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  • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
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  • C12N9/10—Transferases (2.)
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  • C12P7/00—Preparation of oxygen-containing organic compounds
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  • C12Y203/00—Acyltransferases (2.3)
  • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
  • C12Y203/0118—Beta-ketoacyl-acyl-carrier-protein synthase III (2.3.1.180)
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  • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
  • C12P7/6436—Fatty acid esters

Definitions

• Fatty acid and fatty acid derivatives are important precursors to manufacturing many consumer products, industrial chemicals and fuels.
• fatty acids and fatty acid derivatives are used to make detergents, cleaners, plastics, paper, paints, lubricants, waxes, coatings and surfactants. They can also be used as flavor and fragrance agents.
• fatty acids and fatty acid derivatives are produced from oleochemical (plant and animal fats) or petrochemical sources. In general, the fatty acids derived from oleochemical sources have aliphatic chains with an even number of carbons, whereas fatty acids derived from petrochemical sources have aliphatic chains with an odd number of carbons.
• the feedstocks used to produce such fatty acids generally include a mixture of fatty acids of varying carbon chain lengths and may include a wide range of chain lengths, as well as saturated and unsaturated fatty acids.
• Figure 1 is a chart that illustrates the fatty acid carbon composition of various common oleochemical feedstocks.
• Many of the commercial applications for fatty acids and fatty acid derivatives require a fatty acid precursor having greater specificity with respect to its aliphatic chain lengths.
• C6-C10 fatty acids are used in the production of jet lubricants
• C12-C14 fatty acids are used to make surfactants and detergents
• C16 - CI 8 fatty acids are used for metal soap production.
• the disclosure provides for a genetically modified organism comprising a heterologous nucleic acid sequence encoding a 3-ketoacyl-CoA synthase, a ketoacyl-CoA reductase, a hydroxyacyl-CoA dehydratase, or an enoyl-CoA reductase; and wherein said microorganism is capable of producing a fatty acid or fatty acid-derived product having a carbon chain length of C4 or greater.
• the 3-ketoacyl-CoA synthase comprises NphT7.
• the 3-ketoacyl-CoA synthase comprises an amino acid sequence of at least 70% homology to any one of SEQ ID NOs.
• the ketoacyl-CoA reductase is selected from the group consisting of a 3- ketobutyryl-CoA reductase, a 3-hydroxybutyryl-CoA dehydrogenase, a 3-ketovaleryl-CoA reductase, and 3-hydroxyvaleryl-CoA dehydrogenase.
• the ketoacyl-CoA reductase comprises an amino acid sequence of at least 70% homology to any one of SEQ ID NO 183 and SEQ ID NO 271.
• the hydroxyacyl-CoA dehydratase is selected from the group consisting of a 3-hydroxybutyryl-CoA dehydratase and an enoyl-CoA hydratase.
• the hydroxyacyl-CoA dehydratase comprises an amino acid sequence of at least 70% homology to any one of SEQ ID NO 183 and SEQ ID NO 272.
• the enoyl-CoA reductase is trans-2-enoyl-reductase.
• the enoyl-CoA reductase comprises an amino acid sequence of at least 70% homology to SEQ ID NO 275.
• the 3-ketoacyl-CoA synthase comprises a modified NphT7 polypeptide comprising one or more amino acid substitutions selected from the group consisting of a PDRP to HFLQ substitution for amino acids 86-89,F217A, F217E, F217G, F217I, F217L, F217M, F217P, F217S, F217T, F217V, F217W, G288S, G309S, I147A, I147C, I147D, I147E, I147F, I147G, I147H, I147K, I147L, I147M, I147N, I147P, I147Q, I147R, I147S, I147T, I147V, I147W, I147Y, V157F, V196G, and Y144L.
• the 3-ketoacyl-CoA synthase comprises a modified NphT7 polypeptide comprising two amino acid substitutions selected from the group consisting of I147T and F217V, I147T and Y144L, I147T and V196G, I147F and F217V, I147M and F217V, I147S and F217V, I147T and HFLQ, I147T and V157F, I147T and F217G, I147T and F217A, I147T and F217L, I147T and F217I, I147T and F217M, I147T and F217P, I147T and F217S, I147T and F217E, I147S and F217G, I147S and F217A, I147S and F217L, I147S and F217I, I147S and F217M, I147S and F217W, I147S and F217S, I147S and F217S, I147
• the 3-ketoacyl-CoA synthase comprises a modified NphT7 polypeptide comprising three amino acid substitutions selected from the group consisting of Y144L, I147T, and F217V; I147T, F217V, and HFLQ; I147T, V147F, and F217V; and Y144L, I147T, and V157F.
• the 3-ketoacyl-CoA synthase comprises a modified NphT7 polypeptide comprising one or more amino acid substitutions at a position selected from the group consisting of Ser84, Vail 14, Gly288, Ilel94, Gly318, Thr85, Gln90, Vall96, Tyrl44, Phel59, Ilel47, and Phe217.
• the ketoacyl-CoA reductase is selected from the group consisting 3-ketoacyl-CoA reductase and 3-hydroxyacyl-CoA dehydrogenase.
• the ketoacyl-CoA reductase comprises an amino acid sequence of at least 70% homology to any one of SEQ ID NO 183 and SEQ ID NO 271.
• the hydroxyacyl-CoA dehydratase is selected from the group consisting of a 3- hydroxyacyl-CoA dehydratase and enoyl-CoA hydratase.
• the hydroxyacyl-CoA dehydratase comprises an amino acid sequence of at least 70% homology to any one of SEQ ID NO 183, and SEQ ID NO 272.
• the enoyl-CoA reductase is trans-2-enoyl-reductase.
• the enoyl-CoA reductase comprises an amino acid sequence of at least 70% homology to SEQ ID NO 275.
• the genetically modified organism further comprises a heterologous nucleic acid sequence encoding a thioesterase or a wax ester synthase.
• the genetically modified organism further comprises a heterologous nucleic acid sequence encoding a termination enzyme that catalyzes the production of a fatty acid-derived product selected from the group comprising a fatty alcohol, a fatty aldehyde, a fatty alkene, a fatty amide, a fatty alkane, and a fatty diacid.
• the thioesterase is an acyl-CoA esterase and the organism is capable of producing a fatty acid.
• the thioesterase is selected from the group comprising tesA, 'tesA, tesB, yciA, ybgC, ybfF, fadM, AtTE, CpTE, Cperf E, LpTE, and PA2801TE.
• the thioesterase comprises an amino acid sequence of at least 70% homology to any one of SEQ ID NO 277, SEQ ID NO 278, SEQ ID NO 279, SEQ ID NO 280, SEQ ID NO 281, SEQ ID NO 282, SEQ ID NO 283, SEQ ID NO 284, SEQ ID NO 285, SEQ ID NO 286, SEQ ID NO 287, and SEQ ID NO 288.
• the wax ester synthase is selected from the group comprising Maql, Pcryl, Rjosl, and Aborkl, and wherein said organism is capable of producing a fatty ester.
• the wax ester synthase comprises an amino acid sequence of at least 70% homology to any one of SEQ ID NO 289, SEQ ID NO 290, SEQ ID NO 291, and SEQ ID NO 292, and wherein said organism is capable of producing a fatty ester.
• the 3-ketoacyl-CoA synthase is NphT7; the keto-CoA reductase is selected from the group consisting of hbd and fadB; the 3-hydroxy-acyl- CoA dehydratase is selected from the group consisting of crt and fadB; the enoyl-CoA reductase is ter; and the thioesterase is selected from the group consisting of CpTE, fadM, PA2801TE, tesB, ybgC, ybfF, and yciA, and wherein the proteins encoded by the polynucleotides are capable of producing a four or five carbon fatty acid or fatty acid derived product.
• the thioesterase is selected from the group consisting of tesB and yciA.
• one or more 3-ketoacyl-CoA synthases are selected from the group consisting of NphT7, NphT7 I147T, NphT7 F217V, and NphT7 I147T,F217V;the keto-CoA reductase is fadB; the 3-hydroxy- acyl-CoA dehydratase is fadB; the enoyl-CoA reductase is ter; and the thioesterase is selected from the group consisting of AtTE, CpTE, Cperf E, PA2801TE, tesA, tesB, ybfF, ybgC, and yciA, and wherein the proteins encoded by the polynucleotides are capable of producing a six or seven carbon fatty acid or fatty acid derived product.
• the thioesterase is selected from the group consisting of PA2801TE, tesB, and yciA.
• one or more 3-ketoacyl-CoA synthases are selected from the group consisting of NphT7, NphT7 I147T, NphT7 F217V, NphT7 I147T and F217V, Npth7 I147S, Npth7 I147S and F217V, and synthase III;
• the keto-CoA reductase is selected from the group consisting of fadB and fabG;
• the 3- hydroxy-acyl-CoA dehydratase is selected from the group consisting of fadB, ech and ech2;
• the enoyl-CoA reductase is ter;
• the thioesterase is selected from the group consisting of AtTE, CpTE, CperfTE, fadM, PA2801TE, tesA, tes
• the thioesterase is selected from the group consisting of PA2801TE, tesB, and yciA.
• one or more 3-ketoacyl-CoA synthases are selected from the group consisting of NphT7, NphT7 I147T, NphT7 F217V, NphT7 I147T and F217V, Npth7 I147S, Npth7 I147S and F217V, synthase III, synthase IV, and synthase V;
• the keto-CoA reductase is selected from the group consisting of fadB and fabG;
• the 3-hydroxy-acyl-CoA dehydratase is selected from the group consisting of fadB, ech and ech2;
• the enoyl-CoA reductase is ter;
• the thioesterase is selected from the group consisting of AtTE, CpTE, fadM, PA2801TE,
• the thioesterase is selected from the group consisting of tesB and yciA.
• one or more 3-ketoacyl-CoA synthases are selected from the group consisting of NphT7, NphT7 I147T, NphT7 F217V, NphT7 I147T and F217V, Npth7 I147S, Npth7 I147S and F217V, synthase III, synthase IV, synthase V, and synthase VI;
• the keto- CoA reductase is selected from the group consisting of fadB, fabG, and fadJ
• the 3-hydroxy-acyl- CoA dehydratase is selected from the group consisting of fadB, ech, and fadJ;
• the enoyl-CoA reductase is ter; and the thioesterase is selected from the group consisting of AtTE, CpTE, C
• the thioesterase is selected from the group consisting of fadM, PA2801TE, tesA, tesB, and yciA.
• one or more 3- ketoacyl-CoA synthases are selected from the group consisting of NphT7, NphT7 I147T, NphT7 F217V, NphT7 I147T and F217V, Npth7 I147S, Npth7 I147S and F217V, synthase III, synthase IV, synthase V, and synthase VI;
• the keto-CoA reductase is selected from the group consisting of fadB, and fadJ;
• the 3-hydroxy-acyl-CoA dehydratase is selected form the group consisting of fadB, and fadJ;
• the enoyl-CoA reductase is selected from the group consisting of ter, ydiO and f
• the thioesterase is selected from the group consisting of fadM, tesA, tesB, and yciA, and the proteins encoded by the polynucleotides are capable of producing a fourteen or fifteen carbon fatty acid or fatty acid derived product.
• the thioesterase is selected from the group consisting of AtTE, CpTE, CperfTE, fadM, Pa2801TE, tesA, tesB, ybfF, ybgC, and yciA, and the proteins encoded by the polynucleotides are capable of producing a sixteen or seventeen carbon fatty acid or fatty acid derived product.
• the thioesterase is selected from the group consisting of AtTE, fadM, tesA, tesB, ybfF, ybgC, and yciA, and the proteins encoded by the polynucleotides are capable of producing a sixteen or seventeen carbon fatty acid or fatty acid derived product.
• the organism is capable of using acetyl-CoA as a primer and malonyl- CoA as the extender molecule to produce a fatty acid or fatty acid derived product have a carbon chain length selected from 4, 6, 8, 10, 12, 14, 16, 18 and 20.
• the organism is capable of using propionyl-CoA as a primer and malonyl-CoA as the extender molecule to produce a fatty acid or fatty acid derived product have a carbon chain length selected from 5, 7, 9, 11, 13, 15, 17, 19, and 21.
• the disclosure provides for a modified NphT7 polypeptide, comprising an amino acid sequence having at least 70% homology to SEQ ID NO: l and one or more amino acid substitutions, deletions, or insertions, wherein the modified NphT7 polypeptide is capable of accepting an acyl-CoA substrate having a carbon chain length of C4 or greater.
• the modified NphT7 polypeptide is capable of catalyzing a condensation reaction to condense an acyl-CoA substrate with a malonyl-CoA to produce a 3-keto-acyl-CoA having a carbon chain length of C6 or greater.
• a modified NphT7 polypeptide comprises one or more amino acid substitutions selected from the group consisting of I147T, F217V, Y144L, V157F, G309S, G288S, a PDRP to HFLQ substitution for amino acids 86-89, 1147F, I147M, I147Q, I147S, I147C, I147E, I147N, I147W, I147D, I147R, I147P, I147L, V196G, I147G, I147H, I147K, 1147V, 1147 A, I147Y, F217G, F217A, F217L, F217I, F217M, F217T, F217P, F217S, F217E, F217L, F217W, and any combination thereof
• a modified NphT7 polypeptide comprises one amino acid substitution selected from the group consisting of I147V, I147F, I147M, I147Q
• a modified NphT7 polypeptide comprises two amino acid substitutions selected from the group consisting of I147T and F217V, I147T and Y144L, I147T and V196G, I147F and F217V, I147M and F217V, I147S and F217V, I147T and HFLQ, I147T and V157F, I147T and F217G, I147T and F217A, I147T and F217L, I147T and F217I, I147T and F217M, I147T and F217P, I147T and F217S, I147T and F217E, I147S and F217G, I147S and F217A, I147S and F217L, I147S and F217I, I147S and F217M, I147S and F217W, I147S and F217S, I147S and F217E, I147S and F217K, I147F and
• a modified NphT7 polypeptide comprises three amino acid substitutions selected from the group consisting of (Y144L, I147T, and F217V), (I147T, F217V, and HFLQ), (I147T, V147F, and F217V), and (Y144L, I147T, and V157F).
• a modified NphT7 polypeptide comprises one or more amino acid substitutions at a position selected from the group consisting of Ser84, Vail 14, Gly288, Ilel94, Gly318, Thr85, Gln90, Vall96, Tyrl44, Phel59, Ilel47, Phe217, and any combination thereof
• a modified NphT7 polypeptide comprises an I147T amino acid substitution.
• a modified NphT7 polypeptide comprises an F217V amino acid substitution.
• a modified NphT7 polypeptide comprises two or more amino acid substitutions, deletions, or insertions.
• a modified NphT7 polypeptide comprises an I147T amino acid substitution and an F217V amino acid substitution.
• a modified polypeptide of is isolated and purified.
• the disclosure provides for an isolated and purified polynucleotide encoding a modified NphT7 polypeptide of the disclosure.
• the disclosure provides for an isolated and purified polynucleotide comprising a nucleic acid sequence having at least 70% but less than 100% or about 100% homology or complementarity to SEQ ID NO:2, wherein the polynucleotide encodes a modified NphT7 polypeptide of SEQ ID NO: l having one or more amino acid substitutions, wherein the modified NphT7 polypeptide is capable of accepting an acyl-CoA substrate having a carbon chain length of C4 or greater.
• an isolated and purified polynucleotide of encodes a modified NphT7 polypeptide capable of catalyzing a condensation reaction to condense an acyl-CoA substrate with a malonyl-CoA to produce a 3-ketoacyl-CoA having a carbon chain length of C6 or greater.
• an isolated and purified polynucleotide encodes a modified NphT7 polypeptide comprising one or more amino acid substitutions selected from the group consisting of I147T, F217V, Y144L, V157F, G309S, G288S, a PDRP to HFLQ substitution for amino acids 86-89, I147F, I147M, I147Q, I147S, I147C, I147E, I147N, I147W, I147D, I147R, I147P, I147L, V196G, I147G, I147H, I147K, I147V, I147A, I147Y, F217G, F217A, F217L, F217I, F217M, F217T, F217P, F217S, F217E, F217L, F217W, and any combination thereof.
• an isolated and purified polynucleotide encodes a modified NphT7 polypeptide comprising one amino acid substitution selected from the group consisting of I147V, I147F, I147M, I147Q, I147S, I147C, I147E, I147N, I147W, I147D, I147R, I147P, I147L, I147G, I147H, I147K, I147A, I147Y, and F217V.
• an isolated and purified polynucleotide encodes a modified NphT7 polypeptide comprising two amino acid substitutions selected from the group consisting of I147T and F217V, I147T and Y144L, I147T and V196G, I147F and F217V, I147M and F217V, I147S and F217V, I147T and HFLQ, I147T and V157F, I147T and F217G, I147T and F217A, I147T and F217L, I147T and F217I, I147T and F217M, I147T and F217P, I147T and F217S, I147T and F217E, I147S and F217G, I147S and F217A, I147S and F217L, I147S and F217I, I147S and F217M, I147S and F217W, I147S and F217S, I147S and F217S, I147
• an isolated and purified polynucleotide encodes a modified NphT7 polypeptide comprising three amino acid substitutions selected from the group consisting of (Y144L, I147T, and F217V), (I147T, F217V, and HFLQ), (I147T, V147F, and F217V), and (Y144L, I147T, and V157F).
• an isolated and purified polynucleotide encodes a modified NphT7 polypeptide comprising one or more amino acid substitutions at a position selected from the group consisting of Ser84, Vail 14, Gly288, Ilel94, Gly318, Thr85, Gln90, Vall96, Tyrl44, Phel59, Ilel47, Phe217, and any combination thereof.
• an isolated and purified polynucleotide encodes a modified NphT7 polypeptide comprising an I147T amino acid substitution.
• an isolated and purified polynucleotide encodes a modified NphT7 polypeptide comprising an F217V amino acid substitution.
• an isolated and purified polynucleotide encodes a modified NphT7 polypeptide comprising two or more amino acid substitutions. In some embodiments, an isolated and purified polynucleotide encodes a modified NphT7 polypeptide, comprising an I147T amino acid substitution and an F217V amino acid substitution. In some embodiments, an isolated and purified polynucleotide is RNA. In some embodiments, an isolated and purified polynucleotide is an mRNA. In some embodiments, an isolated and purified polynucleotide is a DNA. In some embodiments, an isolated and purified polynucleotide is a cDNA. In some embodiments, vector comprises a polynucleotide of the disclosure. In some embodiments, a plasmid comprises a polynucleotide of the disclosure.
• the disclosure provides for a method of selecting a 3-ketoacyl-CoA synthase as a candidate for condensing a malonyl-CoA with an acyl-CoA having a carbon chain length greater than C2, comprising: identifying a 3-ketoacyl-CoA synthase polypeptide comprising an amino acid sequence having at least 70% but less than 100% or about 100% homology to SEQ ID NO: l; and selecting the 3-ketoacyl-CoA synthase as a candidate for condensing an acyl-CoA having a carbon chain length greater than C2 with a malonyl-CoA, if the 3-ketoacyl-CoA synthase comprises one or more features selected from the group consisting of an (A/G)GGSR sequence motif, lack of a STPDXPQ sequence motif, and solely hydrophobic residues in the substrate binding site.
• the method comprises selecting at least two 3-ketoacyl-CoA syntha
• the disclosure provides for a library of NphT7 homologs selected by a method of the disclosure.
• the disclosure provides for an isolated NphT7 homolog, comprising an amino acid sequence having at least 70% but less than 100% homology to any one of SEQ ID NOs. 1-120.
• the disclosure provides for an isolated polynucleotide encoding a selected 3-ketoacyl-CoA synthase of the disclosure. [0017] In one aspect the disclosure provides for a genetically modified organism expressing a selected 3-ketoacyl-CoA synthase of the disclosure.
• the disclosure provides for a method of producing a genetically modified organism that expresses a selected 3-ketoacyl-CoA synthase of the disclosure, comprising transforming a microorganism with a polynucleotide of the disclosure.
• the disclosure provides for a genetically modified organism capable of producing a fatty acid or fatty acid-derived product having a carbon chain length of C6 or greater at a rate or titer above a control organism lacking the genetic modification, wherein the genetically modified organism does not comprise any one of SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123.
• a genetically modified organism comprises a vector or plasmid of the disclosure.
• the genetically modified organism is transformed with a vector or plasmid.
• the genetically modified organism comprises a polynucleotide of the disclosure.
• the genetically modified organism expresses a modified polypeptide and/or polynucleotide of the disclosure. In some embodiments, the genetically modified organism, expresses an NphT7 homolog or a modified NphT7 polypeptide of the disclosure. In some embodiments, the genetically modified organism comprises a heterologous polypeptide capable of condensing a malonyl-CoA with an acyl-CoA having a carbon chain length greater than C2 to produce a 3-ketoacyl-CoA having a carbon chain length greater than C4. In some embodiments, the acyl-CoA is acetyl-CoA and the 3-ketoacyl- CoA is 3-ketobutyryl-CoA.
• the acyl-CoA is acetyl-CoA and the 3- ketoacyl-CoA is 3-ketobutyryl-CoA. In some embodiments, the acyl-CoA is propionyl-CoA and the 3-ketoacyl-CoA is 3-ketovaleryl-CoA. In some embodiments, the genetically modified organism is capable of producing a free fatty acid or fatty acid-derived product with a carbon chain length C6, C8, CIO, C12, C14, C16, C18, C20, or greater with > 20, 30, 40, 50, 60, 70, 80, or 90% purity.
• the genetically modified organism is capable of producing a free fatty acid or fatty acid-derived product at a rate of about 0.1 g/gDCW*hr, about 0.2 g/gDCW*hr, or greater.
• the genetically modified organism further comprises an additional genetic modification that increases production rate of acyl-CoA.
• the genetically modified organism further comprises an additional genetic modification that increases production rate of malonyl-CoA.
• the genetically modified organism further comprise an additional genetic modification that inhibits a malonyl-ACP fatty acid synthesis pathway.
• the genetically modified organism further comprises an additional genetic modification reduces the conversion of malonyl-CoA to malonyl ACP.
• the genetically modified organism further comprises an additional genetic modification reduces the rate of condensation of malonyl- ACP with acetyl-ACP.
• the genetically modified organism further comprises one or more additional genetic modifications that fully or partially inhibit one or more reactions selected from the group consisting of glucose to methylglyoxal conversion, pyruvate to lactate conversion, acetyl-CoA to acetate conversion, acetyl-CoA to ethanol conversion, fatty acyl to acetyl-CoA conversion, and any combination thereof.
• the genetically modified organism comprises a polynucleotide encoding a 3-ketoacyl-CoA synthase that comprises an amino acid sequence of at least 70% but less than 100% or about 100% homology to any one of SEQ ID NOs. 1-120.
• the genetically modified organism comprises one or more heterologous polypeptides selected from the group consisting of keto-CoA reductase (KCR), 3-hydroxy-acyl-CoA dehydratase (3HDh), enoyl CoA reductase (EnCR), thioesterase enzymes, and any combination thereof.
• the genetically modified organism comprises one or more heterologous KCR selected from the group consisting of fadB, fabG, fadJ, ech2, PhaB, PaFabG, and any combination thereof.
• the genetically modified organism comprises one or more heterologous 3HDh selected from the group consisting of fadB, fadJ, ech, ech2, crt, and any combination thereof.
• the genetically modified organism comprises one or more heterologous EnCR selected from the group consisting of ter, ccr, fadE, ydiO and any combination thereof.
• the genetically modified organism comprises one or more heterologous thioesterases selected from the group consisting of yciA, PA2801TE, ATTE, YbgC, tesA, YbfF, fadM, LpTE, CpTE ( or CperfTE), and any combination thereof.
• the genetically modified organism comprises one or more heterologous 3- ketoacyl-CoA synthases selected from the group consisting of WT NphT7, NphT7 mutated at I147T, and NphT7 mutated at I147T and F217V, and any combination thereof, and at least one of: a heterologous fadB; a heterologous ter; and/or one or more thioesterases selected from the group consisting of tesA, yciA, PA2801TE, and any combination thereof.
• the genetically modified organism comprises one or more heterologous 3-ketoacyl-CoA synthases selected from the group consisting of WT NphT7, NphT7 mutated at I147T, NphT7 mutated at I147T and F217V, synthase III, and any combination thereof; and at least one of: one or more heterologous KCR selected from the group consisting of fadB and fabG; one or more heterologous 3HDh selected from the group consisting of fadB, ech and ech2; a heterologous ter; and/or one or more thioesterases selected from the group consisting of tesA, yciA, PA2801TE, and any combination thereof.
• heterologous 3-ketoacyl-CoA synthases selected from the group consisting of WT NphT7, NphT7 mutated at I147T, NphT7 mutated at I147T and F217V, synthase III
• the genetically modified organism comprises one or more heterologous 3-ketoacyl-CoA synthases selected from the group consisting of WT NphT7, NphT7 mutated at I147T, NphT7 mutated at I147T and F217V, synthase III, synthase IV, synthase V, and any combination thereof; and at least one of: one or more heterologous KCR selected from the group consisting of fadB and fabG; one or more heterologous 3HDh selected from the group consisting of fadB, ech and ech2; a heterologous ter; and/or one or more thioesterases selected from the group consisting of tesA, ATTE, YbgC, and any combination thereof.
• heterologous 3-ketoacyl-CoA synthases selected from the group consisting of WT NphT7, NphT7 mutated at I147T, NphT7 mutated at I147T and
• the genetically modified organism comprises one or more heterologous 3-ketoacyl-CoA synthases selected from the group consisting of WT NphT7, NphT7 mutated at I147T, NphT7 mutated at I147T and F217V, synthase III, synthase IV, synthase V, synthase VI, and any combination thereof; and at least one of: one or more heterologous KCR selected from the group consisting of fadB, fabG, fadJ, and any combination thereof; one or more heterologous 3HDh selected from the group consisting of fadB, fadJ, ech, and any combination thereof; a heterologous ter; and/or one or more thioesterases selected from the group consisting of tesA, ybgC, ybFF, and any combination thereof.
• the genetically modified organism comprises one or more heterologous 3-ketoacyl-CoA synthases selected from the group consisting of WT NphT7, NphT7 mutated at I147T, NphT7 mutated at I147T and F217V, synthase III, synthase IV, synthase V, synthase VI, and any combination thereof; and at least one of: one or more heterologous KCR selected from the group consisting of fadB and fadJ; one or more heterologous 3HDh selected from the group consisting of fadB and fadJ; one or more heterologous EnCR selected from the group consisting of ter, ydiO and fadE; and/or one or more thioesterases selected from the group consisting of tesA, fadM and any combination thereof.
• heterologous 3-ketoacyl-CoA synthases selected from the group consisting of WT NphT7, N
• the genetically modified organism further comprises an additional genetic modification that reduces activity of one or more endogenous polypeptides selected from the group consisting of KCR, hbd, enoyl CoA reductase, thioesterase, and any combination thereof.
• the genetically modified organism further comprises an additional genetic modification that reduces activity of a temperature sensitive version of one or more endogenous polypeptides.
• the genetically modified organism comprises one or more vectors encoding a second genetic modification of the disclosure.
• the genetically modified organism comprises a heterologous transporter that can transport past a cell membrane a free fatty acid having a carbon chain length of C6 or greater.
• the genetically modified organism comprises a heterologous transporter that is an ABC transporter.
• the genetically modified organism comprises a fatty acid-derived product that is a fatty alcohol, a fatty aldehyde, a fatty alkene, a fatty amide, a fatty ester, a fatty alkane, or fatty diacid.
• one or more of thioesterases are fully or partially knocked out, the thioesterases being selected from the group consisting of tesB, YciA, AtTE, CpTE, and any combination thereof.
• the genetically modified organism is isolated and purified.
• the disclosure provides for a genetically modified organism having a genetic modification selected from the group consisting of F-, A(araD-araB)567, AlacZ4787(::rrnB-3), LAM-, rph-1, A(rhaD-rhaB)568, hsdR514, AldhA::frt, ApflB::frt, AmgsA::frt, ApoxB::frt, Apta-ack::frt, fabI(ts)-(S241F)-zeoR, Atig::frt, AatoDAEB::frt, and AfadD::frt, and an additional genetic modification that increases synthesis of fatty acid from Co A substrates.
• a genetic modification selected from the group consisting of F-, A(araD-araB)567, AlacZ4787(::rrnB-3), LAM-, rph-1, A(rhaD
• the genetically modified organism comprises a deletion of a host gene, wherein the deletion results in increased malonyl-CoA production.
• the genetically modified organism comprises a deletion of one or more genes selected from the group consisting of lactate dehydrogenase, pyruvate formate lyase, methylglyoxal synthase, pyruvate oxidase, phosphotransacetylase acetate kinase, bifunctional acetyl-CoA reductase/alcohol dehydrogenase, and any combination thereof.
• the genetically modified organism of the disclosure further comprises an additional genetic modification that is associated with one or more enzymes selected from the group consisting of ACP, fabi, fabB, fabH, fabD, fabF, fabG, fabA, fabZ, fabR, and any combination thereof.
• he genetically modified organism of the disclosure further comprises an additional genetic modification that is associated with one or more enzymes selected from the group consisting of udhA, pntAB, PDH, CoAA, panD, aceA, aceB, aceK, GAPDH, pyk, pyk, gltA, CS, bicA, GOGAT, gdh, can, cynT, cynS, puuC, aldA, aldB, yieP, yibD, pstS, BAAT, rhtA, mdtM, yddG, yebS, yeeO, dedA, ycaP, ytfL, ybbP, yegH, ykgH, ytfF, eamB, ydhP, ypjD, mdlB, acrD, ydcO
• the genetically modified organism of the disclosure further comprises an additional genetic modification associated with an ACCase enzyme. In some embodiments, the genetically modified organism of the disclosure, further comprises an additional genetic modification that is associated with one or more enzymes selected from the group consisting of cscA, cscB, cscK, galP, galKf, and any combination thereof.
• the genetically modified organism further comprises an additional genetic modification that is associated with one or more enzymes selected from the group consisting of fadE, fadD, fadA, fadB, fadl, fadJ, ydiO, paaJ, yqeF, tig, atoD, atoA, atoE, atoB, and any combination thereof.
• the genetically modified organism further comprises an additional genetic modification that is associated with one or more enzymes selected from the group consisting of NphT7, SaFabH, BsFabH, PaFabH, MtFabH, FabH, PaFabG, fabG, hbd, crt, ech, ech2, ter, ccr, and any combination thereof.
• the genetically modified organism further comprises an additional genetic modification resulting in expression of a heterologous thioesterase.
• the genetically modified organism any claim comprises one or more heterologous thioesterases selected from the group consisting of tesA, 'tesA, tesB, yciA, ybgC, ybfF, fadM, AtTE, CpTE (or CperfTE), LpTE, Pa2801TE, and any combination thereof.
• the genetically modified organism further comprises an additional genetic modification resulting in expression of a heterologous wax ester synthase.
• the genetically modified organism comprises one or more heterologous wax ester synthases selected from the group consisting of Maql, Pcryl, Rjosl, Aborkl, and any combination thereof.
• the genetically modified organism further comprises an additional genetic modification that results in expression of one or more heterologous proteins selected from the group consisting of prpE, phaA, phaB, phaC, TFINS, TFINS", and any combination thereof.
• the genetically modified organism is a microorganism. In some embodiments, the genetically modified organism is E. Coli.
• the disclosure provides for a method of producing from malonyl-
• CoA a free fatty acid that has a carbon chain length of C6 or greater comprising culturing a transformed microorganism with a carbon feed source, thereby producing the free fatty acid.
• the disclosure provides for a method of producing from malonyl-
• CoA a free fatty acid that has a carbon chain length of C6 or greater comprising: inducing expression of a polypeptide in a microorganism; and culturing a transformed microorganism with a carbon feed source, thereby producing the free fatty acid.
• the disclosure provides for a method of producing from malonyl-
• CoA a free fatty acid that has a carbon chain length of C6 or greater comprising: providing a genetically modified microorganism; and culturing a transformed microorganism with a carbon feed source, thereby producing the free fatty acid.
• the disclosure provides for a method of producing a free fatty acid that has a carbon chain length of C6 or greater comprising culturing a microorganism under conditions sufficient to increase acyl-CoA and malonyl-CoA production.
• the disclosure provides for a method of producing a free fatty acid that has a carbon chain length of C6 or greater, comprising culturing a microorganism under conditions sufficient to enable condensation of a malonyl-CoA and an acyl-CoA of a carbon chain length of C2 or greater, whereby the condensation results in production of a keto-acyl CoA product having a chain length of C6 or greater.
• the disclosure provides for a method of producing a free fatty acid that has a carbon chain length of C6 or greater, comprising culturing a microorganism under conditions sufficient to reduce a keto group in a keto-acyl CoA product having a carbon chain length of C6 or greater, hereby producing a hydroxyl-acyl-CoA product having a carbon chain length of C6 or greater.
• the disclosure provides for a method of producing a free fatty acid that has a carbon chain length of C6 or greater, comprising culturing a microorganism under conditions sufficient to perform a dehydratase reaction of a hydroxyl-acyl-CoA producing having a carbon chain length of C6 or greater to produce an enoyl-acyl-CoA product having a carbon chain length of C6 or greater.
• the disclosure provides a method of producing a free fatty acid that has a carbon chain length of C6 or greater, comprising culturing a microorganism under conditions sufficient to reduce an enoyl group of an enoyl-acyl-CoA product having a carbon chain length of C6 or greater to produce an acyl-CoA product having a carbon chain length of C6 or greater.
• the disclosure provides a method for a method of producing a free fatty acid that has a carbon chain length of C6 or greater, comprising culturing a microorganism under conditions sufficient to remove a CoA group from an acyl-CoA product having a carbon chain length of C6 or greater to produce a free fatty acid or fatty acid-derived product having a carbon chain length of C6 or greater.
• the disclosure provides for a method of producing a free fatty acid or fatty acid-derived product of chain length of C6 or greater from malonyl-CoA, comprising: culturing a genetically modified organism under conditions sufficient to increase acyl CoA and malonyl-CoA production, condensing the acyl CoA and malonyl-CoA in the genetically modified organism to produce a keto-acyl CoA product having a carbon chain length of C6 or greater; reducing a keto-group in the keto-acyl CoA product to product a hydroxyl-acyl-CoA product having a carbon chain length of C6 or greater; performing a dehydratase reaction on the hydroxyl-acyl-CoA product to produce an enoyl-acyl-CoA product having a carbon chain length of C6 or greater; and reducing an enoyl group of the enoyl-acyl-CoA product to produce an acyl- CoA product having a carbon chain length of C6 or greater
• the method comprises culturing a genetically modified organism that comprises one or more heterologous 3-ketoacyl-CoA synthases selected from the group consisting of WT NphT7, NphT7 mutated at I147T, NphT7 mutated at I147T and F217V, and any combination thereof; and at least one of: a heterologous fadB; a heterologous ter; and/or one or more thioesterases selected from the group consisting of yciA and PA2801TE.
• the method further comprises culturing a genetically modified organism that comprises one or more heterologous 3-ketoacyl-CoA synthases selected from the group consisting of WT NphT7, NphT7 mutated at I147T, NphT7 mutated at I147T and F217V, synthase III, and any combination thereof; and at least one of: one or more heterologous KCR selected from the group consisting of fadB and fabG; one or more heterologous 3HDh selected from the group consisting of fadB, ech and ech2; a heterologous ter; and/or one or more thioesterases selected from the group consisting of tesA, yciA, PA2801TE, and any combination thereof.
• the method further comprises culturing a genetically modified organism that comprises one or more heterologous 3-ketoacyl-CoA synthases selected from the group consisting of WT NphT7, NphT7 mutated at I147T, NphT7 mutated at I147T and F217V, synthase III, synthase IV, synthase V, and any combination thereof; at least one of: one or more heterologous KCR selected from the group consisting of fadB and fabG; one or more heterologous 3HDh selected from the group consisting of fadB, ech and ech2; a heterologous ter; and/or one or more thioesterases selected from the group consisting of tesA, ATTE
• the method further comprises culturing a genetically modified organism that comprises one or more heterologous 3- ketoacyl-CoA synthases selected from the group consisting of WT NphT7, NphT7 mutated at I147T, NphT7 mutated at I147T and F217V, synthase III, synthase IV, synthase V, synthase VI, and any combination thereof; and at least one of: one or more heterologous KCR selected from the group consisting of fadB, fabG, fadJ, and any combination thereof; one or more heterologous 3HDh selected from the group consisting of fadB, fadJ, ech, and any combination thereof; a heterologous ter; and/or one or more thioesterase
• the method further comprises culturing a genetically modified organism that comprises one or more heterologous 3-ketoacyl-CoA synthases selected from the group consisting of WT NphT7, NphT7 mutated at I147T, NphT7 mutated at I147T and F217V, synthase III, synthase IV, synthase V, synthase VI, and any combination thereof; and at least one of: one or more heterologous KCR selected from the group consisting of fadB and fadJ; one or more heterologous 3HDh selected from the group consisting of fadB and fadJ; one or more heterologous EnCR selected from the group consisting of ter, ydiO and fadE; and/or one or
• the method further comprises a cycle that comprises reactions, wherein the cycle comprises reactions employing: a NphT7, a KCR, a 3HDh, and an EnCR, wherein at least one, two, three, four, five, six, seven, eight, or nine cycles are conducted, and at least one of the NphT7, KCR, 3HDh, and/or EnCR is modified.
• the disclosure provides for a free fatty acid or fatty acid-derived product produced from a genetically modified organism.
• the disclosure provides for a free fatty acid or fatty acid-derived product produced by a method of the disclosure.
• the fatty acid-derived product is a fatty alcohol, fatty amide, fatty ester, fatty aldehyde, fatty alkene, fatty alkane, or fatty diacid, each of which is substituted or unsubstituted.
• the disclosure provides for use of a genetically modified organism for producing a fatty acid having a carbon chain length of C6 or greater.
• the disclosure provides for a system for producing a free fatty acid or fatty acid-derived product comprising a carbon chain length of C6 or greater comprising: one or more genetically modified organisms and/or modified polypeptides; and an incubator configured for culturing the one or more genetically modified organisms.
• the system comprises a culture medium that comprises a carbon feed source.
• the system comprises a purification system for purifying a free fatty acid or fatty acid-derived product.
• the system comprises at least two strains of genetically modified organisms.
• the system comprises at least three strains of genetically modified organisms.
• the system is capable of producing a free fatty acid or fatty acid-derived product at a titer of about 5 g/L, about 10 g/L, or greater. In some embodiments, the system is capable of producing a free fatty acid or fatty acid- derived product comprising a carbon chain of C6 or greater at a concentration of about 0.5 g/L or greater. In some embodiments, the system is capable of producing a free fatty acid or fatty acid- derived product comprising a C12 carbon chain at a concentration of about 0.7 g/L or greater.
• the system is capable of producing a free fatty acid or fatty acid-derived product comprising a C14 carbon chain at a concentration of about 0.7 g/L or greater. In some embodiments, the system is capable of producing a free fatty acid or fatty acid-derived product comprising a C16 carbon chain at a concentration of about 0.8 g/L or greater. In some embodiments, the system is capable of yielding a free fatty acid or fatty acid-derived product at about 0.125 g/g, about 0.16 g/g, or greater. In some embodiments, the system further comprises a mixing apparatus. In some embodiments, the system further comprises a heating apparatus, wherein the incubator comprises the heating apparatus. In some embodiments, the system further comprises a reservoir.
• the system further comprises a pump.
• the system the reservoir is operably connected to the incubator, and wherein the pump is operably configured to pump material from the reservoir to the incubator.
• the system further comprises a lysing apparatus.
• the system further comprises an extracting apparatus.
• the system further comprises a distillation apparatus.
• the disclosure provides for a genetically modified organism that is
• the genetically modified organism is a prokaryotic cell. In some embodiments, the genetically modified organism is a eukaryotic cell. In some embodiments, the genetically modified organism is a yeast cell.
• the genetically modified organism is a bacteria cell. In some embodiments, the genetically modified organism is a fungi cell. In some embodiments, the genetically modified organism is a microalgae cell. In some embodiments, the genetically modified organism is an algae cell.
• the disclosure provides for a carbon source comprising a C6 carbon source.
• the carbon source comprises a C3 carbon source.
• the carbon source comprises one or more cellulosic sugars.
• the carbon source comprises glucose, sucrose, fructose, dextrose, lactose, xylose, or any combination thereof.
• the carbon source comprises less than about 50%, 40%, 30%, 20%, 10%, or 5% by mass of glycerol.
• the disclosure provides for a biomass comprising a genetically modified organism.
• the biomass comprises a lysed genetically modified organism.
• the biomass comprises a modified NphT7 polypeptide.
• the biomass comprises a modified polypeptide.
• the biomass comprises a polynucleotide.
• the biomass comprises a free fatty acid or fatty acid-derived product.
• the biomass is dehydrated.
• the disclosure provides for a broth comprising a genetically modified organism. In one aspect the disclosure provides for a broth comprising a lysed genetically modified organism. In one aspect the disclosure provides for a broth comprising a modified NphT7 polypeptide. In one aspect the disclosure provides for a broth comprising a modified polypeptide. In one aspect the disclosure provides for a broth comprising a polynucleotide of the disclosure. In one aspect the disclosure provides for a broth comprising a free fatty acid or fatty acid-derived product of the disclosure.
• an acyl-CoA product comprising about
• acyl-CoA product comprising about 40%> to 50%> by mass of acyl- CoA having a carbon chain length of C6. In one aspect the disclosure provides for an acyl-CoA product, comprising about 5% to 30%> by mass of acyl-CoA having a carbon chain length of C8. In one aspect the disclosure provides for an acyl-CoA product, comprising about 1% to 20%> by mass of acyl-CoA having a carbon chain length of C12.
• the disclosure provides for an acyl-CoA product, wherein the mass ratio of acyl-CoA having a carbon chain length of C4, acyl-CoA having a carbon chain length of C6, acyl-CoA having a carbon chain length of C8, and acyl-CoA having a carbon chain length of CI 2, is about 2:4:2: 1. In one aspect the disclosure provides for an acyl-CoA product, wherein the mass ratio of acyl-CoA having a carbon chain length of C4, acyl-CoA having a carbon chain length of C6, and acyl-CoA having a carbon chain length of C8, is about 7:8: 1.
• the disclosure provides for an acyl-CoA product, wherein the mass ratio of acyl-CoA having a carbon chain length of C4, acyl-CoA having a carbon chain length of C6, acyl-CoA having a carbon chain length of C8, and acyl-CoA having a carbon chain length of C12, is about 2: 1 : 1 : 1.
• the disclosure provides for an acyl- CoA product, wherein the mass ratio of acyl-CoA having a carbon chain length of C4, acyl-CoA having a carbon chain length of C6, acyl-CoA having a carbon chain length of C8, and acyl-CoA having a carbon chain length of C12, is about 8:2:3: 1.
• the acyl-CoA product is selected from the group consisting of 3-ketoacyl-CoA, 3-hydroxyacyl-CoA, and enoyl- CoA.
• the disclosure provides for a free fatty acid or fatty acid-derived product, comprising about 15% to 50 by mass of a free fatty acid or fatty acid-derivative having a carbon chain length of C4. In one aspect the disclosure provides for a free fatty acid or fatty acid- derived product, comprising about 40% to 50%> by mass of a free fatty acid or fatty acid- derivative having a carbon chain length of C6. In one aspect the disclosure provides for a free fatty acid or fatty acid-derived product, comprising about 5% to 30%> by mass of a free fatty acid or fatty acid-derivative having a carbon chain length of C8.
• the disclosure provides for a free fatty acid or fatty acid-derived product, comprising about 1% to 20%> by mass of a free fatty acid or fatty acid-derivative having a carbon chain length of CI 2.
• the disclosure provides for a free fatty acid or fatty acid-derived product, wherein the mass ratio of a free fatty acid or fatty acid-derivative having a carbon chain length of C4, a free fatty acid or fatty acid-derivative having a carbon chain length of C6, a free fatty acid or fatty acid-derivative having a carbon chain length of C8, and acyl-CoA having a carbon chain length of C12, is about 2:4:2: 1.
• the disclosure provides for a free fatty acid or fatty acid-derived product, wherein the mass ratio of a free fatty acid or fatty acid-derivative having a carbon chain length of C4, a free fatty acid or fatty acid-derivative having a carbon chain length of C6, and a free fatty acid or fatty acid-derivative having a carbon chain length of C8, is about 7:8: 1.
• the disclosure provides for a free fatty acid or fatty acid-derived product, wherein the mass ratio of a free fatty acid or fatty acid-derivative having a carbon chain length of C4, a free fatty acid or fatty acid-derivative having a carbon chain length of C6, a free fatty acid or fatty acid-derivative having a carbon chain length of C8, and a free fatty acid or fatty acid-derivative having a carbon chain length of CI 2, is about 2: 1 : 1 : 1.
• the disclosure provides for a free fatty acid or fatty acid-derived product, wherein the mass ratio of a free fatty acid or fatty acid-derivative having a carbon chain length of C4, a free fatty acid or fatty acid-derivative having a carbon chain length of C6, a free fatty acid or fatty acid-derivative having a carbon chain length of C8, and a free fatty acid or fatty acid-derivative having a carbon chain length of C12, is about 8:2:3: 1.
• the disclosure provides for a free fatty acid or fatty acid-derived product, comprising about 16% or greater mass of a free fatty acid or fatty acid-derivative having a carbon chain length of CI 4.
• the disclosure provides for a free fatty acid or fatty acid- derived product, comprising about 20% or greater mass of a free fatty acid or fatty acid- derivative having a carbon chain length of CI 6. In one aspect the disclosure provides for a free fatty acid or fatty acid-derived product, comprising about 36% or greater mass of a free fatty acid or fatty acid-derivative having a carbon chain length of C 14 or CI 6. In one aspect the disclosure provides for a free fatty acid or fatty acid-derived product, comprising about 60%> or greater mass of a free fatty acid or fatty acid-derivative having a carbon chain length of C 14 or CI 6.
• the disclosure provides for a free fatty acid or fatty acid-derived product, wherein the mass ratio of a free fatty acid or fatty acid-derivative having a carbon chain length of C4, a free fatty acid or free fatty acid-derivative having a carbon chain length of C6, a free fatty acid or fatty acid-derivative having a carbon chain length of C8, a free fatty acid or fatty acid-derivative having a carbon chain length of CIO, a free fatty acid or fatty acid-derivative having a carbon chain length of C12, a free fatty acid having a carbon chain length of CI 4, a free fatty acid or fatty acid-derivative having a carbon chain length of CI 6, and a free fatty acid or fatty acid- derivative having a carbon chain length of C18, is about 10:20: 12:7:8: 16:20:7, or about 1 :2: 1 : 1 : 1 :2:2: 1.
• the disclosure provides for an
• the disclosure provides for a method of making one or more fatty acid-derived products selected from the group consisting of fatty ester, fatty amide, fatty alcohol, fatty aldehyde, fatty alkene, fatty alkane, fatty diacid, and any combination thereof, comprising: contacting a carbon source with a microorganism to form a free fatty acid having a carbon chain length of C6 or greater; and converting the free fatty acid to the fatty acid-derived product, wherein the fatty acid-derived product comprises a carbon chain length of C6 or greater.
• the disclosure provides for a method of making an ester of a fatty acid, comprising esterifying a fatty acid produced by a genetically modified organism.
• the disclosure provides for a method of making an amide of a fatty acid, comprising forming an amide of a fatty acid produced by a genetically modified organism of the disclosure. In one aspect the disclosure provides for a method of making a fatty alcohol, comprising forming the fatty alcohol from the fatty acid produced by a genetically modified organism of the disclosure. In one aspect the disclosure provides for a method of making an aldehyde of a fatty acid, comprising forming an aldehyde of a fatty acid produced by a genetically modified organism of the disclosure.
• the disclosure provides for a fuel comprising the acyl-CoA product, free fatty acid product, or fatty-acid derived product of the disclosure.
• a lotion comprising the acyl-CoA product, free fatty acid product, or fatty-acid derived product of the disclosure.
• a soap comprising the acyl-CoA product, free fatty acid product, or fatty-acid derived product of the disclosure.
• a food comprising the acyl-CoA product, free fatty acid product, or fatty-acid derived product of the disclosure.
• the disclosure provides for a cream comprising the acyl-CoA product, free fatty acid product, or fatty-acid derived product of the disclosure.
• the disclosure provides for a shampoo comprising the acyl-CoA product, free fatty acid product, or fatty-acid derived product of the disclosure.
• the disclosure provides for a conditioner comprising the acyl-CoA product, free fatty acid product, or fatty-acid derived product of the disclosure.
• a cleaner comprising the acyl-CoA product, free fatty acid product, or fatty-acid derived product of the disclosure.
• the disclosure provides for a detergent comprising the acyl-CoA product, free fatty acid product, or fatty-acid derived product of the disclosure. In one aspect the disclosure provides for a lubricant comprising the acyl-CoA product, free fatty acid product, or fatty-acid derived product of the disclosure. In one aspect the disclosure provides for a paint comprising the acyl-CoA product, free fatty acid product, or fatty- acid derived product of the disclosure. In one aspect the disclosure provides for a stain comprising the acyl-CoA product, free fatty acid product, or fatty-acid derived product of the disclosure.
• the disclosure provides for an ink comprising the acyl-CoA product, free fatty acid product, or fatty-acid derived product of the disclosure. In one aspect the disclosure provides for a pharmaceutical formulation comprising the acyl-CoA product, free fatty acid product, or fatty-acid derived product of the disclosure. In some embodiments, the product further comprises one or more active agents. In some embodiments, the product further comprises an excipient.
• the disclosure provides for one or more isolated and purified polynucleotides comprising exogenous nucleic acid molecules encoding proteins comprising an acetoacetyl Co A synthase, a keto-CoA reductase, a 3-hydroxy-acyl-CoA dehydratase, an enoyl- CoA reductase, and a thioesterase, wherein the 3-ketoacyl-CoA synthase is selected from the group consisting of NphT7, NphT7 I147T, NphT7 F217V, NphT7 I147T and F217V, Npth7 I147S, Npth7 I147S and F217V, synthase III, synthase IV, synthase V, and synthase VI; the keto- CoA reductase is selected from the group consisting of hbd, fadB, fabG and fadJ the 3-ketoacyl-
• the 3-ketoacyl-CoA synthase is NphT7; the keto-CoA reductase is selected from the group consisting of hbd and fadB; the 3-hydroxy-acyl- CoA dehydratase is selected from the group consisting of crt and fadB; the enoyl-CoA reductase is ter; and the thioesterase is yciA, and wherein the proteins encoded by the polynucleotides are capable of producing a four carbon fatty acid or fatty acid derived product.
• the thioesterase is selected from the group consisting of tesB and yciA.
• the 3-ketoacyl-CoA synthase is NphT7 and wherein the proteins encoded by the polynucleotides are capable of producing a four carbon fatty acid or fatty acid derived product.
• the keto-CoA reductase is selected from the group consisting of hbd and fadB, and wherein the proteins encoded by the polynucleotides are capable of producing a four carbon fatty acid or fatty acid derived product.
• the 3-hydroxy-acyl-CoA dehydratase is selected from the group consisting of crt and fadB, and wherein the proteins encoded by the polynucleotides are capable of producing a four carbon fatty acid or fatty acid derived product.
• the enoyl-CoA reductase is ter, and wherein the proteins encoded by the polynucleotides are capable of producing a four carbon fatty acid or fatty acid derived product.
• the thioesterase is selected from the group consisting of CpTE, fadM, PA2801TE, tesB, ybgC, ybfF, and yciA, and wherein the proteins encoded by the polynucleotides are capable of producing a four carbon fatty acid or fatty acid derived product.
• the thioesterase is selected from the group consisting of tesB and yciA, and wherein the proteins encoded by the polynucleotides are capable of producing a four carbon fatty acid or fatty acid derived product.
• one or more 3-ketoacyl-CoA synthases are selected from the group consisting of NphT7, NphT7 I147T, NphT7 F217V, and NphT7 I147T,F217V; the keto-CoA reductase is fadB; the 3-hydroxy-acyl-CoA dehydratase is fadB; the enoyl-CoA reductase is ter; and the thioesterase is selected from the group consisting of AtTE, CpTE (or CperfTE), PA2801TE, tesA, tesB, ybfF, ybgC, and yciA, and wherein the proteins encoded by the polynucleotides are capable of producing a six carbon fatty acid or fatty acid derived product.
• the keto-CoA reductase is fadB
• the 3-hydroxy-acyl-CoA dehydratase is fadB
• the thioesterase is selected from the group consisting of PA2801TE, tesB, and yciA.
• one or more 3-ketoacyl-CoA synthases are selected from the group consisting of NphT7, NphT7 I147T, NphT7 F217V, and NphT7 I147T and F217V, and wherein the proteins encoded by the polynucleotides are capable of producing a six carbon fatty acid or fatty acid derived product.
• the keto-CoA reductase is fadB, and wherein the proteins encoded by the polynucleotides are capable of producing a six carbon fatty acid or fatty acid derived product.
• the 3-hydroxy-acyl-CoA dehydratase is fadB, and wherein the proteins encoded by the polynucleotides are capable of producing a six carbon fatty acid or fatty acid derived product.
• the enoyl- CoA reductase is ter, and wherein the proteins encoded by the polynucleotides are capable of producing a six carbon fatty acid or fatty acid derived product.
• the thioesterase is selected form the group consisting of AtTE, CpTE (or Cperf E), PA2801TE, tesA, tesB, ybfF, ybgC, and yciA, and wherein the proteins encoded by the polynucleotides are capable of producing a six carbon fatty acid or fatty acid derived product.
• the thioesterase is selected form the group consisting of PA2801TE, tesB, and yciA, and wherein the proteins encoded by the polynucleotides are capable of producing a six carbon fatty acid or fatty acid derived product.
• one or more 3-ketoacyl-CoA synthases are selected from the group consisting of NphT7, NphT7 I147T, NphT7 F217V, NphT7 I147T and F217V, Npth7 I147S, Npth7 I147S and F217V, and synthase III;
• the keto-CoA reductase is selected from the group consisting of fadB and fabG;
• the 3-hydroxy-acyl-CoA dehydratase is selected from the group consisting of fadB, ech and ech2;
• the enoyl-CoA reductase is ter;
• the thioesterase is selected from the group consisting of AtTE, CpTE (or CperfTE), fadM, PA2801TE, tesA, tesB, ybfF, ybgC, and yciA; and wherein the proteins encoded by the polyn
• the thioesterase is selected from the group consisting of PA2801TE, tesB, and yciA.
• one or more 3-ketoacyl-CoA synthases are selected from the group consisting of NphT7, NphT7 I147T, NphT7 F217V, NphT7 I147T and F217V, Npth7 I147S, Npth7 I147S and F217V, and synthase III, and wherein the proteins encoded by the polynucleotides are capable of producing an eight carbon fatty acid or fatty acid derived product.
• the keto-CoA reductase is selected from the group consisting of fadB and fabG, and wherein the proteins encoded by the polynucleotides are capable of producing an eight carbon fatty acid or fatty acid derived product.
• the 3-hydroxy-acyl-CoA dehydratase is selected form the group consisting of fadB, ech and ech2, and wherein the proteins encoded by the polynucleotides are capable of producing an eight carbon fatty acid or fatty acid derived product.
• the enoyl-CoA reductase is ter, and wherein the proteins encoded by the polynucleotides are capable of producing an eight carbon fatty acid or fatty acid derived product.
• the thioesterase is selected from the group consisting of AtTE, CpTE (or CperfTE), fadM, PA2801TE, tesA, tesB, ybfF, ybgC, and yciA, and wherein the proteins encoded by the polynucleotides are capable of producing an eight carbon fatty acid or fatty acid derived product.
• the thioesterase is selected from the group consisting of PA2801TE, tesB, and yciA, and wherein the proteins encoded by the polynucleotides are capable of producing an eight carbon fatty acid or fatty acid derived product.
• one or more 3-ketoacyl-CoA synthases are selected from the group consisting of NphT7, NphT7 I147T, NphT7 F217V, NphT7 I147T and F217V, Npth7 I147S, Npth7 I147S and F217V, synthase III, synthase IV, and synthase V;
• the keto-CoA reductase is selected from the group consisting of fadB and fabG;
• the 3-hydroxy-acyl-CoA dehydratase is selected from the group consisting of fadB, ech and ech2;
• the enoyl-CoA reductase is ter;
• the thioesterase is selected from the group consisting of AtTE, CpTE, fadM, PA2801TE, tesA, tesB, ybfF, ybgC, and yciA, and wherein the proteins
• the thioesterase is selected from the group consisting of tesB and yciA.
• one or more 3-ketoacyl-CoA synthases are selected from the group consisting of NphT7, NphT7 I147T, NphT7 F217V, NphT7 I147T and F217V, Npth7 I147S, Npth7 I147S and F217V, synthase III, synthase IV, and synthase V, and wherein the proteins encoded by the polynucleotides are capable of producing a ten carbon fatty acid or fatty acid derived product.
• the keto-CoA reductase is selected from the group consisting of fadB and fabG, and wherein the proteins encoded by the polynucleotides are capable of producing a ten carbon fatty acid or fatty acid derived product.
• the 3-hydroxy-acyl-CoA dehydratase is selected from the group consisting of fadB, ech and ech2, and wherein the proteins encoded by the polynucleotides are capable of producing a ten carbon fatty acid or fatty acid derived product.
• the enoyl-CoA reductase is ter, and wherein the proteins encoded by the polynucleotides are capable of producing a ten carbon fatty acid or fatty acid derived product.
• the thioesterase is selected from the group consisting of AtTE, CpTE, fadM, PA2801TE, tesA, tesB, ybfF, ybgC, and yciA, and wherein the proteins encoded by the polynucleotides are capable of producing a ten carbon fatty acid or fatty acid derived product.
• the thioesterase is selected from the group consisting of tesB and yciA, and wherein the proteins encoded by the polynucleotides are capable of producing a ten carbon fatty acid or fatty acid derived product.
• one or more 3- ketoacyl-CoA synthases are selected from the group consisting of NphT7, NphT7 I147T, NphT7 F217V, NphT7 I147T and F217V, Npth7 I147S, Npth7 I147S and F217V, synthase III, synthase IV, synthase V, and synthase VI;
• the keto-CoA reductase is selected from the group consisting of fadB, fabG, and fadJ
• the 3-hydroxy-acyl-CoA dehydratase is selected from the group consisting of fadB, ech, and fadJ;
• the enoyl-CoA reductase is ter;
• the thioesterase is selected from the group consisting of AtTE, CpTE (or CperfTE), fadM, LpTE, PA2801TE, tesA, tesB,
• the thioesterase is selected from the group consisting of fadM, PA2801TE, tesA, tesB, and yciA.
• one or more 3-ketoacyl-CoA synthases are selected from the group consisting of NphT7, NphT7 I147T, NphT7 F217V, NphT7 I147T and F217V, Npth7 I147S, Npth7 I147S and F217V, synthase III, synthase IV, synthase V, and synthase VI, and wherein the proteins encoded by the polynucleotides are capable of producing a twelve carbon fatty acid or fatty acid derived product.
• the keto-CoA reductase is selected form the group consisting of fadB, fabG, and fadJ, and wherein the proteins encoded by the polynucleotides are capable of producing a twelve carbon fatty acid or fatty acid derived product.
• the 3- hydroxy-acyl-CoA dehydratase is selected from the group consisting of fadB, ech, and fadJ, and wherein the proteins encoded by the polynucleotides are capable of producing a twelve carbon fatty acid or fatty acid derived product.
• the enoyl-CoA reductase is ter, and wherein the proteins encoded by the polynucleotides are capable of producing a twelve carbon fatty acid or fatty acid derived product.
• the thioesterase is selected from the group consisting of AtTE, CpTE (or CperfTE), fadM, LpTE, PA2801TE, tesA, tesB, ybfF, ybgC, and yciA, and wherein the proteins encoded by the polynucleotides are capable of producing a twelve carbon fatty acid or fatty acid derived product.
• the thioesterase is selected from the group consisting of fadM, PA2801TE, tesA, tesB, and yciA, and wherein the proteins encoded by the polynucleotides are capable of producing a twelve carbon fatty acid or fatty acid derived product.
• one or more 3-ketoacyl-CoA synthases are selected from the group consisting of NphT7, NphT7 I147T, NphT7 F217V, NphT7 I147T and F217V, Npth7 I147S, Npth7 I147S and F217V, synthase III, synthase IV, synthase V, and synthase VI;
• the keto-CoA reductase is selected from the group consisting of fadB, and fadJ;
• the 3-hydroxy-acyl-CoA dehydratase is selected form the group consisting of fadB, and fadJ;
• the enoyl-CoA reductase is selected from the group consisting of ter, ydiO and fadE;
• the thioesterase is selected from the group consisting of AtTE, CpTE (or CperfTE), fadM, LpTE
• the thioesterase is selected from the group consisting of fadM, tesA, tesB, and yciA, and the proteins encoded by the polynucleotides are capable of producing a fourteen carbon fatty acid or fatty acid derived product.
• the thioesterase is selected from the group consisting of AtTE, CpTE (or CperfTE), fadM, Pa2801TE, tesA, tesB, ybfF, ybgC, and yciA, and the proteins encoded by the polynucleotides are capable of producing a sixteen carbon fatty acid or fatty acid derived product.
• the thioesterase is selected from the group consisting of AtTE, fadM, tesA, tesB, ybfF, ybgC, and yciA, and the proteins encoded by the polynucleotides are capable of producing a sixteen carbon fatty acid or fatty acid derived product.
• one or more 3- ketoacyl-CoA synthases are selected from the group consisting of NphT7, NphT7 I147T, NphT7 F217V, NphT7 I147T and F217V, Npth7 I147S, Npth7 I147S and F217V, synthase III, synthase IV, synthase V, and synthase VI, and wherein the proteins encoded by the polynucleotides are capable of producing a fatty acid with a carbon chain length of greater than or equal to fourteen carbons.
• the keto-CoA reductase is selected form the group consisting of fadB, and fadJ, and wherein the proteins encoded by the polynucleotides are capable of producing a fatty acid with a carbon chain length of greater than or equal to fourteen carbons.
• the 3-hydroxy-acyl-CoA dehydratase is selected form the group consisting of fadB, and fadJ, and wherein the proteins encoded by the polynucleotides are capable of producing a fatty acid with a carbon chain length of greater than or equal to fourteen carbons.
• the enoyl-CoA reductase is selected form the group consisting of ter, ydiO and fadE, and wherein the proteins encoded by the polynucleotides are capable of producing a fatty acid with a carbon chain length of greater than or equal to fourteen carbons.
• the thioesterase is selected form the group consisting of AtTE, CpTE (or CperfTE), fadM, LpTE, PA2801TE, tesA, tesB, ybfF, ybgC, and yciA, and wherein the proteins encoded by the polynucleotides are capable of producing a fatty acid with a carbon chain length of greater than or equal to fourteen carbons.
• the disclosure provides for one or more isolated and purified polynucleotides comprising exogenous nucleic acid molecules encoding proteins comprising a 3- oxoacyl-(acyl carrier protein) synthase III from a species selected from the group consisting of Alishewanella aestuarii Bl l, Arcobacter butzleri ED-1, Clostridiales bacterium 1_7_47_FAA, Gluconacetobacter oboediens 174Bp2, Gordonia aichiensis NBRC 108223, Mesorhizobium sp.
• a species selected from the group consisting of Alishewanella aestuarii Bl l, Arcobacter butzleri ED-1, Clostridiales bacterium 1_7_47_FAA, Gluconacetobacter oboediens 174Bp2, Gordonia aichiensis NBRC 108223, Mesorhizobium sp.
• the 3-oxoacyl-(acyl carrier protein) synthase III is from a species selected from the group consisting of Pelosinus fermentans DSM 17108, Saccharomonospora glauca K62, Verrucosispora maris AB- 18-032, and Clostridiales bacterium 1 7 47 FAA, and wherein the proteins encoded by the polynucleotides are capable of producing an acetyl-CoA.
• the 3-oxoacyl-(acyl carrier protein) synthase III is from a species selected from the group consisting of Saccharomonospora glauca K62, Saccharomonospora azurea NA-128, Mesorhizobium sp. STM 4661, and Clostridiales bacterium 1 7 47 FAA, and wherein the proteins encoded by the polynucleotides are capable of producing a four carbon fatty acid or fatty acid derived product.
• the 3-oxoacyl-(acyl carrier protein) synthase III is from a species selected from the group consisting of Gordonia aichiensis NBRC 108223, Arcobacter butzleri ED-1, Clostridiales bacterium 1_7_47_FAA, Saccharomonospora glauca K62, and Ralstonia solanacearum Po82, and wherein the proteins encoded by the polynucleotides are capable of producing a six carbon fatty acid or fatty acid derived product.
• the 3-oxoacyl-(acyl carrier protein) synthase III is from a species selected from the group consisting of Gordonia aichiensis NBRC 108223, Gluconacetobacter oboediens 174Bp2, Arcobacter butzleri ED-1, Ralstonia solanacearum Po82, and Phaeobacter gallaeciensis 2.10, and wherein the proteins encoded by the polynucleotides are capable of producing an eight carbon fatty acid or fatty acid derived product.
• the 3-oxoacyl-(acyl carrier protein) synthase III is from Alishewanella aestuarii Bl l, and wherein the proteins encoded by the polynucleotides are capable of producing a ten carbon fatty acid or fatty acid derived product.
• the proteins encoded by the polynucleotides further comprise a 3-ketoacyl-CoA synthase from Streptomyces sp. (strain CL190).
• Figure 1 is a chart showing the carbon chain length distribution in oleochemical feedstocks used to make fatty acids and fatty acid derivatives.
• Figure 2 is a diagram that illustrates various complete bioproduction pathways of the present invention, and provides a representative example of the conversion of various carbon sources to a CIO fatty acid or CIO fatty ester.
• Figure 3 is a diagram showing production of even chain fatty acids using acetyl-
• CoA as a primer and malonyl-CoA (MCA) as the extender molecule.
• Figure 4 is a diagram showing production of even chain fatty acid esters using acetyl-CoA as a primer and malonyl-CoA (MCA) as the extender molecule.
• Figure 5 is a diagram showing production of odd chain fatty acids using propionyl-CoA as a primer and malonyl-CoA (MCA) as the extender molecule.
• Figure 6 is a diagram showing production of odd chain fatty acid esters using propionyl-CoA as a primer and malonyl-CoA (MCA) as the extender molecule.
• Figure 7 - Figure 14 are a series of various reaction pathways in accordance with the present invention.
• Figure 15 is a bar chart showing the formation of acyl-CoA products produced with NphT7 variants and NphT7 mutants acting (100 ⁇ g, 30 min. assays) on Malonyl-CoA and C4-, C6-, or ClO-CoA to generate the corresponding C6-, C8- and C12-hydroxyacyl-CoA in the presence of PaFabG hydroxyacyl-CoA reductase.
• Figure 16 is a bar chart showing activity of thioesterases on acyl-CoA substrates of different carbon chain lengths.
• Figure 17 is a bar chart showing production rates of C8-C18 free fatty acids produced in different bacteria strains.
• Figure 18 is a bar chart showing titers of C6-C18 free fatty acids produced in different bacteria strains.
• Figure 19 is a pie chart showing the chain length specificity distribution of free fatty acids produced in strain sample 3.
• Figure 20 is a bar chart showing the chain length specificity preference of different 3HDh.
• Figure 21 is a graph showing the fatty acid carbon chain length distribution produced by various microorganisms in accordance with the present invention.
• Figure 22 includes a series of pie charts showing the fatty acid carbon chain length distribution produced by various microorganisms in accordance with the present invention.
• Figure 23 is a bar chart showing the amounts of C4, C6, and C8 free fatty acid produced by various genetically modified microorganisms in accordance with the present invention.
• Figure 24 is a bar chart showing the amounts of total fatty acids (C4-C18) produced by various genetically modified microorganisms in accordance with the present invention.
• Figure 25 includes a series of pie charts showing the distribution of free fatty acids produced by various genetically modified microorganisms in accordance with the present invention.
• Figure 26 shows a fatty acid pathway that comprises four steps which utilizes a pathway that is similar to the type II fatty acid synthesis (FAS) system utilized by bacteria. Both fatty acid syntheses are shown in Figure 26.
• A. In step 1, 3-ketoacyl-CoA synthase catalyzes the condensation of acyl-CoA (or acetyl-CoA at initial step of chain elongation) with malonyl-CoA to yield ⁇ -ketoacyl-CoA.
• ⁇ -ketoacyl-CoA undergoes reduction by ⁇ - ketoacyl-CoA reductase (step 2), dehydration by ⁇ -hydroxyacyl-CoA dehydratase (step 3), and a final reduction by enoyl-CoA reductase (step 4). Reactions are repeated and each cycle adds two carbons to the acyl-CoA chain.
• B Type II FAS System. Fatty acid synthesis is initiated by ⁇ - ketoacyl-ACP synthase (KASIII) (step la) which catalyzes the condensation of acetyl-CoA with malonyl-ACP to yield ⁇ -ketoacyl-ACP.
• ⁇ -ketoacyl-ACP undergoes reduction (step 2), dehydration (step 3), and reduction (step 4) similar to CoA specific pathway.
• Further elongation steps are initiated by KASI or KASII (step lb) which catalyzes the condensation of acyl-ACP with malonyl-ACP.
• Figure 27 depicts the novel CoA dependent fatty acid pathway and the key enzymes associated therewith.
• Figure 27 shows the novel CoA dependent fatty acid pathway and the key enzymes associated therewith.
• the present invention relates generally to various production methods and/or genetically modified microorganisms that have utility for fermentative production of various chemical products, to methods of making such chemical products that utilize populations of these microorganisms in vessels, and to systems for chemical production that employ these microorganisms and methods.
• Among the benefits of the present invention is increased specific productivity when such microorganisms produce a chemical product during a fermentation event or cycle.
• the present invention provides production techniques and/or genetically modified microorganisms to produce a chemical product of interest, such as a fatty acid or fatty acid derived product.
• the invention provides for one or more means for modulating conversion of malonyl-CoA to fatty acyl molecules, wherein the production pathway comprises a malonyl-CoA dependent pathway that includes an enzymatic conversion step that uses malonyl-CoA as a substrate.
• the malonyl-CoA dependent pathway is also a malonyl-ACP independent pathway, and is used in combination with the inhibition of a microorganism's native malonyl-ACP dependent fatty acid synthase pathway.
• fatty acid or fatty acid derived products are produced in a manner dependent on both a malonyl-CoA dependent pathway and a malonyl- ACP dependent pathway.
• the genetically modified microorganisms of the invention are metabolically engineered to increase utilization of malonyl-CoA for production of a fatty acid or fatty acid derived product, through a metabolic pathway that is at least in part malonyl-CoA dependent.
• the fatty acid derived products may include esters, aldehydes, alcohols, alkanes, alkenes, and diacids, with various degrees of desaturation and chain branching, and further downstream products made from such chemical products. Also, genetic modifications may be made to provide one or more chemical products.
• the present invention also relates to genetically engineered microorganisms having encoded therein unique enzymes and combinations of enzymes that function within the malonyl-CoA dependent pathway to produce fatty acids or fatty acid derived products of specific chain lengths.
• the microorganisms and methods provide a cost-competitive means of producing relatively high concentrations of fatty acids or fatty acid derived products of specific chain lengths or products having a relatively narrow carbon chain length distribution (i.e., 2, 3, 4 or less than 5 different carbon chain lengths with in the fatty acid product, e.g. C8/C10 or C8/C10/C12).
• R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V represent the twenty amino acids commonly found in peptides synthesized in nature.
• the convention LetterlNumberLetter2 when applied to polypeptides, means that the amino acid having the one letter code Letter 1, at Number position in the polypeptide, is substituted with the amino acid having the one letter code Letter2.
• I147T means that the amino acid I, found at position 147 in the peptide, is substituted with the amino acid T.
• the symbol CNumber means a carbon backbone chain length having the indicated number of carbon atoms.
• C20 means a chemical backbone having a 20 carbon chain length. Note that the number of carbons included in the carbon backbone does not include carbon contained in functional units attached to the backbone (e.g., a functional unit in a fatty acid derived product).
• enzymatic conversion of the indicated substrate(s) to indicated product(s) under known standard conditions for that enzyme is at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 percent less than the enzymatic activity for the same biochemical conversion by a native (non-modified) enzyme under a standard specified condition.
• enzymatic conversion of the indicated substrate(s) to indicated product(s) under known standard conditions for that enzyme is at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 percent less than the enzymatic activity for the same biochemical conversion by a native (non-modified) enzyme under a standard specified condition.
• These terms also can include elimination of that enzymatic activity.
• a decrease in enzymatic activity may be achieved in variety a ways known to those skilled in the art, including for example, a gene disruption or a gene deletion.
• a decrease in enzymatic activity may be temporal, be controlled through the expression of various genetic elements, or decrease in response to the cultivation conditions of the cell.
• a cell having reduced enzymatic activity of an enzyme can be identified using any method known in the art. For example, enzyme activity assays can be used to identify cells having reduced enzyme activity. See, for example, Enzyme Nomenclature, Academic Press, Inc., New York 2007.
• enzymatic activity As used herein, “increase enzymatic activity,” “increasing enzymatic activity,” and the like is meant to indicate that a microorganism cell's, or an isolated enzyme, exhibits a higher level of activity than that measured in a comparable cell of the same species or its native enzyme. That is, enzymatic conversion of the indicated substrate(s) to indicated product(s) under known standard conditions for that enzyme is at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 percent greater than the enzymatic activity for the same biochemical conversion by a native (non-modified) enzyme under a standard specified condition. These terms also can include addition of an exogenous enzymatic activity.
• An increase in enzymatic activity may be temporal, be controlled through the expression of various genetic elements, or increase in response to the cultivation conditions of the cell.
• a cell having increased enzymatic activity of an enzyme can be identified using any method known in the art, including the enzyme activity assays noted above used to identify cells having reduced enzyme activity.
• the genetic modification can be, for example, deletion of the entire gene, deletion or other modification of a regulatory sequence required for transcription or translation, deletion of a portion of the gene which results in a truncated gene product (e.g., enzyme) or by any of various mutation strategies that reduces activity (including to no detectable activity level) of the encoded gene product.
• a disruption may broadly include a deletion of all or part of the nucleic acid sequence encoding the enzyme, and also includes, but is not limited to other types of genetic modifications, e.g., introduction of stop codons, frame shift mutations, introduction or removal of portions of the gene, and introduction of a degradation signal, those genetic modifications affecting mRNA transcription levels and/or stability, and altering the promoter or repressor upstream of the gene encoding the enzyme.
• a gene disruption is taken to mean any genetic modification to the DNA, mRNA encoded from the DNA, and the corresponding amino acid sequence that results in reduced polypeptide activity.
• Many different methods can be used to make a cell having reduced polypeptide activity.
• a cell can be engineered to have a disrupted regulatory sequence or polypeptide-encoding sequence using common mutagenesis or knock-out technology. See, e.g., Methods in Yeast Genetics (1997 edition), Adams et al., Cold Spring Harbor Press (1998).
• One particularly useful method of gene disruption is complete gene deletion because it reduces or eliminates the occurrence of genetic reversions in the genetically modified microorganisms of the invention.
• antisense technology can be used to reduce the activity of a particular polypeptide.
• a cell can be engineered to contain a cDNA that encodes an antisense molecule that prevents a polypeptide from being translated.
• gene silencing can be used to reduce the activity of a particular polypeptide.
• heterologous is intended to include the term “exogenous” as the latter term is generally used in the art.
• Heterologous can refer to polypeptides and/or nucleic acids which are not ordinarily produced by the host cell.
• Such heterologous polypeptides and/or nucleic acid thus may comprise polypeptides which either do not have substantial amino acid sequence homology with those proteins produced by the host cell or may comprise polypeptides with substantial but incomplete homology to proteins produced by the host cell or the cell line from which the host cell is derived.
• heterologous DNA refers to a nucleic acid sequence wherein at least one of the following is true: (a) the sequence of nucleic acids is foreign to (i.e., not naturally found in) a given host microorganism; (b) the sequence may be naturally found in a given host microorganism, but in an unnatural amount (e.g., greater than expected) or position; or (c) the sequence of nucleic acids comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, regarding instance (c), a heterologous nucleic acid sequence that is recombinantly produced will have two or more sequences from unrelated genes arranged to make a new functional nucleic acid.
• antisense molecule encompasses any nucleic acid molecule or nucleic acid analog (e.g., peptide nucleic acids) that contains a sequence that corresponds to the coding strand of an endogenous polypeptide.
• An antisense molecule also can have flanking sequences (e.g., regulatory sequences).
• antisense molecules can be ribozymes or antisense oligonucleotides.
• a ribozyme can have any general structure including, without limitation, hairpin, hammerhead, or axhead structures, provided the molecule cleaves RNA.
• Bio-production may be aerobic, microaerobic, or anaerobic.
• the language "sufficiently homologous" refers to proteins or portions thereof that have amino acid sequences that include a minimum number of identical or equivalent amino acid residues when compared to an amino acid sequence of the amino acid sequences provided in this application (including the SEQ ID Nos./sequence listings) such that the protein or portion thereof is able to achieve the respective enzymatic reaction and/or other function.
• To determine whether a particular protein or portion thereof is sufficiently homologous may be determined by an assay of enzymatic activity, such as those commonly known in the art.
• nucleic acid sequences may be varied and still encode an enzyme or other polypeptide exhibiting a desired functionality, and such variations are within the scope of the present invention.
• hybridization refers to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide.
• the term “hybridization” may also refer to triple-stranded hybridization.
• the resulting (usually) double-stranded polynucleotide is a “hybrid” or “duplex.”
• “Hybridization conditions” will typically include salt concentrations of less than about 1M, more usually less than about 500 mM and less than about 200 mM.
• Hybridization temperatures can be as low as 5°C, but are typically greater than 22°C, more typically greater than about 30°C, and often are in excess of about 37°C.
• Hybridizations are usually performed under stringent conditions, i.e. conditions under which a probe will hybridize to its target subsequence. Stringent conditions are sequence-dependent and are different in different circumstances. Longer fragments may require higher hybridization temperatures for specific hybridization. As other factors may affect the stringency of hybridization, including base composition and length of the complementary strands, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone.
• stringent conditions are selected to be about 5°C lower than the T m (temperature at which half the DNA is present in a single-stranded (denatured) form) for the specific sequence at a defined ionic strength and pH.
• Exemplary stringent conditions include salt concentration of at least 0.01 M to no more than 1 M Na ion concentration (or other salts) at a pH 7.0 to 8.3 and a temperature of at least 25°C.
• salt concentration at least 0.01 M to no more than 1 M Na ion concentration (or other salts) at a pH 7.0 to 8.3 and a temperature of at least 25°C.
• 5 X SSPE 750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4
• a temperature of 25-30°C are suitable for allele-specific probe hybridizations.
• Hybridizing specifically to or “specifically hybridizing to” or like expressions refer to the binding, duplexing, or hybridizing of a molecule substantially to or only to a particular nucleotide sequence or sequences under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
• segment of interest is meant to include both a gene and any other nucleic acid sequence segment of interest.
• One example of a method used to obtain a segment of interest is to acquire a culture of a microorganism, where that microorganism's genome includes the gene or nucleic acid sequence segment of interest.
• the genetic modification of a gene product i.e., an enzyme
• the genetic modification is of a nucleic acid sequence, such as or including the gene, that normally encodes the stated gene product, i.e., the enzyme.
• a truncated respective polypeptide has at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%>, or at least about 97%, or at least about 98%, or at least about 99% of the full length of a polypeptide encoded by a nucleic acid sequence encoding the respective native enzyme, and more particularly at least 95% of the full length of a polypeptide encoded by a nucleic acid sequence encoding the respective native enzyme.
• a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a polypeptide is intended that the amino acid sequence of the claimed polypeptide is identical to the reference sequence except that the claimed polypeptide sequence can include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the polypeptide.
• up to 5% of the amino acid residues in the reference sequence can be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence can be inserted into the reference sequence.
• alterations of the reference sequence can occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. In other embodiments truncation may be more substantial, as described elsewhere herein. [0092] Species and other phylogenic identifications are according to the classification known to a person skilled in the art of microbiology.
• DCW means dry cell weight
• “s” means second(s)
• “min” means minute(s)
• “h,” “hr,” or “firs” means hour(s)
• "psi” means pounds per square inch
• “nm” means nanometers
• “d” means day(s)
• " ⁇ ” or “uL” or “ul” means microliter(s)
• “mL” means milliliter(s)
• “L” means liter(s)
• “mm” means millimeter(s)
• “nm” means nanometers
• “mM” means millimolar
• " ⁇ ” or “uM” means micromolar
• “M” means molar
• “mmol” means millimole(s)
• " ⁇ ” or “uMol” means micromole(s)
• "g” means gram(s)
• ' ⁇ g” or “ug” means microgram(s)
• “ng” means nanogram(s)
• “PCR”
• Synthase VI (except in the context of the name of a specific enzyme sequence included in a FASTA header in one of the Tables) shall refer to the third, fourth, fifth and sixth synthase, respectively, that is included in among a group of synthases.
• Synthase III, synthase IV, synthase V, and synthase VI may be any 3-ketoacyl-CoA synthase disclosed herein.
• a reference herein to a genetically modified organism comprising a heterologous nucleic acid sequence encoding a 3-ketoacyl-CoA synthase selected from the group consisting of synthase III and synthase IV means either: (1) a genetically modified organism comprising a heterologous nucleic acid sequence encoding at least three 3-ketoacyl-CoA synthases wherein at least one of such 3-ketoacyl-CoA synthases is a 3-ketoacyl-CoA synthase disclosed herein; or (2) a genetically modified organism comprising a heterologous nucleic acid sequence encoding at least four 3-ketoacyl-CoA synthases wherein at least one of such 3-ketoacyl-CoA synthases is a 3- ketoacyl-CoA synthase disclosed herein.
• the present invention relates to a fatty acid pathway that comprises four steps which utilizes a pathway that is similar to the type II fatty acid synthesis (FAS) system utilized by bacteria.
• FAS fatty acid synthesis
• Both fatty acid syntheses are shown below in Scheme 1.
• both pathways are cyclical processes that involve: 1) condensation of acyl chain, 2) reduction of the condensation product, 3) dehydration, and 4) reduction to produce an acyl chain that is two carbon atoms longer and the process is repeated with each cycle adding two additional carbons.
• most enzymes utilized for the type II FAS system can also function in the propose fatty acid pathway.
• a key step involving the chain elongation of acyl moiety is quite different.
• a condensation step of the proposed fatty acid pathway employs, inter alia, a ketoacyl-CoA synthase that catalyzes the condensation of acyl-CoA with malonyl-CoA, while type II FAS system utilizes ketoacyl-ACP synthases that catalyzes the condensation of acyl-ACP with malonyl-ACP.
• This type of CoA dependent pathway has been previously known for elongation of longer fatty acid chain lengths (e.g., elongation to C14 to C16 or higher).
• applicants have discovered novel genetically modified
• microorganisms capable of producing fatty acids through the elongation pathway illustrated in Scheme 1A and which is capable of elongation of lower carbon chain lengths through this pathway (e.g., elongation of C4 to C6, C6 to C8, C8 to CIO, CIO to C12, and C12 to C14).
• elongation of C4 to C6, C6 to C8, C8 to CIO, CIO to C12, and C12 to C14 elongation of C4 to C6, C6 to C8, C8 to CIO, CIO to C12, and C12 to C14.
• ⁇ -Ketoacyl and 3-ketoacyl are synonymous.
• 0 illustrates the production of even chain fatty acid esters using acetyl-CoA as a primer and malonyl-CoA (MCA) as the extender molecule.
• Figure 5 illustrates the production of odd chain fatty acids using propionyl-CoA as a primer and malonyl-CoA (MCA) as the extender molecule.
• Figure 6 illustrates the production of odd chain fatty acid esters using propionyl-CoA as a primer and malonyl-CoA (MCA) as the extender molecule.
• fatty acid or fatty acid derived products are produced in a manner dependent at least in part on a malonyl-CoA dependent pathway.
• the malonyl-CoA dependent pathway is also a malonyl- ACP independent fatty acid production pathway, and may be used in combination with the inhibition of a microorganism's malonyl-ACP dependent fatty acid synthase pathway.
• fatty acid or fatty acid derived products are produced through a microorganism pathway that is partially malonyl-CoA dependent and partially malonyl-ACP dependent.
• fatty acid or fatty acid derived products are produced through a microorganism pathway that is initiated through the reaction of malonyl-CoA and acetyl-CoA via a CoA dependent pathway.
• FIG. 7 to Figure 14 examples of various malonyl-CoA dependent pathways are illustrated.
• the pathways illustrated are examples of the production of fatty acids or esters having carbon chain lengths of 4, 6, 8, or 10.
• One skilled in the art would appreciate that in view of cyclic nature of the pathways, the pathways could be extended to depict higher carbon chain lengths.
• genetically modified microorganisms are provided that include various combinations of enzymes that determine (1) the carbon chain lengths produced by the organism, and (2) the extent to which the pathway is CoA-dependent or both CoA- and ACP-dependent.
• the acetyl-CoA precursor that initiates the pathways shown in Figure 7Error! Reference source not found. to Figure 14 is changed to propionyl-CoA, then fatty acids and esters having a carbon chain length that is an odd number (i.e., 5, 7, 9, or 1 1) will be made through the pathways.
• the present invention herein provides genetically modified microorganisms that are modified to enable and/or improve a microorganism's ability to produce fatty acids and/or fatty acid derivatives at least in part through a malonyl-CoA dependent pathway.
• the malonyl- CoA dependent pathway may be independent of a malonyl-ACP pathway or may be in combination with a malonyl-ACP pathway.
• the genetically modified organism herein can be Clostridium,
• Zymomonas Escherichia, Salmonella, Rhodococcus, Pseudomonas, Streptomyces, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula, Thraustochytrids, Bacteriophage, Saccharomyces; can be a prokaryotic cell; can be a eukaryotic cell; and/or can be a bacteria, yeast, fungi, microalgae or algae cell.
• the genetically modified organism is Escherichia coli.
• the genetic modifications contemplated by the present invention include enhancing the organism's function in three phases of a CoA-dependent fatty acid pathway contemplated herein: (1) initiation of the fatty acid pathway; (2) chain length extension (or elongation); and (3) termination of the process once a desired chain length is achieved. These three phases are exemplified in Figure 7 to Figure 14.
• the first phase of the malonyl-CoA dependent pathway is reaction initiation.
• the reaction to produce even chain fatty acid products is initiated through the conversion of acetyl- CoA + malonyl-CoA to 3-ketobutyryl-CoA.
• This conversion requires a synthase - a ketobutyryl- CoA synthase.
• the reaction initiation phase is completed by the conversion of ketobutyryl-CoA to butyryl-CoA by three enzymes: a ketoacyl-CoA reductase ("KCPv"), a hydroxyacyl-CoA dehydratase (“3HDh”), and an enoyl-CoA reductase ("EnCr").
• KCPv ketoacyl-CoA reductase
• 3HDh hydroxyacyl-CoA dehydratase
• EnCr enoyl-CoA reductase
• a genetically modified microorganism of the present invention includes native or exogenous enzymes encoded therein that provide these functions.
• NphT7 a 3-ketoacyl-CoA synthase from Streptomyces sp.
• Strain CL190 acts as the ketobutyryl-CoA synthase that initiates fatty acid synthesis by catalyzing the condensation of acetyl-CoA with malonyl-CoA to 3- ketobutyryl-CoA and with reduction dehydration reduction to butyryl-CoA (C4-C0A).
• NphT7 acts as the 3-ketovaleryl-CoA synthase that initiates fatty acid synthesis by catalyzing the condensation of propionyl-CoA with malonyl-CoA to 3-ketovaleryl-CoA and with reduction dehydration reduction to valeryl- CoA (C5-C0A).
• the protein sequence for NphT7 (BAJ10048.1 GL299758082) and its nucleotide sequence (AB540131.1 GL299758081) are provided below (SEQ ID NO: l; SEQ ID NO:2).
• the 3-ketobutyryl-CoA synthase of the present invention is a homolog to a synthase comprising a protein sequence of any one of SEQ ID NOs. 1-120, as shown in Table 1 below.
• the 3-ketobutyryl-CoA synthase of the present invention is a 3-ketoacyl-CoA synthase that comprises an amino acid sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 94%, about 96%, about 98%, or about 99%, but less than 100% or about 100% homology to any one of SEQ ID NOs. 1-120.
• the method herein comprises selecting at least two of 3- ketoacyl-CoA synthases, wherein each synthase occupies a different branch of a phylogenetic tree.
• the present invention provides a library of NphT7 homologs herein selected by a method herein.
• the 3-ketovaleryl-CoA synthase of the present invention is a homolog to a synthase comprising a protein sequence of any one of SEQ ID NOs. 1-120, as shown in Table 1 below.
• the 3-ketovaleryl-CoA synthase of the present invention is a 3-ketoacyl-CoA synthase that comprises an amino acid sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 94%, about 96%, about 98%, or about 99%, but less than 100% or about 100% homology to any one of SEQ ID NOs. 1-120.
• the method herein comprises selecting at least two of 3- ketoacyl-CoA synthases, wherein each synthase occupies a different branch of a phylogenetic tree.
• the present invention provides a library of NphT7 homologs herein selected by a method herein.
• VQKHVA SEQ ID NO FASTA Header Protein sequence
• reaction initiation phase for even chain fatty acid products is completed by the conversion of 3-ketobutyryl-CoA to butyryl-CoA by three enzymes: a ketoacyl- CoA reductase, a hydroxyacyl-CoA dehydratase, and an enoyl-CoA reductase.
• the ketoacyl-CoA reductase may be selected from the group consisting of 3-ketobutyryl-CoA reductase (e.g., fadB, a bifunctional enzyme - SEQ ID NO 183) and 3-hydroxybutyryl-CoA dehydrogenase (e.g., hbd - SEQ ID NO 271); the hydroxyacyl-CoA dehydratase may be selected from the group consisting of 3-hydroxybutyryl-CoA dehydratase (e.g., fadB, a bifunctional enzyme - SEQ ID NO 183) and enoyl-CoA hydratase (e.g., crt - SEQ ID NO 272); and the enoyl-CoA reductase may be trans-2-enoyl-reductase (e.g., ter - SEQ ID NO 275).
• 3-ketobutyryl-CoA reductase e.g.,
• the bifunctional fadB is both the ketoacyl-CoA reductase and the hydroxyacyl-CoA dehydratase, or the ketoacyl-CoA reductase is hbd and the hydroxyacyl-CoA dehydratase is crt.
• the reaction initiation phase for odd chain fatty acid products is completed by the conversion of 3-ketovaleryl-CoA to valeryl-CoA by three enzymes: a ketoacyl- CoA reductase, a hydroxyacyl-CoA dehydratase, and an enoyl-CoA reductase.
• the ketoacyl-CoA reductase may be selected from the group consisting of 3-ketovaleryl-CoA reductase (e.g., fadB, a bifunctional enzyme - SEQ ID NO 183) and 3-hydroxyvaleryl-CoA dehydrogenase (e.g., hbd - SEQ ID NO 271); the hydroxyacyl-CoA dehydratase may be selected from the group consisting of 3-hydroxyvaleryl-CoA dehydratase (e.g., fadB, a bifunctional enzyme - SEQ ID NO 183) and enoyl-CoA hydratase (e.g., crt - SEQ ID NO 272); and the enoyl-CoA reductase may be trans-2-enoyl-reductase (e.g., ter - SEQ ID NO 275).
• 3-ketovaleryl-CoA reductase e.g., f
• the bifunctional fadB is both the ketoacyl-CoA reductase and the hydroxyacyl-CoA dehydratase, or the ketoacyl-CoA reductase is hbd and the hydroxyacyl-CoA dehydratase is crt.
• the initiation phase may be achieved through a malonyl-ACP dependent pathway with at least a portion of one or more subsequent phases (i.e., elongation phase and/or termination phase) relying upon the malonyl-CoA dependent pathway.
• the reaction to produce even chain fatty acid products is initiated through conversion of acetyl-CoA + malonyl-ACP to 3-ketobutyryl-ACP.
• the reaction to produce even chain fatty acid products is initiated through conversion of propionyl-CoA + malonyl-ACP to 3-ketovaleryl-ACP.
• a genetically modified microorganism having encoded therein one or more enzymes described herein that catalyze reactions along the malonyl-CoA dependent pathway, and wherein native enzymes facilitate the initiation phase through the native malonyl-ACP pathway.
• a CoA-dependent elongation phase immediately follows an ACP-dependent initiation phase (see for example Figure 13)
• the microorganism must also encode for a transacylase, such as butyryl-ACP:CoA transacylase, which will convert butyryl-ACP to butyryl- CoA or valeryl-ACP:CoA transacylase, which will convert valeryl-ACP to valeryl-CoA.
• a transacylase such as butyryl-ACP:CoA transacylase, which will convert butyryl-ACP to butyryl- CoA or valeryl-ACP:CoA transacylase, which will convert valeryl-ACP to valeryl-CoA.
• the microorganism must also encode for a transacylase, such as butyryl-CoA:ACP transacylase, which will convert butyryl-CoA to butyryl- ACP or valeryl- CoA:ACP transacylase, which will convert valeryl-CoA to valeryl-ACP.
• a transacylase such as butyryl-CoA:ACP transacylase, which will convert butyryl-CoA to butyryl- ACP or valeryl- CoA:ACP transacylase, which will convert valeryl-CoA to valeryl-ACP.
• Suitable butyryl-CoA:ACP transacylase include fabH, preferably from E. Coli, FASN, preferably from Homo sapiens, and FAS1, preferably from Saccharomyces cerevisiae.
• Additional transacylases include enzymes of the class 2.3.1.38, such as from Brassica juncea, Euglena gracilis, and ACT from Streptomyces collinus.
• a genetically modified microorganism may be encoded for a gene that transitions a fatty acid production pathway from an ACP-dependent pathway to a CoA- dependent pathway, or conversely from a CoA-dependent pathway to an ACP-dependent pathway, by converting any ACP intermediate to its corresponding CoA intermediate, or vice versa.
• the genetically modified microorganism may be encoded for phaG, preferably from Pseudomonas putida KT2440, which converts 3-hydroxyacyl-ACP to 3- hydroxyacyl-CoA.
• the second phase of the malonyl-CoA dependent pathway involves a cyclic process wherein the length of the carbon chain is extended by two carbons with each cycle. As illustrated in Figure 7, this phase requires a ketoacyl-CoA synthase, a ketoacyl-CoA reductase, a hydroxyacyl-CoA dehydratase, and an enoyl-CoA reductase. Accordingly, a genetically modified microorganism of the present invention includes native or exogenous enzymes encoded therein that provide these functions.
• NphT7 exhibits significant specificty for acetyl-CoA and propionyl-CoA as primers in the initiation phase, and it shows minimal activity with larger acyl-CoA chains during the elongation phase.
• Most 3-ketoacyl-CoA synthases that are capable of catalyzing the condensation of longer acyl-CoA chains are found in plants, mammals, yeast and other lower eukaryotes.
• the present invention provides a modified NphT7 polypeptide that functions as the ketoacyl-CoA synthase during the elongation phase in the malonyl-CoA dependent pathway.
• the modified NphT7 comprises an amino acid sequence having at least 70% but less than 100% or about 100% homology to SEQ ID NO: l and one or more amino acid substitutions, deletions, or insertions, wherein the modified NphT7 polypeptide is capable of accepting an acyl-CoA substrate having a carbon chain length of C4 or greater, for example C5, C6, C7, C8, C9, CIO, Cl l, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21 or C22.
• the modified NphT7 polypeptide is capable of catalyzing a condensation reaction to condense an acyl-CoA substrate with a malonyl-CoA to produce a 3-ketoacyl-CoA having a carbon chain length of C6 or greater, for example C7, C8, C9, CIO, Cl l, C12, C13, C14, C15, CI 6, CI 7, CI 8, CI 9, C20, C21 or C22.
• the modified NphT7 polypeptide comprises one or more amino acid substitutions selected from the group consisting of I147T, F217V, Y144L, V157F, G309S, G288S, a PDRP to HFLQ substitution for amino acids 86-89, I147F, I147M, I147Q, I147S, I147C, I147E, I147N, I147W, I147D, I147R, I147P, I147L, V196G, I147G, I147H, I147K, 1147V, 1147 A, I147Y, F217G, F217A, F217L, F217I, F217M, F217T, F217P, F217S, F217E, F217L, F217W, and any combination thereof
• the modified NphT7 polypeptide comprises one amino acid substitution selected from the group consisting of I147V, I147F, I147M, I147Q, I
• the modified NphT7 polypeptide comprises two amino acid substitutions selected from the group consisting of I147T and F217V, I147T and Y144L, I147T and V196G, I147F and F217V, I147M and F217V, I147S and F217V, I147T and HFLQ, I147T and V157F, I147T and F217G, I147T and F217A, I147T and F217L, I147T and F217I, I147T and F217M, I147T and F217P, I147T and F217S, I147T and F217E, I147S and F217G, I147S and F217A, I147S and F217L, I147S and F217I, I147S and F217M, I147S and F217W, I147S and F217S, I147S and F217E, I147S and F217K, I147F and F
• the modified NphT7 polypeptide of any embodiment comprising three amino acid substitutions selected from the group consisting of (Y144L, I147T, and F217V), (I147T, F217V, and HFLQ), (I147T, V147F, and F217V), and (Y144L, I147T, and V157F).
• the modified NphT7 polypeptide comprises one or more amino acid substitutions at a position selected from the group consisting of Ser84, Vail 14, Gly288, Ilel94, Gly318, Thr85, Gln90, Vall96, Tyrl44, Phel59, Ilel47, Phe217, and any combination thereof.
• the modified NphT7 polypeptide comprises an I147T amino acid substitution. In some embodiments, the modified NphT7 polypeptide comprises an F217V amino acid substitution. In some embodiments, the modified NphT7 polypeptide comprises two or more amino acid substitutions, deletions, or insertions, such as an I147T amino acid substitution and an F217V amino acid substitution. In some embodiments, the modified polypeptide is isolated and purified.
• the present invention provides an isolated and purified polynucleotide encoding a modified NphT7 polypeptide.
• the isolated and purified polynucleotide comprises a nucleic acid sequence having at least 70% but less than 100% or about 100% homology or complementarity to SEQ ID NO:2, wherein the polynucleotide encodes a modified NphT7 polypeptide of SEQ ID NO: l having one or more amino acid substitutions, deletions, or insertions, wherein the modified NphT7 polypeptide is capable of accepting an acyl-CoA substrate having a carbon chain length of C4 or greater, for example C5, C6, C7, C8, C9, CIO, Cl l, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21 or C22.
• the isolated and purified polynucleotide encodes a modified NphT7 polypeptide capable of catalyzing a condensation reaction to condense an acyl-CoA substrate with a malonyl-CoA to produce a 3-ketoacyl-CoA having a carbon chain length of C6 or greater, for example C7, C8, C9, CIO, Cl l, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21 or C22.
• the isolated and purified polynucleotide encodes a modified NphT7 polypeptide comprising one amino acid substitution selected from the group consisting of 1147V, I147F, I147M, I147Q, I147S, I147C, I147E, I147N, I147W, I147D, I147R, I147P, I147L, I147G, I147H, I147K, I147A, I147Y, and F217V.
• the isolated and purified polynucleotide encodes a modified NphT7 polypeptide comprising two amino acid substitutions selected from the group consisting of I147T and F217V, I147T and Y144L, I147T and V196G, I147F and F217V, I147M and F217V, I147S and F217V, I147T and HFLQ, I147T and V157F, I147T and F217G, I147T and F217A, I147T and F217L, I147T and F217I, I147T and F217M, I147T and F217P, I147T and F217S, I147T and F217E, I147S and F217G, I147S and F217A, I147S and F217L, I147S and F217I, I147S and F217M, I147S and F217W, I147S and F217S, I147S and F217S, I147
• the isolated and purified polynucleotide herein encodes a modified NphT7 polypeptide comprising three amino acid substitutions selected from the group consisting of (Y144L, I147T, and F217V), (I147T, F217V, and HFLQ), (I147T, V147F, and F217V), and (Y144L, I147T, and V157F).
• the isolated and purified polynucleotide herein encodes a modified NphT7 polypeptide comprising one or more amino acid substitutions at a position selected from the group consisting of Ser84, Vail 14, Gly288, Ilel94, Gly318, Thr85, Gln90, Vall96, Tyrl44, Phel59, Ilel47, Phe217, and any combination thereof.
• the isolated and purified polynucleotide herein encodes a modified NphT7 polypeptide comprising an I147T amino acid substitution.
• the isolated and purified polynucleotide herein encodes a modified NphT7 polypeptide comprising an F217V amino acid substitution.
• the isolated and purified polynucleotide herein encodes a modified NphT7 polypeptide comprising two or more amino acid substitutions, deletions, or insertions. In some aspects, the isolated and purified polynucleotide herein encodes a modified NphT7 polypeptide, comprising an I147T amino acid substitution and an F217V amino acid substitution. In some embodiments, the isolated and purified polynucleotide herein is a R A, mRNA, DNA, or cDNA. In some embodiments, the isolated and purified polynucleotide herein is a synthetic polynucleotide. In some embodiments, the isolated and purified polynucleotide herein is synthetic: RNA, mRNA, DNA, or cDNA.
• the present invention provides for a ketoacyl-CoA synthase that is active during the elongation phase in the malonyl-CoA dependent pathway, wherein the ketoacyl-CoA synthase is selected from the group consisting of npht7 F217V, npht7 I147T, synthase III, synthase IV, synthase V, and synthase VI; wherein npht7 F217V and/or npht7 I147T catalyzes a reaction adding the 5 th and/or 6 th carbon in the elongation, npht7 F217V, npht7 I147T, and/or synthase III catalyzes a reaction adding the 7 th and/or 8 th carbon in the elongation, npht7 F217V, npht7 I147T, synthase III, synthase IV, and
• each cycle of the malonyl-CoA dependent elongation phase requires a 3-ketoacyl-CoA reductase ("KCR”), a hydroxyacyl-CoA dehydratase (“3HDh”), and an enoyl-CoA reductase ("EnCr").
• KCR 3-ketoacyl-CoA reductase
• 3HDh hydroxyacyl-CoA dehydratase
• EnCr enoyl-CoA reductase
• the ketoacyl-CoA reductase may be selected from the group consisting of 3-ketoacyl-CoA reductase (e.g., fadB, a bifunctional enzyme - SEQ ID NO 183) and 3-hydroxyacyl-CoA dehydrogenase (e.g., hbd - SEQ ID NO 271); the hydroxyacyl- CoA dehydratase may be selected from the group consisting of 3-hydroxyacyl-CoA dehydratase (e.g., fadB, a bifunctional enzyme - SEQ ID NO 183) and enoyl-CoA hydratase (e.g., crt - SEQ ID NO 272); and the enoyl-CoA reductase may be trans-2-enoyl-reductase (e.g., ter - SEQ ID NO 275).
• 3-ketoacyl-CoA reductase e.g., fad
• the bifunctional fadB is both the ketoacyl-CoA reductase and the hydroxyacyl-CoA dehydratase, or the ketoacyl-CoA reductase is hbd and the hydroxyacyl-CoA dehydratase is crt.
• the elongation phase ends with a termination step once the desired chain length is achieved.
• the genetically modified microorganism encodes an enzyme capable of terminating an acyl elongation cycle substantially at a desired chain length or substantially within a relatively narrow distribution of chain lengths (i.e., a distribution of 2-4 carbons - e.g., C8-C10, C8-C12, CIO- C12, C10-C14, etc.).
• the termination enzyme is a thioesterase such as an acyl-CoA esterase, and the microorganism produces a fatty acid.
• Suitable thioesterases include tesA - SEQ ID NO 277, 'tesA - SEQ ID NO 278, tesB - SEQ ID NO 279, yciA - SEQ ID NO 280, ybgC - SEQ ID NO 281, ybfF - SEQ ID NO 282, fadM - SEQ ID NO 283, AtTE - SEQ ID NO 284, CpTE - SEQ ID NO 285, CperfTE - SEQ ID NO 286, LpTE - SEQ ID NO 287, and PA2801TE - SEQ ID NO 288, and combinations thereof.
• the termination enzyme is a wax ester synthase and the microorganism produces a fatty ester.
• Suitable wax ester synthase include Maql - SEQ ID NO 289, Pcryl - SEQ ID NO 290, Rjosl - SEQ ID NO 291, and Aborkl - SEQ ID NO 292.
• other known termination enzyme(s) that will enable the genetically modified microorganism to produce alternative fatty acid derivatives such as, for example, a fatty alcohol, a fatty aldehyde, a fatty alkene, a fatty amide, a fatty alkane, or a fatty diacid.
• the termination enzyme may be a fatty acid or acyl-CoA reductase that catalyzes the production of a fatty alcohol or fatty aldehyde, or an aldehyde decarbonylase that catalyzes the production of fatty aldehyde, or an aldehyde decarbonylase together with an acyl-ACP reductase or an acyl-CoA reductase that catalyzes the production of an alkane.
• the genetically modified microorganism is engineered to produce a fatty acid or fatty acid derivative product having substantially a specific chain length or having a substantially narrow distribution of chain lengths (i.e., 2-4 carbons).
• a fatty acid or fatty acid derivative product having substantially a specific chain length or having a substantially narrow distribution of chain lengths (i.e., 2-4 carbons).
• at least 50%, 60%, 70%, 80%, or 90% of the fatty acids or fatty acid derivative produced by the genetically modified microorganism of the present invention is of a desired chain length or within a desired narrow distribution of chain lengths.
• Table 2 sets forth certain unique combinations of genes that lead to the production of products having the carbon chain lengths indicated in Table 2.
• a genetically modified microorganism having encoded therein the genes included in Table 2 for a given pathway, wherein such microorganism is capable of producing a fatty acid or fatty acid derivative having a carbon chain length indicated in Table 2 for such pathway.
• a genetically modified microorganism having encoded therein the genes included in Table 2 above for a combination of pathways wherein the microorganism is capable of producing fatty acids or fatty acid derivatives within a substantially narrow distribution of chain lengths corresponding to the carbon chain lengths indicated in Table 2 for such combination of pathways.
• a genetically modified microorganism comprising NphT7, fadB, ter, AtTE, and tesA (CIO pathway A and C12 pathway A), wherein said microorganism is capable of producing a fatty acid composition comprising CIO and C12 fatty acids.
• the present invention provides one or more isolated and purified polynucleotides comprising exogenous nucleic acid molecules encoding proteins comprising a 3-oxoacyl-(acyl carrier protein) synthase III from a species selected from the group consisting of Alishewanella aestuarii Bl l, Arcobacter butzleri ED-1, Clostridiales bacterium 1_7_47_FAA, Gluconacetobacter oboediens 174Bp2, Gordonia aichiensis NBRC 108223, Mesorhizobium sp.
• the 3-oxoacyl-(acyl carrier protein) synthase III is from a species selected from the group consisting of Pelosinus fermentans DSM 17108, Saccharomonospora glauca K62, Verrucosispora maris AB-18-032, and Clostridiales bacterium 1 7 47 FAA, and wherein the proteins encoded by the polynucleotides are capable of producing an acetyl-CoA.
• the 3-oxoacyl-(acyl carrier protein) synthase III is from a species selected from the group consisting of Saccharomonospora glauca K62, Saccharomonospora azurea NA-128, Mesorhizobium sp. STM 4661, and Clostridiales bacterium 1 7 47 FAA, and wherein the proteins encoded by the polynucleotides are capable of producing a four carbon fatty acid.
• the 3-oxoacyl-(acyl carrier protein) synthase III is from a species selected from the group consisting of Gordonia aichiensis NBRC 108223, Arcobacter butzleri ED-1, Clostridiales bacterium 1_7_47_FAA, Saccharomonospora glauca K62, and Ralstonia solanacearum Po82, and wherein the proteins encoded by the polynucleotides are capable of producing a six carbon fatty acid.
• the 3- oxoacyl-(acyl carrier protein) synthase III is from a species selected from the group consisting of Gordonia aichiensis NBRC 108223, Gluconacetobacter oboediens 174Bp2, Arcobacter butzleri ED-1, Ralstonia solanacearum Po82, and Phaeobacter gallaeciensis 2.10, and wherein the proteins encoded by the polynucleotides are capable of producing an eight carbon fatty acid.
• the 3-oxoacyl-(acyl carrier protein) synthase III is from Alishewanella aestuarii Bl 1, and wherein the proteins encoded by the polynucleotides are capable of producing a ten carbon fatty acid.
• the present invention relate to microorganisms that are genetically modified to produce fatty acids and fatty acid derivatives through a malonyl- CoA dependent pathway that is also a malonyl-ACP independent pathway.
• This aspect of the invention may be used in combination with the inhibition of a microorganism's malonyl-ACP dependent fatty acid synthase pathway through one or more genetic modifications to reduce the activity of enzymes encoded by one or more of the microorganism's malonyl-ACP dependent fatty acid synthase system genes.
• the compositions may be used in the methods and systems of the present invention.
• the fatty acid synthase system comprises polypeptides that have the following enzymatic activities: malonyl-CoA-acyl carrier protein (ACP) transacylase; 3-ketoacyl-ACP synthase; 3-ketoacyl-ACP reductase; 3-hydroxyacyl-ACP dehydratase; 3-hydroxyacyl-ACP dehydratase; and enoyl-ACP reductase.
• ACP malonyl-CoA-acyl carrier protein
• nucleic acid sequences that encode temperature-sensitive forms of these polypeptides may be introduced in place of the native enzymes, and when such genetically modified microorganisms are cultured at elevated temperatures (at which these thermolabile polypeptides become inactivated, partially or completely, due to alterations in protein structure or complete denaturation), there is observed an increase in flux through the malonyl-CoA dependent pathway and a decrease in flux through the malonyl-ACP dependent pathway.
• these temperature-sensitive mutant genes could include fabI ts (S241F), fabB ts (A329V) or fabD ts (W257Q).
• fabI ts S241F
• fabB ts A329V
• fabD ts W257Q
• other types of genetic modifications may be made to otherwise modulate, such as lower, enzymatic activities of one or more of these polypeptides.
• a result of such genetic modifications is to shift malonyl- CoA utilization so that there is a reduced conversion of malonyl-CoA to fatty acids via the native pathway, overall biomass, and proportionally greater conversion of carbon source to a chemical product including a fatty acid or fatty acid derived product via a malonyl-CoA dependent, and in some cases a malonyl-ACP independent route.
• the specific productivity for the microbially produced chemical product is unexpectedly high.
• additional genetic modifications, such as to increase malonyl-CoA production may be made for certain embodiments.
• enoyl-acyl carrier protein reductase (EC No. 1.3.1.9, also referred to as enoyl-ACP reductase) is a key enzyme for fatty acid biosynthesis from malonyl-CoA.
• this enzyme, Fabl is encoded by the gene fabl ⁇ See "Enoyl-Acyl Carrier Protein (fabl) Plays a Determinant Role in Completing Cycles of Fatty Acid Elongation in Escherichia coli," Richard J. Heath and Charles O. Rock, J. Biol. Chem. 270:44, pp. 26538-26543 (1995), incorporated by reference for its discussion of fabl and the fatty acid synthase system).
• the present invention may utilize a microorganism that is provided with a nucleic acid sequence (polynucleotide) that encodes a polypeptide having enoyl-ACP reductase enzymatic activity that may be modulated during a fermentation event.
• a nucleic acid sequence encoding a temperature-sensitive enoyl-ACP reductase may be provided in place of the native enoyl-ACP reductase, so that an elevated culture temperature results in reduced enzymatic activity, which then results in a shifting utilization of malonyl-CoA to production of a desired chemical product.
• One such sequence is a mutant temperature-sensitive fabl (fabl ) of E.
• This enzyme may exhibit reduced enzymatic activity at temperatures above 30°C but normal enzymatic activity at 30°C, so that elevating the culture temperature to, for example to 34°C, 35°C, 36°C, 37°C or even 42°C, reduces enzymatic activity of enoyl-ACP reductase.
• nucleic acid and amino acid sequences for enoyl-ACP reductase in species other than E. coli are readily obtained by conducting homology searches in known genomics databases, such as BLASTN and BLASTP. Approaches to obtaining homologues in other species and functional equivalent sequences are described herein. Accordingly, it is appreciated that the present invention may be practiced by one skilled in the art for many microorganism species of commercial interest.
• a genetic modification may be made to reduce the enzymatic activity of the enoyl-ACP reductase gene (e.g., fabl in E. coli).
• the promoter may be induced (such as with isopropyl ⁇ -D-thiogalactopyranoiside (IPTG)) during a first phase of a method herein, and after the IPTG is exhausted, removed or diluted out the second step, of reducing enoyl-ACP reductase enzymatic activity, may begin.
• IPTG isopropyl ⁇ -D-thiogalactopyranoiside
• Other approaches may be applied to control enzyme expression and activity such as are described herein and/or known to those skilled in the art.
• promoters that are turned on in response to phosphate depletion may be used to controllably express desired genes.
• Such promoters could include the yibD or pstS gene promoters in E. coli.
• compositions, methods and systems of the present invention the reduction of enzymatic activity of enoyl-ACP reductase (or, more generally, of the fatty acid synthase system) is made to occur after a sufficient cell density of a genetically modified microorganism is attained.
• This bi-phasic culture approach balances a desired quantity of biocatalyst, in the cell biomass which supports a particular production rate, with yield, which may be partly attributed to having less carbon be directed to cell mass after the enoyl-ACP reductase activity (and/or activity of other enzymes of the fatty acid synthase system) is/are reduced. This results in a shifting net utilization of malonyl-CoA, thus providing for greater carbon flux to a desired chemical product.
• each respective enzymatic activity so modulated may be reduced by at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 percent compared with the activity of the native, non-modulated enzymatic activity (such as in a cell or isolated).
• the conversion of malonyl-CoA to fatty acyl-ACP or molecules may be reduced by at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 percent compared with such conversion in a non-modulated cell or other system.
• the conversion of malonyl-CoA to fatty acid molecules through its native pathway may be reduced by at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 percent compared with such conversion in a non-modulated cell or other system.
• the genetic modifications described hereinabove may be combined with various additional genetic modifications to further enhance production of a desired chemical product.
• additional genetic modifications may result in a variety of beneficial attributes, such as increasing glucose uptake, decreasing consumption of key intermediates by alternative reaction pathways leading to undesirable by-products, and driving carbon flux to malonyl-CoA. Certain of these additional genetic modifications are set forth in Table 3.
• transacylase including by mutation enoyl acyl carrier protein 1.3.1.9, Decrease or increase function, fabl
• 3-ketoacyl-acyl carrier protein Decrease or increase function
• pantothenate kinase 2.7.1.33 coaA Increase function
• aldehyde dehydrogenase A 1.2.1.21 Decrease via deletion or aldA
• 3-ketoacyl-CoA thiolase 2.3.1.16 fadA Increase or decrease function dodecenoyl-CoA ⁇ - isomerase, enoyl-CoA 5.3.3.8
• thioesterase III 3.1.2.- fadM Increase or decrease function hydroxyphenylacetyl-CoA Increase or decrease function paal
• esterase/thioesterase 3.1.2.28 ybgC Increase or decrease function proofreading thioesterase in Increase or decrease function entH
• genetic modifications also are provided to increase the pool and availability of the cofactor NADPH, and/or, consequently, the NADPH/NADP + ratio.
• this may be done by increasing activity, such as by genetic modification, of one or more of the following genes: pgi (in a mutated form), pntAB, overexpressed, gapA:gapN substitution/replacement, and disrupting or modifying a soluble transhydrogenase such as sthA, and/or genetic modifications of one or more of zwf, gnd, and edd.
• any subgroup of genetic modifications may be made to decrease cellular production of fermentation product(s) selected from the group consisting of acetate, acetoin, acetone, acrylic, malate, fatty acid ethyl esters, glycerolipids, lipids, isoprenoids, glycerol, ethylene glycol, ethylene, propylene, butylene, isobutylene, ethyl acetate, vinyl acetate, other acetates, 1 ,4-butanediol, 2,3-butanediol, butanol, isobutanol, sec- butanol, butyrate, isobutyrate, 2-OH-isobutryate, 3-OH-butyrate, ethanol, isopropanol, D-lactate, L-lactate, pyruvate, itaconate, levulinate, glucar
• additional genetic modification is associated with a genotype or an enzyme selected from the group listed in Table 4 - Table 13 below.
• the amino acid sequences of these enzymes are shown in Table 14.
• Table 4. Base strain E. coli K12 BW25113

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