatty alcohols, fatty diols (e.g., 1,3-fatty diols or alpha, omega-diols), fatty aldehydes, fatty amines, fatty amides, fatty acid esters, fatty alcohol acetate esters, hydroxy-fatty acids (including omega-hydroxylated and subterminally -hydroxylated versions), hydroxy-fatty acid esters (including omega-hydroxylated and subterminally-hydroxylated versions), alpha, omega (a,co)-fatty acid diesters, a, co-diacid, co- carboxy fatty ester, alpha, omega (a,co)-free fatty acids, derivatives with a free fatty acid on one end and a fatty acid ester on the other end (i.e., fatty acid half-esters or fatty diacid half-esters), derivatives with a free fatty acid on one end and an amine on the other end, and/or derivatives with a free fatty acid ester on one end and an amine on the other end. The fatty acid ester derivatives can be fatty acid methyl esters (FAMEs), fatty acid ethyl esters (FAEEs), or fatty acid acetate, propyl, isopropyl, butyl, or isobutyl esters. Any of the saturated or monounsaturated fatty acids or fatty acid derivatives described herein can be a straight chain fatty acid or derivative thereof, or can be a branched chain fatty acid or derivative thereof. Any of the monounsaturated free fatty acids or monounsaturated fatty acid derivatives described herein can contain a double bond at the omega-3 (co-3), omega-5 (co-5), omega-7 (co-7), omega-9 (co-9), or omega- 11 (co- 11 ) position. [0025] In certain embodiments, the recombinant cell, microorganism, or microbe, or the cell culture, produces or is capable of producing one or more monounsaturated FAMEs or FAEEs, or produces or is capable of producing a composition comprising one or more monounsaturated FAMEs or FAEEs. In certain embodiments, the monounsaturated FAME or FAEE is a C16:l or a C18:l monounsaturated FAME or FAEE, or a combination of C16:l and C18:l FAMEs or FAEEs. Any of the monounsaturated free fatty acids or monounsaturated fatty acid derivatives described herein can be a straight chain fatty acid or derivative thereof, or can be a branched chain fatty acid or derivative thereof. Any of the monounsaturated free fatty acids or monounsaturated fatty acid derivatives described herein can contain a double bond at the omega- 3 (co-3), omega-5 (co-5), omega-7 (co-7), omega-9 (co-9), or omega-11 (co-11) position.

[0026] Also provided herein are methods for producing one or more saturated and/or monounsaturated fatty acids or derivatives thereof, or for producing a composition comprising one or more saturated and/or monounsaturated fatty acids or derivatives thereof, said methods comprising culturing any of the recombinant cells, microbes, microorganisms, or cell cultures, provided herein. For example, described herein are methods for producing one or more saturated and/or monounsaturated free fatty acids or derivatives thereof, or for producing a composition comprising one or more saturated and/or monounsaturated fatty acids or derivatives thereof, said methods comprising culturing a recombinant cell, microorganism, or microbe, comprising a heterologous or a variant acyl-ACP thioesterase having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:22, or comprising a variant acyl-ACP thioesterase with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more) amino acid modifications or mutations (such as substitutions) relative to SEQ ID NO:2 or SEQ ID NO:3. For example, provided herein are methods for producing one or more saturated or monounsaturated free fatty acids or derivatives thereof, or a composition comprising the same, said methods comprising culturing a recombinant cell, microorganism, or microbe comprising a variant acyl-ACP thioesterase having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2 or SEQ ID NO:3, or to any one of SEQ ID NOs:4-21. Also provided herein are methods for producing one or more saturated or monounsaturated free fatty acids or derivatives thereof, or a composition comprising the same, said methods comprising culturing a recombinant cell, microorganism, or microbe comprising a variant acyl-ACP thioesterase of SEQ ID NO:4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21. [0027] Also provided herein are methods for producing one or more saturated and/or monounsaturated free fatty acids or derivatives thereof, or a composition comprising one or more saturated and/or monounsaturated free fatty acids or derivatives thereof, whereby the methods comprise culturing or growing a cell culture comprising any of the recombinant cells, microorganisms, or microbes provided herein.

[0028] Also provided herein are nucleotide sequences encoding the variant acyl-ACP thioesterase polypeptides described herein, including nucleotide (or nucleic acid) sequences operably linked to one or more regulatory elements. For example, provided herein is an exogenous nucleic acid sequence encoding the variant acyl-ACP thioesterase of any one of SEQ ID NOs:4-21, or encoding a variant acyl-ACP thioesterase variant with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. Also provided herein are vectors comprising the nucleotide sequences.

[0029] Also disclosed herein are nucleotide sequences encoding a heterologous or variant acyl-ACP thioesterase, which are operably linked to one or more heterologous regulatory elements. The nucleotide sequence can be in a vector.

[0030] Also provided herein are uses of the variant acyl-ACP thioesterases, the nucleic acids encoding the variants, the vectors comprising the nucleic acids, the recombinant cells, microbes, or microorganisms, and the cell cultures, described herein, for the preparation or production of the saturated and/or monounsaturated fatty acids and derivatives thereof described herein, or for the preparation or production of compositions comprising the saturated and/or monounsaturated fatty acids and derivatives thereof. For example, provided are uses of the variant acyl-ACP thioesterases, nucleic acid sequences, vectors, recombinant cells, microbes, or microorganisms, and/or cell cultures, for the production of one or more monounsaturated C14-C20 fatty acids or derivatives thereof, such as one or more C14: l, C16:l, or C18:l fatty acids or derivatives thereof, or compositions comprising the same. In particular embodiments, provided are uses of the variants, nucleic acids, vectors, recombinant cells, microbes, or microorganisms, and/or cell cultures, for the preparation of palmitoleic acid ethyl ester, and/or a composition comprising palmitoleic acid ethyl ester. Also provided herein are uses of the variants, nucleic acids, vectors, recombinant cells, microbes, or microorganisms, and/or cell cultures, for the preparation of saturated C16 (C16:0) and/or monounsaturated C16 (C16:l) fatty acids and derivatives thereof, such as, for example, hexadecanoic acid and its derivative 16-hydroxy-hexadecanoic acid; and/or A7-hexadecenoic acid and/or its derivatives 16-hydroxy-7(Z)-hexadecenoic acid and/or 16-hydroxy-(7Z)-hexadecenoic acid methyl or ethyl ester; and/or A9-hexadecenoic acid and/or its derivatives 16-hydroxy-9(Z)-hexadecenoic acid, and/or 16-hydroxy-9(Z)-hexadecenoic acid methyl or ethyl ester, and/or A9-hexadecenol; and/or All-hexadecenoic acid and/or its derivatives Al l-hexadecenol (e.g., Zl l-hexadecenol), Zl l-hexadecenal, Zl l-hexadecenyl- acetate; and/or A9-tetradecenoic acid and/or its derivatives Z9-tetradecenol, Z9-tetradecenal, and/or Z9-tetradecenyl acetate; and/or A13-octadecenoic acid and/or its derivatives Z13- octadecenol, Z13-octadecenal, and/or Z13-octadecenyl acetate; and/or All-octadecenoic acid and/or its derivatives Zl l -octadecenol, Zl l-octadecenal, and/or Zl l-octadecenyl acetate; as well as compositions comprising the same. Also provided herein are uses of the variant acyl- ACP thioesterases, nucleic acid sequences, vectors, recombinant cells, microbes, or microorganisms, and/or cell cultures, for the production of one or more of the compositions described below and elsewhere herein.

[0031] Also provided herein are compositions comprising the saturated and/or monounsaturated fatty acids and derivatives thereof. For example, provided herein are compositions comprising one or more monounsaturated C14-C20 fatty acids or derivatives thereof, such as one or more C14:l, C16:l, or C18:l fatty acids or derivatives thereof. For example, provided herein is a composition comprising at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, by weight of the total composition, of a monounsaturated C16:l fatty acid or derivative thereof. Also provided herein is a composition comprising at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, by weight of the total composition, of a monounsaturated C16:l fatty acid or derivative thereof, and 20% or less, such as 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less, by weight of the total composition, of a saturated fatty acid or derivative thereof, such as a saturated C14 (C14:0), C16 (C16:0), and/or C18 (C18:0) fatty acid or derivative thereof. Also provided herein is a composition comprising: (i) at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, by weight of the total composition, of a monounsaturated C16:l fatty acid or derivative thereof; and/or (ii) 20% or less, such as 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, by weight of the total composition, of a saturated fatty acid or derivative thereof, such as a saturated C14 (C14:0), C16 (C16:0), and/or C18 (C18:0) fatty acid or derivative thereof; and/or (iii) one or more co-5 (omega-5) fatty acids or derivatives thereof (such as, for example, a A11-C16:! fatty acid and/or a A13-C18:! fatty acid, or derivatives thereof); and/or (iv) 20% or less (such as 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less), by weight of the total composition, of a monounsaturated C18 (C18:l) fatty acid or derivative thereof (such as oleic acid or a derivative thereof); and/or (v) 20% or less (such as 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less), by weight of the total composition, of one or more polyunsaturated fatty acids (PUFAs). For example, provided herein is a composition comprising at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or more, by weight of the total composition, of palmitoleic acid and/or a palmitoleic acid derivative (e.g., an ester, such as palmitoleic acid ethyl ester (also known as ethyl palmitoleate)). In other embodiments, provided herein is a composition comprising: (i) at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or more, by weight of the total composition, of palmitoleic acid ester, such as palmitoleic acid ethyl ester (also known as ethyl palmitoleate); and/or (ii) 20% or less, such as 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, by weight of the total composition, of palmitic acid and/or palmitic acid ethyl ester; and/or (iii) one or more co-5 (omega-5) fatty acids or derivatives thereof (such as omega-5 fatty acid ethyl esters); and/or (iv) 20% or less (such as 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less), by weight of the total composition, of oleic acid or a derivative thereof (e.g., oleic acid ethyl ester); and/or (v) 20% or less (such as 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less), by weight of the total composition, of one or more polyunsaturated fatty acids (PUFAs) or derivatives thereof (e.g., fatty ethyl ester derivatives of PUFAs). In other embodiments, provided herein is a composition comprising: (i) at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, by weight of the total composition, of palmitoleic acid and/or palmitoleic acid ester, such as palmitoleic acid ethyl ester (also known as ethyl palmitoleate); and/or (ii) one or more co-5 (omega-5) fatty acids or derivatives thereof (e.g., fatty acid ethyl ester derivatives thereof); and/or (iii) 20% or less (such as 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less), by weight of the total composition, of a monounsaturated C18 (C18:l) fatty acid or derivative thereof (such as oleic acid or a derivative thereof). In particular embodiments, the composition comprises (i) at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, by weight of the total composition, of palmitoleic acid ethyl ester (also known as ethyl palmitoleate); and/or (ii) one or more co-5 (omega-5) fatty acids or derivatives thereof. In some embodiments, provided is a composition (or a fatty acid ethyl ester composition, or a fatty acid derivative composition), comprising palmitoleic acid ethyl ester and about 0%, or less than about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%, by weight of the composition, of one or more PUFAs and/or derivatives (e.g., ethyl esters) thereof. In some embodiments, provided is a composition (or a fatty acid ethyl ester composition, or a fatty acid derivative composition), comprising palmitoleic acid ethyl ester; less than 10% (such as less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%), or 0% (i.e., no detectable amount), by weight of the composition, of oleic acid and/or oleic acid ethyl ester; and less than 5% (such as less than 4%, less than 3%, less than 2%, or less than 1%), or 0% (i.e., no detectable amount), by weight of the composition, of one or more PUFAs and/or derivatives thereof (e.g., ethyl esters of PUFAs). Also disclosed herein are compositions (or fatty acid ethyl ester compositions or fatty acid derivative compositions), comprising palmitoleic acid ethyl ester; one or more co-5 (omega-5) fatty acids or derivatives thereof (e.g., ethyl ester derivatives thereof); and less than 10 wt% (e.g., less than 9 wt%, 8 wt%, 7 wt%, 6 wt%, 5 wt%, 4 wt%, 3 wt%, 2 wt%, or 1 wt%), oleic acid and/or oleic acid ethyl ester. Also disclosed herein is a composition (or a fatty acid ethyl ester composition or a fatty acid derivative composition), comprising palmitoleic acid ethyl ester; one or more co-5 (omega-5) fatty acids or derivatives thereof (e.g., ethyl ester derivatives thereof); less than 10 wt% (e.g., up to and including 0 wt%) oleic acid and/or oleic acid ethyl ester; and less than 10 wt% (e.g., up to and including 0 wt%) of one or more PUFAs and/or derivatives thereof (e.g., ethyl esters of PUFAs). Any of the compositions provided herein can also comprise one or more C14-C20 saturated fatty acids, such as, for example, tetradecanoic acid, hexadecenoic acid (palmitic acid), and/or octadecanoic acid (stearic acid), or derivatives thereof, particularly ethyl ester derivatives thereof; and/or can contain one or more C14-C20 monounsaturated fatty acids, such as tetradecenoic acid, hexadecenoic acid (e.g., palmitoleic acid or All -hexadecenoic acid), octadecenoic acid (e.g., Z9-, Z11-, or Z13-octadecenoic acid), or derivatives thereof, particularly ethyl ester derivatives thereof; in an amount of about 20% or less (e.g., 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, or 0.1% or less), by weight of the composition. Monounsaturated fatty acids or derivatives thereof, produced by the described recombinant cells, microorganisms, or microbes comprising a variant or heterologous acyl-ACP thioesterase as described herein, are also provided.

[0032] Also provided are uses of the saturated and/or monounsaturated fatty acids and derivatives thereof, and uses of the compositions described herein, such as use of a composition comprising one or more saturated or monounsaturated fatty acids or fatty acid derivatives, for the preparation of a nutraceutical, nutritional, dietary, pharmaceutical, pheromone, fragrance, or flavor product or ingredient, or a precursor thereof. For example, provided herein is the use of any of the compositions described above and elsewhere herein, particularly any of the compositions comprising palmitoleic acid ethyl ester, for the preparation of a nutraceutical product, or a nutritional supplement, or a dietary supplement.

Also provided herein is a nutraceutical, nutritional, dietary, pharmaceutical, pheromone, fragrance, or flavor product, or a precursor thereof, comprising one or more of the saturated and/or monounsaturated fatty acids or fatty acid derivatives, or one or more of the compositions comprising the saturated and/or monounsaturated fatty acids or derivatives thereof described herein. For example, provided is a nutraceutical or a nutraceutical product, or a nutritional supplement, or a dietary supplement, comprising palmitoleic acid ethyl ester.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1A-1B depict the production of fatty acid ethyl esters (FAEEs) by recombinant bacterial strains expressing exemplary acyl-ACP thioesterase variants. FIG. 1A depicts the FAEE composition produced by strain sKM.309, containing an acyl-ACP thioesterase variant with the mutations D20S/N58G/V147A (SEQ ID NO: 16) with reference to SEQ ID NOG. FIG. IB depicts the FAEE composition produced by strain sKM.348, containing an acyl-ACP thioesterase variant with the mutation V147A (SEQ ID NO:4) with reference to SEQ ID NOG.

[0034] FIG. 2 depicts a GC chromatograph from the broth extract of strain sKM.348, containing an acyl-ACP thioesterase variant with the mutation V147A (SEQ ID NOG) with reference to SEQ ID NOG.

DETAILED DESCRIPTION

I. Definitions

[0035] The following definitions refer to the various terms used above and throughout the disclosure.

[0036] As used herein, singular articles such as “a” and “an” and “the” and similar referents in the context of describing the elements are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. [0037] As used herein, “about” is understood by persons of ordinary skill in the art and may vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which the term “about” is used, “about” will mean up to plus or minus 10% of the particular term. As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence, “about 10%” means “about 10%” and also means “10%. ” [0038] As used herein, the term "or" is used to mean "and/or" unless explicitly indicated to refer to alternatives only or if the alternatives are mutually exclusive.

[0039] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally expressed polypeptide means that the polypeptide is expressed or is not expressed.

[0040] As will be understood by one skilled in the art, for any and all purposes, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Furthermore, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 atoms refers to groups having 1, 2, or 3 atoms. Similarly, a group having 1-5 atoms refers to groups having 1, 2, 3, 4, or 5 atoms, and so forth.

[0041] Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by a person of ordinary skill in the art. In particular, this disclosure utilizes routine techniques in the field of recombinant genetics, organic chemistry, fermentation, and biochemistry.

[0042] As used herein, an “acyl-ACP thioesterase” or “a polypeptide with acyl-ACP thioesterase activity” refers to a polypeptide or enzyme that catalyzes or is capable of catalyzing the hydrolysis of thioester bonds in fatty acyl-ACPs to terminate fatty acyl extension and generate free fatty acids. In other words, an acyl-ACP thioesterase or a polypeptide with acyl- ACP thioesterase activity is capable of converting or hydrolyzing an acyl-ACP to a free fatty acid. The acyl-ACP thioesterase can be described by the number EC 3.1.2.14 or EC 3.1.2.21, and can also be referred to as an acyl-ACP hydrolase.

[0043] As used herein, a “mature” acyl-ACP thioesterase or acyl-ACP thioesterase variant is one lacking the plastid-transit peptide (also referred to herein as the plastid leader sequence) or the transit peptide. The mature thioesterase or variant thereof can lack all or a portion of the plastid-transit peptide, corresponding to amino acid residues 1-38, 2-38, 1-51, 2-51, 1-66, 2-66, 1-67, 2-67, 1-68, or 2-68, of SEQ ID NO:2. [0044] As used herein, a “plastid-transit peptide” (also referred to herein as the plastid leader sequence) is peptide at the N-terminus of an encoded protein, that targets the transport of the protein to a particular organelle (or plastid).

[0045] As used herein, the term “variant” with reference to a polynucleotide, polypeptide, or enzyme, is used interchangeably with “mutant” or “modified”, and refers to a polynucleotide or polypeptide sequence with one or more modifications compared to a reference, typically a wildtype, unmodified, or native sequence. For example, a variant acyl-ACP thioesterase, as provided herein, contains one or more modifications (e.g., amino acid deletions, substitutions, insertions and/or additions), compared to a wild-type or unmodified thioesterase. For example, a variant acyl-ACP thioesterase, as provided herein, contains one or more amino acid substitutions and/or deletions compared to SEQ ID NO:2 or SEQ ID NO:3.

[0046] As used herein, “increased thioesterase activity” or “increased acyl-ACP thioesterase activity” refers to an increased activity in the conversion of an acyl-ACP to a free fatty acid. This increased activity can result in an increased yield, titer, and/or productivity of one or more fatty acids and/or fatty acid derivatives. For example, increased thioesterase activity can result in an increased amount of fatty acids, which are then converted to one or more fatty acid derivatives (e.g., fatty esters, fatty alcohols, fatty aldehydes, etc.); since the amount of fatty acids is increased by the increased thioesterase activity, the amount of the fatty acid derivatives also is increased.

[0047] As used herein, “specificity” for a substrate, refers to a preference of an enzyme, e.g., a variant acyl-ACP thioesterase provided herein, for a specific substrate, e.g., an acyl-ACP of a specific length, or one with a particular double bond position. The term “selectivity” as used herein refers to the preference of an enzyme for one substrate over another. For example, the variant acyl-ACP thioesterases provided herein can have increased specificity and/or selectivity for a C16 (saturated) or a C16:l (monounsaturated) acyl-ACP.

[0048] The term “fatty acid” or “free fatty acid” as used herein, refers to an aliphatic carboxylic acid having the formula RCOOH, wherein R is an aliphatic group having at least 4 carbons, typically between about 4 and about 28 carbon atoms. The aliphatic R group can be saturated or unsaturated, and/or can be branched or unbranched. Branched aliphatic R groups may include branches comprising lower alkyl branches, such as a C1-C4 alkyl, preferably in an co-1 or co-2 position. In some embodiments, the branched aliphatic R group may be a methyl group in the co-1 or co-2 position. Unsaturated fatty acids may be monounsaturated or polyunsaturated. A “3-hydroxy fatty acid” refers to a fatty acid with a hydroxy (OH) group in the 3 position, where the carboxyl group carbon is assigned position number 1. A “3-hydroxy” or “3-OH” fatty acid or fatty acid derivative can also be referred to as a “beta-hydroxy,” “beta- OH”, or “P-hydroxy” or “P-OH” fatty acid or fatty acid derivative.

[0049] The term “omega” or “co” as used herein, with respect to positioning within the carbon chain, refers to the last carbon in the chain, farthest from the carboxyl group, in a fatty acid or fatty acid derivative, or farthest from the thioester group, for example, in a fatty acyl- CoA or fatty acyl-ACP molecule. When a number is appended to the term “omega” or “co,” that number denotes the position with respect to the omega carbon. For example, a substituent at the omega-1 (co-1) position is attached to the penultimate carbon. For example, a C12 fatty acid, with a hydroxy group at the co position can be referred to as 12-hydroxy dodecanoic acid; a C12 fatty acid with a hydroxy group at the co-1 position can be referred to as 11 -hydroxy dodecanoic acid; a C12 fatty acid with a hydroxy group at the co-2 position can be referred to as 10-hydroxy dodecanoic acid, and so forth. The omega (co) numbering of the double bond position in a compound does not indicate the geometric isomerism of the compound; thus, as used herein, co7- hexadecenoic acid can have a cis or a trans double bond, or the term may refer to a mixture of cis and trans isomers thereof.

[0050] The position of a double bond within a carbon chain in any of the fatty acids or derivatives thereof provided herein also can be described by the upper-case Greek letter “A”, or “delta”, followed by a number, which refers to the position of the double bond with respect to the carboxyl group (in a fatty acid or derivative thereof), or with respect to the thioester group (in a fatty acyl-CoA or fatty acyl-ACP), where the carbon of the carboxyl or thioester group is designated as position number 1. For example, A9-hexadecenoic acid refers to a C 16 fatty acid containing a double bond between carbon numbers 9 and 10, where the carboxyl carbon is at position number 1. Similarly, A7-hexadecenoic acid has a double bond between carbon numbers 7 and 8, with the carboxyl carbon having position number 1. A7-hexadecenoic acid and A9- hexadecenoic acid can also be referred to as co9-hexadecenoic acid and co7-hexadecenoic acid, respectively. The delta (A) numbering of the double bond position in a compound does not indicate the geometric isomerism of the compound; thus, as used herein, A9-hexadecenoic acid can refer to Z9-hexadecenoic acid (or cis-9- or (9Z)-hexadecenoic acid), or to E9-hexadecenoic acid (or trans-9- or (9E)-hexadecenoic acid), or to a mixture thereof.

[0051] The fatty acids and derivatives thereof provided herein can be described in terms of their geometric isomerism. Geometric isomers can be represented by the symbol which denotes a bond that can be a single, double, or triple bond as described herein. Provided herein are various geometric isomers and mixtures thereof resulting from the arrangement of substituents around a carbon-carbon double bond. Substituents around a carbon-carbon double bond are designated as being in the "Z" or "E" configuration wherein the terms "Z" and "E" are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting double bonds encompass both the "E" and "Z" isomers.

[0052] Substituents around a carbon-carbon double bond alternatively can be referred to as "cis" or "trans," where "cis" represents substituents on the same side of the double bond and "trans" represents substituents on opposite sides of the double bond. The term "cis" represents substituents on the same side of the plane of the ring, and the term "trans" represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated "cis/trans." [0053] The term “fatty acid derivative” as used herein, refers to a product derived from a fatty acid, or from a fatty acyl thioester, such as a fatty acyl-ACP or a fatty acyl-CoA. Thus, a fatty acid derivative can refer to a compound that includes a fatty acid as defined above with a modification. In general, fatty acid derivatives include malonyl-CoA derived compounds, including acyl-ACP or acyl-CoA derivatives. Thus, a fatty acid derivative includes alkylthioesters and acyl-thioesters. Further, a fatty acid derivative includes a molecule or compound that is derived from a metabolic pathway that includes a fatty acid derivative enzyme. Exemplary fatty acid derivatives include, but are not limited to, for example, fatty acids, fatty acid esters (e.g., waxes), fatty acid alkyl esters, fatty acid methyl esters (FAME), fatty acid ethyl esters (FAEE), fatty alcohol acetate esters (FACE; also referred to herein as fatty alcohol acetates), fatty amines, fatty amides, fatty acetates, 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-l)-hydroxy fatty acids, (co-2)-hydroxy fatty acids, (co-3)-hydroxy fatty acids, co-hydroxy fatty esters, co-carboxy fatty esters, a, co-fatty diacids, a, co-fatty diesters, 1,3 fatty diols, a,co-diols, a,co-3-hydroxy triols, co-hydroxy FAME, co-OH FAEE, etc.), and unsaturated fatty acid derivatives, including unsaturated versions of each of the above mentioned fatty acid derivatives. The fatty acid derivatives can be saturated or unsaturated, and/or can be branched or unbranched. Unsaturated fatty acid derivatives can be monounsaturated or polyunsaturated. The fatty acid derivative typically contains between about 4 and about 28 carbon atoms, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 carbon atoms. A fatty acid alkyl ester can be a methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or other alkyl ester.

[0054] The fatty acids or fatty acid derivatives, as used herein, can be produced within a cell through the process of fatty acid biosynthesis, through the reverse of fatty acid degradation or beta (P)-oxidation, or they can be fed to a cell. As is well known in the art, fatty acid biosynthesis is generally a malonyl-CoA dependent synthesis of acyl-ACPs or acyl-CoAs, while the reverse of beta-oxidation is acetyl-CoA dependent and results in the synthesis of acyl-CoAs. Fatty acids fed to cells are converted to acyl-CoAs and can be converted to acyl-ACPs. Fatty acids can be synthesized in a cell by natural (i.e., native or endogenous) 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.

[0055] The term “malonyl-CoA derived compound” as used herein refers to any compound or chemical entity i.e., intermediate or end product) that is made via a biochemical pathway wherein malonyl-CoA functions as an intermediate and/or is made upstream of the compound or chemical entity. For example, a malonyl-CoA derived compound may include, but is not limited to, a fatty acid derivative such as, for example, a fatty acid; a fatty ester including, but not limited to a fatty acid methyl ester (FAME) and/or a fatty acid ethyl ester (FAEE); a fatty alcohol; a fatty aldehyde; a fatty amine; an alkane; an olefin or alkene; a hydrocarbon; a bifunctional fatty acid derivative; a multifunctional fatty acid derivative; a native or non-native unsaturated fatty acid derivative, etc.

[0056] As used herein, an “alkyl-thioester” or equivalently an “acyl thioester” is a compound in which the carbonyl carbon of an acyl chain and the sulfhydryl group of an organic thiol are joined through a thioester bond. Representative organic thiols include, e.g., cysteine, beta-cysteine, glutathione, mycothiol, pantetheine, Coenzyme A (CoA), and the acyl carrier protein (ACP). An “acyl-ACP” refers to an “alkyl-thioester” formed between the carbonyl carbon of an acyl chain and the sulfhydryl group of the phosphopantetheinyl moiety of an ACP. An “acyl-CoA” refers to an “alkyl-thioester” formed between the carbonyl carbon of an acyl chain and the sulfhydryl group of the phosphopantetheinyl moiety of CoA. In some embodiments an “alkyl-thioester”, such as an acyl-ACP or an acyl-CoA, is an intermediate in the synthesis of fully saturated acyl thioesters. In other embodiments, an “alkyl-thioester”, such as an acyl-ACP or an acyl-CoA, is an intermediate in the synthesis of unsaturated acyl thioesters. In some embodiments, the carbon chain of the acyl group of an acyl thioester has 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 carbons. In other embodiments, the carbon chain of the acyl group of an acyl thioester is a medium-chain and has 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbons. In other exemplary embodiments the carbon chain of the acyl group of an acyl-thioester is 8 carbons in length. In other exemplary embodiments the carbon chain of the acyl group of an acyl-thioester is 10 carbons in length. In still other exemplary embodiments, the carbon chain of the acyl group of an acyl-thioester is 12 carbons in length. In still other exemplary embodiments, the carbon chain of the acyl group of an acyl-thioester is 14 carbons in length. In still other exemplary embodiments, the carbon chain of the acyl group of an acyl-thioester is 16 carbons in length. Alkyl-thioesters are substrates for fatty acid derivative enzymes, such as, e.g., lactonizing enzymes, thioesterases, acyl-ACP reductases, acyl-CoA reductases, and ester synthases, and their engineered variants, that convert the acyl-thioester to fatty acid derivatives such, as e.g., natural lactones, fatty acids, fatty aldehydes, or fatty esters.

[0057] The term “medium- to long-chain” or “medium-chain to long-chain”, with reference to fatty acids or derivatives thereof, is used herein to refer to fatty acids or derivatives thereof, as well as the alkyl- or acyl-thioesters from which they are derived, that contain between 14 and 20 carbon atoms; such as, for example, C14, C16, and/or C18 fatty acids or derivatives thereof (or alkyl- or acyl-thioesters). The term “long-chain fatty acids” as used herein, can be used to refer to fatty acids and derivatives thereof with sixteen or eighteen carbon chain lengths, e.g. hexadecanoic acid (palmitic acid) (C16:0), A9-hexadecenoic acid (palmitoleic acid) (C16:l), octadecanoic acid (stearic acid) (C18:0), All-octadecenoic acid (vaccenic acid) (C18:l), or AO- octadecenoic acid (oleic acid) (C18:l), and derivatives thereof, but it can also be used to refer to fatty acids or derivatives thereof with 14, 15, 17, 19, or 20 carbon chain lengths.

[0058] As used herein, the expression “fatty acid derivative biosynthetic/biosynthesis pathway” refers to a biochemical pathway that produces fatty acid derivatives and/or the precursors thereof. The enzymes that comprise a “fatty acid derivative biosynthetic/biosynthesis pathway” are thus referred to herein as “fatty acid derivative biosynthetic/biosynthesis polypeptides/enzymes” or equivalently “fatty acid derivative polypeptides” or “fatty acid derivative enzymes.” As discussed supra, and elsewhere herein, the term “fatty acid derivative” includes a molecule or compound derived from a biochemical pathway that includes a fatty acid derivative enzyme. Thus, a thioesterase enzyme (e.g., an enzyme having thioesterase activity, such as EC 3.2.1.14) is a “fatty acid derivative biosynthetic/biosynthesis polypeptide” or equivalently, a “fatty acid derivative enzyme.” Thus, the term "fatty acid derivative enzymes" or equivalently "fatty acid derivative biosynthetic/biosynthesis polypeptides" refers, collectively and individually, to enzymes that may be expressed or overexpressed (e.g., in a host cell, microbe, or microorganism) to produce fatty acids and/or fatty acid derivatives, such as, e.g., omega-hydroxy fatty acids or esters; fatty aldehydes; fatty alcohols; fatty esters (e.g., fatty acid methyl esters (FAMEs) or fatty acid ethyl esters (FAEEs)); and other derivatives as described herein and as known in the art. Additional non-limiting examples of "fatty acid derivative enzymes" or equivalently "fatty acid derivative biosynthetic/biosynthesis polypeptides" include, e.g., fatty acid synthases, lactonizing enzymes, thioesterases, acyl-CoA synthetases, acyl-CoA reductases, acyl-ACP reductases, alcohol dehydrogenases, alcohol oxidases, aldehyde dehydrogenases, alcohol O-acyltransferases, fatty alcohol-forming acyl-CoA reductases, fatty acid decarboxylases, fatty aldehyde decarbonylases and/or oxidative deformylases, carboxylic acid reductases, fatty alcohol O-acetyl transferases, hydroxylating enzymes (including, for example omega-hydroxylases, oxygenases, or monooxygenases), hydratases, desaturases, ester synthases, transaminases (aminotransferases), etc. "Fatty acid derivative enzymes" or equivalently "fatty acid derivative biosynthetic/biosynthesis polypeptides" convert substrates into fatty acids or fatty acid derivatives. The substrate for a fatty acid derivative enzyme can be an intermediate of a fatty acid derivative biosynthetic/biosynthesis pathway. For example, a fatty acyl-ACP can be a substrate for a thioesterase, which converts the acyl-ACP to a free fatty acid, and the free fatty acid (as an intermediate), in turn, can be a substrate for a carboxylic acid reductase, which converts the fatty acid to a fatty aldehyde. Further, the fatty aldehyde can act as an intermediate, and can be a substrate for an alcohol dehydrogenase, which converts the fatty aldehyde intermediate into a fatty alcohol product.

[0059] The expression “fatty acid composition” or “fatty acid derivative composition” as used herein, refers to a composition of fatty acids and/or fatty acid derivatives, for example, a composition of monounsaturated fatty acids or derivatives thereof. For example, a fatty acid or fatty acid derivative composition produced by the recombinant cells or microbes described herein, such as a recombinant proteobacterium comprising a heterologous or variant acyl-ACP thioesterase. A fatty acid derivative composition can comprise a single fatty acid derivative species or can comprise a mixture of fatty acid derivative species. In some exemplary embodiments, the mixture of fatty acid derivatives includes more than one type of fatty acid derivative product (e.g., fatty acids, fatty acid esters, fatty alcohols, fatty alcohol acetates, fatty aldehydes, fatty amines, bifunctional fatty acid derivatives, and non-native monounsaturated fatty acid derivatives, etc.). In other exemplary embodiments, the mixture of fatty acid derivatives includes a mixture of monounsaturated fatty acid esters (and/or another fatty acid derivative(s)) with different chain lengths, saturation and/or branching characteristics. In other exemplary embodiments, the mixture of fatty acid derivatives comprises predominantly one type of fatty acid derivative, e.g., a palmitoleic acid or palmitoleic acid alkyl ester, such as a palmitoleic acid ethyl ester. In still other exemplary embodiments, a fatty acid derivative composition comprises a mixture of more than one type of fatty acid derivative product, e.g., fatty acid derivatives with different chain lengths, saturation and/or branching characteristics. In still other exemplary embodiments, a “fatty acid derivative composition” comprises a mixture of fatty esters and 3 -hydroxy esters. In still other exemplary embodiments, a fatty acid derivative composition comprises a mixture of fatty alcohols and fatty aldehydes, for example, a mixture of monounsaturated fatty alcohols or fatty aldehydes. In other exemplary embodiments, the mixture of fatty acid derivatives includes a mixture of saturated and monounsaturated fatty acid derivatives with different chain lengths, saturation levels, branching characteristics, and/or functional group characteristics.

[0060] Sequence Accession numbers throughout this description were obtained from databases provided by the NCBI (National Center for Biotechnology Information) maintained by the National Institutes of Health, U.S.A, (which are identified herein as “NCBI Accession Numbers” or alternatively as “GenBank Accession Numbers” or alternatively as simply “Accession Numbers”), and from the UniProt Knowledgebase (UniProtKB) and Swiss-Prot databases, provided by the Swiss Institute of Bioinformatics (which are identified herein as “UniProtKB Accession Numbers”).

[0061] The term “enzyme classification (EC) number” refers to a number that denotes a specific polypeptide sequence or enzyme. EC numbers classify enzymes according to the reaction they catalyze. EC numbers are established by the nomenclature committee of the international union of biochemistry and molecular biology (IUBMB), a description of which is available on the IUBMB enzyme nomenclature website on the world wide web.

[0062] As used herein, the terms “isolated” and “purified,” with respect to products (such as monounsaturated fatty acids and derivatives thereof disclosed herein), refers to products that are separated from cellular components, cell culture media, fermentation broth, and/or chemical or synthetic precursors. The monounsaturated fatty acids and derivatives thereof disclosed herein, produced by the cells, microbes, cell cultures, and/or methods disclosed herein, can be relatively immiscible in the fermentation broth, as well as in the cytoplasm. Therefore, in exemplary embodiments, the monounsaturated fatty acids and derivatives thereof disclosed herein collect in an organic phase extracellularly and are thereby “isolated”.

[0063] As used herein, the terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues that is typically 12 or more amino acids in length. Polypeptides less than 12 amino acids in length are referred to herein as “peptides.” The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The term “recombinant polypeptide” refers to a polypeptide that is produced by recombinant techniques, wherein generally DNA or RNA encoding the expressed protein is inserted into a suitable expression vector that is in turn used to transform a host cell to produce the polypeptide. In some exemplary embodiments, DNA or RNA encoding an expressed peptide, polypeptide, or protein is inserted into the host chromosome via homologous recombination or other means well known in the art, and is so used to transform a host cell to produce the peptide, polypeptide, or protein. Similarly, the terms “recombinant polynucleotide” or “recombinant nucleic acid” or “recombinant DNA” are produced by recombinant techniques that are known to those of skill in the art (see, e.g., methods described in Sambrook et al., Molecular Cloning— A Laboratory Manual, Cold Spring Harbor Press 4^th Edition (Cold Spring Harbor, N.Y. 2012) and/or in Current Protocols in Molecular Biology (Volumes 1-3, John Wiley & Sons, Inc. (1994-1998) and Supplements 1-115 (1987-2016)).

[0064] As used herein, a “modification” refers to modification of a sequence of amino acid residues of a polypeptide, or a sequence of nucleotides in a nucleic acid molecule, and includes deletions, insertions, additions, and replacements (substitutions) of amino acids and nucleotides, respectively. Modifications also can include post-translational modifications or other changes to the molecule that can occur due to conjugation or linkage, directly or indirectly, to another moiety. Methods of modifying a polypeptide are routine to those of skill in the art, such as by using recombinant DNA methodologies.

[0065] As used herein, “deletion,” when referring to a modification of a nucleic acid or polypeptide sequence, refers to the removal of one or more nucleotides or amino acids compared to a sequence, such as a target or reference polynucleotide or polypeptide, or a native or wildtype sequence. Thus, an amino acid sequence or nucleic acid molecule that contains one or more deletions compared to a wild-type sequence, contains one or more fewer amino acids or nucleotides within the linear length of the sequence.

[0066] As used herein, “insertion” when referring to modification of a nucleic acid or amino acid sequence, describes the inclusion of one or more additional nucleotides or amino acids, within a target, native, wild-type or other related sequence. Thus, an amino acid or nucleic acid molecule that contains one or more insertions compared to a wild-type sequence, contains one or more additional amino acids or nucleotides within the linear length of the sequence.

[0067] As used herein, “additions” to nucleic acid and amino acid sequences describe addition of nucleotides or amino acids onto either termini compared to another sequence.

[0068] As used herein, “substitution” or “replacement” with respect to a modification refers to the replacing of one or more nucleotides or amino acids in a native, target, wild-type or other nucleic acid or polypeptide sequence, with an alternative nucleotide or amino acid, without changing the length (as described in numbers of residues) of the molecule. Thus, one or more substitutions in a molecule does not change the number of amino acid residues or nucleotides of the molecule. Amino acid replacements compared to a particular polypeptide can be expressed in terms of the number of the amino acid residue along the length of the polypeptide sequence or a reference polypeptide sequence. For example, a modified polypeptide having a modification in the amino acid at the 19^th position of the amino acid sequence that is a substitution of Isoleucine (He; I) for cysteine (Cys; C) can be expressed as "replacement with Cys or C at a position corresponding to position 19," I19C, Ilel9Cys, or simply C19, to indicate that the amino acid at the modified 19^th position is a cysteine. In this example, the molecule having the substitution has a modification at He 19 of the unmodified polypeptide.

[0069] Amino acid substitutions contemplated include conservative substitutions, such as those set forth in the Table below, which do not eliminate the desired activity (e.g., thioesterase activity). As used herein, suitable conservative substitutions of amino acids are known to those of skill in this art and can be made generally without altering the biological activity of the resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. Co., p.224). Such substitutions can be made in accordance with those set forth in the Table below, as follows:

[0070] Other substitutions also are permissible and can be determined empirically or in accord with known conservative substitutions. [0071] As use herein, “sequence identity” refers to the number of identical amino acids (or nucleotide bases) in a comparison between a test and a reference polypeptide or polynucleotide. When referring to two nucleotide or polypeptide sequences, the “percentage of sequence identity” between the two sequences is determined by comparing the two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The “percentage of sequence identity” is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0072] Thus, the expression “percent identity,” or equivalently “percent sequence identity,” “homology, or “homologous” in the context of two or more nucleic acid sequences or peptides or polypeptides, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (e.g., about 50% identity, preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured e.g., using a BLAST or BLAST 2.0 sequence comparison algorithm with default parameters (see e.g., Altschul et al. (1990) J. Mol. Biol. 215(3):403-410 and/or the NCBI web site at ncbi.nlm.nih.gov/BLAST/), or by manual alignment and visual inspection. Percent sequence identity between two nucleic acid or amino acid sequences also can be determined using e.g., the Needleman and Wunsch algorithm that has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 (see, e.g., Needleman and Wunsch (1970) J. Mol. Biol. 48:444-453). The percent sequence identity between two nucleotide sequences also can be determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6. One of ordinary skill in the art can perform initial sequence identity calculations and adjust the algorithm parameters accordingly. A set of parameters that may be used if a practitioner is uncertain about which parameters should be applied to determine if a molecule is within a sequence identity limitation of the claims, are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. Additional methods of sequence alignment are known in the biotechnology arts (see, e.g., Rosenberg (2005) BMC Bioinformatics 6:278; Altschul et al. (2005) FEBS J. 272(20):5101- 5109).

[0073] Two or more nucleic acid or amino acid sequences are said to be “substantially identical,” when they are aligned and analyzed as discussed above and are found to share about 50% identity, preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region. Two nucleic acid sequences or polypeptide sequences are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences are the same when aligned for maximum correspondence as described above. This definition also refers to, or may be applied to, the compliment of a test sequence. Identity is typically calculated over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length, or over the entire length of a given sequence.

[0074] The term “endogenous” as used herein refers to a substance, e.g., a nucleic acid, protein, enzyme, etc., that is produced from within a cell and/or that is naturally occurring or naturally found inside a cell. Similarly, an endogenous pathway (such as a fatty acid biosynthesis pathway or a fatty acid derivative pathway) is one that is naturally occurring or naturally found inside a cell. Thus, an endogenous nucleic acid sequence, gene, polynucleotide, or polypeptide refers to a nucleic acid sequence, gene, polynucleotide, or polypeptide produced by and found inside the cell. In some exemplary embodiments, an endogenous polypeptide or polynucleotide is encoded by the genome of the parental cell (or host cell). In other exemplary embodiments, an endogenous polypeptide or polynucleotide is encoded by an autonomously replicating plasmid carried by the parental cell (or host cell). In some exemplary embodiments, an endogenous gene or nucleic acid sequence is a gene or nucleic acid sequence that was present in the cell when the cell was originally isolated from nature, i.e., the gene is native to the cell.

[0075] In contrast, an “exogenous” nucleic acid sequence, gene, polynucleotide, or polypeptide (e.g., an enzyme), or other substance (e.g., fatty acid derivative, small molecule compound, etc.), as used herein, refers to a nucleic acid sequence, gene, polynucleotide, or polypeptide or other substance that is not encoded by or produced by the cell, and which is therefore added to a cell, a cell culture, or assay, from outside of the cell. A nucleic acid sequence encoding a variant (i.e., mutant) polypeptide, when added to the cell, is one example of an exogenous nucleic acid sequence. Similarly, a nucleic acid sequence encoding a fatty acid biosynthesis enzyme or fatty acid derivative enzyme, when introduced into a cell (e.g., in a vector, such as a plasmid), is considered an exogenous nucleic acid sequence. The exogenous nucleic acid sequence can encode a polypeptide or an enzyme that is also otherwise endogenous or native to the cell. Such an encoded polypeptide or enzyme can be considered “exogenously expressed.” For example, to achieve overexpression of an endogenous gene, additional copies of the gene can be introduced into the cell (e.g., in a vector, such as a plasmid); such additional copies of the endogenous gene can be considered as “exogenous” (e.g., exogenous gene(s) or an exogenous nucleic acid sequence(s)), because the additional copies are introduced into the cell from outside the cell. An “exogenous gene” or “exogenous nucleic acid sequence” also refers to a native (or endogenous) gene or nucleic acid sequence that is deregulated (e.g., upregulated or attenuated) or otherwise altered or modified, for example, by operably linking it to a regulatory element, such as a heterologous, or non-native, or non-naturally occurring, regulatory element (e.g., a promoter, enhancer, 5’-UTR, ribosome binding site, etc.); such a deregulated or altered gene or nucleic acid sequence can be on a chromosome or can be on a plasmid. An exogenous nucleic acid sequence or exogenous gene can also be used to express or overexpress a heterologous polypeptide or enzyme in a cell. Thus, an exogenous nucleic acid sequence or an exogenous gene can encode a polypeptide (e.g., an enzyme) that is native to the cell, that is otherwise endogenous to the cell, or that is heterologous to the cell.

[0076] The term “heterologous” as used herein refers to a polypeptide or polynucleotide which is in a non-native state. Thus, a polynucleotide or a polypeptide is “heterologous” to a cell when the polynucleotide and/or the polypeptide and the cell are not found in the same relationship to each other in nature. Therefore, a polynucleotide or polypeptide sequence is “heterologous” to an organism or a second sequence if it originates from a different organism, different cell type, or different species, or, if from the same species, it is modified from its original form. Thus, in an exemplary embodiment, a polynucleotide or polypeptide is “heterologous” when it is not naturally present in a given organism. For example, a polynucleotide sequence that is native to cyanobacteria can be introduced into a host cell of E. coli (a proteobacterium) by recombinant methods, and the polynucleotide from cyanobacteria is then heterologous to the E. coli cell (i.e., the now recombinant E.coli cell).

[0077] Similarly, a polynucleotide or polypeptide is heterologous when it is modified from its native form or from its relationship with other polynucleotide sequences or is present in a recombinant host cell in a non-native state. Thus, in an exemplary embodiment, a heterologous polynucleotide or polypeptide comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, a promoter operably linked to a nucleotide coding sequence derived from a species different from that from which the promoter was derived. Alternatively, in another example, if a promoter is operably linked to a nucleotide coding sequence derived from a species that is the same as that from which the promoter was derived, then the operably-linked promoter and coding sequence are “heterologous” if the coding sequence is not naturally associated with the promoter (e.g. a constitutive promoter operably linked to a developmentally regulated coding sequence that is derived from the same species as the promoter). In other exemplary embodiments, a heterologous polynucleotide or polypeptide is modified relative to the wild type sequence naturally present in the corresponding wild type host cell, e.g., an intentional modification e.g., an intentional mutation in the sequence of a polynucleotide or polypeptide or a modification in the level of expression of the polynucleotide or polypeptide. Typically, a heterologous nucleic acid or polynucleotide is recombinantly produced. A heterologous polynucleotide, polypeptide, or enzyme, for example, is typically exogenous to the cell, or exogenously expressed (or overexpressed) in the cell, i.e., is introduced into or added to the cell from outside the cell.

[0078] As used herein, the term “native” refers to the form of a nucleic acid, protein, polypeptide, or a fragment thereof, that is isolated from nature, or to a nucleic acid, protein, polypeptide or a fragment thereof that is in its natural state without intentionally introduced mutations in the structural sequence and/or without any engineered changes in expression, such as e.g., changing a developmentally regulated gene to a constitutively expressed gene. As used herein, “native” also refers to “wildtype” or “wild-type,” in which the nucleic acid, protein, polypeptide, or a fragment thereof, is present in both sequence, quantity, and relative quantity, as typically found in the organism as naturally found. Wild-type organisms may serve as a control and/or reference for determination of cellular functions, such as to identity and/or quantity monounsaturated fatty acid(s) and/or derivatives thereof produced. A native gene, nucleic acid sequence, polypeptide, or enzyme, for example, is typically endogenous to a cell, i.e., found in or produced by the cell. An exogenous nucleic acid sequence or an exogenous gene can encode a native polypeptide or enzyme, for example, where additional copies of a native gene or nucleic acid sequence are added to the cell from outside the cell, or where a native gene or nucleic acid sequence is deregulated or altered, e.g., by operably coupling it to a regulatory element that is not native or endogenous to the cell.

[0079] The term “non-native” is used herein to refer to nucleic acid sequences, amino acid sequences, polypeptide sequences, enzymes, fatty acids and derivatives thereof, and/or small molecules that do not occur naturally in the host. Heterologous genes and polypeptides are considered “non-native.” A nucleic acid sequence or amino acid sequence that has been removed from a host cell, subjected to laboratory manipulation, and introduced or reintroduced into a host cell, is also considered “non-native.” Synthetic or partially synthetic genes introduced into a host cell are “non-native.” Non-native genes further include genes that are endogenous and/or native to the host microorganism but that are operably linked to one or more heterologous regulatory sequences that have been recombined into the host genome. A naturally occurring gene under the control of a heterologous regulatory sequence is considered “non-native.” In some embodiments, an organism comprising a non-native gene may be utilized as a control and/or reference for an organism having additional and/or different variations from wildtype organisms.

[0080] Additionally, the term “non-native monounsaturated fatty acid or derivative thereof’ as used herein, refers to any monounsaturated fatty acid derivative derived from an acylthioester where the double bond position is non-native to the producing cell (e.g., recombinant proteobacterium). For example, in E. coli, the native double-bond position in monounsaturated fatty acids is omega-7 (co-7). Therefore, for example in E. coli, a monounsaturated fatty acid or derivative thereof with a double bond in a position other than the co-7 position is defined as a “non-native” monounsaturated fatty acid or derivative thereof for this bacterium. Examples of non-native monounsaturated fatty acids and derivatives thereof have double bonds at co-3, co-5, co-6, co-8, co-9, co-11, co-12, and/or co-13 positions.

[0081] The term “gene” as used herein, refers to a nucleic acid sequence e.g., a DNA sequence, which encodes either an RNA product or a protein product, as well as operably-linked nucleic acid sequences that affect expression of the RNA or protein product (e.g., expression control sequences, such as, e.g., promoters, enhancers, ribosome binding sites, translational control sequences, etc.). The term “gene product” refers to either the RNA (e.g., tRNA, mRNA) and/or protein expressed from a particular gene. Nucleic acid sequences can include those with degenerate codon sequences. For example, reference to any nucleic acid sequences encoding the variant acyl-ACP thioesterases provided herein also include nucleic acid sequences with degenerate codon sequences that encode the same variant acyl-ACP thioesterases.

[0082] The term “expression” or “expressed” as used herein in reference to a gene, refers to the production of one or more transcriptional and/or translational product(s) of a gene. In exemplary embodiments, the level of expression of a DNA molecule in a cell is determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell. The term “expressed genes” refers to genes that are transcribed into messenger RNA (mRNA) and then translated into protein, as well as genes that are transcribed into other types of RNA, such as e.g., transfer RNA (tRNA), ribosomal RNA (rRNA), and regulatory RNA, which are not translated into protein. [0083] The level of expression of a nucleic acid molecule in a cell or cell-free system is influenced by “expression control sequences” or equivalently “regulatory sequences” or “regulatory elements.” Expression control sequences, regulatory sequences, or regulatory elements are known in the art and include, for example, promoters, enhancers, polyadenylation signals, transcription terminators, nucleotide sequences that affect RNA stability, internal ribosome entry sites (IRES), and the like, that provide for the expression of the polynucleotide sequence in a host cell. In exemplary embodiments, “expression control sequences” interact specifically with cellular proteins involved in transcription (see e.g., Maniatis el al., Science, 236: 1237-1245 (1987); Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990)). In exemplary methods, an expression control sequence, regulatory sequence, or regulatory element is operably linked to a polynucleotide sequence. By “operably linked” is meant that a polynucleotide sequence and an expression control sequence(s) or regulatory element(s) are functionally connected so as to permit expression of the polynucleotide sequence when the appropriate molecules (e.g., transcriptional activator proteins) contact the expression control sequence(s). In exemplary embodiments, operably linked promoters are located upstream of the selected polynucleotide sequence in terms of the direction of transcription and translation. In some exemplary embodiments, operably linked enhancers can be located upstream, within, or downstream of the selected polynucleotide. [0084] As used herein, the phrase “expression of said nucleotide sequence is modified relative to the wild-type nucleotide sequence,” refers to a change, e.g., an increase or decrease in the level of expression of a native nucleotide sequence, or a change, e.g., an increase or decrease in the level of the expression of a heterologous or non-native polypeptide-encoding nucleotide sequence as compared to a control nucleotide sequence e.g., wild-type control. In some exemplary embodiments, the phrase “the expression of said nucleotide sequence is modified relative to the wild type nucleotide sequence,” refers to a change in the pattern of expression of a nucleotide sequence as compared to a control pattern of expression e.g., constitutive expression as compared to developmentally timed expression.

[0085] A “control” sample (e.g., a control nucleotide sequence, a control polypeptide sequence, a control cell, etc., or value) refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample. For example, in an exemplary embodiment, a test sample comprises a monounsaturated fatty acid derivative composition made by a recombinant microbe that comprises a heterologous acyl-ACP thioesterase as disclosed herein, while the control sample comprises a monounsaturated free fatty acid or derivative thereof composition made by the corresponding or designated microbe that does not comprise a heterologous acyl-ACP thioesterase. Additionally, a control cell or microorganism may be referred to as a corresponding wild type or host cell. One of skill will recognize that controls can be designed for assessment of any number of parameters. Furthermore, one of skill in the art will understand which controls are valuable in a given situation and will be able to analyze data based on comparisons to control values.

[0086] The term “overexpressed” or “up-regulated” as used herein, refers to a gene whose expression is elevated in comparison to a control level of expression. In exemplary embodiments, overexpression of a gene is caused by an elevated rate of transcription as compared to the native transcription rate for that gene. In other exemplary embodiments, overexpression is caused by an elevated rate of translation of the gene compared to the native translation rate for that gene. Methods of testing for overexpression are well known in the art, for example transcribed RNA levels can be assessed using rtPCR and protein levels can be assessed using SDS page gel analysis.

[0087] In other embodiments, the polypeptide (e.g., enzyme), polynucleotide, or gene having an altered level of expression is “attenuated” or has a “decreased level of expression” or is “down-regulated.” As used herein, these terms mean to express or cause to be expressed a polynucleotide, polypeptide (e.g., enzyme), or gene in a cell at a lesser concentration than is normally expressed in a corresponding control cell (e.g., wild type cell) under the same conditions. In other words, the term “attenuate” means to weaken, reduce, or diminish. For example, a polypeptide can be attenuated by modifying the polypeptide to reduce its activity e.g., by modifying a nucleotide sequence that encodes the polypeptide).

[0088] A polynucleotide or polypeptide can be attenuated using any method known in the art. For example, in some exemplary embodiments, the expression of a gene or polypeptide encoded by the gene is attenuated by mutating the regulatory polynucleotide sequences which control expression of the gene. In other exemplary embodiments, the expression of a gene or polypeptide encoded by the gene is attenuated by overexpressing a repressor protein, or by providing an exogenous regulatory element that activates a repressor protein. In still other exemplary embodiments, DNA- or RNA-based gene silencing methods are used to attenuate the expression of a gene or polynucleotide. In some embodiments, the expression of a gene or polypeptide is completely attenuated, e.g., by deleting all or a portion of the polynucleotide sequence of a gene.

[0089] The degree of overexpression or attenuation can be 1.5-fold or more, e.g., 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, or 15-fold or more. Alternatively, or in addition, the degree of overexpression or attenuation can be 500-fold or less, e.g., 100-fold or less, 50-fold or less, 25-fold or less, or 20-fold or less. Thus, the degree of overexpression or attenuation can be bounded by any two of the above endpoints. For example, the degree of overexpression or attenuation can be 1.5-500-fold, 2-50-fold, 10-25-fold, or 15-20-fold.

[0090] As used herein, “substantially free” refers to a condition wherein the recombinant microbe comprises none or almost none of the component it is deemed to be “substantially free” of. For example, the recombinant microbe would be substantially free of the component if it contained less than about 5 wt%, less than about 4 wt%, less than about 3 wt%, less than about 2 wt%, less than about 1 wt%, less than about 0.5 wt%, less than about 0.1 wt%, less than about 0.05 wt%, less than about 0.01 wt%, or about 0 wt% of the component normally found in the microbe. Alternatively, the term “substantially free” can refer to a low amount of the component in relation to another component within the recombinant microbe. For example, a recombinant E. coli is substantially free of polyunsaturated fatty acids or derivatives thereof if the polyunsaturated fatty acids or derivatives thereof comprise about 5 wt% or less of the total amount of fatty acids and derivatives thereof within the E coli. Alternatively, the recombinant E. coli would be considered substantially free of polyunsaturated fatty acids or derivatives thereof if the polyunsaturated fatty acids or derivatives thereof comprise less than about 4 wt%, less than about 3 wt%, less than about 2 wt%, less than about 1 wt%, less than about 0.5 wt%, less than about 0.1 wt%, less than about 0.05 wt%, less than about 0.01 wt%, or about 0 wt% of the total amount of fatty acids and derivatives thereof within the E coli.

[0091] As used herein, “modified activity” or an “altered level of activity” of a protein/polypeptide in a recombinant host cell refers to a difference in one or more characteristics in the activity the protein/polypeptide as compared to the characteristics of an appropriate control protein e.g., the corresponding parent protein or corresponding wild type protein. Thus, in exemplary embodiments, a difference in activity of a protein having “modified activity” as compared to a corresponding control protein is determined by measuring the activity of the modified protein in a recombinant host cell and comparing that to a measure of the same activity of a corresponding control protein in an otherwise isogenic host cell. Modified activities can be the result of, for example, changes in the structure of the protein (e.g., changes to the primary structure, such as e.g., changes to the protein’s nucleotide coding sequence that result in changes in substrate specificity, changes in observed kinetic parameters, changes in solubility, etc.); changes in protein stability (e.g., increased or decreased degradation of the protein) etc.

[0092] The term “recombinant” as used herein, refers to a genetically modified polynucleotide, polypeptide, cell, tissue, or organism. When used with reference to a cell, the term “recombinant” indicates that the cell has been modified by the introduction of a heterologous nucleic acid or protein, or has been modified by alteration of a native nucleic acid or protein, or that the cell is derived from a cell so-modified and that the derived cell comprises the modification. Thus, for example, “recombinant cells” or equivalently “recombinant host cells” may be modified to express genes that are not found within the native (non-recombinant) form of the cell or may be modified to abnormally express native genes e.g., native genes may be overexpressed, under expressed, or not expressed at all. In exemplary embodiments, a “recombinant cell” or “recombinant host cell” is engineered to express a heterologous enzyme pathway capable of producing a bifunctional fatty acid derivative molecule. A recombinant cell can be derived from a microorganism or microbe such as a bacterium, proteobacterium, archaea, a virus, algae, or a fungus. In addition, a recombinant cell can be derived from a plant or an animal cell. In exemplary embodiments, a “recombinant host cell” or “recombinant cell” is used to produce one or more saturated and/or monounsaturated fatty acids or derivatives thereof including, but not limited to, palmitic acid, palmitoleic acid, oleic acid, and/or derivatives thereof. Therefore, in some exemplary embodiments, a “recombinant host cell” is a “production host” or equivalently, a “production host cell”. In some exemplary embodiments, the recombinant cell includes one or more polynucleotides, each polynucleotide encoding a polypeptide having fatty acid biosynthetic enzyme activity, wherein the recombinant cell produces a saturated and/or monounsaturated fatty acid or derivative thereof when cultured in the presence of a (simple) carbon source under conditions effective to express the polynucleotides.

[0093] When used with reference to a polynucleotide, the term “recombinant” indicates that the polynucleotide has been modified by comparison to the native or naturally occurring form of the polynucleotide or has been modified by comparison to a naturally occurring variant of the polynucleotide. In an exemplary embodiment, a recombinant polynucleotide (or a copy or complement of a recombinant polynucleotide) is one that has been manipulated by the hand of man to be different from its naturally occurring form. Thus, in an exemplary embodiment, a recombinant polynucleotide is a mutant form of a native gene or a mutant form of a naturally occurring variant of a native gene wherein the mutation is made by intentional human manipulation e.g., made by saturation mutagenesis using mutagenic oligonucleotides, through the use of UV radiation, mutagenic chemicals, chemical synthesis etc. Such a recombinant polynucleotide might comprise one or more point mutations, deletions and/or insertions relative to the native or naturally occurring variant form of the gene. Similarly, a polynucleotide comprising a promoter operably linked to a second polynucleotide (e.g., a coding sequence) is a “recombinant” polynucleotide. Thus, a recombinant polynucleotide comprises polynucleotide combinations that are not found in nature. A recombinant protein (discussed supra) is typically one that is expressed from a recombinant polynucleotide, and recombinant cells, tissues, and organisms are those that comprise recombinant sequences (polynucleotide and/or polypeptide).

[0094] The term “vector,” as used herein, refers to a polynucleotide sequence that contains a gene of interest (e.g., it encodes one or more proteins or enzymes described herein) and a promoter operably linked to the fatty acid biosynthetic polynucleotide sequence of interest. Once a polynucleotide sequence(s) encoding a fatty acid biosynthetic pathway polypeptide has been prepared and isolated, various methods may be used to construct expression cassettes, vectors and other DNA constructs. The skilled artisan is well aware of the genetic elements that must be present on an expression construct/vector in order to successfully transform, select, and propagate the expression construct in host cells. Techniques for manipulation of nucleic acids such as subcloning nucleic acid sequences into expression vectors, labeling probes, DNA hybridization are well known in the art.

[0095] As used herein, the term “microbe” or “microorganism” refers generally to a microscopic organism. Microbes can be prokaryotic or eukaryotic. Exemplary prokaryotic microbes include e.g., bacteria (including y-proteobacteria), archaea, cyanobacteria, etc. An exemplary proteobacterium is Escherichia coli. Exemplary eukaryotic microorganisms include e.g., yeast, protozoa, algae, etc. In exemplary embodiments, a “recombinant microbe” is a microbe that has been genetically altered and thereby expresses or encompasses an exogenous and/or a heterologous nucleic acid sequence and/or an exogenous and/or a heterologous peptide, polypeptide, or protein.

[0096] A microbe as used herein, can grow on a carbon source e.g., a simple carbon source. Typically, as used herein, a recombinant microbe, including a recombinant proteobacterium, comprises at least an acyl-ACP thioesterase variant having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO: 22. The recombinant microbe may be a gamma-proteobacterium (also known as a y-proteobacterium), a cyanobacterium, a yeast, or an algae. In some embodiments, the recombinant proteobacterium may be Escherichia coli, Salmonella spp., Vibrio natriegens, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Xanthomonas axonopodis, Pseudomonas syringae, Pseudomonas citronellolis , Pseudomonas mendocina, Pseudomonas plecoglossicida, Pseudomonas mosselii, Pseudomonas fulva, Xyella fastidiosa, Marinobacter aquaeolei, Yersinia pestis, or Vibrio cholerae. In some embodiments, the recombinant cyanobacterium may be Synechococcus elongatus PCC7942, or Synechocystis sp. PCC6803. In some embodiments, the recombinant yeast may be Saccharomyces cerevisiae, Scheffersomyces stipitis, Schizosaccharomyces pombe, Kluyveromyces marxianus, K. lactis, Pichia pastoris, Hansenula polymorpha, or Yarrowia lipolytica. In some embodiments, the recombinant algae may be Botryococcus braunii, Nannochloropsis gaditina, Chlamydomonas reinhardtii, Chlorella vulgaris, Spirulina platensis, Ostreococcus tauri, Phaeodactylum tricornutum, Symbiodinium spp., algal phytoplanktons, Saccharina japonica, Chlorococcum spp., or Spiro gyra spp.

[0097] As used herein, the term “culture” typically refers to a liquid media comprising viable cells. In one embodiment, a culture comprises cells reproducing in a predetermined culture media under controlled conditions, for example, a culture of recombinant host cells grown in liquid media comprising a selected carbon source and nitrogen.

[0098] “Culturing” or “cultivation” refers to growing a population of recombinant host cells (e.g., recombinant microbes) under suitable conditions in a liquid or on a solid medium. In particular embodiments, culturing refers to the fermentative bioconversion of a substrate to an end-product. Culturing media are well-known and individual components of such culture media are available from commercial sources, e.g., under the Difco™ and BBL™ trademarks. In one non-limiting example, the aqueous nutrient medium is a “rich medium” comprising complex sources of nitrogen, salts, and carbon, such as YP medium, comprising 10 g/L of peptone and 10 g/L yeast extract of such a medium.

[0099] Typically, a ‘ ‘recombinant microbe” as disclosed herein will comprise within its cellular fatty acids/membrane phospholipids the monounsaturated fatty acids or derivatives thereof, or the compositions comprising the monounsaturated fatty acids or derivatives thereof, produced by the microbe that has the characteristic double bond structure. In some embodiments, the monounsaturated fatty acids or derivatives thereof, comprise at least 5% of the membrane phospholipids. In other embodiments, the monounsaturated fatty acids or derivatives thereof, comprise at least 10% of the membrane phospholipids. In still other embodiments, the monounsaturated fatty acids or derivatives thereof, comprise at least 11%, at least 12%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, of the membrane phospholipids. In another embodiment, the recombinant microbe will comprise at least 11%, at least 12%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, or at least 70%, monounsaturated fatty acids or derivatives thereof.

[00100] A “production host” or equivalently a “production host cell” is a cell used to produce products. As disclosed herein, a production host is typically modified to express or overexpress selected genes, or to have attenuated expression of selected genes. Thus, a production host or a “production host cell” is a recombinant host or equivalently a recombinant host cell. Non- limiting examples of production hosts include e.g., recombinant microbes as disclosed above. An exemplary production host is a recombinant proteobacterium comprising a variant acyl-ACP thioesterase.

[00101] As used herein, the terms “purify,” “purified,” or “purification” mean the removal or isolation of a molecule from its environment by, for example, isolation or separation. “Substantially purified” molecules are at least about 60% free e.g., at least about 65% free, at least about 70% free, at least about 75% free, at least about 80% free, at least about 85% free, at least about 90% free, at least about 95% free, at least about 96% free, at least about 97% free, at least about 98% free, at least about 99% free) from other components with which they are associated. As used herein, these terms also refer to the removal of contaminants from a sample. [00102] As used herein, the term “carbon source” refers to a substrate or compound suitable to be used as a source of carbon for prokaryotic or simple eukaryotic cell growth. Carbon sources can be in various forms, including, but not limited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, and gases (e.g., CO and CO2). Exemplary carbon sources include, but are not limited to, monosaccharides, such as glucose, fructose, mannose, galactose, xylose, and arabinose; oligosaccharides, such as fructo-oligosaccharide and galacto-oligosaccharide; polysaccharides such as starch, cellulose, pectin, and xylan; disaccharides, such as sucrose, maltose, cellobiose, and turanose; cellulosic material and variants such as hemicelluloses, methyl cellulose and sodium carboxymethyl cellulose; succinate, lactate, and acetate; alcohols, such as ethanol, methanol, and glycerol, or mixtures thereof. The carbon source can also be a product of photosynthesis, such as glucose. In certain embodiments, the carbon source is a biomass. In other embodiments, the carbon source is glucose. In other embodiments the carbon source is sucrose. In other embodiments the carbon source is glycerol. In other embodiments, the carbon source is a simple carbon source such as e.g., glucose. In other embodiments, the carbon source is a renewable carbon source. In other embodiment, the carbon source is natural gas. In other embodiments the carbon source comprises one or more components of natural gas, such as methane, ethane, or propane. In other embodiments, the carbon source is flu gas or synthesis gas. In still other embodiments, the carbon source comprises one or more components of flu or synthesis gas such as carbon monoxide, carbon dioxide, hydrogen, etc. As used herein, the term “carbon source” or “simple carbon source” specifically excludes oleochemicals such as e.g., saturated or unsaturated fatty acids.

II. Enzymes

[00103] Improved variants of acyl-ACP thioesterase are disclosed herein for producing fatty acids or derivatives thereof in recombinant microbes. As used herein, the term “acyl-ACP thioesterase” refers to an enzyme that catalyzes the hydrolysis of thioester bonds in fatty acyl- ACPs to terminate fatty acyl extension and generate free fatty acids. The acyl-ACP thioesterase is sometimes referred to herein as “FatA” or a “FatA thioesterase.” In some embodiments, variants of the wild-type (unmodified) plant acyl-ACP thioesterase FatA from Arabidopsis thaliana with improved properties are provided. The improved properties of the variant thioesterases provided herein are in comparison to the corresponding wild-type or unmodified thioesterase, including full-length and mature sequences thereof. In some embodiments, the improved properties of the variant acyl-ACP thioesterases provided herein include increased activity (thioesterase activity), for example, resulting in an increased production (or productivity), amount, yield, or titer, of free fatty acids, and also, of the derivatives of the free fatty acids. The increased production, amount, titer, or yield of free fatty acids in turn results in an increased production (or productivity), amount, yield, or titer, of derivatives of the fatty acids. In some embodiments, the improved properties include increased specificity and/or selectivity for a fatty acyl-ACP substrate, such as a long-chain fatty acyl-ACP substrate (e.g., C16 and/or C18). In some embodiments, the increased specificity and/or selectivity is for a monounsaturated long-chain fatty acyl-ACP substrate, such as, for example, a monounsaturated C16:l or C18:l acyl-ACP substrate. In particular embodiments, the increased specificity and/or selectivity of the variant acyl-ACP thioesterase is towards palmitoleoyl-ACP (Z9-hexadecenoyl- ACP or cis-9-hexadecenoyl-ACP), resulting in the increased production (or productivity), yield, or titer of palmitoleic acid (9Z-hexadecenoic acid). In other embodiments, the increased specificity and/or selectivity of the variant acyl-ACP thioesterase is towards one or more monounsaturated C16 or C18 acyl-ACP substrates, for example, Z7-tetradecenoyl-ACP, Z9- tetradecenoyl-ACP, Z7-hexadecenoyl-ACP, Z9-hexadecenoyl-ACP, Zl l-hexadecenoyl-ACP, Z13-hexadecenoyl-ACP, Z7-octadecenoyl-ACP, Z9-octadecenoyl-ACP, Zl l-octadecenoyl- ACP, Z13-octadecenoyl-ACP, or Z15-octadecenoyl-ACP, or various combinations thereof. In some embodiments, the variant acyl-ACP thioesterases or the variant FatA thioesterases provided herein have increased thioesterase activity and/or increased specificity and/or increased selectivity for a monounsaturated long-chain fatty acyl-ACP substrate, such as, for example, a monounsaturated C16:l or C18:l acyl-ACP substrate. In some embodiments, the variant acyl- ACP thioesterases or the variant FatA thioesterases provided herein have increased thioesterase activity and/or increased selectivity and/or increased specificity for a saturated or monounsaturated medium-chain to long-chain fatty acyl-ACP substrate, such as for example, a saturated or monounsaturated C14, C15, C16, C17, C18, C19, or C20 acyl-ACP. For example, the variant acyl-ACP thioesterases can have increased activity and/or specificity and/or selectivity towards Z9-tetradecenoyl-ACP. In some embodiments, the variant acyl-ACP thioesterases or the variant FatA thioesterases provided herein have increased thioesterase activity and increased selectivity for a monounsaturated long-chain fatty acyl-ACP substrate.

[00104] The sequence of acyl-ACP thioesterase from Arabidopsis thaliana was first described in 1992. SEQ ID NO: 2 represents the full-length amino acid sequence, including the plastid transit peptide, of the wild-type acyl-ACP thioesterase (FatA) of Arabidopsis thaliana. SEQ ID NO:3 represents the mature amino acid sequence (/'.<?., without the plastid transit peptide) of the wild-type Arabidopsis thaliana acyl-ACP thioesterase, while SEQ ID NO:1 represents the nucleotide sequence encoding the mature wild-type acyl-ACP thioesterase from A. thaliana. SEQ ID NO:22 represents the amino acid sequence of a variant (or modified), non- naturally occurring acyl-ACP thioesterase, having locations (i.e., amino acid positions) of potential substitutions. The positions for potential substitutions in SEQ ID NO:22 (“Xaa”) are numbered with reference to the mature sequence set forth in SEQ ID NO:3 (i.e., SEQ ID NO:22 does not include the plastid transit peptide). In one embodiment, the acyl-ACP thioesterase variant has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% sequence identity to SEQ ID NO:22. In another embodiment, the acyl-ACP thioesterase variant has at least 85% sequence identity to SEQ ID NO:22. In a further embodiment, the acyl-ACP thioesterase variant has at least a 90% sequence identity to SEQ ID NO: 22, at least a 95% sequence identity to SEQ ID NO: 22, at least a 99% sequence identity to SEQ ID NO: 22, or is SEQ ID NO: 22 (e.g., has 100% sequence homology or identity to SEQ ID NO:22).

[00105] In some embodiments, the acyl-ACP thioesterase variant has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity to the wild-type thioesterase sequence set forth in SEQ ID NO:2. In some embodiments, the acyl-ACP thioesterase variant comprises:

(i) at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 86%, 87%, 98%, or 99% sequence identity to the wild-type thioesterase of SEQ ID NO:2; and/or

(ii) one or more amino acid substitutions at a position corresponding to 70, 90, 97, 100, 108, 132, 133, 197, 236, 342, 349, 353, or 355, or a combination thereof, with reference to SEQ ID NO:2; and/or (iii) a deletion of all or a portion of the plastid transit peptide, for example, a deletion of positions corresponding to amino acid residues 1-38, 2-38, 1-51, 2-51, 1-67, 2-67, 1-68, or 2-68, or any portions thereof, of SEQ ID NO:2. In certain embodiments, the acyl-ACP thioesterase variant comprises (i), (ii), and (iii). In other embodiments, the acyl-ACP thioesterase variant comprises (i) and (ii), or the acyl-ACP thioesterase variant comprises (i) and (iii), or the acyl- ACP thioesterase variant comprises (ii) and (iii). In certain embodiments, the acyl-ACP thioesterase variant comprises one or more amino acid substitutions corresponding to D70S, V90M, S97E, T100R, N108G, T132D, T133C, T133K, V197A, S236L, L342G, I349T, I349V, T353Q, or L355R, or a combination thereof, with reference to SEQ ID NO:2.

[00106] In particular embodiments, the acyl-ACP thioesterase variant has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NOG. In other embodiments, the acyl-ACP thioesterase variant has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 86%, 87%, 98%, or 99% sequence identity to the wild-type thioesterase sequence set forth in SEQ ID NOG and/or has one or more amino acid substitutions at a position corresponding to 20, 40, 47, 50, 58, 82, 83, 147, 186, 292, 299, 303, or 305, or a combination thereof, with reference to SEQ ID NOG. In certain embodiments, the acyl-ACP thioesterase variant comprises one or more amino acid substitutions corresponding to D20S, V40M, S47E, T50R, N58G, T82D, T83C, T83K, V147A, S186L, L292G, I299T, I299V, T3O3Q, or L305R, or combinations thereof, with reference to SEQ ID NOG.

[00107] In a specific embodiment, the acyl-ACP thioesterase variant has a sequence having at least 70%, at least 75%, at least 80%, or at least 85% sequence identity to a sequence of SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21. In a further specific embodiment, the acyl-ACP thioesterase variant comprises a sequence having at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to, or is (e.g., having 100% sequence homology or identity to) a sequence set forth in SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.

[00108] In a further embodiment, the variant acyl-ACP thioesterase can comprise a sequence having a substitution at one or more of amino acid positions of SEQ ID NO:3, such as at positions 20, 40, 50, 83, 147, 292, 299, 303, or 305, or a combination thereof, of SEQ ID NO:3. In a still further embodiment, the acyl-ACP thioesterase variant comprises one or more amino acid substitutions, including, for example, D20S, V40M, T50R, T83C, T83K, V147A, L292G, I299T, I299V, T3O3Q, or L305R, or a combination thereof, with reference to SEQ ID NO:3. In some embodiments, the acyl-ACP thioesterase variant comprises a sequence having a substitution at one or more of positions 20, 40, 47, 50, 58, 82, 83, 147, 186, 292, 299, 303, or 305, or a combination thereof, with respect to SEQ ID NO:3, or at one or more of positions 70, 90, 97, 100, 108, 132, 133, 197, 236, 342, 349, 353, or 355, or a combination thereof, with respect to SEQ ID NO:2. In yet another embodiment, the acyl-ACP thioesterase variant comprises one or more amino acid substitutions corresponding to D20S, V40M, S47E, T50R, N58G, T82D, T83C, T83K, V147A, S186L, L292G, I299T, I299V, T3O3Q, or L305R, or a combination thereof, with reference to SEQ ID NOG, or corresponding to D70S, V90M, S97E, T100R, N108G, T132D, T133C, T133K, V197A, S236L, L342G, I349T, I349V, T353Q, or L355R, or a combination thereof, with reference to SEQ ID NOG.

[00109] Where the acyl-ACP thioesterase variant comprises two amino acid substitutions, exemplary combinations of positions that can be substituted, with reference to SEQ ID NOG, are listed in Table 1.

[00110] Table 1

[00111] Where the acyl-ACP thioesterase variant comprises three substitutions, exemplary combinations of positions that can be substituted, with reference to SEQ ID NO:3, are listed in Table 2. [00112] Table 2

[00113] Where the acyl-ACP thioesterase variant comprises four substitutions, exemplary combinations of positions that can be substituted, with reference to SEQ ID NO:3, are listed in Table 3. [00114] Table 3

[00115] Where the acyl-ACP thioesterase variant comprises five substitutions, exemplary combinations of positions that can be substituted, with reference to SEQ ID NO:3, are listed in Table 4. [00116] Table 4

substitutions

[00117] Where the acyl-ACP thioesterase variant comprises six substitutions, exemplary combinations of positions that can be substituted, with reference to SEQ ID NO:3, are listed in Table 5.

[00118] Table 5

[00119] Where the acyl-ACP thioesterase variant comprises seven substitutions, exemplary combinations of positions that can be substituted, with reference to SEQ ID NO:3, are listed in Table 6.

[00120] Table 6

[00121] Where the acyl-ACP thioesterase variant comprises eight substitutions, exemplary combinations of positions that can be substituted, with reference to SEQ ID NO:3, are listed in Table 7. [00122] Table 7

[00123] The positions listed in Tables 1-7 above are with respect to SEQ ID NO:3. The same combinations of substitutions can be made in the corresponding positions of SEQ ID NO:2, whereby positions 20, 40, 47, 50, 58, 82, 83, 147, 186, 292, 299, 303, and 305, with reference to SEQ ID NOG correspond to positions 70, 90, 97, 100, 108, 132, 133, 197, 236, 342, 349, 353, and 355, respectively, with reference to SEQ ID NO:2.

[00124] Alternatively, the acyl-ACP thioesterase variant can comprise substitutions at each of positions 20, 40, 50, 83, 147, 292, 299, 303, and 305, with reference to SEQ ID NO:3 (e.g., the acyl-ACP thioesterase variant comprises nine substitutions), or can comprise substitutions at each of positions 20, 40, 47, 50, 58, 82, 83, 147, 186, 292, 299, 303, and 305, with reference to SEQ ID NO:3 (e.g., the acyl-ACP thioesterase variant comprises 13 substitutions).

[00125] In a particular embodiment, the acyl-ACP thioesterase variant has a combination of two or more substitutions with reference to, or in the sequence of SEQ ID NO:3, at amino acid positions such as 147 and 292; 147 and 299; 147 and 303; 147 and 305; 20, 58, and 147; 50, 58, and 147; 20, 47, and 147; 20, 50, and 147; 20, 58, 82, and 147; 47, 82, 147, and 186; and 47, 50, 58, 82, and 147. In a further particular embodiment, the acyl-ACP thioesterase variant comprises amino acid substitutions corresponding to, for example, V147A/L292G; V147A/I299T; V147A/I299V; V147A/T303Q; V147A/L305R; D20S/S47E/V147A; D20S/T50R/V147A; D20S/N58G/V147A; T50R/N58G/V147A; D20S/N58G/T82D/V147A;

S47E/T82D/V147A/S186L; or S47E/T50R/N58G/T82D/V147A, with reference to SEQ ID NOG.

[00126] Additionally, the variant acyl-ACP thioesterase (alternatively the variant FatA, the variant FatA thioesterase, the FatA variant, the acyl-ACP thioesterase variant, or the FatA thioesterase variant) described herein may belong to EC 3.1.2.14.

[00127] As used herein, the term “0-ketoacyl-ACP synthase,” or a polypeptide having “0- ketoacyl-ACP synthase activity,” which includes P-ketoacyl-ACP synthase I, e.g., “FabB,” and/or 0-ketoacyl-ACP synthase II, e.g., “FabF,” refers to enzymes that catalyze the condensation reaction to elongate the fatty acid chain. The 0-ketoacyl-ACP synthase may be native to the recombinant cell or microbe, z.e., from or derived from the same species as the recombinant cell or microbe, or may be heterologous, z.e., from or derived from an organism or species that is different from the recombinant cell or microbe. The native or heterologous 0- ketoacyl-ACP synthase can be expressed, or can be overexpressed, in the recombinant cell or microbe. In some embodiments, the native 0-ketoacyl-ACP-synthase may be endogenous, wherein the enzyme or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA) is produced by the cell. For example, the recombinant cell or microbe can comprise an endogenous nucleic acid sequence or endogenous gene encoding the native 0-ketoacyl-ACP-synthase. In other embodiments, the native 0-ketoacyl-ACP-synthase can be encoded by an exogenous nucleic acid sequence or an exogenous gene, such that the encoding nucleic acid sequence or gene is added to the cell from outside the cell, for example, where overexpression of the native enzyme is desired. Overexpression of a native enzyme, such as a P-ketoacyl-ACP-synthase, can also be achieved by other methods known in the art, such as, for example, by placing the encoding nucleic acid sequence or gene under control of a different (e.g., a more active, or constitutively active, or stronger) promoter, or by modifying the native or endogenous promoter, or by modifying other associated regulatory elements. In such a case, the encoding nucleic acid sequence with the modified or altered regulatory element(s) is considered an exogenous nucleic acid sequence. In another embodiment, the P-ketoacyl-ACP-synthase is heterologous (to the recombinant cell or microbe), , and the polynucleotide, nucleic acid sequence, or gene, encoding the enzyme, is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell. P-ketoacyl-ACP-synthase or P-ketoacyl-ACP-synthase activity may be described by EC 2.3.1.41 (P-ketoacyl-ACP synthase I) or EC 2.3.1.179 (P-ketoacyl-ACP synthase II).

[00128] As used herein, the term “acyl-CoA synthetase” (alternatively “acyl-CoA synthase”) or a polypeptide having “acyl-CoA synthetase activity,” refers to an enzyme or polypeptide that can convert free fatty acids (e.g., C14-C20, such as C16 and/or C18 free fatty acids) to their corresponding fatty acyl-CoAs. The acyl-CoA synthetase may be native to the recombinant cell or microbe, z.e., from or derived from the same species as the recombinant cell or microbe, or may be heterologous, z.e., from or derived from an organism or species that is different from the recombinant cell or microbe. The native or heterologous acyl-CoA synthetase can be expressed, or can be overexpressed, in the recombinant cell or microbe. In some embodiments, the native acyl-CoA synthetase may be endogenous, wherein the enzyme or a polynucleotide encoding the enzyme (e.g., RNA, mRNA, or DNA) is produced by the cell. For example, the recombinant cell or microbe can comprise an endogenous nucleic acid sequence or endogenous gene encoding the native acyl-CoA synthetase. In other embodiments, the native P-ketoacyl-ACP-synthase can be encoded by an exogenous nucleic acid sequence or an exogenous gene, such that the encoding nucleic acid sequence or gene is added to the cell from outside the cell, for example, where overexpression of the native enzyme is desired. In another embodiment, the acyl-CoA synthetase is heterologous (to the recombinant cell or microbe), and the polynucleotide, nucleic acid sequence, or gene, encoding the enzyme, is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell. Acyl-CoA synthetase or acyl-CoA synthetase activity may be described by EC 6.2.1.3, and can be alternatively referred to as a fatty acid-CoA ligase, or a long-chain-fatty-acid-CoA ligase. A native, endogenous, or heterologous acyl-CoA synthetase can be expressed or overexpressed in the recombinant cell or microbe. In some embodiments, the acyl-CoA synthetase is native to the cell and is overexpressed. In other embodiments, the acyl-CoA synthetase is heterologous to the cell and is expressed in the cell.

[00129] As used herein, the term “acyl-CoA reductase” refers to an enzyme that catalyzes the reduction of saturated and/or unsaturated fatty acyl-CoAs to fatty alcohols. For example, the fatty acyl-CoA can be a saturated or unsaturated C14, C15, C16, C17, C18, C19, or C20 fatty acyl-CoA, such as a monounsaturated C16 and/or C18 fatty acyl-CoA. The acyl-CoA reductase may be native to the recombinant cell or microbe, z.e., from or derived from the same species as the recombinant cell or microbe, or may be heterologous, z.e., from or derived from an organism or species that is different from the recombinant cell or microbe. The native or heterologous acyl-CoA reductase can be expressed, or can be overexpressed, in the recombinant cell or microbe. In some embodiments, the native acyl-CoA reductase may be endogenous, wherein the enzyme or a polynucleotide encoding the enzyme (e.g., RNA, mRNA, or DNA) is produced by the cell. For example, the recombinant cell or microbe can comprise an endogenous nucleic acid sequence or endogenous gene encoding the native acyl-CoA reductase. In other embodiments, the native P-ketoacyl-ACP-synthase can be encoded by an exogenous nucleic acid sequence or an exogenous gene, such that the encoding nucleic acid sequence or gene is added to the cell from outside the cell, for example, where overexpression of the native enzyme is desired. In another embodiment, the acyl-CoA reductase is heterologous (to the recombinant cell or microbe), and the polynucleotide, nucleic acid sequence, or gene, encoding the enzyme, is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell. Acyl-CoA reductase or acyl-CoA reductase activity may be described by EC 1.2.1.50, and the acyl-CoA reductase can be alternatively referred to as a long-chain-fatty-acyl-CoA reductase.

[00130] As used herein, the term “fatty alcohol forming acyl-CoA reductase” refers to an enzyme or polypeptide that catalyzes the reduction of saturated and/or unsaturated fatty acyl- CoAs to fatty aldehydes, and catalyzes the subsequent reduction of the fatty aldehydes to fatty alcohols. For example, the fatty acyl-CoA, fatty aldehyde, or fatty alcohol can be a saturated or unsaturated C14, C15, C16, C17, C18, C19, or C20 fatty acyl-CoA, fatty aldehyde, or fatty alcohol, particularly a monounsaturated C16 and/or C18 fatty acyl-CoA, fatty aldehyde, or fatty alcohol. The fatty alcohol forming acyl-CoA reductase may be native to the recombinant cell or microbe, z.e., from or derived from the same species as the recombinant cell or microbe, or may be heterologous, z.e., from or derived from an organism or species that is different from the recombinant cell or microbe. The native or heterologous fatty alcohol forming acyl-CoA reductase can be expressed, or can be overexpressed, in the recombinant cell or microbe. In some embodiments, the native fatty alcohol forming acyl-CoA reductase (FAR) may be endogenous, wherein the enzyme or a polynucleotide encoding the enzyme (e.g., RNA, mRNA, or DNA) is produced by the cell. For example, the recombinant cell or microbe can comprise an endogenous nucleic acid sequence or endogenous gene encoding the native FAR. In other embodiments, the native FAR can be encoded by an exogenous nucleic acid sequence or an exogenous gene, such that the encoding nucleic acid sequence or gene is added to the cell from outside the cell, for example, where overexpression of the native enzyme is desired.

[00131] In another embodiment, the fatty alcohol forming acyl-CoA reductase is heterologous (to the recombinant cell or microbe), and the polynucleotide, nucleic acid sequence, or gene, encoding the enzyme, is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell. Fatty alcohol forming acyl-CoA reductase may be described by EC 1.2.1.84 and can be alternatively referred to as alcohol-forming fatty acyl-CoA reductase. In some embodiments, the fatty alcohol forming acyl-CoA reductase is native to the cell and is overexpressed. In other embodiments, the fatty alcohol forming acyl-CoA reductase is heterologous to the cell and is expressed in the cell.

[00132] As used here, the term “ester synthase” refers to an enzyme that catalyzes the reaction (condensation) of a free fatty acid and an alcohol to form a fatty acid ester. The alcohol can be, but is not limited to, for example, methanol or ethanol, whereby the resulting fatty acid ester is a fatty acid methyl ester (FAME) or a fatty acid ethyl ester (FAEE), respectively. In some embodiments, the ester synthase can also convert a fatty acid ester to a free fatty acid and an alcohol, e.g., can convert an FAEE to a free fatty acid and ethanol. The ester synthase may be native to the recombinant cell or microbe, z.e., from or derived from the same species as the recombinant cell or microbe, or may be heterologous, z.e., from or derived from an organism or species that is different from the recombinant cell or microbe. The native or heterologous ester synthase can be expressed, or can be overexpressed, in the recombinant cell or microbe.

[00133] In some embodiments, the native ester synthase may be endogenous, wherein the enzyme or a polynucleotide encoding the enzyme (e.g., RNA, mRNA, or DNA) is produced by the cell. For example, the recombinant cell or microbe can comprise an endogenous nucleic acid sequence or endogenous gene encoding the native ester synthase. In other embodiments, the native ester synthase can be encoded by an exogenous nucleic acid sequence or an exogenous gene, such that the encoding nucleic acid sequence or gene is added to the cell from outside the cell, for example, where overexpression of the native enzyme is desired.

[00134] In another embodiment, the ester synthase is heterologous (to the recombinant cell or microbe), and the polynucleotide, nucleic acid sequence, or gene, encoding the enzyme, is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell. The ester synthase may be described by EC 2.3.1.20 or EC 2.3.1.75.

[00135] As used herein, the term “co-hydroxylase” or “omega-hydroxylase” refers to an enzyme or polypeptide that hydroxylates a fatty acid or fatty acid derivative in the co-position (omega-position), i.e., adds a hydroxy (-OH) group to the co-position of the fatty acid or derivative thereof. The omega- (co)-position indicates the reduced end of a fatty acid derivative, or the position of the last carbon along the fatty acid derivative chain (farthest from the carboxyl group, for example). The co-hydroxylase may be native to the recombinant cell or microbe i.e., from or derived from the same species as the recombinant cell or microbe, or may be heterologous, i.e., from or derived from an organism or species that is different from the recombinant cell or microbe. The native or heterologous P-ketoacyl-ACP synthase can be expressed, or can be overexpressed, in the recombinant cell or microbe. In some embodiments, the native co-hydroxylase may be endogenous, wherein the enzyme, or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA) is produced by the cell. For example, the recombinant cell or microbe can comprise an endogenous nucleic acid sequence or endogenous gene encoding the native co-hydroxylase.

[00136] In another embodiment, the co-hydroxylase is heterologous (to the recombinant cell or microbe), and the polynucleotide, nucleic acid sequence, or gene, encoding the enzyme, is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell. In some embodiments, the co-hydroxylase may belong to EC 1.14.15.3 or 1.14.14.80, and can alternatively be referred to as a monooxygenase, an alkane 1 -monooxygenase, an alkane 1- hydroxylase, a fatty acid omega-hydroxylase, or a long chain fatty acid omega-monooxygenase.

[00137] As used herein, the term “carboxylic acid reductase” refers to an enzyme or polypeptide that converts (or reduces) a fatty acid to its corresponding fatty aldehyde. The carboxylic acid reductase (CAR) may be native to the recombinant cell or microbe, i.e., from or derived from the same species as the recombinant cell or microbe, or may be heterologous, i.e., from or derived from an organism or species that is different from the recombinant cell or microbe. The native or heterologous CAR can be expressed, or can be overexpressed, in the recombinant cell or microbe. In some embodiments, the native carboxylic acid reductase may be endogenous, wherein the enzyme, or a polynucleotide encoding the enzyme (e.g., RNA, mRNA, or DNA), is produced by the cell. For example, the recombinant cell or microbe can comprise an endogenous nucleic acid sequence or endogenous gene encoding the native CAR. In other embodiments, the native CAR can be encoded by an exogenous nucleic acid sequence or an exogenous gene, such that the encoding nucleic acid sequence or gene is added to the cell from outside the cell, for example, where overexpression of the native enzyme is desired.

[00138] In another embodiment, the carboxylic acid reductase is heterologous (to the recombinant cell or microbe), and the polynucleotide, nucleic acid sequence, or gene, encoding the enzyme, is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell. In some embodiments, the carboxylic acid reductase described herein may belong to EC 1.2.1.30, and can be alternatively referred to as a carboxylate reductase. In some embodiments, the carboxylic acid reductase is native to the cell and is overexpressed. In other embodiments, the carboxylic acid reductase is heterologous to the cell and is expressed in the cell.

[00139] As used herein, the term “desaturase” refers to an enzyme that belongs in the oxidoreductase family of enzymes and catalyzes the conversion of a saturated fatty acid to its corresponding monounsaturated fatty acid (which can be cis or trans). In embodiments herein, the desaturase is an acyl-ACP desaturase, whereby it converts a saturated fatty acyl-ACP to its corresponding monounsaturated fatty acyl-ACP. The desaturase determines the position of the double bond by its distance from the carboxylic acid end of the fatty acid (or the thioester end of the fatty acyl-ACP). The saturated acyl-ACP may be a palmitoyl-ACP (C16-ACP; hexadecanoyl-ACP) or a stearoyl- ACP (C18-ACP; octadecanoyl-ACP), and the corresponding cis-monounsaturated acyl-ACP may be palmitoleyl-ACP ((Z9)-C16:1-ACP; (Z9)-hexadecenoyl- ACP) or oleoyl-ACP ((Z9)-C18:1-ACP; (Z9)-octadecenoyl-ACP). The desaturase may be native to the recombinant cell or microbe, z.e., from or derived from the same species as the recombinant cell or microbe, or may be heterologous, z.e., from or derived from an organism or species that is different from the recombinant cell or microbe. The native or heterologous desaturase can be expressed, or can be overexpressed, in the recombinant cell or microbe.

[00140] In some embodiments, the native desaturase may be endogenous, wherein the enzyme, or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA) is produced by the cell. For example, the recombinant cell or microbe can comprise an endogenous nucleic acid sequence or endogenous gene encoding the native desaturase. In other embodiments, the native desaturase can be encoded by an exogenous nucleic acid sequence or an exogenous gene, such that the encoding nucleic acid sequence or gene is added to the cell from outside the cell, for example, where overexpression of the native enzyme is desired.

[00141] In another embodiment, the desaturase is heterologous (to the recombinant cell or microbe), and the polynucleotide, nucleic acid sequence, or gene, encoding the enzyme, is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell. In some embodiments, the acyl-ACP desaturases described herein may belong to EC 1.14.19.2. The particular acyl-ACP desaturase determines where the (cis) double bond in the monounsaturated fatty acid or derivative thereof is located in the carbon chain. In some embodiments, the acyl-ACP desaturase is native to the cell and is overexpressed. In other embodiments, the acyl-ACP desaturase is heterologous to the cell and is expressed in the cell. [00142] As used herein, the term “aldehyde dehydrogenase” refers to enzymes that convert aldehydes to carboxylic acids. The aldehyde dehydrogenase may be native to the recombinant cell or microbe, i.e., from or derived from the same species as the recombinant cell or microbe, or may be heterologous, i.e., from or derived from an organism or species that is different from the recombinant cell or microbe. The native or heterologous aldehyde dehydrogenase can be expressed, or can be overexpressed, in the recombinant cell or microbe.

[00143] In some embodiments, the native aldehyde dehydrogenase may be endogenous, wherein the enzyme, or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA) is produced by the cell. For example, the recombinant cell or microbe can comprise an endogenous nucleic acid sequence or endogenous gene encoding the native aldehyde dehydrogenase.

[00144] In other embodiments, the native aldehyde dehydrogenase can be encoded by an exogenous nucleic acid sequence or an exogenous gene, such that the encoding nucleic acid sequence or gene is added to the cell from outside the cell, for example, where overexpression of the native enzyme is desired.

[00145] In another embodiment, the aldehyde dehydrogenase is heterologous (to the recombinant cell or microbe), and the polynucleotide, nucleic acid sequence, or gene, encoding the enzyme, is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell. Aldehyde dehydrogenases may be described by EC 1.2.1.3. In some embodiments, the aldehyde dehydrogenase is native to the cell and is overexpressed. In other embodiments, the aldehyde dehydrogenase is heterologous to the cell and is expressed in the cell.

[00146] As used herein, the term “alcohol dehydrogenase” refers to an enzyme that catalyzes the interconversion between aliphatic alcohols (e.g., aliphatic medium-chain alcohols) and their corresponding aldehydes. In some embodiments, and under some conditions, the alcohol dehydrogenase converts an alcohol into an aldehyde. In some embodiments and under some conditions, the alcohol dehydrogenase converts an aldehyde into an alcohol. The alcohol dehydrogenase may be native to the recombinant cell or microbe, i.e., from or derived from the same species as the recombinant cell or microbe, or may be heterologous, i.e., from or derived from an organism or species that is different from the recombinant cell or microbe. The native or heterologous alcohol dehydrogenase can be expressed, or can be overexpressed, in the recombinant cell or microbe.

[00147] In some embodiments, the native alcohol dehydrogenase may be endogenous, wherein the enzyme, or a polynucleotide encoding the enzyme (e.g., RNA, DNA, mRNA), is produced by the cell. For example, the recombinant cell or microbe can comprise an endogenous nucleic acid sequence or endogenous gene encoding the native alcohol dehydrogenase. In other embodiments, the native alcohol dehydrogenase can be encoded by an exogenous nucleic acid sequence or an exogenous gene, such that the encoding nucleic acid sequence or gene is added to the cell from outside the cell, for example, where overexpression of the native enzyme is desired.

[00148] In another embodiment, the alcohol dehydrogenase is heterologous (to the recombinant cell or microbe), and the polynucleotide, nucleic acid sequence, or gene, encoding the enzyme, is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell. In some embodiments, the alcohol dehydrogenase may belong to EC 1.1.1.1 or EC 1.1.1.2, or EC 1.1.1.-, and can be alternatively referred to as an aldehyde reductase. In some embodiments, the alcohol dehydrogenase is native to the cell and is overexpressed. In other embodiments, the alcohol dehydrogenase is heterologous to the cell and is expressed in the cell.

[00149] In any of the embodiments described herein, any one or more of the fatty acid biosynthesis enzymes and/or fatty acid derivative enzymes described herein, can be native or heterologous to the recombinant cell or microbe (or microorganism). For example, a native enzyme or polypeptide is from or derived from the same species as the recombinant cell or microbe. A heterologous enzyme or polypeptide is from or derived from an organism or species that is different from the recombinant cell or microbe. Any of the native or heterologous enzymes or polypeptides described herein can be expressed, or can be overexpressed, in the recombinant cell or microbe.

[00150] The native enzyme or polypeptide, or the encoding polynucleotide sequence or gene, can be endogenous, i.e., found in and produced within the cell. For example, the recombinant cell or microbe or microorganism can comprise an endogenous nucleic acid sequence or endogenous gene encoding the native enzyme or polypeptide. In other embodiments, the native enzyme or polypeptide can be encoded by an exogenous nucleic acid sequence or an exogenous gene, such that the encoding nucleic acid sequence or gene is added to the cell from outside the cell, for example, where overexpression of the native enzyme or polypeptide is desired. Overexpression of a native enzyme or polypeptide, such as any described herein, can also be achieved by other methods known in the art, such as, for example, by placing the encoding nucleic acid sequence or gene under control of a different (e.g., a more active, or constitutively active, or stronger) promoter, or by modifying the native or endogenous promoter, or by modifying other associated regulatory elements. In such a case, the encoding nucleic acid sequence with the modified or altered regulatory element(s) is considered an exogenous nucleic acid sequence.

[00151] The gene or nucleic acid sequence encoding a native enzyme or polypeptide can be a non-native variant, for example, where the gene or nucleic acid sequence is operably linked to a non-native regulatory element; in such a case, the non-native gene or nucleic acid sequence typically is referred to herein as an exogenous gene or nucleic acid sequence, even though it can encode a native polypeptide or enzyme.

[00152] In other embodiments, any of the enzymes or polypeptides described herein can be a heterologous enzyme or polypeptide, and the polynucleotide, nucleic acid sequence, or gene, encoding the enzyme, is exogenous and is not produced by the cell, but instead is added to the cell from outside the cell.

[00153] A native, endogenous, or heterologous enzyme or polypeptide can be expressed or overexpressed in the recombinant cell or microbe or microorganism. For example, in some embodiments, an enzyme or polypeptide is native and is expressed in the recombinant cell or microbe by an endogenous nucleic acid sequence or gene. In other embodiments, the polypeptide or enzyme is native to the cell and is overexpressed, for example, where the recombinant cell or microbe contains an exogenous nucleic acid sequence encoding the native enzyme or polypeptide. In other embodiments, the enzyme or polypeptide is heterologous to the recombinant cell or microbe, and can be expressed or overexpressed in the recombinant cell or microbe by an exogenous nucleic acid sequence.

III. Recombinant microbes comprising the variant acyl-ACP thioesterases

[00154] As discussed above, there is a need for new and efficient recombinant microbes (also referred to herein as recombinant microorganisms) for producing fatty acids and derivatives thereof, particularly monounsaturated fatty acids and derivatives thereof, such as palmitoleic acid and palmitoleic acid esters (e.g., methyl or ethyl esters). Thus, in one embodiment, a recombinant microbe comprising a heterologous or variant acyl-ACP thioesterase, having at least 70%, 75%, 80%, or 85% sequence identity to SEQ ID NO:22, is disclosed herein. The heterologous or variant acyl-ACP thioesterase may have at least a 90% sequence identity, at least a 95% sequence identity, or at least a 99% sequence identity to SEQ ID NO: 22. The recombinant microbe may comprise any one of the variant acyl-ACP thioesterases (also referred to herein as acyl-ACP thioesterase variants) described above and elsewhere herein. In a specific embodiment, the acyl-ACP thioesterase variant comprises a sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21. In particular, the acyl-ACP thioesterase variant comprises a sequence corresponding to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21. The recombinant cell or microbe comprising any of the heterologous and/or variant acyl-ACP thioesterases described herein can further comprise one or more additional fatty acid derivative enzymes, and/or one or more additional fatty acid biosynthetic enzymes, which can be native or heterologous to the cell or microbe, or a combination thereof. For example, the recombinant microbe may also comprise a P-ketoacyl-ACP synthase (e.g., FabB and/or FabF). The recombinant microbe can further comprise at least one additional native or heterologous gene or nucleic acid sequence that encodes, for example, an acyl-CoA synthetase (ACS), an acyl-CoA reductase (ACR), a fatty alcohol forming acyl-CoA reductase (FAR), an ester synthase, an omega-hydroxylase (cohydroxylase), a carboxylic acid reductase (CAR), a desaturase, an aminotransferase or transaminase or amine dehydrogenase, a CoA-ligase/transferase, an alcohol-O-acetyl transferase, an aldehyde decarbonylase, an aldehyde oxidative deformylase, a decarboxylase, one or more subunits (e.g., AccA, AccB, AccC, and/or AccD) of an acetyl-CoA carboxylase (AccABCD), an OleA, an OleBCD, an OleABCD, an OleACD, an aldehyde dehydrogenase, and/or an alcohol dehydrogenase.

[00155] The recombinant cell or microbe described herein may be a bacterium, a yeast, or an algae. In one embodiment, the recombinant microbe is a recombinant proteobacterium, such as a y-proteobacterium (gamma-proteobacterium). The y-proteobacterium may be Escherichia coli, Salmonella spp., Vibrio natriegens, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Xanthomonas axonopodis, Pseudomonas syringae, Pseudomonas citronellolis , Pseudomonas mendocina, Pseudomonas plecoglossicida, Pseudomonas mosselii, Pseudomonas fulva, Xyella fastidiosa, or Marinobacter aquaeolei. In a particular embodiment, the y-proteobacterium can be Escherichia coli.

[00156] Additionally or alternatively, the recombinant cell or microbe may be a cyanobacterium, such as, for example, Synechococcus elongatus PCC7942 or Synechocystis sp. PCC6803.

[00157] Additionally or alternatively, the recombinant cell or microbe may be a yeast, such as, for example, Saccharomyces cerevisiae, Scheffersomyces stipitis, Schizosaccharomyces pombe, Kluyveromyces marxianus, K. lactis, Pichia pastoris, Hansenula polymorpha, or Yarrowia lipolytica, or the recombinant microbe can be an algae, such as, for example, Botryococcus braunii, Nannochloropsis gaditina, Chlamydomonas reinhardtii, Chlorella vulgaris., Spirulina platensis, Ostreococcus tauri, Phaeodactylum tricornutum, Symbiodinium sp., algal phytoplanktons, Saccharina japonica, Chlorococum spp., or Spirogyra spp.

[00158] As discussed herein, the recombinant cell or microbe can produce at least one monounsaturated free fatty acid or derivative thereof, or a composition comprising at least one monounsaturated free fatty acid or derivative thereof, such as, but not limited to, for example, Z7-tetradecenoic acid, Z9-tetradecenoic acid, Z13-hexadecenoic acid, Z11 -hexadecenoic acid, Z7-hexadecenoic acid, Z9-hexadecenoic acid, Z15-octadecenoic acid, Z13-octadecenoic acid, Z9-octadecenoic acid, Z7-octadecenoic acid, or Zl l-octadecenoic acid, and/or derivatives thereof. The recombinant cell or microbe may produce an ester derivative, such as a monounsaturated fatty acid alkyl ester. In certain embodiments, the fatty acid alkyl ester is a methyl or ethyl fatty acid ester. In some embodiments, the recombinant cell or microbe produces an ester of palmitoleic acid, such as, for example, palmitoleic acid ethyl ester or palmitoleic acid methyl ester. In some embodiments, the recombinant microbe (or microorganism or cell) comprising an acyl-ACP thioesterase variant provided herein produces a greater or higher amount, titer, and/or yield of a fatty acid and/or a derivative thereof, such as a monounsaturated fatty acid and/or derivative thereof, compared to a corresponding microbe (or microorganism or cell) comprising a wild-type or unmodified thioesterase, such as, for example, the thioesterase of SEQ ID NO:2 or SEQ ID NO:3. For example, the recombinant microbe (or microorganism or cell) produces an amount, titer, and/or yield of a (monounsaturated) fatty acid and/or derivative thereof that is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, greater than a corresponding microbe (or microorganism or cell) comprising a wild-type or unmodified thioesterase, such as, for example, the thioesterase of SEQ ID NO:2 or SEQ ID NO:3. In particular, the recombinant cell or microbe may produce 5 weight (wt)% or more, 10 wt% or more, 15 wt% or more, 20 wt% or more, 25 wt% or more, 30 wt% or more, 35 wt% or more, 40 wt% or more, 45 wt% or more, or 50 wt% or more of a fatty acid and/or derivative thereof, such as palmitoleic acid and/or a derivative thereof, e.g., palmitoleic acid ethyl ester, more than a control recombinant microbe comprising SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 (where the weight % is relative to the total weight of the fatty acids and/or derivatives thereof produced by the cell or microbe). In some embodiments, the recombinant microbe (or microorganism or cell), comprising a variant acyl-ACP thioesterase provided herein, produces an amount, titer, and/or yield of a (monounsaturated) fatty acid or derivative thereof that is at least about 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-, 2.1-, 2.2-, 2.3-, 2.4-, 2.5-, 2.6-, 2.7-, 2.8-, 2.9-, or 3-fold, or more, greater than a corresponding or otherwise isogenic microbe (or microorganism or cell), comprising a wild-type or unmodified thioesterase, such as, for example, the thioesterase of SEQ ID NO:2 or SEQ ID NO:3.

[00159] In some recombinant cells or microbes, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the total fatty acids or derivatives thereof, produced by the recombinant microbes, is palmitoleic acid ethyl ester.

[00160] Additionally or alternatively, one or more co-5 (omega-5) monounsaturated fatty acids or derivatives thereof may also be produced by the recombinant cell or microbe, such as, for example, (Z9)-tetradecenoic acid, (Z9)-tetradecenoic acid ethyl ester, (Zl l)-hexadecenoic acid, (Zl l)-hexadecenoic acid ethyl ester, Z13-octadecenoic acid, or Z13-octadecenoic acid ethyl ester, or a combination thereof.

[00161] Additionally or alternatively, the recombinant cell or microbe may produce a plurality of fatty acids or derivatives thereof, or a composition comprising the same, that is/are substantially free, as defined herein, of polyunsaturated fatty acids (PUFAs) or derivatives thereof, such as ethyl esters thereof. Additionally or alternatively, the recombinant cell or microbe may produce a plurality of fatty acids or derivatives thereof or a composition comprising the same, that is/are substantially free, as defined herein, of oleic acid and/or derivatives thereof, such as oleic acid ethyl ester.

[00162] Additionally or alternatively, the recombinant cell or microbe may produce one or more free fatty acids, such as hexadecanoic acid, tetradecanoic acid, tetradecenoic acid, All- hexadecenoic acid, octadecanoic acid, and All-octadecenoic acid. In a particular embodiment, hexadecanoic acid may comprise more than 1 wt%, more than 2 wt%, more than 3 wt%, more than 4 wt%, more than 5 wt%, more than 10 wt%, more than 15 wt%, more than 20 wt%, more than 25 wt%, more than 30 wt%, more than 35 wt%, more than 40 wt%, more than 45 wt%, or more than 50 wt%, of the total weight of a composition that comprises fatty acids. Compositions comprising fatty acids and/or derivatives thereof (e.g., monounsaturated fatty acid alkyl esters, one or more co-5 monounsaturated fatty acids or derivatives thereof, and a limited amount of oleic acid and/or PUFAs) are described at length in Section IV.

[00163] In some exemplary embodiments, the host cell (e.g., a recombinant microbe; or a recombinant bacterium, proteobacterium, cyanobacterium, yeast, or algae) may further comprise genetic manipulations and alterations to enhance or otherwise fine tune the production of saturated and/or monounsaturated free fatty acids or derivatives thereof. The optional genetic manipulations can be used interchangeably from one host cell to another, depending on what other heterologous enzymes and what native enzymatic pathways are present in the host cell. Some optional genetic manipulations include one or more of the following modifications described below.

[00164] The gene encoding acyl-CoA dehydrogenase (e.g., FadE) can optionally be attenuated or deleted in the recombinant cells, microbes, or microorganisms provided herein. FadE (Acyl-CoA dehydrogenase) catalyzes the first step in fatty acid utilization/degradation (P- oxidation cycle), which is the oxidation of acyl-CoA to 2-enoyl-CoA (see e.g., Campbell, J.W. and Cronan, J.E. Jr (2002) J. Bacteriol. 184(13):3759-3764; and Lennen, R.M. and Pfleger, B.F (2012) Trends Biotechnol. 30(12):659-667). Since FadE initiates the P-oxidation cycle, when E. coli lacks FadE, it cannot grow on fatty acids as a carbon source (see e.g., Campbell, J.W. and Cronan supra). The same effect can be achieved by attenuating or deleting other enzymes from the P-oxidation cycle, e.g., FadA, which is a 3-ketoacyl-CoA thiolase, or FadB, which is a dual 3-hydroxyacyl-CoA-dehydrogenase/dehydratase.

[00165] However, when a microbe such as E. coli is grown on a carbon source other than fatty acids, e.g., when it is grown on sugar, acetate, etc., FadE attenuation is optional, because under such conditions, FadE expression is repressed by FadR. Therefore, when cells are grown on a simple carbon source, such as, e.g., glucose, the FadE gene product is already attenuated. Accordingly, when grown on a carbon source other than fatty acids, a FadE mutation/deletion or attenuation is optional.

[00166] In some embodiments, the fatty acid biosynthetic pathway in the production host uses the precursors acetyl-CoA and malonyl-CoA. E. coli or other host organisms engineered to overproduce these components can serve as the starting point for subsequent genetic engineering steps to provide the specific output product (such as, fatty acids, fatty esters, hydrocarbons, fatty alcohols, etc.). Several different modifications can be made, either in combination or individually, to the host cell or strain, to obtain increased acetyl-CoA, malonyl-CoA, fatty acid, and/or fatty acid derivative production. See, for example, U.S. Patent Application Publication 2010/0199548, which is incorporated herein by reference in its entirety. For example, to increase malonyl-CoA production, one or more of the acetyl-CoA carboxylase subunits, including AccA, AccB, AccC, and/or AccD, can be expressed or overexpressed in the recombinant cell or microbe.

[00167] Other exemplary modifications of a host cell include, e.g., overexpression of nonnative and/or native and/or variants of genes involved in the synthesis of acyl-ACP. In general, increasing acyl-ACP synthesis increases the amount of acyl-ACP, which is the substrate of thioesterases, ester synthases, and acyl-ACP reductases. Exemplary enzymes that increase acyl- ACP production include, e.g., enzymes that make up the “fatty acid synthase” (FAS). As is known in the art (see e.g., U.S. 2010/0199548) FAS enzymes are a group of enzymes that catalyze the initiation and elongation of acyl chains. The acyl carrier protein (ACP) along with the enzymes in the FAS pathway control the length, degree of saturation, and branching of the fatty acids produced. FAS pathway enzymes include, for example, AccABCD, FabD, FabH, FabG, FabA, FabB, FabZ, FabF, FabI, FabK, FabU, FabM, FabQ, FabV, FabX, FabR, and FadR(see, e.g., Table A below for a description of these and other enzymes),, and homologs thereof and corresponding enzymes with the same activities that are derived from other organisms or species. Depending upon the desired product, one or more of these genes can be attenuated, deleted, downregulated, expressed, upregulated, or over-expressed, or otherwise modified or deregulated. The functions or exemplary uses for FAS genes (e.g., accA, accB, accC, accD, fabA, fabB, fabD, fabF, fabG, fabH, fabl, fabR, fabV, fabZ, fabK, fabL, fabM, fabX) are provided in Table A below. Table A also provides the functions or exemplary uses genes encoding other enzymes, including, for example, certain fatty acid derivative genes (e.g., acyl- CoA synthetases, thioesterases, ester synthases, alcohol dehydrogenases, acyl-CoA reductases, etc.). Any one or more of the genes listed in Table A can be expressed or overexpressed in the recombinant cells or microbes provided herein, including heterologously expressed or overexpressed. Additionally or alternatively, the expression or activity of any one or more of the genes listed in Table A can be altered, deregulated, or modified, for example, by attenuation, downregulation, or deletion of one or more genes and their encoded products.

[00168] In some embodiments, the recombinant cells, microbes, or microorganisms provided herein contain pathways that use a renewable feedstock, such as glucose, to produce fatty acids and derivatives thereof. Glucose is converted to an acyl-ACP by the native organism. Polynucleotides that code for polypeptides with fatty acid degradation enzyme activity can be optionally attenuated depending on the desired product. Non-limiting examples of such polypeptides are acyl-CoA synthetase (FadD) and acyl-CoA dehydrogenase (FadE). The Table below (Table A) provides a comprehensive list of enzymatic activity (infra) within the metabolic pathway, including various fatty acid degradation enzymes that can be optionally attenuated according to methods known in the art (see, e.g., U.S. Patent No. 8,283,143). [00169] For example, FadR (see Table A, infra) is a key regulatory factor involved in fatty acid degradation and fatty acid biosynthetic pathways (Cronan et al., Mol. Microbiol., 29(4): 937-943 (1998)). The E. coli enzyme FadD (see Table 1, infra) and the fatty acid transport protein FadL are components of a fatty acid uptake system. FadL mediates transport of fatty acids into the bacterial cell, and FadD mediates formation of acyl-CoA esters. When no other carbon source is available, exogenous fatty acids are taken up by bacteria and converted to acyl- CoA esters, which can bind to the transcription factor FadR and depress the expression of the fad genes that encode proteins responsible for fatty acid transport (FadL), activation (FadD), and P-oxidation (FadA, FadB, and FadE,). When alternative sources of carbon are available, bacteria synthesize fatty acids as acyl-ACPs, which are used for phospholipid synthesis, but are not substrates for P-oxidation. Thus, acyl-CoA and acyl-ACP are both independent sources of fatty acids that can result in different end-products (Caviglia et al., J. Biol. Chem., 279(12): 1163-1169 (2004)).

Table A: Enzymatic Activities

[00170] In some embodiments, a host strain may overexpress one or more of the FAS genes (e.g., any one or more of those described above and/or listed in Table A). Exemplary FAS genes that may be overexpressed include, e.g., FadR from Escherichia coli (see, e.g., GenBank Accession No. NP_415705.1), FabB from Escherichia coli (see, e.g., UniProtKB Accession No. P0A953), or FabZ from Escherichia coli (see, e.g., UniProtKB Accession No. P0A6Q6) or FabZ Acinetobacter baylyi (see, e.g., UniProtKB Accession No. Q6FCG4), as well as homologs thereof and corresponding enzymes, with the same activities, that are derived from other organisms or species. In another embodiment, the host strain encompasses optional overexpression of one or more genes, including, for example, fadR, fabA, fabD, fabG, fabH, fabV, and/or fabF. Examples of such genes are fadR from Escherichia coli, fabA from Salmonella typhimurium (NP_460041),/a/zD from Salmonella typhimurium (NP_460164), fabG from Salmonella typhimurium (NP_460165), /h/?/7 from Salmonella typhimurium (NP_460163), fabV from Vibrio cholera (YP_001217283), and tabF from Clostridium acetobutylicum (NP_350156). In some exemplary embodiments, the overexpression of one or more of these genes, which code for enzymes and regulators in fatty acid biosynthesis, serves to further increase the titer of fatty acids and fatty acid derivative compounds under particular culture conditions.

IV. Compositions

[00171] Further provided herein are compositions comprising saturated and/or monounsaturated fatty acids and/or derivatives thereof (e.g., fatty acid alkyl esters), and a limited amount of oleic acid and/or derivatives thereof, and/or a limited amount of polyunsaturated fatty acids (PUFAs) and/or derivatives thereof. Additionally, the composition may comprise one or more co-5 monounsaturated fatty acids or derivatives thereof (i.e., where the double bond is at the omega-5 position). All percentages used herein are with respect to the total weight of the composition. In a particular embodiment, the composition may comprise palmitoleic acid ethyl ester, one or more co-5 monounsaturated fatty acids or derivatives thereof, and less than 10 wt% of oleic acid and/or oleic acid ethyl ester. Alternatively, the composition may comprise less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or 0% (e.g., no) oleic acid and/or oleic acid ethyl ester, relative to the total weight of the composition. In a further embodiment, the composition comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% palmitoleic acid ethyl ester, relative to the total fatty acids or derivatives thereof, or relative to the total weight of the composition. In a particular embodiment, the composition may comprise at least 60% (at least 60 wt%) palmitoleic acid ethyl ester of the total fatty acids or derivatives thereof. [00172] The composition may comprise one or more monounsaturated fatty acids or derivatives thereof, such as, but not limited to, for example, Z7-tetradecenoic acid, Z9- tetradecenoic acid, Z7-hexadecenoic acid, Z9-hexadecenoic acid (palmitoleic acid), Zl l- hexadecenoic acid, Z13-hexadecenoic acid, Z9-octadecenoic acid, Zl l-octadecenoic acid, Z13- octadecenoic acid, Z15-octadecenoic acid, Z7 -hexadecenoic acid ester, Z9 -hexadecenoic acid ester (palmitoleic acid ester), Zl l -hexadecenoic acid ester, Z13-hexadecenoic acid ester, Z9- octadecenoic acid ester, Zl l-octadecenoic acid ester, Z13-octadecenoic acid ester, Z15- octadecenoic acid ester, 16-hydroxy-7(Z)-hexadecenoic acid, 16-hydroxy-9(Z)-hexadecenoic acid, 16-hydroxy-7(Z)-hexadecenoic acid ester, 16-hydroxy-9(Z)-hexadecenoic acid ester, Z9- hexadecenol, Zl l -hexadecenol, Zl l-hexadecenal, Zl l -hexadecenyl acetate, Z9-tetradecenol, Z9-tetradecenal, Z9-tetradecenyl acetate, Zl l-octadecenol, Zl l-octadecenal, Zl l-octadecenyl acetate, Z13-octadecenol, Z13-octadecenal, and/or Z13-octadecenyl acetate. As discussed above, the composition may comprise one or more co-5 monounsaturated fatty acids or derivatives thereof. Examples of such co-5 monounsaturated fatty acids or derivatives thereof include, without limitation, (Z9)-tetradecenoic acid, (Z9)-tetradecenoic acid ethyl ester, (Zl l)- hexadecenoic acid, (Zl l)-hexadecenoic acid ethyl ester, (Z13)-octadecenoic acid, (Z13)- octadecenoic acid ethyl ester, or a combination thereof. The co-5 monounsaturated fatty acids or derivatives thereof may be present in an amount of about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 30% or about 40%, or more, of the total weight of the composition.

[00173] Additionally or alternatively, the composition may be free or substantially free of polyunsaturated fatty acids (PUFAs) and/or derivatives thereof. For example, the composition may comprise less than about 10 wt%, less than about 9 wt%, less than about 8 wt%, less than about 7 wt%, less than about 6 wt%, less than about 5 wt%, less than about 4 wt%, less than about 3 wt%, less than about 2 wt%, less than about 1 wt%, less than about 0.5%, less than about 0.1 wt%, less than about 0.05 wt%, or less than about 0.01 wt%, or about 0 wt%, polyunsaturated fatty acids (PUFAs) and/or derivatives thereof.

[00174] Additionally or alternatively, the composition may further comprise one or more free fatty acids, such as, for example, hexadecanoic acid, tetradecanoic acid, tetradecenoic acid, octadecanoic acid, All-hexadecenoic acid, A9-octadecenoic acid, A13-octadecenoic acid, and/or Al l-octadecenoic acid. For example, the composition may comprise more than 1 wt%, more than 2 wt%, more than 3 wt%, more than 4 wt%, more than 5 wt%, more than 10 wt%, more than 15 wt%, more than 20 wt%, more than 25 wt%, more than 30 wt%, more than 35 wt%, more than 40 wt%, more than 45 wt%, or more than 50 wt%, hexadecanoic acid, relative to the total weight of the composition. Moreover, the composition may comprise more than 1 wt% hexadecanoic acid relative to the total weight of the composition. The composition can comprise at least 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, or more, of one or more saturated and/or monounsaturated free fatty acids, such as, for example, hexadecanoic acid, tetradecanoic acid, tetradecenoic acid, octadecanoic acid, Al l-hexadecenoic acid, A9-octadecenoic acid, A13- octadecenoic acid, and/or All-octadecenoic acid. Additionally or alternatively, the composition can comprise less than 30 wt% of saturated fatty acids and/or derivatives thereof.

[00175] Additionally or alternatively, the composition can comprise predominantly palmitoleic acid ethyl ester, and may be free or substantially free of both oleic acid ethyl ester and ethyl esters of polyunsaturated fatty acids. For example, the composition may comprise at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% palmitoleic acid ethyl ester, of the total fatty acids or derivatives thereof, and may comprise less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% oleic acid ethyl ester, and less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% ethyl esters of polyunsaturated fatty acids, each by weight of the composition. In particular, the composition may comprise at least 60% palmitoleic acid ethyl ester, less than 10% oleic acid ethyl ester, and less than 10% ethyl esters of polyunsaturated fatty acids, each by weight of the total composition. The composition may comprise more than about 80% (80 wt%) palmitoleic acid ethyl ester. In particular, the composition may be free or substantially free of oleic acid ethyl ester and/or ethyl esters of polyunsaturated fatty acids.

[00176] Additionally or alternatively, the composition may be a fermentation broth, prepared by culturing a recombinant cell or microbe described herein, such as a recombinant cell or microbe comprising a heterologous and/or variant acyl-ACP thioesterase, wherein the heterologous and/or variant acyl-ACP thioesterase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO: 22, or wherein the heterologous and/or variant acyl-ACP thioesterase has have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4-21, or wherein the variant thioesterase comprises the sequence of any one of SEQ ID NOs:4-21. The fermentation broth may comprise palmitoleic acid ethyl ester and/or one or more co-5 monounsaturated fatty acids or derivatives thereof, and/or one or more saturated fatty acids or derivatives thereof. [00177] Additionally, the fermentation broth may comprise one or more co-5 monounsaturated fatty acids or derivatives thereof. All percentages used herein are with respect to the total weight of the composition (comprising the fatty acids and/or derivatives thereof). In a particular embodiment, the fermentation broth may comprise palmitoleic acid ethyl ester, one or more co-5 monounsaturated fatty acids or derivatives thereof, and less than 10 wt% of oleic acid and/or derivatives thereof. Alternatively, the fermentation broth may comprise less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.1%, or 0% (e.g., no) oleic acid and/or derivatives thereof, relative to the total weight of the composition. In a further embodiment, the fermentation broth comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% palmitoleic acid ethyl ester, of the total fatty acids and/or derivatives thereof. In a particular embodiment, the fermentation broth may comprise at least 60% palmitoleic acid ethyl ester of the total fatty acids and/or derivatives thereof.

[00178] As discussed above, the fermentation broth may comprise one or more co-5 monounsaturated fatty acids or derivatives thereof. Examples of such co-5 monounsaturated fatty acids or derivatives thereof include, without limitation, (Z9)-tetradecenoic acid, (Z9)- tetradecenoic acid ethyl ester, (Zl l)-hexadecenoic acid, (Zl l)-hexadecenoic acid ethyl ester, (Z13)-octadecenoic acid, (Z13)-octadecenoic acid ethyl ester, or a combination thereof. The co-5 monounsaturated fatty acids or derivatives thereof may be present in an amount of about 1%, about 2%, about 3%, about 4%, 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 30%, or about 40%, or more, of the total weight of the composition.

[00179] Additionally or alternatively, the fermentation broth may be free or substantially free of polyunsaturated fatty acids (PUFAs) and/or derivatives thereof. For example, the fermentation broth may comprise less than about 5 wt%, less than about 4 wt%, less than about 3 wt%, less than about 2 wt%, less than about 1 wt%, less than about 0.5 wt%, less than about 0.1 wt%, less than about 0.05 wt%, less than about 0.01 wt%, or about 0 wt%, polyunsaturated fatty acids and/or derivatives thereof.

[00180] Additionally or alternatively, the fermentation broth may further comprise one or more free fatty acids, such as hexadecanoic acid, tetradecanoic acid, tetradecenoic acid, All- hexadecenoic acid, octadecanoic acid, A9-octadecenoic acid, A13-octadecenoic acid, and/or Al l-octadecenoic acid. For example, the fermentation broth may comprise more than 1 wt%, more than 2 wt%, more than 3 wt%, more than 4 wt%, more than 5 wt%, more than 10 wt%, more than 15 wt%, more than 20 wt%, more than 25 wt%, more than 30 wt%, more than 35 wt%, more than 40 wt%, more than 45 wt%, or more than 50 wt% hexadecanoic acid, relative to the total weight of the composition. Moreover, the fermentation broth may comprise more than 1 wt% hexadecanoic acid relative to the total weight of the fermentation broth. The fermentation broth can comprise at least 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, or more, by weight of the composition, of one or more saturated and/or monounsaturated free fatty acids, such as, for example, hexadecanoic acid, tetradecanoic acid, tetradecenoic acid, octadecanoic acid, Al l-hexadecenoic acid, A13-octadecenoic acid, and/or Al l-octadecenoic acid.

[00181] Additionally or alternatively, the fermentation broth can comprise predominantly palmitoleic acid ethyl ester, and can be or is free or substantially free of both oleic acid ethyl ester and ethyl esters of polyunsaturated fatty acids (PUFAs). For example, the fermentation broth may comprise at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% palmitoleic acid ethyl ester; less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% oleic acid ethyl ester; and less than less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% ethyl esters of polyunsaturated fatty acids, each by weight of the total fatty acids and/or derivatives thereof (or by weight of the composition). In particular, the fermentation broth may comprise at least 60% palmitoleic acid ethyl ester, less than 10% oleic acid ethyl ester, and less than 10% ethyl esters of polyunsaturated fatty acids, by weight of the composition. The fermentation broth may comprise more than about 80% (80 wt%) palmitoleic acid ethyl ester. In particular, the fermentation broth may be free or substantially free of oleic acid ethyl ester and/or ethyl esters of polyunsaturated fatty acids. Additionally or alternatively, the composition can comprise less than 30 wt% of saturated fatty acids and/or derivatives thereof.

V. Nucleotide sequences and vectors

[00182] Also described herein are nucleotide sequences encoding the acyl-ACP thioesterase variants having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or 100% sequence identity to SEQ ID NO:22, and degenerates thereof, and vectors comprising the nucleotide sequences and degenerates thereof that encode the acyl-ACP thioesterase variants having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or 100% sequence identity to SEQ ID NO:22. For example, the nucleotide sequence (or degenerates thereof) encoding the acyl-ACP thioesterase variant having at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:22 and/or a vector comprising the nucleotide sequence (or the degenerates thereof) encoding the acyl-ACP thioesterase having at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:22, may be constructed by methods well known in the art. Also provided herein are nucleotide sequences, and degenerates thereof, encoding the acyl-ACP thioesterase variants having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2 or SEQ ID NO:3, as well as vectors comprising such nucleotide sequences and degenerates thereof. In some embodiments, the nucleotide sequences (or degenerates thereof) encode acyl-ACP variants having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, or having 100% sequence identity, to any one of SEQ ID NOs:4-21. A degenerate nucleotide or nucleotide sequence (or nucleic acid sequence) is one that can perform the same function or yield the same product or output as a structurally different nucleotide or nucleotide sequence (or nucleic acid sequence). In some embodiments, the nucleic acid molecule or nucleic acid sequence comprises a sequence of nucleotides set forth in any one of SEQ ID NOs: 32-49. The nucleic acid sequences set forth in SEQ ID NOs:32-49 encode the amino acid sequences corresponding to SEQ ID NOs:4-21. In some embodiments, the variant acyl-ACP thioesterases provided herein are encoded by the nucleic acid sequences set forth in SEQ ID NOs:32-49, and/or by degenerates of the sequences of SEQ ID NOs:32-49, whereby a degenerate nucleic acid sequence encodes the same polypeptide (e.g., a variant acyl-ACP thioesterase provided herein), as the original or reference nucleic acid sequence it is a degenerate of.

[00183] The nucleotide sequence (or degenerates thereof) encoding the acyl-ACP thioesterase or acyl-ACP thioesterase variant having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:22, or to SEQ ID NO:2, or to SEQ ID NO:3, or to any one of SEQ ID NOs:4-21, such as, for example, any of the nucleic acid sequences set forth in SEQ ID NOs:32-49 and degenerates thereof, may be operably linked to one or more regulatory elements, including one or more heterologous regulatory elements. Where the vector comprises a nucleotide sequence (or degenerates thereof) encoding any of the acyl-ACP thioesterases or thioesterase variants recited above, and one or more nucleotide sequences (or degenerates thereof) encoding one or more additional proteins, polypeptides, or enzymes (e.g., fatty acid biosynthetic, or fatty acid derivative enzymes), the vector may comprise a single regulatory element, or a single heterologous regulatory element, that directs expression of both the acyl-ACP thioesterase (variant) and the additional protein(s)/polypeptide(s)/enzyme(s), or the vector may comprise additional elements or multiple heterologous regulatory elements that each independently directs expression of each of the acyl-ACP thioesterase (variant) and the one or more of the additional protein(s)/polypeptide(s)/enzyme(s) encoded by the vector.

[00184] As noted above, a polynucleotide or polypeptide can be overexpressed using methods well known in the art. In some embodiments, overexpression of a polypeptide is achieved by the use of an exogenous regulatory element. The term “exogenous regulatory element” generally refers to a regulatory element originating outside of the host cell. However, in certain embodiments, the term “exogenous regulatory element” can refer to a regulatory element derived from the host cell whose function is replicated or usurped for the purpose of controlling the expression of an endogenous polypeptide. For example, if the host cell is an E. coli cell, and the acyl-ACP thioesterase polypeptide is encoded by an endogenous gene, then expression of the endogenous gene can be controlled by a promoter derived from another E. coli gene or from another species entirely.

[00185] In some embodiments, the exogenous regulatory element is a chemical compound, such as a small molecule. As used herein, the term “small molecule” refers to a substance or compound having a molecular weight of less than about 1,000 g/mol.

[00186] In some embodiments, the exogenous regulatory element is an expression control sequence which is operably linked to the endogenous gene by recombinant integration into the genome of the host cell. In certain embodiments, the expression control sequence is integrated into a host cell chromosome by homologous recombination using methods well known in the art (see, e.g., Datsenko et al., Proc. Natl. Acad. Sci. U.S.A., 97(12) 6640-6645 (2000)).

[00187] In some embodiments, a vector described herein comprises a promoter operably linked to the polynucleotide sequence. 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 cell- specific promoter.

[00188] In some embodiments, a vector described herein comprises at least one sequence such as (a) an expression control sequence (or regulatory element) operatively coupled to the polynucleotide sequence; (b) a selection marker operatively coupled to the polynucleotide sequence; (c) a marker sequence operatively coupled to the polynucleotide sequence; (d) a purification moiety operatively coupled to the polynucleotide sequence; (e) a secretion sequence operatively coupled to the polynucleotide sequence; and/or (f) a targeting sequence operatively coupled to the polynucleotide sequence.

[00189] The expression vectors described herein include a polynucleotide sequence described herein in a form suitable for expression of the polynucleotide sequence in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. The expression vectors described herein can be introduced into host cells to produce polypeptides, including fusion polypeptides, encoded by the polynucleotide sequences as described herein.

[00190] Expression of genes encoding polypeptides in prokaryotes, for example, E. coli, is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino- or carboxy- terminus of the recombinant polypeptide. Such fusion vectors typically 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/or (3) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced 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.

[00191] 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 l id (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA, pp. 60-89 (1990)). In certain embodiments, a polynucleotide sequence of the invention 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. Cell Biol., 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 (Kaufinan et al., EMBO J., 6: 187-195 (1987)).

[00192] Vectors 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-precipitation, 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).

[00193] 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 host cells along with the gene of interest. Selectable markers include those that confer resistance to drugs such as, but not limited to, ampicillin, kanamycin, chloramphenicol, spectinomycin, or tetracycline. Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a polypeptide described 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.

[00194] Similarly, for stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to an antibiotic) can be introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin, and methotrexate. Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a polypeptide described herein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by growth in the presence of an appropriate selection drug.

VI. Methods of producing monounsaturated free fatty adds or derivatives thereof and cell cultures

[00195] Methods of producing saturated and/or monounsaturated free fatty acids or derivatives thereof, compositions comprising saturated and/or monounsaturated free fatty acids or derivatives thereof, cell cultures, and additional fatty acid compositions are also described herein. [00196] The recombinant cell or microbe described herein can be used to produce saturated and/or monounsaturated free fatty acids or derivatives thereof, particularly palmitoleic acid ethyl esters. Thus, in one embodiment, a method is provided herein, comprising culturing a recombinant cell or microbe comprising a heterologous acyl-ACP thioesterase, or an acyl-ACP thioesterase variant, having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:22, or any one of SEQ ID NOs:4-21, in or on a suitable carbon source. In some embodiments, the acyl- ACP thioesterase variant comprises the amino acid sequence set forth in any one of SEQ ID NOs:4-21. As described above, the recombinant cell or microbe may further comprise one or more enzymes, such as one or more fatty acid biosynthetic or fatty acid derivative enzymes, such as a P-ketoacyl-ACP synthase (e.g., FabB and/or FabF), an acyl-CoA synthetase, an acyl- CoA reductase, a fatty alcohol forming acyl-CoA reductase, an ester synthase, an omega- hydroxylase, a carboxylic acid reductase, a desaturase, an aminotransferase or transaminase or amine dehydrogenase, a CoA-ligase/transferase, an alcohol-O-acetyl transferase, an aldehyde decarbonylase, an aldehyde oxidative deformylase, a decarboxylase, one or more subunits (e.g., AccA, AccB, AccC, and/or AccD) of an acetyl-CoA carboxylase (AccABCD), an OleA, an OleBCD, an OleABCD, an OleACD, an aldehyde dehydrogenase, or an alcohol dehydrogenase, or combinations thereof. These enzymes may be native or heterologous, endogenous or exogenous, to the recombinant cell or microbe, and any of the enzymes can be expressed or overexpressed in the recombinant cell or microbe.

[00197] In general, saturated and/or monounsaturated free fatty acids and/or derivatives thereof are prepared by growing and/or fermenting the recombinant cell or microbe on or in a suitable carbon source. The recombinant cells or microbes are grown and/or fermented under appropriate conditions for a sufficient period of time to produce the saturated and/or monounsaturated free fatty acids and/or derivatives thereof. The carbon source may be culture media that comprises carbohydrates (e.g., monosaccharides, oligosaccharides, and/or polysaccharides), supplements (e.g., amino acids, antibiotics, polymers, acids, alcohols, aldehydes, ketones, peptides, and/or gases), and/or mineral salts. In a particular embodiment, the carbon source is LB media or nitrogen (N)-mineral media with glucose as a carbon source. In a further embodiment, the method further comprises isolating the saturated and/or monounsaturated free fatty acid(s) and/or derivative(s) thereof. The monounsaturated free fatty acid may be Z7-tetradecenoic acid, Z9 -tetradecenoic acid, Z7-hexadecenoic acid, Z9- hexadecenoic acid (palmitoleic acid), Zl l-hexadecenoic acid, Z13-hexadecenoic acid, Z9- octadecenoic acid, Zl l-octadecenoic acid, Z13 -octadecenoic acid, Z15-octadecenoic acid, Z7- hexadecenoic acid ester, Z9-hexadecenoic acid ester (palmitoleic acid ester), Zl l -hexadecenoic acid ester, Z13-hexadecenoic acid ester, Z9-octadecenoic acid ester, Zl l-octadecenoic acid ester, Z13-octadecenoic acid ester, Z15-octadecenoic acid ester, 16-hydroxy-7(Z)-hexadecenoic acid, 16-hydroxy-9(Z)-hexadecenoic acid, 16-hydroxy-7(Z)-hexadecenoic acid ester, 16- hydroxy-9(Z)-hexadecenoic acid ester, Z9-hexadecenol, Zl l -hexadecenol, Zll-hexadecenal, Zl l -hexadecenyl acetate, Z9-tetradecenol, Z9-tetradecenal, Z9-tetradecenyl acetate, Zl l- octadecenol, Zl l-octadecenal, Zl l-octadecenyl acetate, Z13-octadecenol, Z13-octadecenal, or Z13-octadecenyl acetate, and/or derivatives thereof.

[00198] In another embodiment, a saturated and/or monounsaturated free fatty acid or derivative thereof is prepared by culturing the recombinant cell or microbe, or a cell culture comprising the recombinant cell or microbe, whereby the recombinant cell or microbe comprises a heterologous or variant acyl-ACP thioesterase having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% sequence identity to SEQ ID NO:22, in or on a suitable carbon source. In certain embodiments, a saturated and/or monounsaturated free fatty acid or derivative thereof is prepared by culturing the recombinant cell or microbe, or a cell culture comprising the recombinant cell or microbe, whereby the recombinant cell or microbe comprises an acyl-ACP thioesterase variant having least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, sequence identity to any one of SEQ ID NOs:2-22, or an acyl-ACP thioesterase variant comprising the sequence set forth in any one of SEQ ID NOs:4-21, in or on a suitable carbon source. In some embodiments, the saturated and/or monounsaturated free fatty acid or derivative thereof is isolated from the cell culture or fermentation broth. In a further embodiment, the saturated and/or monounsaturated free fatty acid or derivative thereof is purified. In a still further embodiment, the saturated and/or monounsaturated free fatty acid or derivative thereof is purified by a method such as a two-step centrifugation and water- washing; decanting centrifugation and solvent extraction from a biomass; and/or whole broth extraction with a water immiscible solvent.

[00199] Thus, also provided herein is a cell culture comprising any of the recombinant cells or microbes described herein, and one or more saturated and/or monounsaturated free fatty acids or derivatives thereof. Additionally provided herein is a fatty acid or fatty acid derivative composition produced by the recombinant microbe or cell culture as described herein.

[00200] In some embodiments, the saturated and/or monounsaturated free fatty acids or derivatives thereof are placed in a composition comprising the saturated and/or monounsaturated free fatty acids or derivatives thereof, wherein the monounsaturated free fatty acids or derivatives thereof are prepared by culturing and/or fermenting the recombinant cell or microbe (or cell culture comprising the recombinant cell or microbe). In some embodiments, the composition comprises one or more than one (e.g., two, three, four, five, or more) monounsaturated free fatty acids or derivatives thereof. In a preferred embodiment, the monounsaturated free fatty acid or derivative thereof is palmitoleic acid ethyl ester.

[00201] Additionally or alternatively, a method for making a product, wherein the method comprises adding palmitoleic acid, palmitoleic acid ethyl ester, or a combination thereof to a product precursor, wherein the palmitoleic acid or palmitoleic acid ethyl ester is obtained from a recombinant cell or microbe (or cell culture comprising the recombinant cell or microbe) comprising a heterologous acyl-ACP thioesterase or an acyl-ACP thioesterase variant having at 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:22, SEQ ID NO:3, SEQ ID NO:2, or to any one of SEQ ID NOs:4-21, or having 100% sequence identity to any one of SEQ ID NOs:4-21, is provided herein. Additionally or alternatively, a method for making a product, wherein the method comprises adding derivatives of (Z7)-hexadecenoic acid, 16-hydroxy-7(Z)-hexadecenoic acid, , 16- hydroxy-9(Z)-hexadecenoic acid, Z9-hexadecenoic acid (palmitoleic acid), Zl l-hexadecenoic acid, Z7-tetradecenoic acid, Z13-hexadecenoic acid, Z9-octadecenoic acid, Z9-tetradecenoic acid, Zl l -octadecenoic acid, Z13-octadecenoic acid (e.g., Z7 -hexadecenolide, Z9- hexadecenolide, Z9-hexadecenol, Zl l-hexadecenol, Zl l-hexadecenal, Z11 -hexadecenyl acetate, Z9-tetradecenol, Z9-tetradecenal, Z9 -tetradecenyl acetate, Zl l -octadecenol, Zl l-octadecenal, Zl l -octadecenyl acetate, Z13-octadecenol, Z13-octadecenal, and/or Z13 -octadecenyl acetate), Z15-octadecenoic acid, Z7-hexadecenoic acid ester, Z9-hexadecenoic acid ester (palmitoleic acid ester), Zl l-hexadecenoic acid ester, Z13-hexadecenoic acid ester, Z9-octadecenoic acid ester, Zl l-octadecenoic acid ester, Z13-octadecenoic acid ester, Z15-octadecenoic acid ester, 16-hydroxy-7(Z)-hexadecenoic acid ester, and/or 16-hydroxy-9(Z)-hexadecenoic acid ester, to a product precursor, wherein the compounds or the derivatives thereof, are obtained from a recombinant cell or microbe (or cell culture comprising the recombinant cell or microbe) comprising an acyl-ACP thioesterase variant having at 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:22, SEQ ID NO:3, SEQ ID NO:2, or to any one of SEQ ID NOs:4-21, or having 100% sequence identity to any one of SEQ ID NOs:4-21, is provided herein. The product precursor may be a fragrance precursor, a flavor precursor, a pheromone precursor, a nutraceutical precursor, a nutritional or dietary supplement precursor, or a pharmaceutical precursor, or a combination thereof. In some embodiments, the product is a fragrance, flavor, nutraceutical, nutritional, dietary, pheromone, or pharmaceutical product, or a precursor thereof, or a combination thereof. V. Uses

[00202] The variant acyl-ACP thioesterases, recombinant cells or microbes, and cell cultures described herein can be used for a variety of purposes. In particular, the variant acyl-ACP thioesterases, recombinant cells or microbes, and cell cultures may be used to produce one or more of a saturated and/or monounsaturated free fatty acid or a derivative thereof, or a composition comprising one or more of a saturated and/or monounsaturated free fatty acid or a derivative thereof, or a product or product precursor or ingredient comprising one or more of a saturated and/or monounsaturated free fatty acid or a derivative thereof or a composition comprising the same.

[00203] In some embodiments, the saturated and/or monounsaturated free fatty acid and/or derivative thereof, prepared by the cultured and/or fermented recombinant cell or microbe, is used in a composition. In some embodiments, the saturated and/or monounsaturated free fatty acid and/or derivative thereof is a fermentation product of the recombinant microbe or cell culture. In other embodiments, the composition comprises one or more than one (e.g., two, three, four, five, or more) particular species of monounsaturated free fatty acids and/or derivatives thereof. In a particular embodiment, the composition is a fragrance, flavor, pheromone, nutraceutical, nutritional, dietary, or pharmaceutical composition, or a precursor thereof.

[00204] In some embodiments, the saturated and/or monounsaturated free fatty acid and/or derivative thereof is prepared at a time and/or location that is different than when the composition is prepared. For example, the saturated and/or monounsaturated free fatty acid and/or derivative thereof may be produced by the recombinant microbe or cell culture in one location (e.g., a first facility, city, state, or country), transported to another location (e.g., a second facility, city, state, or country), and then incorporated into the composition comprising the saturated and/or monounsaturated free fatty acid(s) and/or derivative(s) thereof.

[00205] In some embodiments, the saturated and/or monounsaturated free fatty acid and/or derivative thereof is purified. In some instances, the saturated and/or monounsaturated free fatty acid and/or derivative thereof is purified prior to its use in the composition. The saturated and/or monounsaturated free fatty acid and/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.

[00206] In some embodiments, the saturated and/or monounsaturated free fatty acids and/or derivatives thereof are insoluble or highly insoluble in water. In such cases, the saturated and/or monounsaturated free fatty acids and/or derivatives thereof are in a separate phase from the environment in which the recombinant microbes (or cell culture) reside (e.g., fermentation broth). In some embodiments, the saturated and/or monounsaturated free fatty acids and/or derivatives thereof are solid at room temperature. In another embodiment, the saturated and/or monounsaturated free fatty acids and/or derivatives thereof (e.g., alcohol derivatives) are liquid. [00207] Additional purification steps may be required depending on the final product’s applications and specifications. These steps may include saponification, bleaching, and eventually distillation if high purity of a single chain length is required. All these are standard unit operations that are used regularly in the industry.

[00208] In another particular embodiment, purification of the saturated and/or monounsaturated free fatty acids and/or derivatives thereof involves isolating and recovering fatty acids. Purification of fatty acids differs from the separation of alcohols in that the fatty acids mixed with the biomass are both solids.

[00209] Two different approaches can be applied:

[00210] One approach includes recovery of the solid phase of biomass plus product via decanting centrifugation, followed by solvent extraction of the product from the biomass with an appropriate solvent (e.g., methanol or ethanol). The fatty acids dissolve in the solvent and the biomass is removed either by centrifugation or filtration, or a combination thereof. The recovery of the fatty acids is then completed by evaporating the solvent. Depending on the application, the product can be further used as a solution in the solvent, or as a solid. Other purification steps, including distillation, could be applied to meet final specifications.

[00211] Another approach includes recovery of the product via whole broth extraction with a water immiscible solvent. In this approach, the fermentation broth is contacted in either batch or continuous schemes with an appropriate solvent (e.g., butyl acetate, medium chain alcohols, or esters) to allow for the complete dissolution of the product in the solvent. The light organic solvent phase can be separated from the water phase in a similar way as those described for the recovery of the long chain alcohols. Once a clear solvent phase has been obtained, the final product is again recovered by solvent evaporation.

[00212] In another embodiment, the saturated and/or monounsaturated free fatty acids or derivatives thereof prepared by the recombinant microbe (or cell culture), or a composition comprising the saturated and/or monounsaturated free fatty acids or derivatives thereof prepared by the recombinant microbe (or cell culture), is/are incorporated into a product. This product is made by combining, mixing, or otherwise using the saturated and/or monounsaturated free fatty acid(s) or derivative(s) thereof produced by the recombinant microbe (or cell culture), in combination with other or more additional components, to prepare the product. The product may comprise one or more than one (e.g., two, three, four, five, or more) saturated and/or monounsaturated free fatty acids or derivatives thereof prepared by the recombinant microbe (or cell culture). In a particular embodiment, the product is a pheromone or a precursor thereof, a fragrance or a precursor thereof, a pharmaceutical agent or a precursor thereof, a flavor or a precursor thereof, a nutraceutical or a precursor thereof, or a nutritional supplement or a precursor thereof.

EXAMPLES

[00213] The following examples are provided to further illustrate the invention disclosed herein but should not be construed as in any way limiting its scope.

[00214] Example 1: General Protocols

[00215] (A) Small Scale Fermentation:

[00216] 40 pL LB culture (from an LB culture growing in a 96 well plate) was used to inoculate 360 pL LB media, which was then incubated with shaking for approximately 4 hours at 32°C. 80 pL of the LB seed was used to inoculate 320 pL N-LIM media (see, Table 8), optionally containing an alcohol (e.g., methanol or ethanol; 0.5-2% v/v) when fatty acid ethyl esters and not free fatty acids were the target products. After growing at 32°C for 2 hours, the cultures were induced with isopropyl P-D-l -thiogalactopyranoside (IPTG) (final concentration 1 mM). The cultures were then incubated at 32°C with shaking for 20 hours (unless otherwise noted), after which they were extracted with the extraction protocol detailed below.

[00217] Table 8: N-LIM Media Formulation

* Aminolevulinic acid is only added for fatty acid derivatives, not for production of free fatty acids. [00218] (B) Free Fatty Add Species Extraction and Analytical Protocols:

[00219] To each well to be extracted, 80 pL of IM HC1, followed by 400 pL of butyl acetate containing 500 mg/L 1 -undecanol as an internal standard, was added. The 96 well plates were then heat-sealed and shaken for 30 minutes at 2000 rpm. After shaking, the plates were centrifuged for 10 minutes at 4500 rpm at room temperature to separate the aqueous and organic layers. 50 pL of the organic layer was transferred to a 96 well plate and derivatized with 50 pL of TMS/BSTFA. The plate was subsequently heat-sealed and stored at -20°C until evaluated by either GC-FID or GC-MS.

[00220] Fatty Acid Analytics: [00221] The GC-MS parameters used to generate chromatograms and mass spectra for compound identification were as follows:

[00222] Table 9. GC-MS Parameters

[00223] The mass spectrometry parameters are shown in Table 10. [00224] Table 10. Mass Spectrometry Parameters

[00225] The GC-FID parameters used to quantify each compound are shown in Table 11:

[00226] Table 11. GC-FID Parameters

[00227] The protocols detailed above represent standard conditions, which can be modified as necessary.

[00228] Example 2: Identification of Engineered FatA Thioesterases with Improved Activity in Comparison to SEQ ID NO:3 from a Site Saturation Library

[00229] This example describes the generation/engineering and identification of variant acyl- ACP thioesterases (variant FatA thioesterases), containing single amino acid substitutions that confer improved activity in comparison to the corresponding wild-type thioesterase (SEQ ID NO:3). The variants have improved activity for producing long-chain fatty acids and improved selectivity for production of palmitoleic acid (A9-hexadecenoic acid; C16:l) in particular.

[00230] The gene (SEQ ID NO:1) encoding mature wild-type acyl-ACP thioesterase (FatA) (with the amino acid sequence set forth in SEQ ID NO:3) was cloned into a pCL1920-derivative vector (SC 101 replicon, spectinomycin resistance marker), such that its transcription was controlled by the IPTG-inducible Ptrc promoter. The plasmid (p AS.040) was transformed into an E. coli MG1655 derivative strain. The resulting strain produced over 1 g/L of free fatty acids (FFA) from glucose as assayed by the analytical protocol described above. This transformed E. coli served as the control for evaluating the engineered acyl-ACP thioesterase variants described herein. The FFA produced by the control strain included mainly hexadecanoic acid (C16:0; also known as palmitic acid) and A9-hexadecenoic acid (Cl 6:1; also known as palmitoleic acid). The strain also produced tetradecanoic acid (C14:0; also known as myristic acid) and All- octadecenoic acid (Cl 8:1; also known as vaccenic acid).

[00231] Standard techniques known to those of skill in the art were used to prepare a site saturation library of FatA in plasmid pAS.040. The library was transformed into the same E. coli MG1655 derivative strain described above and screened for FatA variants with increased/improved enzyme activity (z.e., increased free fatty acid titer) and/or increased/improved palmitoleyl acyl-ACP specificity and/or selectivity (z.e., increased percentage of palmitoleic acid produced), in comparison to that of the control strain comprising the wild-type FatA of SEQ ID NO:3.

[00232] The library was screened as described in Example 1 above. FatA variants with improved enzyme activity and/or specificity/selectivity were identified and are shown in Table 12 below.

[00233] Table 12: Summary of thioesterase variants with improved properties from a site saturation library of FatA (SEQ ID NO:3).

* FIOC: Fold Improvement Over Control (wild-type FatA)

[00234] The amino acid mutations/substitutions shown in Table 12 above are listed with respect to residue positioning in the mature FatA thioesterase of SEQ ID NO:3. The same mutations also can be introduced into the full-length FatA thioesterase of SEQ ID NO:2. For example, the mutations D20S, V40M, T50R, T83C, T83K, and V147A, with reference to SEQ ID NO:3, correspond to the mutations D70S, V90M, T100R, T133C, T133K, and V197A, respectively, with reference to SEQ ID NO:2.

[00235] As shown in Table 12, variants with the mutations D20S, V40M, T50R, T83C, T83K, and V147A, with reference to SEQ ID NO:3, all resulted in the production of increased amounts of free fatty acids,

improved enzyme activity, in comparison to the corresponding unmodified (wild-type) thioesterase. The variants produced similar amounts of free fatty acids with a 16-carbon chain length (Cl 6 Production in Table 12), which included both saturated and unsaturated C16 FFAs. However, the variants all produced a higher percentage of C16:l FFA (palmitoleic acid) compared to the control, indicating that the mutations conferred improved specificity and/or selectivity for the C16:l acyl-ACP substrate.

[00236] Example 3: Identification of Engineered FatA Thioesterases with Improved Activity in Comparison to SEQ ID NO:4 from a Site Saturation Library

[00237] This example illustrates the generation and characterization of variant acyl-ACP thioesterases (variant FatA thioesterases) that contain a single V147A substitution (with reference to SEQ ID N0:3) and an additional amino acid substitution. The double substitution variants conferred improved production of long-chain length fatty acids (i.e., improved enzyme activity) and/or improved specificity and/or selectivity for production of palmitoleic acid (C16:l) in particular, as compared to the variant acyl-ACP thioesterase comprising a single V147A substitution (i.e., SEQ ID NO:4).

[00238] The gene coding for the acyl-ACP thioesterase variant of SEQ ID NO:3, having the V147A substitution (SEQ ID NO:4), was cloned into a pCL1920-derivative vector (SC101 replicon, spectinomycin resistance marker), and its transcription was controlled by the IPTG- inducible Ptrc promoter. The variant acyl-ACP thioesterase gene formed an operon with a gene coding for a P-ketoacyl-ACP synthase (FabB). This plasmid was named pLKW.071.

[00239] Plasmid pLKW.071 was transformed into the E. coli MG1655 derivative strain described above. The resulting strain was screened for fatty acid derivative production as described in Example 1 and was capable of producing over 2 g/L of free fatty acids from glucose and served as the control for evaluating novel, dual substitution FatA thioesterase variants. The fatty acid derivatives produced by the control strain included mainly derivatives of hexadecanoic acid (C16:0; also known as palmitic acid) and A9-hexadecenoic acid (C16:l; also known as palmitoleic acid), but also included derivatives of tetradecanoic acid (C14:0; also known as myristic acid) and Al l-octadecenoic acid (Cl 8:1; also known as vaccenic acid).

[00240] A partial site saturation library of the acyl-ACP thioesterase variant with the V147A substitution (SEQ ID NO:4) was prepared in the pLKW.071 plasmid. The library was transformed into the E. coli MG1655 derivative strain and screened for FatA variants with improved or increased enzyme activity, as measured by an increased FFA titer, and/or with improved or increased palmitoleyl acyl-ACP selectivity or specificity (such as an increased percentage of palmitoleic acid production), as compared to the control strain with SEQ ID NO:4. [00241] The library was screened as described above. FatA variants with improved enzyme activity and/or specificity/selectivity for C16:l acyl-ACP substrate are shown in table 13.

[00242] Table 13: Summary of improved variants from a partial saturation library of FatA(V147A) (SEQ ID NO:4).

* FIOC: Fold improvement over FatA(V147A) control

[00243] The amino acid mutations/substitutions shown in Table 13 above are listed with respect to residue positioning in the mature FatA thioesterase of SEQ ID NO:3. The same mutations also can be introduced into the full-length FatA thioesterase of SEQ ID NO:2. For example, the mutations V147A, L292G, I299T, I299V, T3O3Q, and L305R, with reference to SEQ ID NOG, correspond to the mutations V197A, L342G, I349T, I349V, T353Q, and L355R, respectively, with reference to SEQ ID NO:2.

[00244] Example 4: Identification of Engineered FatA Thioesterases with Improved Activity in Comparison to SEQ ID NO:4 from a Combination Library

[00245] This example illustrates the generation and characterization of variant acyl-ACP thioesterases (variant FatA thioesterases) that contain two or more amino acid substitutions compared to the corresponding wild-type FatA thioesterase. The variants comprising multiple mutations in acyl-ACP thioesterase showed improved production of long-chain length fatty acids and/or improved selectivity and/or specificity for production of palmitoleic acid (C16:l) and its derivatives in particular, as compared to the variant acyl-ACP thioesterase comprising a single V147A substitution (i.e., a control of SEQ ID NO:4).

[00246] Omega-hydroxy fatty acids were produced by the recombinant strains in this Example, by engineering the strains to encode an omega-hydroxylase, which converts free fatty acids, made by the variant thioesterases provided herein, to the corresponding omega-hydroxy fatty acids. Omega-hydroxy fatty acids are exemplary fatty acid derivatives; as described elsewhere herein and as known in the art, other fatty acid derivatives can be made from the fatty acids produced by the variant thioesterases provided herein, by expressing the appropriate fatty acid derivative enzyme(s). For example, strains expressing a carboxylic acid reductase (CAR) can produce fatty aldehydes; strains expressing a CAR and an alcohol dehydrogenase can produce fatty alcohols; strains expressing an acyl-CoA synthetase and an ester synthase can produce fatty esters; strains expressing a CAR and a transaminase or aminotransferase can produce fatty amines, and so forth.

[00247] The gene coding for acyl-ACP thioesterase having the V147A substitution (with the amino acid sequence set forth in SEQ ID NO:4) was cloned into a pCL1920-derivative vector (SC 101 replicon, spectinomycin resistance marker), and its transcription was controlled by the IPTG-inducible Ptrc promoter. The variant acyl-ACP thioesterase gene formed an operon with a gene coding for a P-ketoacyl-ACP synthase (FabB). In addition, the plasmid contained a fatty acid derivative enzyme (an omega-hydroxylase), controlled by an IPTG-inducible PT5 promoter. This plasmid was named pEP.362.

[00248] Plasmid pEP.362 was transformed into the E. coli MG1655 derivative strain described above. The resulting strain was screened for fatty acid derivative production as described in Example 1. The strain comprising the thioesterase variant with the V147A substitution (i.e., SEQ ID NO:4) was capable of producing over 3 g/L of total fatty acid derivatives/species (including C14-C18 free fatty acids and C14-C18 omega-hydroxy fatty acids) from glucose and served as the control for evaluating novel, FatA variants with multiple amino acid substitutions. The fatty acid derivatives produced by the control strain included mainly derivatives (i.e., free fatty acids and omega-hydroxy fatty acids) of hexadecanoic acid (C16:0; also known as palmitic acid) and A9-hexadecenoic acid (C16:l; also known as palmitoleic acid), but also included derivatives (i.e., free fatty acids and omega-hydroxy fatty acids) of tetradecanoic acid (C14:0; also known as myristic acid) and All-octadecenoic acid (Cl 8:1; also known as vaccenic acid).

[00249] A combination library of FatA(V147A) was generated using the transfer PCR (tPCR) protocol (see, Erijman et al. (2011) J. Structural Bio. 175:171-177) in plasmid pEP.362. The library was transformed into the same E. coli MG 1655 derivative strain described above and screened for FatA variants with increased/improved enzyme activity i.e., increased total fatty acid derivative titer), in comparison to that of the control strain with SEQ ID NO:4.

[00250] The library was screened as described above. FatA variants with improved enzyme activity were identified and are shown in Table 14.

[00251] Table 14: Summary of improved variants from a combination library of FatA(V147A) (SEQ ID NO:4).

* FIOC: Fold improvement over FatA(V147A) control [00252] The amino acid mutations/substitutions shown in Table 14 above are listed with respect to residue positioning in the mature FatA thioesterase of SEQ ID NO:3. The same mutations also can be introduced into the full-length FatA thioesterase of SEQ ID NO:2. For example, the mutations D20S, S47E, T50R, N58G, T82D, V147A, and S186L, with reference to SEQ ID NOG, correspond to the mutations D70S, S97E, T100R, N108G, T132D, V197A, and S236L, respectively, with reference to SEQ ID NO:2.

[00253] Example 5: Production of Fatty Acid Esters with High Percentage of Palmitoleic Acid Ester by Strains Expressing Variant FatA Thioesterases

[00254] This example describes the production of fatty acid esters by recombinant microorganisms expressing the FatA thioesterase variants provided herein. The production of fatty acid ethyl esters (FAEEs) is exemplified herein, however, similar results are expected for the production of other fatty acid esters, such as, for example, fatty acid methyl esters (FAMEs), fatty acid propyl esters, and others, for example, by substitution of ethanol with the appropriate alcohol (e.g., methanol for the production of FAMEs, or propanol for the production of fatty acid propyl esters, etc.).

[00255] More specifically, described herein are recombinant E. coli strains that express selected FatA variants and produce fatty acid ethyl ester (FAEE) compositions which are high in palmitoleic acid ethyl ester (Z9-C16:l FAEE) and do not contain oleic acid ethyl ester (Z9- C18:l FAEE) or ethyl esters of polyunsaturated fatty acids (PUFAs).

[00256] The gene encoding the acyl-ACP thioesterase FatA(V147A) variant (amino acid sequence set forth in SEQ ID NO:4), or the FatA(D20S, N58G, V147A) variant (amino acid sequence set forth in SEQ ID NO: 16), was cloned into a pCL1920-derivative vector (SC101 replicon, spectinomycin resistance marker), such that transcription of each gene was controlled by the IPTG-inducible Ptrc promoter, and such that each FatA variant gene formed an operon with a P-ketoacyl-ACP synthase I gene (JabB). The vector backbones also contained a second operon containing a gene encoding an ester synthase from Limnobacter (UniProtKB Accession No. A6GSQ9; SEQ ID NO:27) controlled by an IPTG-inducible PT5 promoter.

[00257] The resulting plasmids (pKM.038 with FatA(V147A) and pKM.026 with FatA(D20S, N58G, V147A)) were transformed into an E. coli MG1655 derivative strain comprising the following genomic modifications: (i) the genes encoding acyl-CoA dehydrogenase (FadE) and the transcriptional regulator FabR were deleted; (ii) a variant of the transcriptional regulator FadR was overexpressed; and (iii) the gene encoding acyl-CoA synthetase (FadD) was overexpressed. In addition, an operon containing (i) a A9-hexadecanoyl- ACP desaturase gene (without the plastid targeting leader sequence) from Arabidopsis thaliana (encoding amino acids 38-401 of SEQ ID NO:24 and of UniProtKB Accession No. Q9LF05 with a Met added at position 1); (ii) a flavodoxin reductase gene (fpf) from E. coli (see, e.g., UniProtKB Accession No. P28861; SEQ ID NO:25); and (iii) a ferredoxin gene (petF) from Nostoc punctiforme PCC 73102 (UniProtKB Accession No. B2J0U5; SEQ ID NO:30), all controlled by a constitutive PT5 promoter, was integrated into the bacterial chromosome. The resulting strains were designated sKM.309 (containing plasmid pKM0.26) and sKM.348 (containing plasmid pKMO.38).

[00258] The two strains were subjected to small scale fermentation in the presence of ethanol (2%, v/v) as described in Example 1 and product analysis was carried out as described in Example 1. Strain sKM.309, containing the FatA thioesterase variant with the mutations D20S, N58G, and V147A (SEQ ID NO: 16), produced 1597 mg/L fatty acid ethyl ester (FAEE) and no detectable free fatty acids (FFA). Strain sKM.348, containing the FatA thioesterase variant with the V147A mutation (SEQ ID NO:4), produced 2445 mg/L FAEE and 13 g/L FFA. The FAEE compositions produced by strains sKM.309 and sKM.348 are shown in FIGs. 1A and IB, respectively. A GC chromatograph from the broth extract of strain sKM.348 is shown in FIG. 2. [00259] As shown in FIGs. 1A and IB, respectively, and in Table 15 below, strains sKM.309 and sKM.348 produced FAEE compositions with 82.7 wt% and 86.2 wt% palmitoleic acid ethyl ester (Z9-C16:l FAEE), respectively. The FAEE compositions produced by strains sKM.309 and sKM.348 contained 6.9 wt% and 5.6 wt%, respectively, of palmitic acid ethyl ester (C16:0 FAEE). Small amounts of omega-5 (co-5) unsaturated ethyl esters, for example, Zl l- hexadecenoic acid ethyl ester (Zl l-C16:l FAEE) and/or Z13-octadecenoic acid ethyl ester (Z13-C18:l FAEE), were also detected in the FAEE compositions of both strains. On the other hand, oleic acid ethyl ester (Z9-C18:l FAEE), and ethyl esters of polyunsaturated fatty acids (PUFAs), were undetectable in the broth from both strains.

[00260] Table 15: Compositions Produced by Recombinant Strains Expressing the Variant Acyl-ACP Thioesterases

[00261] This example, thus, demonstrates that recombinant cells/microbes (exemplified herein by E. coli strains) that express the variant acyl-ACP thioesterases provided herein, are capable of producing fatty acid ethyl ester compositions that are very high in palmitoleic acid ethyl ester content, and that do not contain detectable amounts of oleic acid ethyl ester (Z9- C18:l FAEE) or ethyl esters of polyunsaturated fatty acids (PUFAs).

[00262] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[00263] Particular embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those particular embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[00264] Sequence Table

Claims

CLAIMS What is claimed is:

1. A variant acyl-ACP thioesterase, comprising one or more amino acid substitutions in the sequence of amino acids of an unmodified acyl-ACP thioesterase polypeptide, wherein: the unmodified acyl-ACP thioesterase polypeptide comprises the sequence of amino acids set forth in SEQ ID NO:3, or a sequence of amino acids having at least about 70% sequence identity to the sequence of amino acids set forth in SEQ ID NO:3; and the variant acyl-ACP thioesterase comprises one or more amino acid substitutions at one or more amino acid positions corresponding to positions 20, 40, 47, 50, 58, 82, 83, 147, 186, 292, 299, 303, or 305, or a combination thereof, with reference to SEQ ID NO:3.

2. The variant acyl-ACP thioesterase of claim 1, wherein the variant acyl-ACP thioesterase has 1 or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions, insertions, or deletions, compared to the unmodified acyl-ACP thioesterase polypeptide of SEQ ID NO:3.

3. The variant acyl-ACP thioesterase of claim 1 or claim 2, wherein the variant acyl-ACP thioesterase comprises one or more amino acid substitutions corresponding to D20S, V40M, S47E, T50R, N58G, T82D, T83C, T83K, V147A, S186L, L292G, I299T, I299V, T3O3Q, or L305R, or a combination thereof, with reference to SEQ ID NOG.

4. The variant acyl-ACP thioesterase of claim 1 or claim 2, comprising one or more substitutions at amino acid positions corresponding to:

147;

147 and 292;

147 and 299;

147 and 303;

147 and 305;

20, 47, and 147;

20, 50, and 147;

20, 58, and 147;

50, 58, and 147;

20, 58, 82, and 147;

47, 82, 147, and 186; or

47, 50, 58, 82, and 147, wherein the positions are listed with reference to SEQ ID NO:3.

5. The variant acyl-ACP thioesterase of claim 4, comprising amino acid substitutions corresponding to: V147A; V147A/L292G; V147A/I299T; V147A/I299V; V147A/T303Q; V147A/L305R; D20S/S47E/V147A; D20S/T50R/V147A; D20S/N58G/V147A;

T50R/N58G/V147A; D20S/N58G/T82D/V147A; S47E/T82D/V147A/S186L; or

S47E/T50R/N58G/T82D/V147A

6. The variant acyl-ACP thioesterase of any one of claims 1-5, comprising an amino acid sequence having at least about 70% sequence identity to the sequence set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.

7. The variant acyl-ACP thioesterase of any one of claims 1-5, comprising an amino acid sequence corresponding to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, or SEQ ID NO:21.

8. A variant acyl-ACP thioesterase, comprising one or more amino acid modifications in the sequence of amino acids of an unmodified acyl-ACP thioesterase polypeptide, wherein: the unmodified acyl-ACP thioesterase polypeptide comprises the sequence of amino acids set forth in SEQ ID NO:2, or a sequence of amino acids having at least 70% sequence identity to the sequence of amino acids set forth in SEQ ID NO:2; the amino acid modification(s) are selected from among amino acid substitution(s), deletion(s), and/or insertion(s) in the unmodified acyl-ACP thioesterase polypeptide; and the variant acyl-ACP thioesterase comprises one or more amino acid substitutions at one or more amino acid positions corresponding to positions 70, 90, 97, 100, 108, 132, 133, 197, 236, 342, 349, 353, or 355, or a combination thereof, with reference to SEQ ID NO:2.

9. The variant acyl-ACP thioesterase of claim 8, comprising a deletion of all or a portion of the plastid transit peptide, corresponding to amino acid residues 1-38, 2-38, 1-51, 2-51, 1-66, 2- 66, 1-67, 2-67, 1-68, or 2-68, of SEQ ID NO:2.

10. The variant acyl-ACP thioesterase of claim 8 or claim 9, comprising wherein the variant acyl-ACP thioesterase has 1 or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions, insertions, or deletions, compared to the unmodified acyl-ACP thioesterase polypeptide of SEQ ID NO:2.

11. The variant acyl-ACP thioesterase of any one of claims 8-10, wherein the variant acyl- ACP thioesterase comprises one or more amino acid substitutions corresponding to D70S, V90M, S97E, T100R, N108G, T132D, T133C, T133K, V197A, S236L, L342G, I349T, I349V, T353Q, or L355R, or a combination thereof, with reference to SEQ ID NO:2.

12. The variant acyl-ACP thioesterase of any one of claims 1-11, wherein the variant acyl- ACP thioesterase, when expressed in a cell, results in the production of a saturated and/or monounsaturated medium-chain to long-chain fatty acid or derivative thereof, or a composition comprising a saturated and/or monounsaturated medium-chain to long-chain fatty acid or derivative thereof.

13. The variant acyl-ACP thioesterase of any one of claims 1-12, wherein the variant acyl- ACP thioesterase exhibits one or more improved properties selected from increased thioesterase activity, increased specificity for a substrate, and/or increased selectivity for a substrate, in comparison to a corresponding wild-type, unmodified, or reference acyl-ACP thioesterase polypeptide.

14. The variant acyl-ACP thioesterase of any one of claims 1-7, wherein the variant acyl- ACP thioesterase exhibits one or more improved properties selected from increased thioesterase activity, increased specificity for a substrate, and/or increased selectivity for a substrate, compared to SEQ ID NOG.

15. The variant acyl-ACP thioesterase of claim 13 or claim 14, wherein: the increased thioesterase activity results in an increased amount, titer, yield, and/or productivity of a saturated or monounsaturated medium-chain to long-chain fatty acid or derivative thereof; and the increased specificity and/or selectivity is towards a saturated or monounsaturated medium-chain to long-chain acyl-ACP substrate.

16. The variant acyl-ACP thioesterase of claim 15, wherein the saturated or monounsaturated medium-chain to long-chain fatty acid or derivative thereof, or the saturated or monounsaturated medium-chain to long-chain acyl-ACP substrate is a C14-C20 fatty acid or derivative thereof, or a C14-C20 acyl-ACP substrate.

17. The variant acyl-ACP thioesterase of claim 15, wherein: the increased thioesterase activity results in an increased amount, titer, yield, and/or productivity of a monounsaturated long-chain fatty acid or derivative thereof; and the increased specificity and/or selectivity is towards a monounsaturated long-chain acyl- ACP substrate.

18. The variant acyl-ACP thioesterase of claim 17, wherein: the monounsaturated long-chain fatty acid or derivative thereof is a C16:l or C18:l fatty acid or derivative thereof, or a combination thereof; and the monounsaturated long-chain acyl-ACP substrate is a C16:l or C18:l acyl-ACP, or a combination thereof.

19. The variant acyl-ACP thioesterase of any one of claims 12-18 wherein the fatty acid or derivative thereof is a free fatty acid, a fatty alcohol, a fatty diol, a 1,3-fatty diol, an alpha, omega(a,co)-diol, a fatty aldehyde, a fatty amine, a fatty amide, a fatty acid ester, a fatty acid methyl ester (FAME), a fatty acid ethyl ester (FAEE), a fatty acid acetate ester, a fatty alcohol acetate ester, a hydroxylated fatty acid, a hydroxylated fatty acid ester, an omegahydroxy (co-hydroxy) fatty acid, an co-hydroxy fatty acid ester, an a,co-diester, an a, co-diacid, an co-carboxy fatty ester, a derivative with a free fatty acid on one end and a fatty acid ester on the other end, a derivative with a free fatty acid on one end and an amine on the other end, a derivative with a fatty acid ester on one end and an amine on the other end, or a combination thereof.

20. The variant acyl-ACP thioesterase of any one of claims 12-19, wherein the monounsaturated fatty acid or derivative thereof is Z7-tetradecenoic acid, Z9-tetradecenoic acid, Z7-hexadecenoic acid, Z9-hexadecenoic acid (palmitoleic acid), Zl l -hexadecenoic acid, Z13- hexadecenoic acid, Z9-octadecenoic acid, Zl l-octadecenoic acid, Z13-octadecenoic acid, Z15- octadecenoic acid, Z7 -hexadecenoic acid ester, Z9-hexadecenoic acid ester (palmitoleic acid ester), Zl l-hexadecenoic acid ester, Z13 -hexadecenoic acid ester, Z9-octadecenoic acid ester, Zl l-octadecenoic acid ester, Z13-octadecenoic acid ester, Z15-octadecenoic acid ester, 16- hydroxy-7(Z)-hexadecenoic acid, 16-hydroxy-9(Z)-hexadecenoic acid, 16-hydroxy-7(Z)- hexadecenoic acid ester, 16-hydroxy-9(Z)-hexadecenoic acid ester, Z9-hexadecenol, Zl l- hexadecenol, Zl l-hexadecenal, Zl l -hexadecenyl acetate, Z9-tetradecenol, Z9-tetradecenal, Z9- tetradecenyl acetate, Zl l-octadecenol, Zl l-octadecenal, Zl l -octadecenyl acetate, Z13- octadecenol, Z13-octadecenal, or Z13-octadecenyl acetate, or a combination thereof.

21. The variant acyl-ACP thioesterase of claim 20, wherein the monounsaturated fatty acid or derivative thereof is palmitoleic acid, or palmitoleic acid ethyl ester, or a combination thereof.

22. A nucleic acid molecule, encoding the variant acyl-ACP thioesterase of any one of claims 1-21.

23. The nucleic acid molecule of claim 22, comprising a sequence of nucleotides set forth in any one of SEQ ID NOs: 32-49, or degenerate sequences thereof.

24. The nucleic acid molecule of claim 22 or claim 23, wherein the nucleic acid molecule is operably linked to one or more heterologous regulatory elements

25. A vector, comprising the nucleic acid molecule of any one of claims 22-24.

26. An isolated cell or cell culture, comprising the variant acyl-ACP thioesterase of any one of claims 1-21, or the nucleic acid molecule of claim 22 or claim 23, or the vector of claim 24 or claim 25.

27. A recombinant cell, comprising the variant acyl-ACP thioesterase of any one of claims 1-21, or the nucleic acid molecule of claim 22 or claim 23, or the vector of claim 24 or claim 25.

28. A recombinant microbe, comprising the variant acyl-ACP thioesterase of any one of claims 1-21, or the nucleic acid molecule of claim 22 or claim 23, or the vector of claim 24 or claim 25.

29. A recombinant microbe, comprising a variant acyl-ACP thioesterase, wherein: variant acyl-ACP thioesterase comprises an amino acid sequence having at least about 70% sequence identity to the sequence of amino acids set forth in SEQ ID NO:3; and the variant acyl-ACP thioesterase comprises one or more amino acid substitutions at one or more amino acid positions corresponding to positions 20, 40, 47, 50, 58, 82, 83, 147, 186, 292, 299, 303, or 305, or a combination thereof, with reference to SEQ ID NO:3.

30. The recombinant microbe of claim 29, wherein the variant acyl-ACP thioesterase has 1 or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions, insertions, or deletions, compared to the unmodified acyl-ACP thioesterase polypeptide of SEQ ID NO:3.

31. The recombinant microbe of claim 29 or claim 30, wherein the variant acyl-ACP thioesterase comprises one or more amino acid substitutions corresponding to D20S, V40M, S47E, T50R, N58G, T82D, T83C, T83K, V147A, S186L, L292G, I299T, I299V, T3O3Q, or L305R, or a combination thereof, with reference to SEQ ID NOG.

32. The recombinant microbe of claim 31, wherein the variant acyl-ACP thioesterase comprises amino acid substitutions corresponding to V147A; V147A/L292G; V147A/I299T; V147A/I299V; V147A/T303Q; V147A/L305R; D20S/S47E/V147A; D20S/T50R/V147A; D20S/N58G/V147A; T50R/N58G/V147A; D20S/N58G/T82D/V147A;

S47E/T82D/V147A/S186L; or S47E/T50R/N58G/T82D/V147A.

33. The recombinant microbe of any one of claims 29-32, comprising an amino acid sequence having at least about 70% sequence identity to the sequence set forth in SEQ ID NO:4, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.

34. The isolated cell, recombinant cell, or recombinant microbe of any one of claims 26-33, further comprising at least one fatty acid biosynthesis enzyme, or at least one fatty acid derivative enzyme, or a combination thereof.

35. The isolated cell, recombinant cell, or recombinant microbe of claim 34, wherein the fatty acid biosynthesis enzyme and/or the fatty acid derivative enzyme is heterologous to the cell or microbe.

36. The isolated cell, recombinant cell, or recombinant microbe of claim 34 or claim 35, wherein the at least one fatty acid biosynthesis enzyme or the at least one fatty acid derivative enzyme is a P-ketoacyl-ACP synthase I, a P-ketoacyl-ACP synthase II, an acyl-CoA synthetase, an acyl-CoA reductase, a fatty alcohol forming acyl-CoA reductase, an ester synthase, an omega-hydroxylase (co-hydroxylase), a carboxylic acid reductase, a desaturase, a transaminase, an aminotransferase, an amine dehydrogenase, a CoA-ligase/transferase, an alcohol-O-acetyl transferase, an aldehyde decarbonylase, an aldehyde oxidative deformylase, a decarboxylase, one or more subunits of an acetyl-CoA carboxylase (AccABCD), an OleA, an OleBCD, an OleABCD, an OleACD, an aldehyde dehydrogenase, or an alcohol dehydrogenase, or a combination thereof.

37. The isolated cell, recombinant cell, or recombinant microbe of any one of claims 26-36, wherein the cell or microbe produces one or more saturated and/or monounsaturated mediumchain to long-chain fatty acids or derivatives thereof, or produces a composition comprising one or more saturated and/or monounsaturated medium-chain to long-chain fatty acids or derivatives thereof.

38. The isolated cell, recombinant cell, or recombinant microbe of any one of claims 26-36, wherein the cell or microbe produces at least one monounsaturated free fatty acid or a derivative thereof, or produces a composition comprising at least one monounsaturated free fatty acid or a derivative thereof.

39. The isolated cell, recombinant cell, or recombinant microbe of claim 37 or claim 38 wherein: the saturated and/or monounsaturated fatty acid or derivative thereof is a C14-C20 saturated and/or monounsaturated fatty acid or derivative thereof; or the monounsaturated long-chain fatty acid or derivative thereof is a C16:l or a C18:l fatty acid or derivative thereof, or a combination thereof.

40. The isolated cell, recombinant cell, or recombinant microbe of any one of claims 37-39, wherein the fatty acid or derivative thereof is a free fatty acid, a fatty alcohol, a fatty diol, a 1,3- fatty diol, an alpha, omega(a,co)-diol, a fatty aldehyde, a fatty amine, a fatty amide, a fatty acid ester, a fatty acid methyl ester (FAME), a fatty acid ethyl ester (FAEE), a fatty acid acetate ester, a fatty alcohol acetate ester, a hydroxylated fatty acid, a hydroxylated fatty acid ester, an omegahydroxy (co-hydroxy) fatty acid, an co-hydroxy fatty acid ester, an a,co-diester, an a, co-diacid, an co-carboxy fatty ester, a derivative with a free fatty acid on one end and a fatty acid ester on the other end, a derivative with a free fatty acid on one end and an amine on the other end, a derivative with a fatty acid ester on one end and an amine on the other end, or a combination thereof.

41. The isolated cell, recombinant cell, or recombinant microbe of any one of claims 37-40, wherein the monounsaturated fatty acid or derivative thereof is Z7-tetradecenoic acid, Z9- tetradecenoic acid, Z7-hexadecenoic acid, Z9-hexadecenoic acid (palmitoleic acid), Zl l- hexadecenoic acid, Z13-hexadecenoic acid, Z9-octadecenoic acid, Zl l-octadecenoic acid, Z13- octadecenoic acid, Z15-octadecenoic acid, Z7 -hexadecenoic acid ester, Z9 -hexadecenoic acid ester (palmitoleic acid ester), Zl l -hexadecenoic acid ester, Z13-hexadecenoic acid ester, Z9- octadecenoic acid ester, Zl l-octadecenoic acid ester, Z13-octadecenoic acid ester, Z15- octadecenoic acid ester, 16-hydroxy-7(Z)-hexadecenoic acid, 16-hydroxy-9(Z)-hexadecenoic acid, 16-hydroxy-7(Z)-hexadecenoic acid ester, 16-hydroxy-9(Z)-hexadecenoic acid ester, Z9- hexadecenol, Zl l -hexadecenol, Zl l-hexadecenal, Zl l -hexadecenyl acetate, Z9-tetradecenol, Z9-tetradecenal, Z9-tetradecenyl acetate, Zl l-octadecenol, Zl l-octadecenal, Zl l-octadecenyl acetate, Z13-octadecenol, Z13-octadecenal, or Z13-octadecenyl acetate, or a combination thereof.

42. The isolated cell, recombinant cell, or recombinant microbe of any one of claims 37-40, wherein the monounsaturated fatty acid or derivative thereof is palmitoleic acid (Z9- hexadecenoic acid), or palmitoleic acid ethyl ester (Z9-hexadecenoic acid ethyl ester), or a combination thereof.

43. The isolated cell, recombinant cell, or recombinant microbe of any one of claims 37-42, wherein the cell or microbe produces a higher amount of one or more saturated and/or monounsaturated fatty acids or derivatives thereof, compared to a corresponding cell or microbe that comprises the thioesterase of SEQ ID NO:2 or SEQ ID NO:3.

44. The isolated cell, recombinant cell, or recombinant microbe of claim 43, wherein the cell or microbe produces one or more monounsaturated fatty acids or derivatives thereof in an amount that is 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more, higher than the amount produced by a corresponding cell or microbe that comprises the thioesterase of SEQ ID NO:2 or SEQ ID NO:3.

45. The isolated cell, recombinant cell, or recombinant microbe of any one of claims 26-44, wherein the cell or microbe is a bacterium, a cyanobacterium, a yeast, or an algae.

46. The isolated cell, recombinant cell, or recombinant microbe of claim 45 that is a y- proteobacterium.

47. The isolated cell, recombinant cell, or recombinant microbe of claim 46, wherein the y- proteobacterium is selected from Escherichia coli, Salmonella spp., Vibrio natriegens, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Xanthomonas axonopodis, Pseudomonas syringae, Xyella fastidiosa, and Marinobacter aquaeolei.

48. The isolated cell, recombinant cell, or recombinant microbe of claim 47, wherein the y- proteobacterium is Escherichia coli.

49. The isolated cell, recombinant cell, or recombinant microbe of any one of claims 26-44, wherein the cell or microbe is a cyanobacterium selected from Synechococcus elongatus PCC7942 and Synechocystis sp. PCC6803.

50. The isolated cell, recombinant cell, or recombinant microbe of claim 45, wherein the cell or microbe is a yeast selected from Saccharomyces cerevisiae, Scheffersomyces stipitis, Schizosaccharomyces pombe, Kluyveromyces marxianus, K. lactis, Pichia pastoris, Hansenula polymorpha, and Yarrowia lipolytica or an algae selected from the group consisting of Botryococcus braunii, Nannochloropsis gaditina, Chlamydomonas reinhardtii, Chlorella vulgaris., Spirulina platensis, Ostreococcus tauri, Phaeodactylum tricornutum, Symbiodinium sp., algal phytoplanktons, Saccharina japonica, Chlorococum spp., and Spirogyra spp.

51. A composition, comprising one or more monounsaturated fatty acids or derivatives thereof selected from Z7-tetradecenoic acid, Z9-tetradecenoic acid, Z7 -hexadecenoic acid, Z9- hexadecenoic acid (palmitoleic acid), Zl l-hexadecenoic acid, Z13-hexadecenoic acid, Z9- octadecenoic acid, Zl l-octadecenoic acid, Z13 -octadecenoic acid, Z15-octadecenoic acid, Z7- hexadecenoic acid ester, Z9-hexadecenoic acid ester (palmitoleic acid ester), Zl l-hexadecenoic acid ester, Z13-hexadecenoic acid ester, Z9-octadecenoic acid ester, Zl l-octadecenoic acid ester, Z13-octadecenoic acid ester, Z15-octadecenoic acid ester, 16-hydroxy-7(Z)-hexadecenoic acid, 16-hydroxy-9(Z)-hexadecenoic acid, 16-hydroxy-7(Z)-hexadecenoic acid ester, 16- hydroxy-9(Z)-hexadecenoic acid ester, Z9-hexadecenol, Zl l -hexadecenol, Zll-hexadecenal, Zl l -hexadecenyl acetate, Z9-tetradecenol, Z9-tetradecenal, Z9-tetradecenyl acetate, Zl l- octadecenol, Zl l-octadecenal, Zl l-octadecenyl acetate, Z13-octadecenol, Z13-octadecenal, and Z13-octadecenyl acetate, or a combination thereof.

52. A composition, comprising palmitoleic acid ethyl ester and one or more co-5 fatty acids or derivatives thereof.

53. The composition of claim 52, further comprising less than 10 wt% oleic acid and/or derivatives thereof.

54. The composition of claim 52 or claim 53, further comprising less than 10 wt% of polyunsaturated fatty acids and/or derivatives thereof.

55. The composition of any one of claims 52-54, further comprising less than 30 wt% of saturated fatty acids and/or derivatives thereof.

56. The composition of any one of claims 52-55, wherein the composition comprises at least 60 wt%, 65 wt%, 70 wt%, 75 wt%, or 80 wt%, of palmitoleic acid ethyl ester.

57. The composition of any one of claims 52-56, wherein the one or more co-5 fatty acids or derivatives thereof are selected from (Z9)-tetradecenoic acid, (Z9)-tetradecenoic acid ethyl ester, (Zl l)-hexadecenoic acid, (Zl l)-hexadecenoic acid ethyl ester, (Z13)-octadecenoic acid, (Z13)- octadecenoic acid ethyl ester, and a combination thereof.

58. The composition of any one of claim 52-57, wherein the composition is free or substantially free of polyunsaturated fatty acids and/or derivatives thereof.

59. The composition of any one of claims 52-58, further comprising one or more free fatty acids selected from palmitoleic acid, hexadecanoic acid, tetradecanoic acid, tetradecenoic acid, Al l-hexadecenoic acid, octadecanoic acid, and Al l-octadecenoic acid.

60. The composition of any one of claims 52-59, comprising at least 1 wt% hexadecanoic acid, relative to the total weight of the composition.

61. A composition, comprising at least 60 wt% palmitoleic acid ethyl ester, less than 10 wt% oleic acid ethyl ester, and less than 10 wt% of ethyl esters of polyunsaturated fatty acids.

62. The composition of claim 61, wherein the composition comprises at least about 80 wt% palmitoleic acid ethyl ester.

63. The composition of claim 61 or 62, wherein the composition is free or substantially free of ethyl esters of polyunsaturated fatty acids and/or is free or substantially free of oleic acid ethyl ester.

64. A composition, comprising a fermentation broth prepared by culturing the isolated cell, recombinant cell, or recombinant microbe of any one of claims 26-50, wherein the fermentation comprises palmitoleic acid ethyl ester and one or more co-5 fatty acids or derivatives thereof.

65. The composition of claim 64, comprising:

(i) at least 60 wt% palmitoleic acid ethyl ester;

(ii) one or more co-5 fatty acids or derivatives thereof;

(iii) less than 10 wt% oleic acid ethyl ester;

(iv) less than 10 wt% of ethyl esters of polyunsaturated fatty acids; and

(v) less than 30 wt% of saturated fatty acids and/or derivatives thereof.

66. A cell culture, comprising the isolated cell, recombinant cell, or recombinant microbe, of any one of claims 26-50.

67. Use of the composition of any one of claims 51-65 for the preparation of a nutraceutical, nutritional, dietary, pharmaceutical, pheromone, fragrance, or flavor product or ingredient, or a precursor thereof.

68. Use of variant acyl-ACP thioesterase of any one of claims 1-21, or the isolated cell, recombinant cell, or recombinant microbe of any one of claims 26-50, or the cell culture of claim 66, for producing a monounsaturated free fatty acid or derivative thereof, or for producing a composition comprising a monounsaturated free fatty acid or derivative thereof.

69. A method for producing a monounsaturated fatty acid or derivative thereof, or for producing a composition comprising a monounsaturated fatty acid or derivative thereof, the method comprising culturing the isolated cell, recombinant cell, or recombinant microbe of any one of claims 26-50, or the cell culture of claim 66, in or on a carbon source.

70. The method of claim 69, further comprising isolating the monounsaturated fatty acid or derivative thereof, or the composition comprising the monounsaturated fatty acid or derivative thereof.

71. The method of claim 69 or 70, wherein the monounsaturated fatty acid or derivative thereof is Z7-tetradecenoic acid, Z9-tetradecenoic acid, Z7 -hexadecenoic acid, Z9-hexadecenoic acid (palmitoleic acid), Zl l-hexadecenoic acid, Z13-hexadecenoic acid, Z9-octadecenoic acid, Zl l -octadecenoic acid, Z13-octadecenoic acid, Z15-octadecenoic acid, Z7-hexadecenoic acid ester, Z9 -hexadecenoic acid ester (palmitoleic acid ester), Zl l-hexadecenoic acid ester, Z13- hexadecenoic acid ester, Z9-octadecenoic acid ester, Zl l -octadecenoic acid ester, Z13- octadecenoic acid ester, Z15-octadecenoic acid ester, 16-hydroxy-7(Z)-hexadecenoic acid, 16- hydroxy-9(Z)-hexadecenoic acid, 16-hydroxy-7(Z)-hexadecenoic acid ester, 16-hydroxy-9(Z)- hexadecenoic acid ester, Z9-hexadecenol, Zl l -hexadecenol, Zl l-hexadecenal, Zl l-hexadecenyl acetate, Z9-tetradecenol, Z9-tetradecenal, Z9-tetradecenyl acetate, Z11 -octadecenol, Zl l- octadecenal, Zl l -octadecenyl acetate, Z13-octadecenol, Z13-octadecenal, or Z13-octadecenyl acetate, or a combination thereof

72. The method of any one of claims 69-71, wherein the monounsaturated fatty acid or derivative thereof is palmitoleic acid or palmitoleic acid ethyl ester, or a combination thereof.

73. A monounsaturated fatty acid or derivative thereof, or a composition comprising a monounsaturated fatty acid or derivative thereof, prepared by the method of any one of claims 69-72.

74. A monounsaturated fatty acid or derivative thereof, or a composition comprising a monounsaturated fatty acid or derivative thereof, produced by the isolated cell, recombinant cell, or recombinant microbe of any one of claims 26-50.

75. A monounsaturated fatty acid or derivative thereof, or a composition comprising a monounsaturated fatty acid or derivative thereof, produced by the cell culture of claim 66.

76. The monounsaturated fatty acid or derivative thereof, or the composition comprising the monounsaturated fatty acid or derivative thereof, of any one of claims 73-75, that is purified.

77. The monounsaturated fatty acid or derivative thereof, or the composition, of claim 76, wherein the monounsaturated fatty acid or derivative thereof, or the composition, is purified by a two-step centrifugation and water- washing; decanting centrifugation and solvent extraction from a biomass; or whole broth extraction with a water immiscible solvent.

78. Use of monounsaturated fatty acid or derivative thereof, or the composition, of any one of claims 73-77, for the preparation of a nutraceutical, nutritional, dietary, pharmaceutical, pheromone, fragrance, or flavor product or ingredient, or a precursor thereof.

79. A method for making a product, the method comprising: adding palmitoleic acid or palmitoleic acid ethyl ester, or a combination thereof, to a product precursor; wherein the palmitoleic acid or palmitoleic acid ethyl ester is obtained from or produced by the isolated cell, recombinant cell, or recombinant microbe of any one of claims 26-50.

80. A method for making a product, the method comprising adding a monounsaturated fatty acid and/or a derivative thereof to a product precursor, wherein: the monounsaturated fatty acid and/or derivative thereof is obtained from or produced by the isolated cell, recombinant cell, or recombinant microbe of any one of claims 26-50; and the monounsaturated fatty acid and/or derivative thereof is Z7-tetradecenoic acid, Z9- tetradecenoic acid, Z7-hexadecenoic acid, Z9-hexadecenoic acid (palmitoleic acid), Zl l- hexadecenoic acid, Z13-hexadecenoic acid, Z9-octadecenoic acid, Zl l-octadecenoic acid, Z13- octadecenoic acid, Z15-octadecenoic acid, Z7 -hexadecenoic acid ester, Z9 -hexadecenoic acid ester (palmitoleic acid ester), Zl l -hexadecenoic acid ester, Z13-hexadecenoic acid ester, Z9- octadecenoic acid ester, Zl l -octadecenoic acid ester, Z13-octadecenoic acid ester, Z15- octadecenoic acid ester, 16-hydroxy-7(Z)-hexadecenoic acid, 16-hydroxy-9(Z)-hexadecenoic acid, 16-hydroxy-7(Z)-hexadecenoic acid ester, 16-hydroxy-9(Z)-hexadecenoic acid ester, Z9- hexadecenol, Zl l -hexadecenol, Zl l-hexadecenal, Zl l -hexadecenyl acetate, Z9-tetradecenol, Z9-tetradecenal, Z9-tetradecenyl acetate, Zl l-octadecenol, Zl l-octadecenal, Zl l-octadecenyl acetate, Z13-octadecenol, Z13-octadecenal, or Z13-octadecenyl acetate, or derivatives thereof.

81. The method of claim 79 or claim 80, wherein the product precursor is a fragrance precursor, a flavor precursor, a pheromone precursor, a nutraceutical precursor, a nutritional or dietary supplement precursor, or a pharmaceutical precursor.

82. The method of any one of claims 79-81, wherein the product is a fragrance, flavor, nutraceutical, nutritional, dietary, pheromone, or pharmaceutical product.