WO2019236819A1 - Methods and genetically modified cells for producing c8 fatty acids and fatty acid chain products - Google Patents

Methods and genetically modified cells for producing c8 fatty acids and fatty acid chain products Download PDF

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WO2019236819A1
WO2019236819A1 PCT/US2019/035757 US2019035757W WO2019236819A1 WO 2019236819 A1 WO2019236819 A1 WO 2019236819A1 US 2019035757 W US2019035757 W US 2019035757W WO 2019236819 A1 WO2019236819 A1 WO 2019236819A1
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coa
fatty acid
polypeptide
malonyl
recombinant cell
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PCT/US2019/035757
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French (fr)
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David A. Hogsett
Hans H. Liao
Michael T.M. LOUIE
Catherine Poor
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Cargill, Incorporated
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Publication of WO2019236819A1 publication Critical patent/WO2019236819A1/en

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1018—Carboxy- and carbamoyl transferases (2.1.3)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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
    • C12P7/6409—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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
    • C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • This disclosure relates to recombinant cells that produce fatty acids and fatty acid chain products.
  • this disclosure relates to recombinant cells that produce C6- C10 fatty acids and C6-C10 fatty acid chain products such as C6-C10 fatty acid esters and C6- C10 fatty alcohols.
  • Fatty acids and fatty acid chain products have a number of important industrial uses.
  • fatty acids and fatty acid chain products can be utilized as solvents for lacquers, paints, varnishes and other compositions; as plasticizers for organic resins, as fragrances and flavorings, as fuels for jet and other internal combustion engines, and as raw materials for making a variety of downstream products.
  • fatty acids and fatty acid chain products are produced industrially from non-renewable fossil fuels or from plant oils such as palm and coconut oil.
  • these conventional methods of producing fatty acids and fatty acid chain products have a number of drawbacks.
  • a particular drawback is poor selectivity of a specific carbon chain length, and in particular, selectivity of C6-C10 chain lengths.
  • the products of these conventional methods tend to be a mixture of compounds having a range of carbon chain lengths. Separating these mixtures of compounds can be difficult and can often lead to poor yields with much of the non-renewable fossil fuels or plant oils converted to lower- value byproducts that do not have the desired carbon chain lengths.
  • Some biological cells can naturally produce some types of fatty acid chain products. For example, almost all living cells produce triglycerides of fatty acids as well as other fatty acid esters. These triglycerides and other esters play important roles in the metabolism, cellular structure, and other biological processes of the cells, and can perform other useful functions such as storing energy. Therefore, some biological cells can potentially be used to produce fatty acid chain products industrially. Among other potential advantages, biological production of fatty acids and fatty acid products in some cases can rely on annually renewable carbon sources such as sugars, rather than on fossil fuels.
  • Biological cells tend to produce fatty acid groups with chain lengths of 12 carbon atoms or greater.
  • naturally-occurring biological cells are a good source for C12 and higher fatty acids and their chain products.
  • triglycerides produced naturally by such cells can be hydrolyzed to produce C12 or higher fatty acids, which can in turn be converted to other derivatives such as esters or alcohols.
  • few cells naturally produce fatty acids of C6 - C10 chain length in significant quantities.
  • Some biological cells can produce fatty acid chain products through a native metabolic pathway that starts with acetyl-CoA and malonyl-ACP.
  • Acetyl-CoA condenses with malonyl-ACP with loss of carbon dioxide and CoA to produce 3-ketobutyryl-ACP.
  • Subsequent enzymatic reactions convert the 3-ketobutyryl-ACP successively to 3-hydroxybutyryl-ACP, then to trans-2-butenoyl-ACP (with loss of water) and finally to butyryl-ACP.
  • the butyryl-ACP can re-enter this reaction cycle in place of acetyl-CoA to produce hexanoyl-ACP.
  • This cycle repeats itself, producing in each iteration a longer carbon atom chain by adding two carbon atoms at a time, until terminated by some other cellular process.
  • this native metabolic pathway has low termination rates for C6-C10 chain lengths, resulting in low production of C6-C10 fatty acids and fatty acid chain products.
  • Some biological cells can be genetically modified to increase production of C6-
  • C10 chain lengths are C10 chain lengths.
  • These genetic modifications can include insertions of heterologous genes and modification and/or deletions of endogenous genes to increase production of C6-C10 chain lengths.
  • heterologous genes can be inserted to provide enzymes to catalyze specific reactions between chemical intermediates along a pathway that results in production of C6-C10 chain lengths.
  • Okamura et ak in PNAS vol. 107, no. 25, pp. 11265-11270 (2010), reported that an enzyme produced by the nphT7 gene of a soil-isolated Streptomyces sp. strain can catalyze a single condensation of acetyl-CoA and malonyl-CoA to produce acetoacetyl- CoA.
  • U.S. Application No. 2014/0051136 disclosed that fatty acid chain products can be produced by a cell modified to include the nphT7 gene and additional heterologous genes.
  • the amounts of C6 or longer chain fatty acid chain products produced were limited and selectivity of C6-C10 chain lengths was limited.
  • WO 2015/010103 described mutants of the nphT7 gene that code for NphT7 enzymes that more efficiently catalyze the condensation of longer-chain acyl-CoA compounds with malonyl-CoA.
  • Cells modified to include both the wild-type nphT7 gene and the nphT7 mutant gene produced greater relative quantities of longer fatty acids, but selectivity of C6-C10 chain lengths remained poor.
  • C6-C10 fatty acid chain products in particular were made in only small amounts.
  • nucleic acid sequences and proteins as well as biological processes to produce fatty acids and/or fatty acid chain products with selectivity to C6-C10 chain lengths.
  • One aspect provides a recombinant cell comprising a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl- CoA, wherein the polypeptide has SEQ ID NO: 2-9 or has at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 2-9, wherein the recombinant cell produces a fatty acid and/or a fatty acid chain product.
  • the polypeptide catalyzes one or more condensations selected from the group consisting of a) C2- CoA with malonyl-CoA to form 3-keto C4-CoA; b) C4-CoA with malonyl-CoA to form 3-keto C6-C0A; c) C6-C0A with malonyl-CoA to form 3-keto C8-C0A; d) C8-C0A with malonyl-CoA to form 3-keto ClO-CoA; and e) ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
  • the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than C8-C0A with malonyl-CoA to form 3-keto ClO-CoA. In one aspect, the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8- CoA than ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA. In another aspect, the fatty acid and/or fatty acid product comprises a chain length of one or more of C6, C8, and/or C10.
  • the recombinant cell produces more fatty acid and/or fatty acid chain product of C8 chain length than other chain lengths.
  • the total fatty acid and/or total fatty acid chain product comprises 35% or less of C10 chain length and/or the total fatty acid and/or total fatty acid chain product comprises 10% or less of C12 chain length.
  • the total fatty acid and/or total fatty acid chain product comprises 40% or more of C8 chain length.
  • the total fatty acid and/or total fatty acid chain product comprises 45% or less of C6 chain length.
  • the recombinant cell further comprises one or more of a heterologous gene encoding a polypeptide with enoyl-CoA reductase activity; a heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity; a heterologous gene encoding a polypeptide with ester synthase activity; and a heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity.
  • the heterologous gene encoding a polypeptide with enoyl-CoA reductase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17;
  • the heterologous gene encoding a polypeptide with bifunctional 3- hydroxyacyl-CoA dehydrogenase/dehydratase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11;
  • the polypeptide that catalyzes the condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA is under control of a constitutive promoter.
  • the polypeptide that catalyzes the condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA is under control of an inducible promoter.
  • the polypeptide that catalyzes the condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA is under control of an inducible promoter sensitive to lowering phosphate concentration.
  • the polypeptide that catalyzes the condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA is under control of a PpstSIH promoter or a PphoE promoter.
  • the fatty acid chain product comprises one or more products selected from the group consisting of fatty acid ester, fatty alcohol, fatty acid amide, and fatty acid amine.
  • the fatty acid ester comprises one or more esters selected from the group consisting of fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester, fatty acid butyl ester, fatty acid pentyl ester, and fatty acid hexyl ester.
  • the recombinant cell is a fungal cell, a bacterial cell, or a plant cell.
  • the recombinant cell comprises an Escherichia coli species or Bacillus genus.
  • the recombinant cell comprises a yeast cell.
  • ketoacyl-CoA synthase having SEQ ID NO: 10 or being at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 10, wherein amino acid residue 223 is not a valine, amino acid residue 46 is not a threonine, amino acid residue 256 is not a serine, amino acid residue 246 is not a isoleucine, and/or amino acid residue 282 is not a serine.
  • amino acid residue 223 is an alanine.
  • amino acid residue 46 is a methionine.
  • amino acid residue 256 is a glycine.
  • amino acid residue 246 is a proline or leucine.
  • amino acid residue 282 is a threonine.
  • the ketoacyl-CoA synthase catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA.
  • the ketoacyl-CoA synthase catalyzes one or more condensations selected from the group consisting of C2-CoA with malonyl-CoA to form 3-keto C4-CoA; C4-CoA with malonyl-CoA to form 3-keto C6-C0A; C6-C0A with malonyl-CoA to form 3-keto C8-C0A; C8-C0A with malonyl-CoA to form 3-keto ClO-CoA; and ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
  • the ketoacyl-CoA synthase catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than C8-C0A with malonyl-CoA to form 3-keto ClO-CoA.
  • the ketoacyl-CoA synthase catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
  • ketoacyl-CoA synthase having SEQ ID NO: 10 or being at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 10, comprising at least one of the following features a) amino acid residue 223 is not a valine; b) amino acid residue 46 is not a threonine; c) amino acid residue 256 is not a serine; d) amino acid residue 246 is not a isoleucine; e) amino acid residue 282 is not a serine; f) amino acid residue 223 is an alanine; g) amino acid residue 46 is a methionine; h) amino acid residue 256 is a glycine; i) amino acid residue 246 is a proline or leucine; and j) amino acid residue 282 is a threonine.
  • One aspect provides a cell culture comprising a) a recombinant cell comprising a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA, wherein the polypeptide has at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 2-10, wherein the recombinant cell produces a fatty acid and/or a fatty acid chain product; and b) one or more fatty acids or fatty acid chain products produced by the recombinant cell, wherein at least one of the fatty acids or fatty acid chain products is present at a concentration of at least 0.1 g/L of the cell culture.
  • the fatty acid and/or fatty acid product produced comprises a chain length of one or more of C6, C8, and/or C10.
  • the cell culture comprises more fatty acid and/or fatty acid chain product of C8 chain length than other chain lengths.
  • the total fatty acid and/or total fatty acid chain product comprises less than 30% C10.
  • the recombinant cell further comprises one or more of a heterologous gene encoding a polypeptide with enoyl-CoA reductase activity; a heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity; a heterologous gene encoding a polypeptide with ester synthase activity; and a heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity.
  • the heterologous gene encoding a polypeptide with enoyl-CoA reductase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17;
  • the heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11;
  • the heterologous gene encoding a polypeptide with ester synthase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20;
  • the heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in
  • the fatty acid ester comprises one or more of fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester, fatty acid butyl ester, fatty acid pentyl ester, and fatty acid hexyl ester.
  • One aspect provides a method of producing fatty acids and/or fatty acid chain products comprising culturing a recombinant cell in a culture medium, wherein the recombinant cell comprises a heterologous gene encoding a polypeptide that catalyzes condensation of acyl- CoA with malonyl-CoA to form 3-keto acyl-CoA, wherein the polypeptide has SEQ ID NO: 2- 10 or has at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 2-10; and the recombinant cell is grown under conditions in which the heterologous gene is expressed.
  • the polypeptide catalyzes one or more condensations selected from the group consisting of a) C2-CoA with malonyl-CoA to form 3- keto C4-CoA; b) C4-CoA with malonyl-CoA to form 3-keto C6-C0A; c) C6-C0A with malonyl- CoA to form 3-keto C8-C0A; d) C8-C0A with malonyl-CoA to form 3-keto ClO-CoA; and e) ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
  • the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than C8- CoA with malonyl-CoA to form 3-keto ClO-CoA.
  • the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
  • the fatty acid and/or fatty acid product comprises a chain length of one or more of C6, C8, and/or C10.
  • the recombinant cell produces more fatty acid and/or fatty acid chain product of C8 chain length than other chain lengths.
  • the total fatty acid and/or total fatty acid chain product comprises 35% or less of C10 chain length.
  • the total fatty acid and/or total fatty acid chain product comprises 41% or more of C8 chain length.
  • the total fatty acid and/or total fatty acid chain product comprises 44% or less of C6 chain length.
  • the method further comprises one or more of a heterologous gene encoding a polypeptide with enoyl-CoA reductase activity; a heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity; a heterologous gene encoding a polypeptide with ester synthase activity; and a heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity.
  • the heterologous gene encoding a polypeptide with enoyl-CoA reductase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17;
  • the heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11;
  • the heterologous gene encoding a polypeptide with ester synthase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20;
  • the heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in
  • the fatty acid chain product comprises one or more of fatty acid ester, fatty alcohol, fatty acid amide, and fatty acid amine.
  • the fatty acid ester comprises one or more of fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester, fatty acid butyl ester, fatty acid pentyl ester, and fatty acid hexyl ester.
  • the recombinant cell is a fungal cell, a bacterial cell, or a plant cell.
  • the recombinant cell comprises an Escherichia coli species or Bacillus genus.
  • the recombinant cell comprises a yeast cell.
  • One aspect provides a method of producing fatty acid methyl ester comprising culturing a recombinant cell in a culture medium, wherein the recombinant cell comprises a heterologous gene encoding a polypeptide having SEQ ID NO: 10 or being at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 10; and the recombinant cell is grown under conditions in which the heterologous gene is expressed.
  • the polypeptide comprises at least one of the following features a) amino acid residue 223 is an alanine; b) amino acid residue 46 is a methionine; c) amino acid residue 256 is a glycine; d) amino acid residue 246 is a proline or leucine; and/or e) amino acid residue 282 is a threonine.
  • the polypeptide catalyzes one or more condensations selected from the group consisting of a) C2-CoA with malonyl-CoA to form 3-keto C4-CoA; b) C4-CoA with malonyl-CoA to form 3-keto C6-C0A; c) C6-C0A with malonyl-CoA to form 3-keto C8-C0A; d) C8-C0A with malonyl-CoA to form 3-keto ClO-CoA; and/or e) ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
  • the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than C8-C0A with malonyl-CoA to form 3-keto C10- CoA.
  • the polypeptide catalyzes condensation of more C6-C0A with malonyl- CoA to form 3-keto C8-C0A than ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
  • the fatty acid methyl ester comprises a chain length of one or more of C6, C8, and/or C10.
  • the recombinant cell produces more fatty acid methyl ester of C8 chain length than other chain lengths.
  • the total fatty acid methyl ester comprises 35% or less of C10 chain length.
  • the total fatty acid methyl ester comprises 41% or more of C8 chain length.
  • the total fatty acid methyl ester comprises 44% or less of C6 chain length.
  • the recombinant cell further comprises one or more of a heterologous gene encoding a polypeptide with enoyl-CoA reductase activity; a heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity; a heterologous gene encoding a polypeptide with ester synthase activity; and a heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity.
  • the heterologous gene encoding a polypeptide with enoyl-CoA reductase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17;
  • the heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11;
  • the heterologous gene encoding a polypeptide with ester synthase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20;
  • the heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in
  • the recombinant cell is a fungal cell, a bacterial cell, or a plant cell.
  • the recombinant cell comprises an Escherichia coli species or Bacillus genus.
  • the recombinant cell comprises a yeast cell.
  • FIGS. 1A, 1B, and 1C show graphical views of a sequence alignment. Each line corresponds to a 3-ketoacyl-CoA synthase enzyme as indicated by the designation. The amino acid position of the first residue of each row is indicated.
  • FIG. 2 shows the total amount of FAME produced in g/L per strain and the total amount of each of methyl hexanoate (C6), methyl octanoate (C8), methyl decanoate (C10), and methyl dodecanoate (C12) per strain.
  • the strains comprise 3-ketoacyl-CoA synthase enzyme from each of Clostridiales bacterium l_7_47_FAA, Clostridium clostridioforme, Clostridium bolteae 90A9, Clostridium saccharolyticum, Clostridium saccharolyticum, Clostridium clostridioforme 2_l_49FAA, Clostridium asparagiforme DSM 15981, Clostridium hathewayi, and Clostridium hathewayi WAL- 18680, respectively.
  • FIG. 3 shows FAME production by each strain based on the chain length of the product as a percentage of the total FAME produced by that strain.
  • the strains comprise 3- ketoacyl-CoA synthase enzyme from each of Clostridiales bacterium l_7_47_FAA, Clostridium clostridioforme, Clostridium bolteae 90A9, Clostridium saccharolyticum, Clostridium saccharolyticum, Clostridium clostridioforme 2_l_49FAA, Clostridium asparagiforme DSM 15981, Clostridium hathewayi, and Clostridium hathewayi WAL-18680, respectively.
  • FIG. 4 shows specific activity for either a C2-CoA primer, a C4-CoA primer, a
  • C6-C0A primer or a C8-C0A primer with malonyl-CoA for each of the Cbac, Cbac(V223A), Cbac(T46M), and Cbac(S256G) mutant enzymes.
  • FIG. 5 shows total amount of FAME produced in g/L per strain and the total amount of each of methyl hexanoate (C6), methyl octanoate (C8), methyl decanoate (C10), and methyl dodecanoate (C12) per strain.
  • the strains comprise Cbac or a mutant Cbac(V223A), Cbac(T46M), or Cbac(S256G) enzyme.
  • FIG. 6 shows FAME production by each strain based on the chain length of the product as a percentage of the total FAME produced by that strain for each strain comprising Cbac, Cbac(V223A), Cbac(T46M), or Cbac(S256G) mutant enzyme.
  • FIG. 7 shows specific activity for either a C2-CoA primer, a C4-CoA primer, a
  • C6-C0A primer or a C8-C0A primer with malonyl-CoA for each of the Cbac(V223A), Cbac(V223A, I246P), Cbac (V223A, I246L), and Cbac (V223A, S282T) mutant enzymes.
  • FIG. 8 shows total amount of FAME produced in g/L per strain and the total amount of each of methyl hexanoate (C6), methyl octanoate (C8), methyl decanoate (C10), and methyl dodecanoate (C12) per strain.
  • the strains comprise either Cbac or Cbac(V223A, I246L) mutant, with or without NphT7, and with or without NphT7(LSVA).
  • wild type biological cells can naturally produce some types of fatty acid chain products, these wild type biological cells tend to produce fatty acids and fatty acid chain products with chain lengths of C12 or higher. Few wild type biological cells naturally produce fatty acids and/or fatty acid chain products of C6-C10 chain length in significant quantities.
  • recombinant cells can be genetically engineered to express heterologous polypeptides for the biosynthesis of C6-C10 fatty acids and/or fatty acid chain products. These recombinant cells can be genetically modified to express identified enzymes that catalyze condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA (having 3-ketoacyl-CoA synthase activity) to produce C6-C10 fatty acids and/or fatty acid chain products.
  • these identified enzymes having 3-ketoacyl-CoA synthase activity can catalyze the stepwise addition of malonyl-CoA to an acyl-CoA substrate to produce a C6-C10 fatty acid and/or fatty acid chain product.
  • an enzyme having 3-ketoacyl-CoA synthase activity can catalyze the addition of malonyl-CoA to a C2-CoA substrate to lead to a subsequent C4 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C4- CoA substrate to lead to a subsequent C6 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C6-C0A substrate to lead to a subsequent C8 fatty acid and/or fatty acid chain product, and/or the addition of malonyl-CoA to a C8-C0A substrate to lead to a subsequent C10 fatty acid and/or fatty acid chain product.
  • an enzyme having 3-ketoacyl-CoA synthase activity can catalyze the addition of malonyl-CoA to a C2-CoA substrate (acetyl-CoA) to lead to a 3-keto C4-CoA, the addition of malonyl-CoA to a C4-CoA substrate to lead to a 3-keto C6-C0A, the addition of malonyl-CoA to a C6-C0A substrate to lead to a 3-keto C8-C0A, and/or the addition of malonyl-CoA to a C8-C0A substrate to lead to a 3-keto ClO-CoA.
  • the recombinant cells can be genetically modified to express other heterologous polypeptides important for the biosynthesis of C6-C10 fatty acids and/or fatty acid chain products.
  • the recombinant cells can be genetically modified to express one or more heterologous polypeptides capable of converting a 3-keto-CX-CoA substrate (e.g., 3-keto-C4-CoA, 3-keto-C6-CoA, 3- keto-C8-CoA, and/or 3-keto-ClO-CoA) to a 3-OH CX-CoA substrate (e.g., 3-OH-C4-CoA, 3- OH-C6-C0A, 3-OH-C8-CoA, and/or 3-OH-ClO-CoA) (where X represents the carbon chain length).
  • a 3-keto-CX-CoA substrate e.g., 3-keto-C4-CoA, 3-keto-C6-CoA, 3- keto-C8-CoA, and/or 3-keto-ClO-CoA
  • 3-OH CX-CoA substrate e.g., 3-OH-C4-CoA, 3- OH-C6-C0
  • the recombinant cells can be genetically modified to express one or more heterologous polypeptides capable of converting a 3-OH-CX-CoA substrate (e.g., 3-OH-C4-CoA, 3-OH-C6- CoA, 3-OH-C8-CoA, and/or 3-OH-ClO-CoA) to a trans-enoyl-CX-CoA substrate (e.g., enoyl- C4-CoA, enoyl-C6-CoA, enoyl-C8-CoA, and/or enoyl-ClO-CoA) (where X represents the carbon chain length).
  • a 3-OH-CX-CoA substrate e.g., 3-OH-C4-CoA, 3-OH-C6- CoA, 3-OH-C8-CoA, and/or 3-OH-ClO-CoA
  • trans-enoyl-CX-CoA substrate e.g., enoyl-
  • the recombinant cells can be genetically modified to express one or more heterologous polypeptides capable of converting a trans-enoyl-CX-CoA substrate (e.g., enoyl- C4-CoA, enoyl-C6-CoA, enoyl-C8-CoA, and/or enoyl-ClO-CoA) to a CX-CoA substrate (e.g., C4-CoA, C6-C0A, C8-C0A, and/or ClO-CoA) (where X represents the carbon chain length).
  • a trans-enoyl-CX-CoA substrate e.g., enoyl- C4-CoA, enoyl-C6-CoA, enoyl-C8-CoA, and/or enoyl-ClO-CoA
  • CX-CoA substrate e.g., C4-CoA, C6-C0
  • the recombinant cells can be genetically modified to express one or more heterologous polypeptides capable of converting a CX-CoA substrate (e.g., C4-CoA, C6-C0A, C8-C0A, and/or ClO-CoA) to a fatty acid ester.
  • a CX-CoA substrate e.g., C4-CoA, C6-C0A, C8-C0A, and/or ClO-CoA
  • nucleic acid means one or more nucleic acids.
  • Methods well known to those skilled in the art can be used to construct genetic expression constructs and recombinant cells as described. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic DNA techniques, in vivo
  • nucleic acid can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof.
  • Amino acid residues in all amino acid sequences described herein are ordered in the N-terminus to C-terminus direction. “Upstream” means in the direction toward the N- terminus, and“downstream” means toward the C-terminus direction.
  • The“start” of an amino acid sequence is the first amino acid residue in the N-terminus direction.
  • the first amino acid residue (amino acid residue 1) for any sequence or sub-sequence described herein is the amino acid residue at its N-terminus.
  • A“sub-sequence” is a sequence of amino acid residues contained within a larger amino acid sequence.
  • Polypeptides refer to polymeric chains of amino acids connected by peptide bonds.
  • polypeptides can fold into unique three dimensional structures that allow the polypeptides to function as enzymes that catalyze distinct biochemical reactions.
  • a 3-ketoacyl-CoA synthase polypeptide catalyzes the condensation reaction of an acyl- CoA with malonyl-CoA to form a 3-ketoacyl-CoA
  • Identity is used herein to indicate the extent to which two (nucleotide or amino acid) sequences have the same residues at the same positions in side-by-side alignment of their sequences. The identity is expressed herein as a % identity as determined using BLAST (National Center for Biological Information (NCBI) Basic Local Alignment Search Tool) version 2.2.31 software (National Center for Biotechnology Information, U.S. National Library of Medicine, Bethesda, Maryland, USA), using default parameters unless indicated otherwise in this paragraph.
  • NCBI National Center for Biological Information
  • NCBI National Center for Biological Information
  • Nucleic acid % sequence identity between nucleic acid sequences is determined using standard nucleotide BLAST with the following default parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1, -2; Gap costs: Linear; Filter: Low complexity regions; Mask: Mask for lookup table only.
  • a sequence having a % identity score of XX% (for example, 80%) to a reference sequence as determined in this manner is considered to be XX% identical to or, equivalently, have XX% sequence identity to, the reference sequence.
  • the sequence containing the sub-sequence under investigation is aligned with the reference sequence, and the amino acid residue of the sub-sequence under investigation occupies the same position in the alignment as does the amino acid residue of the reference sub-sequence.
  • the 3-ketoacyl-CoA synthase enzyme of wild type Clostridiales bacterium l_7_47_FAA includes the following 9 amino acid residue sub sequence.
  • SEQ ID NO: 2 clostridioforme is aligned with SEQ ID NO: 1 using the BLAST software as described, the following amino acid residues occupy the same positions in the alignment:
  • each of alanine 227, valine 228, lysine 229, lysine 230, valine 231, proline 232, glutamine 233, and cystine 234 of the Clostridium clostridioforme full sequence align to each of alanine 2, valine 3, arginine 4, lysine 5, valine 6, proline 7, glutamic acid 8, and cystine 9 of the above Clostridiales bacterium sub-sequence, respectively.
  • each of alanine 227, valine 228, lysine 229, lysine 230, valine 231, proline 232, glutamine 233, and cystine 234 of the Clostridium clostridioforme full sequence align to each of alanine 219, valine 220, arginine 221, lysine 222, valine 223, proline 224, glutamic acid 225, and cystine 226 of Clostridiales bacterium l_7_47_FAA full sequence, respectively.
  • A“recombinant cell” is a cell whose genetic material has been altered by a human using genetic engineering techniques including molecular cloning.
  • Recombinant cells can comprise cells whose genetic material has been altered by the addition of genetic material from a cell of a different organism by genetic engineering.
  • Recombinant cells can also comprise cells whose genetic material has been altered by the deletion of genetic material by genetic engineering.
  • Recombinant cells can also comprise cells altered by the addition of extra copies of genetic material that is normally native to the recombinant cell by genetic engineering.
  • genetic material such as genes, promoters and terminators is“heterologous” if it is (i) non-native to the recombinant cell and/or (ii) is native to the recombinant cell, but is present at a location different than where that genetic material is present in the wild-type cell and/or (iii) is under the regulatory control of a non-native promoter and/or non-native terminator ⁇ Extra copies of native genetic material are considered as “heterologous” for purposes of this application, even if such extra copies are present at the same locus as that genetic material is present in the wild-type cell.
  • a polypeptide (such as a 3-ketoacyl-CoA synthase enzyme) is“heterologous” if it is non-native to a wild-type version of the recombinant cell, if it is native to the recombinant cell, but is expressed by a gene at a location different than where that gene is present in the wild- type version of the recombinant cell, if it is expressed by a gene that is under the regulatory control of a non-native promoter and/or non-native terminator, and/or if extra copies are present at the same locus where that gene is normally present in the wild-type version of the
  • A“3-ketoacyl-CoA synthase” is an enzyme that catalyzes the condensation reaction of an acyl-CoA with malonyl-CoA to form a 3-ketoacyl-CoA.
  • One method for evaluating the ability of an enzyme to catalyze this reaction is by measuring the release of free CoA-SH using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), malonyl-CoA as the donor substrate, and a C2-C8 acyl-CoA as the primer substrate, in the presence of the enzyme.
  • DTNB 5,5'-dithiobis-(2-nitrobenzoic acid)
  • malonyl-CoA as the donor substrate
  • C2-C8 acyl-CoA as the primer substrate
  • the ability of an enzyme to catalyze this condensation reaction can also be evaluated using C2-C8 acyl-CoA as the primer substrate and malonyl-CoA as the donor substrate in the presence of 5 mM Mg ++ salt by measuring the increase in absorbance at 303 nm as a function of the increase in the formation of a Mg ++ -complex with the 3-ketoacyl-CoA product.
  • 3-ketoacyl-CoA synthase will produce acetoacetyl-CoA from acetyl-CoA primer, 3- ketohexanoyl-CoA from butyryl-CoA primer, 3-ketooctanoyl-CoA from hexanoyl-CoA primer, and 3-ketodecanoyl-CoA from octanoyl-CoA primer.
  • NC-IUBMB Enzyme Nomenclature
  • A“CoA” or“CoA-SH”, as described here refers to Coenzyme A.
  • a CoA substrate can refer to a straight chain carbon chain covalently linked to Coenzyme A.
  • C4-CoA refers to a C4 chain covalently linked to Coenzyme A via a thioester bond between the thiol of the Coenzyme A and a terminal carbon of the C4 chain.
  • A“fatty acid”, as described here, comprises a carboxylic acid with an aliphatic chain, the aliphatic chain having at least four carbon atoms.
  • A“fatty acid chain product” is a compound having a straight carbon chain formed in a series of one or more reactions at the site of the terminal carboxyl group of a fatty acid or thioester bond of a corresponding -CoA compound.
  • a fatty acid chain product can comprise a straight carbon chain with a different end group such as, for example, an ester, an alcohol group, an amino group, an aldehyde group, a ketone, a methyl group, or an alkenyl group.
  • Fatty acid chain product can include fatty acid ester, fatty alcohol, fatty acid amide, fatty acid imide, and fatty acid amine.
  • A“fatty acid ester” is an ester compound corresponding to the reaction product of a fatty acid and an alcohol (with loss of water). Fatty acid ester can also correspond to the reaction product of a fatty acyl CoA and an alcohol. Fatty acid ester can include fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester, fatty acid butyl ester, fatty acid pentyl ester, and fatty acid hexyl ester.
  • Fatty acid methyl ester (FAME) is an ester compound corresponding to the reaction product of a fatty acid or a fatty acyl-CoA and methanol.
  • Chain lengths of fatty acids and fatty acid chain products are sometimes indicated herein by the shorthand“CX”, wherein X is a number designating the number of carbon atoms.
  • the number of carbon atoms designated in each case represents the carbon length of the straight- chain compound (after removal of CoA or ACP coenzymes) formed by the recombinant cell through one or more iterations of the reaction cycle:
  • acyl-CoA (or acyl-ACP) + malonyl-CoA to form a 3-ketoacyl compound
  • CoA or acyl-ACP The number of carbon atoms does not include additional carbon atoms that may be added during the formation of any fatty acid chain products of the fatty acid, such as, for example, carbons included in an ester group following an esterification.
  • hexanoic acid methyl ester is considered as a“C6” fatty acid ester compound, the carbon of the methyl ester group not being counted.
  • The“CX” designation can also apply or refer to fatty acid chain products.
  • C4 FAME refers to methyl butanoate
  • C6 FAME refers to methyl hexanoate
  • C8 FAME refers to methyl octanoate
  • C10 FAME refers to methyl decanoate
  • C12 FAME refers to methyl dodecanoate.
  • The“CX” designation can also be used for fatty acid
  • C2-CoA refers to acetyl-CoA
  • C4-CoA refers to butyryl-CoA
  • C6- CoA refers to hexanoyl-CoA
  • C8-C0A refers to octanoyl-CoA
  • ClO-CoA refers to decanoyl- CoA
  • Cl2-CoA refers to dodecanoyl-CoA.
  • engineered biosynthetic pathway refers to a biosynthetic pathway that is genetically engineered into a recombinant cell and comprises the enzymes to carry out a sequence of steps to produce a desired product such as a fatty acid or fatty acid chain product.
  • one or more of the enzymes of the engineered biosynthetic pathway does not naturally occur in a wild type version of the recombinant cell. These enzymes can be introduced into the recombinant cell by using genetic engineering techniques to introduce one or more heterologous genes to overexpress the enzymes. In some aspects, one or more of the enzymes of the engineered biosynthetic pathway do not naturally occur in sufficient copy number in a wild- type version of the recombinant cell and additional copies of the enzymes must be introduced by using genetic engineering techniques to introduce one or more heterologous genes to overexpress the enzymes. In some cases, the engineered biosynthetic pathway also comprises genetic modifications to the recombinant cell to reduce or eliminate competing metabolic pathways and/or to reduce interfering activities such as degradation of desired products or necessary intermediates.
  • the term“endogenous” gene refers to a gene that is naturally found in a particular cell.
  • the term“overexpress” is used to refer to the expression of a heterologous gene in a recombinant cell at levels higher than the level of gene expression in a wild type cell.
  • the term“overexpress” can also refer to the expression of a polypeptide from a heterologous gene in a recombinant cell.
  • an endogenous gene is deleted.
  • the terms“deletion,” “deleted,”“knockout,” and“knocked out” can be used interchangeably to refer to an endogenous gene that has been engineered to no longer be expressed in a recombinant cell.
  • a deleted/knocked out gene is an endogenous gene that is deleted to increase production of a desired product such as a fatty acid or fatty acid chain product.
  • the terms“variant” and“mutant” are used to describe a protein sequence that has been modified at one or more amino acids, compared to the wild type sequence of a particular protein. Mutations to the amino acid residues encoded by the wild-type genes are designated herein by the shorthand designation for the wild-type protein, followed in parentheses by a 3-, 4- or 5 character code consisting of a first letter designating the amino acid residue in the native enzyme, a 1-, 2- or 3-digit number indicating the position of that amino acid residue in the native enzyme, and a final letter designating the amino acid residue in that position in the mutated enzyme.
  • the single-letter designations are IUPAC amino acid abbreviations as reported, for example, at Eur. J. Biochem.
  • the designation“Cbac(V223A)” indicates that a valine (V) at amino acid residue position 223 in the wild type 3-ketoacyl-CoA synthase enzyme from Clostridiales bacterium l_7_47_FAA (Cbac enzyme) has been replaced with an alanine (A).
  • the designation“Cbac(V223X)” indicates that a valine (V) at amino acid residue position 223 in the wild type 3-ketoacyl-CoA synthase enzyme from Clostridiales bacterium l_7_47_FAA (Cbac enzyme) is replaced with an amino acid other than valine.
  • the recombinant cell is a prokaryotic cell. In some aspects, the recombinant cell is a eukaryotic cell.
  • the recombinant cell is a microorganism, and may be a single- celled microorganism.
  • the recombinant cell may be a plant cell, including a cell from a plant within any of the Chlorophyta, Charophyta, Marchantiophyta, Anthocerotophyta, Bryophyta,
  • Lycopodiophyta Pteridophyta, Cycadophyta, Ginkgophyta, Pinophyta, Gnetophyta or
  • Such a plant cell may be, for example, a cell from a plant within any of the genera Arabidopsis, Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris, Populus, Saccharum, Salix, Simmondsia, and Zea.
  • the recombinant cell may be a fungi, microalgae, algae or red algae (heterokont) cell.
  • the recombinant cell may be a yeast cell.
  • a yeast or fungus cell may be an oleaginous yeast or fungus, and/or may be a Crabtree negative yeast or fungus.
  • oleaginous fungi refers to yeasts or filamentous fungi, which accumulate at least 10%, 12.5%, 15%, 17.5%, preferably at least 20% or even at least 25%
  • biomass (w/w) of their biomass as lipid. They may even accumulate at least 30%, 40%, 50%, 60%, 70%, 80% (w/w) or more of their biomass as lipids.
  • the biomass is usually measured as cell dry weight (CDW).
  • A“Crabtree -positive” organism is one that is capable of producing ethanol in the presence of oxygen, whereas a "Crabtree-negative” organism is not.
  • a yeast cell having a Crabtree-negative phenotype is any yeast cell that does not exhibit the Crabtree effect.
  • the term “Crabtree-negative” refers to both naturally occurring and genetically modified organisms. Briefly, the Crabtree effect is defined as the inhibition of oxygen consumption by a
  • a yeast cell having a Crabtree positive phenotype continues to ferment irrespective of oxygen availability due to the presence of glucose, while a yeast cell having a Crabtree-negative phenotype does not exhibit glucose mediated inhibition of oxygen consumption.
  • Crabtree-positive yeast produce an excess of alcohol rather than biomass production.
  • yeast cells include, Pichia, Candida, Klebsiella, Hansenula,
  • Saccharomyces Schizosaccharomyces, Zygosaccharomyces, Debaryomyces, Cryptoococcus, Rhodotorula, Rhodosporidium, Lipomyces and Yarrowia.
  • yeast cells include C. sonorensis, K. marxianus, K. thermotolerans, C. methane sorbosa, Saccharomyces bulderi (S. bulderi), I. orientalis, C. lambica, C. sorboxylosa, C. zemplinina, C. geochares, P. membranifaciens, Z. kombuchaensis, C. sorbosivorans, C. vanderwaltii, C. sorbophila, Z.
  • K. marxianus and C. sonorensis include those described in WO 00/71738 Al, WO 02/42471 A2, WO 03/049525 A2, WO 03/102152 A2 and WO
  • Suitable strains of I. orientalis are ATCC strain 32196 and ATCC strain PTA- 6648.
  • the recombinant cell is a bacteria cell.
  • the bacteria may be a gram-positive or gram-negative bacteria. It may be a cell within any of the Chlamydiae, green nonsulfur, actinobacteria, planctomycetes, spirochaetes, fusobacteria, cyanobacteria, thermophilic sulphate-reducer, acidobacteria or proteobacteria classifications of bacteria (Ciccarelli et al, Science 311 (5765): 1283-7 (2006).
  • suitable bacteria cells include, for example, those within any of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Streptomyces, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Bacteriophage, Brevibacterium, Acanthoceras, Acanthococcus, Acarvochloris, Achnanthes, Achnanthidiun, Actinastrum, Actinochloris, Actinocyclus,
  • Batrarhospermum Binurlearia, Bitrichia, Blidingia, Botrdiopsis, Botrydium, Botryococcus, Botryosphaerella, Brachiomonas, Brachysira, Brachytrichia, Brebissonia, Bulbochaete, Bumnilleria, Buinilleriopsis, Caloneis, Calothrix, Campylodiscus, Capsosiphon, Carteria, Catena, Cavinula, Cenritractus, Centroniella, Ceratiunt, Chaetoceros, Chaetochloris,
  • Chaetomorpha Chaetonella, Chaetonemna, Chaetopeltis, Chaetophora, Chaetosphaeridium, Chamaesiphion, Chara, Characiochloris, Characiopsis, Characium, Chorales, Chilomonas, Chlainomonas, Chlamydoblepharis, Chlamydocapsa, Chlamydomonas, Chlamydomonopsis, Chlamydomnyxa, Chlamydonephris, Chlorangiella, Chlorangiopsis, Chlorella, Chlorobotrys, Chlorobrachis, Chlorochytrium, Chloroccun, Chlorogloea, Chlorogloeopsis, Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema, Cholorphyta, Cholorosaccus, Cholorosarcina, Choricystis, Chromophyton, Ch
  • Chrysocapsella Chrysochaete, Chrysohromulina, Chrysococcus, Chrysocrinus,
  • Chrynsolepidomonas Chrysolykos, Chrysonebula, Chrysophyta, Chrysopyxis, Chrysosaccus, Chrysophaerella, Chrysotephanosphaera, Clodophora, Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis, Coelastrella, Coelastrum, Coelosphaerium, Coenochloris,
  • Coenococcus Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis,
  • Compsopogon Conjugatophyta, Conoehaete, Coronastrum, Cosmarium, Cosmnioneis, Cosmocladium, Crateriportula, Craticula, Crinalium, Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta, Ctenophora, Cyanodictyon, Cyanonephron, Cyanophora,
  • Cyanophyta Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella,
  • Cylindrocapsa Cylindrocystis, Cylindrospermum, Cylindrotheca, Cymatopleura, Cymbella, Cymbeilonitzschia, Cystodinium Dactylococcopsis, Debarya, Denticula, Dermatochrysis, Dermorarpa, Dermocarpella, Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon, Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula,
  • Dichothrix Dichtotomococcrus, Dicranochaete, Dictyochloris, Dictyococcus, Dictyosphaerium, Didymocystis, Didymogenes, Didymosphenia, Dilabifilum, Dimorphoccus, Dinobryon,
  • Entomorpha Entocladia, Entomoeis, Entophysalis, Ephichrysis, Epipyxis, Epithemia, Eremosphaura, Euastropsis, Euatstrum, Eucapsis, Eucocconeis, Eudorina, Euglena,
  • Euglenophyta Eunotia, Eustigmatophyta, Eutreptia, Fallcia, Ficherella, Fragilaria,
  • Glaucophyta Glenodiniopsis, Glenodinium, Gloeomonas, Gloeoplax, Gloeothece, Geloeotila, Gloeotrichia, Gloiodictyon, Golenkinia, Golenkiniopsis, Gomontia, Gomphocymbella,
  • Gomphonema Gomphosphaeria, Gonatozygon, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum, Granulochloris, Granulocystopsis, Groenbladia, Gymnodiunium, Gymnozyga, Gyrosignma, Haematocuccus, Hafniomonas, Hallassia, Hammatoidea, Hannaea, Hantzchia, Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Hemidinuim, Hemitonia, Heribaudiella, Heteromastix, Heterothrix, Hibberdia, Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema, Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium, Hyalothe
  • mice Micractinium, Micrasterias, Microchaete, Microcoleus, Microcystis, Microglena, Micromonas, Microspora, Microthamnion, Mischococcus, Monocrysis, Monodus, Monomastix,
  • Nephroclamys Nephrocytium, Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia, Nodularia, Nostoc, Ochromonas, Oedogonium, Oligochaetophora, Onychonema, Oocadrium, Oocrystis, Opephora, Ophiocytium, Orthoseira, Oscillartoria, Oxyneis,
  • Pachycladella Palmella, Palmodictyon, Pnadorina, Pannus, Paralia, Pascherina, Paulshulzia, Pediastrum, Pedinella, Pedinomonas, Pedinopera, Pelagodictyon, Penium, Peranema, Peridiniopsis, Peridinium, Peronia, Petroneis, Phacotus, Phacus, Phaeaster, Phaeodermatium, Phaeophyta, Phaeoshaera, Phaeothamnion, Phormidium, Phycopeltis, Phyllariochloris, Phyllocadium, Phyllomitas, Pinnilaria, Pitophora, Placoneis, Planctonema, Planktophaeria, Planothidium, Plectonema, Pleodorina, Pleurastrum, Pleurocapsa, Pleurocladia, Pleurodiscus, Pleurosigma, Pleurosira, Pleurot
  • Pseudophaerocystis Pseudostaurastrum, Pseudostraurosira, Pyrrophyta, Quadrichloris, Quadricoccus, Quadrigula, Radiocuccus, Radiobetalum, Raphidiopsis, Raphidocelis,
  • Raphidonema Raphidophyta
  • Peimeria Rhadorderma, Rhabomonas, Rhizoclonium
  • Rhodomonas Rhodiphyta, Rhoicosenia, Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus, Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix, Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia, Scytonema, Slenastrum,
  • Selenochloris Sellaphora, Semiorbis, Siderocelis, Diderocystopsis, Dimonsenia, Siphononema, Sirocladium, Sirogonium, Skeletonema, Sorastrum, Spermatozopis, Sphaerellocystis, Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma, Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum, Spondylosium, Sporotetras, Spumella, Staurastrum, Stauerodesmus, Stauroneis, Staurosira, Staurrosiella, Stenopterobia, Stephanocostis,
  • bacteria cells include Escherichia coli; Oligotropha carboxidovorans, Pseudomononas sp. Alcaligenes eutrophus ( Cupriavidus necator), Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis, Bacillus subtilis, Cupriavidus basilensis, Cupriavidus campinensis, Cupriavidus gilardi, Cupriavidus laharsis, Cupriavidus metallidurans, Cupriavidus oxalaticus, Cupriavidus pauculus, Cupriavidus pinatubonensis, Cupriavidus respiraculi, Cupriavidus taiwanensis, In some aspects, the
  • the recombinant cell may be a synthetic cell or a cell produced by a synthetic genome, as described in U.S. Patent Publication 2007/0264688, or 2007/0269862.
  • the cell may be a CHO cell, a COS cell, a VERO cell, a BHK cell, a HeLa cell, a Cvl cell, an MDCK cell, a 293 cell, a 3T3 cell, or a PC 12 cell.
  • this application discloses a recombinant cell genetically modified to express one or more heterologous enzymes having 3-ketoacyl-CoA synthase activity.
  • the heterologous enzymes having 3-ketoacyl-CoA synthase activity can comprise enzymes naturally found in Clostridiales bacterium l_7_47_FAA (SEQ ID NO: 1)
  • a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA comprises the amino acid sequence of SEQ ID NO: 1 and corresponds to the polypeptide designated as Cbac.
  • the heterologous enzyme having 3-ketoacyl-CoA synthase activity comprises a polypeptide having more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity to the amino acid sequence of SEQ ID NO: 1.
  • a recombinant cell genetically modified to express the polypeptide designated as Cbac produces fatty acids and/or fatty acid chain products.
  • the recombinant cell expressing heterologous Cbac can also produce FAME.
  • the recombinant cell expressing heterologous Cbac can also produce a mixture of FAMEs with different carbon chain lengths.
  • the recombinant cell expressing heterologous Cbac can produce a FAME mixture comprising C6 FAME, C8 FAME, C10 FAME, and C12 FAME.
  • the recombinant cell expressing heterologous Cbac can also produce a FAME mixture comprising C6 FAME (methyl hexanoate), C8 FAME (methyl octanoate), C10 FAME (methyl decanoate), and C12 FAME (methyl dodecanoate) with C8 FAME (methyl octanoate) as the predominant species.
  • the recombinant cell expressing heterologous Cbac can produce fatty acids and/or fatty acid chain products without the recombinant cell comprising an additional heterologous polypeptide with 3-ketoacyl-CoA synthase activity.
  • the recombinant cell expressing heterologous Cbac can catalyze the addition of malonyl-CoA to a C2-CoA substrate to lead to a C4 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C4-CoA substrate to lead to a C6 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C6-C0A substrate to lead to a C8 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C8-C0A substrate to lead to a C 10 fatty acid and/or fatty acid chain product, and/or the addition of malonyl-CoA to a ClO-CoA substrate to lead to a C12 fatty acid and/or fatty acid chain product.
  • the recombinant cell expressing heterologous Cbac can also comprise one or more additional heterologous polypeptides with 3- ketoacyl-CoA synthase activity (e.g., a NphT7 polypeptide and/or a NphT7 polypeptide mutant as described below) to increase production of fatty acids and/or fatty acid chain products.
  • additional heterologous polypeptides with 3- ketoacyl-CoA synthase activity e.g., a NphT7 polypeptide and/or a NphT7 polypeptide mutant as described below
  • a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA comprises the amino acid sequence of SEQ ID NO: 2 and corresponds to the polypeptide designated as Cclol.
  • the heterologous enzyme having 3-ketoacyl-CoA synthase activity comprises a polypeptide having more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity to the amino acid sequence of SEQ ID NO: 2.
  • a recombinant cell genetically modified to express the polypeptide designated as Cclol produces fatty acids and/or fatty acid chain products.
  • the recombinant cell expressing heterologous Cclol can also produce FAME.
  • the recombinant cell expressing heterologous Cclol can also produce a mixture of FAMEs with different carbon chain lengths.
  • the recombinant cell expressing heterologous Cclol can produce a FAME mixture comprising C6 FAME, C8 FAME, C10 FAME, and C12 FAME.
  • the recombinant cell expressing heterologous Cclol can also produce a FAME mixture comprising C6 FAME (methyl hexanoate), C8 FAME (methyl octanoate), C10 FAME (methyl decanoate), and C12 FAME (methyl dodecanoate) with C8 FAME (methyl octanoate) as the predominant species.
  • the recombinant cell expressing heterologous Cclol can produce fatty acids and/or fatty acid chain products without the recombinant cell comprising an additional heterologous polypeptide with 3-ketoacyl-CoA synthase activity.
  • the recombinant cell expressing heterologous Cclol can catalyze the addition of malonyl-CoA to a C2-CoA substrate to lead to a C4 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C4-CoA substrate to lead to a C6 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C6-C0A substrate to lead to a C8 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C8-C0A substrate to lead to a C 10 fatty acid and/or fatty acid chain product, and/or the addition of malonyl-CoA to a ClO-CoA substrate to lead to a C12 fatty acid and/or fatty acid chain product.
  • the recombinant cell expressing heterologous Cclol can also comprise one or more additional heterologous polypeptides with 3- ketoacyl-CoA synthase activity (e.g., a NphT7 polypeptide and/or a NphT7 polypeptide mutant as described below) to increase production of fatty acids and/or fatty acid chain products.
  • a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA comprises the amino acid sequence of SEQ ID NO: 3 and corresponds to the polypeptide designated as Cbol.
  • the heterologous enzyme having 3-ketoacyl-CoA synthase activity comprises a polypeptide having more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity to the amino acid sequence of SEQ ID NO: 3.
  • a recombinant cell genetically modified to express the polypeptide designated as Cbol produces fatty acids and/or fatty acid chain products.
  • the recombinant cell expressing heterologous Cbol can also produce FAME.
  • the recombinant cell expressing heterologous Cbol can also produce a mixture of FAMEs with different carbon chain lengths.
  • the recombinant cell expressing heterologous Cbol can produce a FAME mixture comprising C6 FAME, C8 FAME, C10 FAME, and C12 FAME.
  • the recombinant cell expressing heterologous Cbol can also produce a FAME mixture comprising C6 FAME (methyl hexanoate), C8 FAME (methyl octanoate), C10 FAME (methyl decanoate), and C12 FAME (methyl dodecanoate) with C8 FAME (methyl octanoate) as the predominant species.
  • the recombinant cell expressing heterologous Cbol can produce fatty acids and/or fatty acid chain products without the recombinant cell comprising an additional heterologous polypeptide with 3-ketoacyl-CoA synthase activity.
  • the recombinant cell expressing heterologous Cbol can catalyze the addition of malonyl-CoA to a C2-CoA substrate to lead to a C4 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C4-CoA substrate to lead to a C6 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C6-C0A substrate to lead to a C8 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C8-C0A substrate to lead to a C 10 fatty acid and/or fatty acid chain product, and/or the addition of malonyl-CoA to a ClO-CoA substrate to lead to a C12 fatty acid and/or fatty acid chain product.
  • the recombinant cell expressing heterologous Cbol can catalyze the addition of malonyl-CoA to a C2-CoA substrate to lead
  • heterologous Cbol can also comprise one or more additional heterologous polypeptides with 3- ketoacyl-CoA synthase activity (e.g., a NphT7 polypeptide and/or a NphT7 polypeptide mutant as described below) to increase production of fatty acids and/or fatty acid chain products.
  • a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA comprises the amino acid sequence of SEQ ID NO: 4 and corresponds to the polypeptide designated as Csacl.
  • the heterologous enzyme having 3-ketoacyl-CoA synthase activity comprises a polypeptide having more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity to the amino acid sequence of SEQ ID NO: 4.
  • a recombinant cell genetically modified to express the polypeptide designated as Csacl produces fatty acids and/or fatty acid chain products.
  • the recombinant cell expressing heterologous Csacl can also produce FAME.
  • the recombinant cell expressing heterologous Csacl can also produce a mixture of FAMEs with different carbon chain lengths.
  • the recombinant cell expressing heterologous Csacl can produce a FAME mixture comprising C6 FAME, C8 FAME, C10 FAME, and C12 FAME.
  • the recombinant cell expressing heterologous Csacl can also produce a FAME mixture comprising C6 FAME (methyl hexanoate), C8 FAME (methyl octanoate), C10 FAME (methyl decanoate), and C12 FAME (methyl dodecanoate) with C8 FAME (methyl octanoate) as the predominant species.
  • the recombinant cell expressing heterologous Csacl can produce fatty acids and/or fatty acid chain products without the recombinant cell comprising an additional heterologous polypeptide with 3-ketoacyl-CoA synthase activity.
  • the recombinant cell expressing heterologous Csacl can catalyze the addition of malonyl-CoA to a C2-CoA substrate to lead to a C4 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C4-CoA substrate to lead to a C6 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C6-C0A substrate to lead to a C8 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C8-C0A substrate to lead to a C 10 fatty acid and/or fatty acid chain product, and/or the addition of malonyl-CoA to a ClO-CoA substrate to lead to a C12 fatty acid and/or fatty acid chain product.
  • the recombinant cell expressing heterologous Csacl can catalyze the addition of malonyl-CoA to a C
  • heterologous Csacl can also comprise one or more additional heterologous polypeptides with 3- ketoacyl-CoA synthase activity (e.g., a NphT7 polypeptide and/or a NphT7 polypeptide mutant as described below) to increase production of fatty acids and/or fatty acid chain products.
  • a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA comprises the amino acid sequence of SEQ ID NO: 5 and corresponds to the polypeptide designated as Csac2.
  • the heterologous enzyme having 3-ketoacyl-CoA synthase activity comprises a polypeptide having more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity to the amino acid sequence of SEQ ID NO: 5.
  • a recombinant cell genetically modified to express the polypeptide designated as Csac2 produces fatty acids and/or fatty acid chain products.
  • the recombinant cell expressing heterologous Csac2 can also produce FAME.
  • the recombinant cell expressing heterologous Csac2 can also produce a mixture of FAMEs with different carbon chain lengths.
  • the recombinant cell expressing heterologous Csac2 can produce a FAME mixture comprising C6 FAME, C8 FAME, C10 FAME, and C12 FAME.
  • the recombinant cell expressing heterologous Csac2 can also produce a FAME mixture comprising C6 FAME (methyl hexanoate), C8 FAME (methyl octanoate), C10 FAME (methyl decanoate), and C12 FAME (methyl dodecanoate) with C8 FAME (methyl octanoate) as the predominant species.
  • the recombinant cell expressing heterologous Csac2 can produce fatty acids and/or fatty acid chain products without the recombinant cell comprising an additional heterologous polypeptide with 3-ketoacyl-CoA synthase activity.
  • the recombinant cell expressing heterologous Csac2 can catalyze the addition of malonyl-CoA to a C2-CoA substrate to lead to a C4 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C4-CoA substrate to lead to a C6 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C6-C0A substrate to lead to a C8 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C8-C0A substrate to lead to a C 10 fatty acid and/or fatty acid chain product, and/or the addition of malonyl-CoA to a ClO-CoA substrate to lead to a C12 fatty acid and/or fatty acid chain product.
  • heterologous Csac2 can also comprise one or more additional heterologous polypeptides with 3- ketoacyl-CoA synthase activity (e.g., a NphT7 polypeptide and/or a NphT7 polypeptide mutant as described below) to increase production of fatty acids and/or fatty acid chain products.
  • a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA comprises the amino acid sequence of SEQ ID NO: 6 and corresponds to the polypeptide designated as Cclo2.
  • the heterologous enzyme having 3-ketoacyl-CoA synthase activity comprises a polypeptide having more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity to the amino acid sequence of SEQ ID NO: 6.
  • a recombinant cell genetically modified to express the polypeptide designated as Cclo2 produces fatty acids and/or fatty acid chain products.
  • the recombinant cell expressing heterologous Cclo2 can also produce FAME.
  • the recombinant cell expressing heterologous Cclo2 can also produce a mixture of FAMEs with different carbon chain lengths.
  • the recombinant cell expressing heterologous Cclo2 can produce a FAME mixture comprising C6 FAME, C8 FAME, C10 FAME, and C12 FAME.
  • the recombinant cell expressing heterologous Cclo2 can also produce a FAME mixture comprising C6 FAME (methyl hexanoate), C8 FAME (methyl octanoate), C10 FAME (methyl decanoate), and C12 FAME (methyl dodecanoate) with C8 FAME (methyl octanoate) as the predominant species.
  • the recombinant cell expressing heterologous Cclo2 can produce fatty acids and/or fatty acid chain products without the recombinant cell comprising an additional heterologous polypeptide with 3-ketoacyl-CoA synthase activity.
  • the recombinant cell expressing heterologous Cclo2 can catalyze the addition of malonyl-CoA to a C2-CoA substrate to lead to a C4 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C4-CoA substrate to lead to a C6 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C6-C0A substrate to lead to a C8 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C8-C0A substrate to lead to a C 10 fatty acid and/or fatty acid chain product, and/or the addition of malonyl-CoA to a ClO-CoA substrate to lead to a C12 fatty acid and/or fatty acid chain product.
  • heterologous Cclo2 can also comprise one or more additional heterologous polypeptides with 3- ketoacyl-CoA synthase activity (e.g., a NphT7 polypeptide and/or a NphT7 polypeptide mutant as described below) to increase production of fatty acids and/or fatty acid chain products.
  • a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA comprises the amino acid sequence of SEQ ID NO: 7 and corresponds to the polypeptide designated as Casp.
  • the heterologous enzyme having 3-ketoacyl-CoA synthase activity comprises a polypeptide having more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity to the amino acid sequence of SEQ ID NO: 7.
  • a recombinant cell genetically modified to express the polypeptide designated as Casp produces fatty acids and/or fatty acid chain products.
  • the recombinant cell expressing heterologous Casp can also produce FAME.
  • the recombinant cell expressing heterologous Casp can also produce a mixture of FAMEs with different carbon chain lengths.
  • the recombinant cell expressing heterologous Casp can produce a FAME mixture comprising C6 FAME, C8 FAME, C10 FAME, and C12 FAME.
  • the recombinant cell expressing heterologous Casp can also produce a FAME mixture comprising C6 FAME (methyl hexanoate), C8 FAME (methyl octanoate), C10 FAME (methyl decanoate), and C12 FAME (methyl dodecanoate) with C8 FAME (methyl octanoate) as the predominant species.
  • the recombinant cell expressing heterologous Casp can produce fatty acids and/or fatty acid chain products without the recombinant cell comprising an additional heterologous polypeptide with 3-ketoacyl-CoA synthase activity.
  • the recombinant cell expressing heterologous Casp can catalyze the addition of malonyl-CoA to a C2-CoA substrate to lead to a C4 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C4-CoA substrate to lead to a C6 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C6-C0A substrate to lead to a C8 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C8-C0A substrate to lead to a C 10 fatty acid and/or fatty acid chain product, and/or the addition of malonyl-CoA to a ClO-CoA substrate to lead to a C12 fatty acid and/or fatty acid chain product.
  • heterologous Casp can also comprise one or more additional heterologous polypeptides with 3- ketoacyl-CoA synthase activity (e.g., a NphT7 polypeptide and/or a NphT7 polypeptide mutant as described below) to increase production of fatty acids and/or fatty acid chain products.
  • a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA comprises the amino acid sequence of SEQ ID NO: 8 and corresponds to the polypeptide designated as Chat2.
  • the heterologous enzyme having 3-ketoacyl-CoA synthase activity comprises a polypeptide having more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity to the amino acid sequence of SEQ ID NO: 8.
  • a recombinant cell genetically modified to express the polypeptide designated as Chat2 produces fatty acids and/or fatty acid chain products.
  • the recombinant cell expressing heterologous Chat2 can also produce FAME.
  • the recombinant cell expressing heterologous Chat2 can also produce a mixture of FAMEs with different carbon chain lengths.
  • the recombinant cell expressing heterologous Chat2 can produce a FAME mixture comprising C6 FAME, C8 FAME, C10 FAME, and C12 FAME.
  • the recombinant cell expressing heterologous Chat2 can also produce a FAME mixture comprising C6 FAME (methyl hexanoate), C8 FAME (methyl octanoate), C10 FAME (methyl decanoate), and C12 FAME (methyl dodecanoate) with C8 FAME (methyl octanoate) as the predominant species.
  • the recombinant cell expressing heterologous Chat2 can produce fatty acids and/or fatty acid chain products without the recombinant cell comprising an additional heterologous polypeptide with 3-ketoacyl-CoA synthase activity.
  • the recombinant cell expressing heterologous Chat2 can catalyze the addition of malonyl-CoA to a C2-CoA substrate to lead to a C4 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C4-CoA substrate to lead to a C6 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C6-C0A substrate to lead to a C8 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C8-C0A substrate to lead to a C 10 fatty acid and/or fatty acid chain product, and/or the addition of malonyl-CoA to a ClO-CoA substrate to lead to a C12 fatty acid and/or fatty acid chain product.
  • heterologous Chat2 can also comprise one or more additional heterologous polypeptides with 3- ketoacyl-CoA synthase activity (e.g., a NphT7 polypeptide and/or a NphT7 polypeptide mutant as described below) to increase production of fatty acids and/or fatty acid chain products.
  • additional heterologous polypeptides with 3- ketoacyl-CoA synthase activity e.g., a NphT7 polypeptide and/or a NphT7 polypeptide mutant as described below
  • a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA comprises the amino acid sequence of SEQ ID NO 9 and corresponds to the polypeptide designated as Chatl.
  • the heterologous enzyme having 3-ketoacyl-CoA synthase activity comprises a polypeptide having more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity to the amino acid sequence of SEQ ID NO: 9.
  • a recombinant cell genetically modified to express the polypeptide designated as Chatl produces fatty acids and/or fatty acid chain products.
  • the recombinant cell expressing heterologous Chatl can also produce FAME.
  • the recombinant cell expressing heterologous Chatl can also produce a mixture of FAMEs with different carbon chain lengths.
  • the recombinant cell expressing heterologous Chatl can produce a FAME mixture comprising C6 FAME, C8 FAME, C10 FAME, and C12 FAME.
  • the recombinant cell expressing heterologous Chatl can also produce a FAME mixture comprising C6 FAME (methyl hexanoate), C8 FAME (methyl octanoate), C10 FAME (methyl decanoate), and C12 FAME (methyl dodecanoate) with C8 FAME (methyl octanoate) as the predominant species.
  • the recombinant cell expressing heterologous Chatl can produce fatty acids and/or fatty acid chain products without the recombinant cell comprising an additional heterologous polypeptide with 3-ketoacyl-CoA synthase activity.
  • the recombinant cell expressing heterologous Chatl can catalyze the addition of malonyl-CoA to a C2-CoA substrate to lead to a C4 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C4-CoA substrate to lead to a C6 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C6-C0A substrate to lead to a C8 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C8-C0A substrate to lead to a C 10 fatty acid and/or fatty acid chain product, and/or the addition of malonyl-CoA to a ClO-CoA substrate to lead to a C12 fatty acid and/or fatty acid chain product.
  • heterologous Chatl can also comprise one or more additional heterologous polypeptides with 3- ketoacyl-CoA synthase activity (e.g., a NphT7 polypeptide and/or a NphT7 polypeptide mutant as described below) to increase production of fatty acids and/or fatty acid chain products.
  • additional heterologous polypeptides with 3- ketoacyl-CoA synthase activity e.g., a NphT7 polypeptide and/or a NphT7 polypeptide mutant as described below
  • V223A can comprise an enzyme with increased specific activity for C2-CoA, C4-CoA, C6-C0A, and C8-C0A substrates when compared to the respective polypeptide with unaltered sequence.
  • the Cbac variant with the identified amino acid substitution of V223A can comprise more than twice the specific activity for C2-CoA, C4-CoA, and C6-C0A substrates when compared to the respective polypeptide with unaltered sequence. In other cases, the Cbac variant with the identified amino acid substitution of V223A can comprise a similar specific activity for C8-C0A substrates when compared to the respective polypeptide with unaltered sequence.
  • a Cbac variant with the identified amino acid substitution of T46M can comprise a similar or slightly reduced specific activity for C2-CoA, C4-CoA, C6-C0A, and C8-C0A substrates when compared to the respective polypeptide with unaltered sequence.
  • a Cbac variant with the identified amino acid substitution of S256G can comprise an increased or similar specific activity for C2-CoA, C4-CoA, C6-C0A, and C8-C0A substrates when compared to the respective polypeptide with unaltered sequence.
  • a recombinant cell with a Cbac(V223A) variant can comprise an increased total FAME production of a mixture of C6-FAME (methyl hexanoate), C8-FAME (methyl octanoate), C10-FAME (methyl decanoate), and C12-FAME (methyl dodecanoate) when compared to a recombinant cell with the respective polypeptide with unaltered sequence.
  • the recombinant cell with the Cbac(V223A) variant can comprise an increased total FAME production when compared to the recombinant cell with respective polypeptide with unaltered sequence. In some cases, the recombinant cell with the Cbac(V223A) variant can comprise an increased total FAME production of 40% or more when compared to the recombinant cell with the respective polypeptide with unaltered sequence. In some cases, the recombinant cell with the Cbac(T46M) variant can comprise an increased total FAME production when compared to the recombinant cell with the respective polypeptide with unaltered sequence.
  • the recombinant cell with the Cbac(T46M) variant can comprise an increased total FAME production of 20% or more when compared to the recombinant cell with the respective polypeptide with unaltered sequence. In some cases, the recombinant cell with the Cbac(S256G) variant can comprise an increased total FAME production when compared to the recombinant cell with the respective polypeptide with unaltered sequence. In some cases, the recombinant cell with the Cbac(S256G) variant can comprise an increased total FAME production of 9% or more when compared to the
  • a recombinant cell with a Cbac(V223A) variant, a Cbac(T46M) variant, or a Cbac(S256G) variant can comprise a similar ratio of FAME production by carbon chain length when compared to the recombinant cell with the Cbac polypeptide with unaltered sequence.
  • C8-FAME methyl octanoate
  • C6-FAME methyl hexanoate
  • C10-FAME methyl decanoate
  • C12-FAME methyl dodecanoate
  • C12-FAME methyl dodecanoate
  • a Cbac variant with more than one identified amino acid substitution can comprise an altered specific activity for C2-CoA, C4-CoA, C6-C0A, and C8- CoA substrates when compared to the respective polypeptide with unaltered sequence.
  • a Cbac(V223A, I246P) variant can comprise similar specific activity for C2-CoA, less specific activity for C4-CoA, less specific activity for C6-C0A substrates, and less specific activity for C8-C0A substrates when compared to the respective polypeptide with unaltered sequence.
  • a Cbac(V223A, I246L) variant can comprise increased specific activity for C2-CoA, increased specific activity for C4-CoA, increased specific activity for C6- CoA substrates, and similar specific activity for C8-C0A substrates when compared to the respective polypeptide with unaltered sequence.
  • a Cbac(V223A, I246L) variant can comprise 30% or greater specific activity for C2-CoA, 30% or greater specific activity for C4-CoA, 100% or greater specific activity for C6-C0A substrates, and similar specificity for C8-C0A substrates when compared to the respective polypeptide with unaltered sequence.
  • a Cbac(V223A, S282T) variant can comprise similar specific activity for C2-CoA, similar specific activity for C4-CoA, increased specific activity for C6-C0A substrates, and similar specific activity for C8-C0A substrates when compared to the respective polypeptide with unaltered sequence.
  • a Cbac(V223A, I246L, S282T) variant can comprise reduced specific activity for C2-CoA, reduced specific activity for C4-CoA, reduced specific activity for C6-C0A substrates, and reduced specific activity for C8-C0A substrates when compared to the respective polypeptide with unaltered sequence.
  • the recombinant cell can comprise heterologous 3-ketoacyl-CoA synthase enzymes having complementary acyl-CoA chain length specificities.
  • a heterologous NphT7 comprising high specific activity for C2-CoA substrates can be combined with another heterologous 3-ketoacyl-CoA synthase enzyme.
  • a heterologous NphT7 LSVA variant comprising high specific activity for C4-CoA substrates can be combined with another heterologous 3-ketoacyl-CoA synthase enzyme.
  • heterologous enzymes with certain high specific activities can be paired with heterologous enzymes having other high specific activities (e.g., for C6-C0A substrates and/or C8-C0A substrates).
  • a recombinant cell comprising heterologous Cbac and heterologous NphT7 comprises increased total FAME production compared to a recombinant cell comprising heterologous Cbac and no heterologous NphT7 or heterologous NphT7 LSVA variant.
  • a recombinant cell comprising heterologous Cbac and heterologous NphT7 comprises 35% or more total FAME production compared to a recombinant cell comprising heterologous Cbac and no heterologous NphT7 or heterologous NphT7 LSVA variant.
  • a recombinant cell comprising heterologous Cbac and heterologous NphT7 LSVA comprises similar total FAME production compared to a recombinant cell comprising heterologous Cbac and no heterologous NphT7 or heterologous NphT7 LSVA variant.
  • a recombinant cell comprising heterologous Cbac, heterologous NphT7, and heterologous NphT7 LSVA variant comprises increased total FAME production compared to a recombinant cell comprising heterologous Cbac and no heterologous NphT7 or heterologous NphT7 LSVA variant.
  • a recombinant cell comprising heterologous Cbac, heterologous NphT7, and heterologous NphT7 LSVA variant comprises 65% or more total FAME production compared to a recombinant cell comprising heterologous Cbac and no heterologous NphT7 or heterologous NphT7 LSVA variant.
  • a recombinant cell comprising heterologous Cbac(V223A,
  • I246L) variant and heterologous NphT7 comprises increased total FAME production compared to a recombinant cell comprising heterologous Cbac(V223A, I246L) variant and no
  • heterologous NphT7 or heterologous NphT7 LSVA variant a recombinant cell comprising heterologous Cbac(V223A, I246L) variant and heterologous NphT7 comprises 65% or more total FAME production compared to a recombinant cell comprising heterologous Cbac(V223A, I246L) variant and no heterologous NphT7 or heterologous NphT7 LSVA variant.
  • a recombinant cell comprising heterologous Cbac(V223A, I246L) variant and heterologous NphT7 LSVA variant comprises increased total FAME production compared to a recombinant cell comprising heterologous Cbac(V223A, I246L) variant and no heterologous NphT7 or heterologous NphT7 LSVA.
  • a recombinant cell comprising heterologous Cbac(V223A, I246L) variant and heterologous NphT7 LSVA variant comprises 25% or more total FAME production compared to a recombinant cell comprising heterologous Cbac(V223A, I246L) variant and no heterologous NphT7 or heterologous NphT7 LSVA variant.
  • a recombinant cell comprising heterologous Cbac(V223A, I246L) variant, heterologous NphT7, and heterologous NphT7 LSVA variant comprises increased total FAME production compared to a recombinant cell comprising heterologous Cbac(V223A, I246L) variant and no
  • heterologous NphT7 or heterologous NphT7 LSVA variant comprises 70% or more total FAME production compared to a recombinant cell comprising heterologous Cbac(V223A, I246L) variant and no heterologous NphT7 or heterologous NphT7 LSVA variant.
  • this application discloses a recombinant cell with an engineered biosynthetic pathway comprising one or more heterologous enzymes that convert chemical precursors and/or substrates into desired chemical products.
  • the engineered biosynthetic pathway comprises heterologous enzymes to synthesize the desired chemical product.
  • individual heterologous enzymes work in a stepwise fashion to convert a precursor into the desired chemical product.
  • the engineered biosynthetic pathway can comprise one or more identified heterologous enzymes having 3-ketoacyl-CoA synthase activity to produce fatty acids and fatty acid chain products of C6-C10 chain length.
  • the engineered biosynthetic pathway can comprise one or more of Cbac, Cclol, Cbol, Csacl,
  • the engineered biosynthetic pathway can also comprise additional heterologous enzyme(s) that work in combination with one or more of Cbac, Cclol, Cbol, Csacl, Csac2, Cclo2, Casp, Chat2, Chatl, and their respective variants to produce fatty acids and fatty acid chain products of C6-C10 chain length.
  • the recombinant cell comprises one or more of Cbac, Cclol,
  • the second reaction is a dehydration to the corresponding trans-2-enoylacyl compound, which is reduced in a third step to the corresponding acyl-CoA.
  • the first reaction step is enzymatically catalyzed by a keto-CoA reductase (KCR) enzyme (EC 1.1.1.35).
  • KCR keto-CoA reductase
  • the second step is enzymatically catalyzed by a 3-hydroxy-acyl-CoA dehydratase (3HDh) enzyme (EC 4.2.1.17).
  • Some bifunctional enzymes catalyze both of the first and second step reactions (EC 1.1.1.35 and EC 4.2.1.55).
  • the third reaction step is enzymatically catalyzed by an enoyl- CoA reductase (ECR) enzyme (EC 1.1.1.32).
  • the engineered biosynthetic pathway preferably further comprises at least one of (1) a heterologous KCR gene that encodes for a KCR enzyme; (2) a heterologous 3HDh gene that encodes for a 3HDh enzyme; (3) a heterologous gene that encodes for a bifunctional enzyme that catalyzes both of the first and second reaction steps (EC 1.1.1.35 and 4.1.2.55) and (4) a heterologous ECR gene that encodes for an ECR enzyme.
  • the recombinant cell contains at least (1), (2) and (4) or at least (3) and (4).
  • the gene preferably is under the control of promoter and/or terminator sequences active in the
  • the KCR enzyme may be, for example, one encoded by a P. aeruginosa fadB gene and/or having an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 11, one encoded by a P. aeruginosa fadG gene and/or having an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 12, one encoded by a C. beijerinckii hbd gene and/or having an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 11, and others as described in WO 2015/010103.
  • the 3HDh enzyme may be, for example, one encoded by a C. acetobutylicum crt
  • short-chain-enoyl-CoA hydratase gene and/or having an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 14, one encoded by a P. putida ech (enoyl-CoA hydratase/aldolase) gene and/or having an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 15, and others as described in WO 2015/010103.
  • Suitable bifunctional enzymes that catalyze both the first and second reactions steps include, for example, one encoded by an E. colifadB gene and/or having an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 16; one encoded by an R. novegicus ech2 gene, and others as described in WO 2015/010103.
  • Suitable ECR enzymes include, for example, one encoded by a T denticola ter gene and/or having an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 17.
  • the recombinant cell can further include at least one heterologous 3-ketobutyryl-
  • CoA synthase gene different from the modified 3-ketoacyl-CoA synthases described above, which encodes for a 3-ketobutyryl-CoA synthase.
  • the heterologous 3-ketobutyryl-CoA synthase gene may encode for a 3-ketobutyryl-CoA synthase enzyme that is at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to any of those identified as SEQ ID NO: 1-120 of WO 2015/10103.
  • heterologous 3-ketobutyryl-CoA synthase gene is a
  • the recombinant cell includes at least one gene that encodes for a modified NphT7 enzyme as described in WO 2015/10103.
  • the modified NphT7 enzyme comprises an amino acid sequence having at least 70% but less than 100% to SEQ ID NO: 18.
  • the modified NphT7 enzyme may have, for example, one or more amino acid substitutions selected from the group consisting of H100L, I147T, F217V, Y144L, V157F, G309S, G288S, a PDRP to HFLQ substitution for amino acid residues 86-89, 1147F, I147M, I147Q, I147S, I147C, 1147E, I147N, I147W, I147D, I147R, I147P, I147L, V196G, I147G, I147H, I147K, I147V,
  • the modified NphT7 enzyme comprises at least one amino acid substitution selected from the group consisting of I147V, I147S, I147T, and at least one additional amino acid substitution selected from H100L, F217V, S323A and S323V.
  • the modified NphT7 enzyme corresponds to SEQ ID NO: 19.
  • the modified NphT7 enzyme comprises an I147V, I147S or I147T amino acid substitution and an S323A amino acid substitution (corresponding to SEQ ID NO: 19 in which amino acid 100 is H, amino acid 147 is V, S or T, amino acid 217 is F and amino acid 323 is A).
  • the modified NphT7 enzyme comprises an H100L substitution, an I147V, I147S or I147T amino acid substitution, an F217V substitution and an S323A amino acid substitution (corresponding to SEQ ID NO: 19 in which amino acid residue 100 is L, amino acid residue 147 is V, S or T, amino acid residue 217 is V and amino acid residue 323 is A).
  • the recombinant cell includes both of (1) a Streptomyces Sp
  • the recombinant cell includes a gene that encodes for an enzyme having SEQ ID NO: 18 and another gene that encodes for an enzyme having SEQ ID NO: 19.
  • the recombinant cell includes a gene that encodes for an enzyme having SEQ ID NO: 18 and another gene that encodes for an enzyme having SEQ ID NO: 19 in which amino acid residue 100 is H or L, amino acid residue 147 is S, T or V, amino acid residue 271 is F or V and amino acid residue 323 is A.
  • the recombinant cell can produce one or more enzymes that terminate the acyl elongation cycle and produce a product having the desired chain length.
  • a termination enzyme may or may not be heterologous.
  • the selection of termination enzyme may depend on whether the desired product is a fatty acid or a derivative thereof such as a fatty alcohol, a fatty aldehyde, a fatty alkene, a fatty amide, a fatty ester or a fatty alkane.
  • the recombinant cell in some aspects includes a heterologous thioesterase gene that encodes for a thioesterase such as an acyl-CoA esterase, in which case the product will be a fatty acid.
  • a thioesterase such as an acyl-CoA esterase
  • Suitable thioesterases include those described in Table 11 of WO 2015/101013.
  • the recombinant cell includes a gene that encodes for an ester synthase, in which case the product typically is a fatty acid ester.
  • Suitable ester synthases have amino acid sequences at least 80%, at least 90%, at least 95%, at least 99% or at least 100% identical to any of the Marinobacter aquacolei Maql enzyme (SEQ ID NO: 289 of WO 2015/10103), the Psychrobacter cryohaloentis Pcryl enzyme (SEQ ID NO: 290 of WO
  • the ester synthase may have an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99% or at least 100% identical to the Hahella chejuensis Hche ester synthase (SEQ ID NO: 20).
  • the recombinant cell may also include one or more genes that encode for one or more of a fatty acyl-CoA reductase (alcohol or aldehyde forming), a fatty aldehyde reductase, an acyl-ACP reductase, an acyl-CoA:ACP acyltransferase, an acyl-CoA hydrolase, a carboxylic acid reductase, an aldehyde dehydrogenase and/or an acyl-ACP reductase.
  • a fatty acyl-CoA reductase alcohol or aldehyde forming
  • a fatty aldehyde reductase an acyl-ACP reductase
  • an acyl-CoA:ACP acyltransferase an acyl-CoA hydrolase
  • carboxylic acid reductase an aldehyde dehydrogenase
  • the recombinant cell also may include (A) one or more genes that encode for a carboxyl transferase subunit a enzyme, (EC 6.3.1.2) such as an E. coli accA enzyme or an enzyme that is at least 80%, at least 90%, at least 95% or at least 99% identical thereto; (B) one or more genes that encode for a biotin carboxyl carrier protein, (EC 6.4.1.2) such as an E. coli accB enzyme or an enzyme that is at least 80%, at least 90%, at least 95% or at least 99% identical thereto; (C) one or more genes that encode for a biotin carboxylase subunit enzyme,
  • (EC 6.3.4.14) such as an E. coli accC enzyme or an enzyme that is at least 80%, at least 90%, at least 95% or at least 99% identical thereto;
  • (D) a carboxyl transferase subunit b (EC 6.4.1.2), such as an E. coli accD enzyme or an enzyme that is at least 80%, at least 90%, at least 95% or at least 99% identical thereto,
  • SEQ ID NO: 26 fused E. coli accD subunit and accA subunit enzyme
  • the recombinant cell described here further comprises one or more additional genetic modifications to reduce or eliminate the expression of certain endogenous enzymes in the recombinant cell. Reducing or eliminating the expression of these certain endogenous enzymes in the recombinant cell can increase the production of desired products such as fatty acids and/or fatty acid chain products.
  • These reduced or eliminated endogenous enzymes include one or more of the following enzymes:
  • Methylglyoxal synthase (EC 4.2.3.3), for example that encoded by the E. coli mgsA gene.
  • Lactate dehydrogenase (EC 1.1.1.27), for example that encoded by the E. coli IdhA gene.
  • Phosphotransacetylase (EC 2.3.1.8), for example that encoded by the E. coli pta gene.
  • Acetate kinase (EC 2.7.2.1), for example that encoded by E. coli ackA gene.
  • Acyl-CoA synthase (EC 6.2.1.3), for example that encoded by the E. colifadD gene.
  • Pyruvate formate lyase (EC 2.3.1.54), for example that encoded by the E.coli pflB gene.
  • Pyruvate oxidase (EC 1.2.2.2), for example that encoded by the E. coli poxB gene.
  • Fused acetaldehyde-CoA dehydrogense (EC 1.2.1.10), for example that encoded by the E. coli adhE gene.
  • Trigger factor (EC 5.2.1.8), for example that encoded by the E. coli tig gene.
  • Restriction endonuclease (EC 3.1.21.3), for example that encoded by the E. coli hsdr514 gene.
  • the atoDAEB operon The atoDAEB operon.
  • Acyl-CoA thioesterase (EC 3.1.2.-), for example that encoded by the E. coli tesB or yciA gene.
  • Acyl-coenzyme A dehydrogenase (EC 1.3.8.7), for example that encoded by the E. coli fadE gene.
  • 3-ketoacyl-CoA thiolase (EC 2.3.1.16), for example that encoded by the E. colifadA gene.
  • L-ribulokinase (EC2.7.1.16), for example that encoded by the E. coli araB gene.
  • L-ribulose-5-phosphate-4-epimerase (EC 5.1.3.4), for example that encoded by the E. coli araD gene.
  • Beta-D-galactosidase (EC 3.2.1.23), for example that encoded by the E.coli lacZ gene.
  • Rhamnulose-l -phosphate aldolase (EC 4.1.2.19), for example that encoded by the E. coli rhaD gene.
  • Rhamnulokinase (EC 2.7.1.5), for example that encoded by the E. coli rhaB gene.
  • Truncated RNase PH (EC2.7.7.56) Rph-1 gene.
  • Any heterologous gene may be operatively linked to a promoter and/or terminator sequence that is functional in the recombinant strain.
  • the promoter may be an inducible promoter that functions only under certain conditions.
  • a low phosphate inducible promoter such as the promoter of the wild-type E. coli phoE gene ( PphoE )
  • PphoE wild-type E. coli phoE gene
  • Such a promoter is active in a low phosphate environment. Accordingly, a recombinant cell in which the 3-ketoacyl synthase gene is under the control of an E.
  • the coli phoE promoter or another low phosphate inducible promoter may be cultivated in a fermentation medium containing no more than 25 mM phosphate, especially no more than 20 mM, no more than 2 mM, no more than lmM, no more than 0.5 mM, or no more than 0.25 mM phosphate.
  • the promoter that is a low phosphate inducible promoter is the promoter for the pstS gene ( PpstS ). This promoter may be constructed to include a binding site for Integration Host Factor and is thus designated PpstSIH (Lyzen et ak, Plasmid 60:125 (2008)).
  • Any heterologous gene may be integrated into the genome of the recombinant strain and/or present in one or more plasmids. If integrated into the genome, the heterologous gene may be inserted at a targeted or random location. Transformation methods such as electroporation and chemical methods (including calcium chloride and/or lithium acetate methods) known in the art are suitable. Examples of suitable transformation methods are described, for example, in Molecular Cloning: A Laboratory Manual, 4 th Ed. Spring Harbor Press 2012. In general, no special transformation methods are necessary to produce the recombinant cells.
  • Deletions and/or disruptions of native genes can be performed by transformation methods, by mutagenesis and/or by forced evolution methods.
  • mutagenesis methods cells are exposed to ultraviolet radiation or a mutagenic substance, under conditions sufficient to achieve a high kill rate (60-99.9%, preferably 90-99.9%) of the cells.
  • Surviving cells are then plated and selected or screened for cells having the deleted or disrupted metabolic activity.
  • Disruption or deletion of the desired native gene(s) can be confirmed through PCR or Southern analysis methods.
  • the recombinant cells described herein are used to produce compounds having a straight-chain alkyl group.
  • the recombinant cells are grown under conditions such that they produce such compounds, and the compounds are recovered.
  • the recombinant cell is a plant cell
  • the plant can be grown and the compound having the straight-chain alkyl group can be recovered from the plant or any portion thereof, such as roots, stems, leaves, flowers, seeds, seed pods and the like, in which the compound accumulates during the growth of the plant.
  • Single-cell and other microcells can be used in a culturing process to produce such compounds.
  • Culturing is performed generally by forming a culture medium that includes at least one carbon source that is capable of being metabolized by the recombinant cell to produce the product compounds and nutrients as may be required by the specific recombinant cell.
  • the nutrients may include, for example, at least one nitrogen source such as yeast extract, peptone, tryptone, soy flour, com steep liquor, or casein, at least one phosphorus source, one or more vitamins such as biotin, vitamin B12 and derivatives of vitamin B 12, thiamin, pantothenate, one or more trace metals and the like.
  • the fermentation medium may also contain additional materials such as anti-foam agents, biocides, buffers and the like.
  • the culture medium may also include a reagent that reacts with the straight-chain compound to produce the desired product.
  • the culture medium preferably contains an alkanol such as methanol, ethanol or a C3-C8 alkanol. The alkanol reacts to produce the corresponding ester.
  • a native or heterologous ester synthase, or other appropriate enzyme, may be expressed by the recombinant cell to catalyze such a reaction.
  • the culture medium is inoculated with the recombinant cell, and the inoculum is cultured in the medium so that the cell density increases to a cell density suitable for production.
  • the culture medium is then maintained at conditions sufficient for the recombinant cells to produce the desired product.
  • Suitable culture conditions will of course depend on the requirements of the particular recombinant strain.
  • the temperature of the culture medium may be, for example from 20°C to 70°C, with a temperature of 25 to 40°C being preferred for most recombinant cells.
  • the pH of the culture medium may be, for example, from 2.0 to 10.0, from 3.0 to
  • the culturing may be performed under aerobic, microaerobic, or anaerobic conditions, as required or can be tolerated by the particular recombinant cell.
  • the equipment may include, for example, a tank suitable for holding the recombinant cell and the culture medium; a line for discharging contents from the culture tank to an extraction and/or separation vessel; and an extraction and/or separation vessel suitable for removal of the chemical product from cell culture waste.
  • the carbon source is one or more carbon-containing compounds that can be metabolized by the recombinant cell as a source of carbon.
  • suitable carbon sources include sugars such as glucose, sucrose, fructose, lactose, C-5 sugars such as xylose and arabinose, glycerol and polysaccharides such as starch and cellulose.
  • Other suitable carbon sources include fermentable sugars as may be obtained from cellulosic and lignocellulosic biomass through processes of pretreatment and saccharification, as described, for example, in U.S. Patent Publication No. 2007/0031918A1, hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides.
  • suitable carbon sources include high-fructose com syrup, cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Still other suitable carbon sources include carbon dioxide, carbon monoxide, methanol, methylamine and glucosamine.
  • the culturing process may be continued until a titer of the desired product reaches at least 0.01, at least 0.05, at least 0.1, at least 0.25, at least 0.5 or at least 1 g per liter of culture medium (g/L).
  • the fermentation process may be continued until the titer reaches, for example, up to 40, up to 45, up to 50, up to 80, up to 100, or up to 120 g/L.
  • the specific productivity may be, for example, from 0.01 and 0.60 grams of the desired product per gram of cells on a dry weight basis per hour (g chemical product/g DCW-hr).
  • the volumetric productivity achieved may be at least 0.005 g of the desired product per liter per hour (g/L-hr), at least 0.01 g/L-hr, at least 0.1 g/L-hr or at least 0.5 g/L-hr, and may be up to, for example, 10 g/L-hr, up to 5 g/L-hr or up to 1 g/L-hr.
  • specific productivity as measured over a 24-hour fermentation (culture) period may be greater than about 0.01, 0.05, 0.10, 0.20, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 or 12.0 grams of chemical product per gram DCW of cells (based on the final DCW at the end of the 24-hour period).
  • Residue 46 Thr or Met
  • Residue 223 Val or Ala
  • Residue 256 Ser or Gly
  • Residue 282 Ser or Thr
  • RESIDUE 99 His or Leu
  • MGFLSGKRIL VTGVASKLSI AYGIAQAMHR EGAELAFTYQ NDKLKGRVEE FAAQLGSDIV 60 LQCDVAEDAS IDTMFAELGK VWPKFDGFVH SIGFAPGDQL DGDYVNAVTR EGFKIAHDIS 120 SYSFVAMAKA CRSMLNPGSA LLTLSYLGAE RAIPNYNVMG LAKASLEANV RYMANAMGPE 180 GVRVNAISAG PIRTLAASGI KDFRKMLAHC EAVTPIRRTV TIEDVGNSAA FLCSDLSAGI 240 FGEWHVDGG FSIAAMNELE LK 262
  • MDIRKIKKLI ELVEESGISE LEISEGEESV RISRAAPAAS FPVMQQAYAA PMMQQPAQSN 60 AAAPATVPSM EAPAAAEISG HIVRSPMVGT FYRTPSPDAK AFIEVGQKVN VGDTLCIVEA 120 MKMMNQIEAD KSGTVKAILV ESGQPVEFDE PLWIE 156
  • Recombinant Strain 1 is a mutant of the E. coli strain designated BW25113, available from the E. coli Genetic Strain Center (CGSC#7636; Dept of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut), having the following additional genetic modifications:
  • Recombinant Strain 2 is a mutant of the BW25113 E. coli strain with the following genetic modifications:
  • Recombinant Strain 3 is a mutant of the BW25113 E. coli strain with the following genetic modifications:
  • Type 1 plasmids are pACYC plasmids containing the pl5a origin of replication and a chloramphenicol resistance marker:
  • Type 1A this plasmid includes an E. coli bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase (fadB) gene (SEQ ID NO: 11) and a T denticola enoyl-CoA (ter) gene (SEQ ID NO: 17) cassette, all under a native E. coli pstSIH promoter (SEQ ID NO: 22) and a native E. coli terminator.
  • This plasmid also contains a Hahella chejuensis ester synthase gene (SEQ ID NO: 20) fused to a DNA sequence encoding a protein fragment containing 6 histidine residues and a protease recognition site under an E. coli phoE promoter (SEQ ID NO: 23).
  • Type 1B this plasmid includes a mutated Streptomyces sp. nphT7 gene encoding for a 3-ketoacyl-CoA synthase having H100L, I147S, F217V and S323A mutations (the “LSVA” NphT7 mutant, SEQ ID NO: 19), an E. coli bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase (fadB) (SEQ ID NO: 11) gene and a T. denticola enoyl-CoA (ter) gene (SEQ ID NO: 17) cassette, all under a native E.
  • This plasmid also contains a Hahella chejuensis ester synthase gene (SEQ ID NO: 20) fused to a DNA sequence encoding a protein fragment containing 6 histidine residues and a protease recognition site under an E. coli phoE promoter (SEQ ID NO: 23), and an ACC (acetyl-CoA carboxylase) cassette including fused E. coli accD and accA genes (SEQ ID NO: 26) with a E. coli tpiA promoter (SEQ ID NO: 27) and a cassette including the E. coli accB (SEQ ID NO: 24) and E. coli accC genes (SEQ ID NO: 25) under an E. coli rpiA promoter (SEQ ID NO: 28).
  • SEQ ID NO: 20 Hahella chejuensis ester synthase gene
  • SEQ ID NO: 23 a DNA sequence encoding a protein fragment containing 6 histidine residue
  • Type 1C this plasmid includes an E. coli bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase (fadB) gene (SEQ ID NO: 11) and a T. denticola enoyl-CoA (ter) gene (SEQ ID NO: 17) cassette, all under a native E. coli pstSIH promoter (SEQ ID NO: 22) and a native E. coli terminator.
  • This plasmid also contains a Hahella chejuensis ester synthase gene (SEQ ID NO: 20) fused to a DNA sequence encoding a protein fragment containing 6 histidine residues and a protease recognition site under an E.
  • Type 2 plasmids are pET Plasmids containing a ColEl origin of replication and a kanamycin resistance marker:
  • Type 2A this plasmid includes the 3-ketoacyl-CoA synthase gene to be evaluated
  • the E. coli promoter is either the promoter for the native pstS gene (PpstSIH promoter) or that for the native phoE gene (PphoE promoter), both of which are induced in response to lowering phosphate concentrations as phosphate is incorporated into cell biomass during growth, or to medium with low phosphate concentrations.
  • 3-ketoacyl-CoA synthase genes are synthesized based on published polypeptide sequence information for various wild type enzymes. Site-specific mutants of the synthesized 3- ketoacyl-CoA synthase genes are generated by oligonucleotide-directed mutagenesis.
  • the sources of the wild- type genes, the short-hand designations used herein for each of them, and the amino acid sequence of the native enzyme produced by the wild-type genes are as follows:
  • GeneInfo Identifier refers to sequence identification numbers used by the GenBank genetic sequence database at the National Center for Biotechnology Information, U.S. National Library of Medicine, Bethesda, Maryland, USA
  • Mutations to the amino acid residues encoded by the wild- type genes are designated herein by the shorthand designation for the wild-type enzyme, followed in parentheses by a 3-, 4- or 5 character code consisting of a first letter designating the amino acid residue in the native enzyme, a 1-, 2- or 3-digit number indicating the position of that amino acid residue in the native enzyme, and a final letter designating the amino acid residue in that position in the mutated enzyme.
  • the single-letter designations are IUPAC amino acid abbreviations as reported, for example, at Eur. J. Biochem. 138:9-37(1984).
  • Cbac(V223A) indicates that a valine (V) at amino acid residue position 147 in the wild type Clostridiales bacterium l_7_47_FAA enzyme has been replaced with an alanine (A).
  • Recombinant E. coli strains are prepared using standard electroporation methods.
  • one of the above described recombinant strains (e.g. Recombinant Strain 1, Recombinant Strain 2, or Recombinant Strain 3) is transformed with a“Type 1” plasmid (e.g. Type 1A, Type 1B, or Type 1C) and an above-described“Type 2” plasmid (e.g. Type 2A).
  • a“Type 1” plasmid e.g. Type 1A, Type 1B, or Type 1C
  • an above-described“Type 2” plasmid e.g. Type 2A
  • the 3-ketoacyl-CoA synthase and the promoter for the 3-ketoacyl-CoA synthase gene are as indicated in the specific examples below.
  • the pelleted cells are resuspended thoroughly in 4 mL of fresh medium containing, 30 g/L glucose, 0.158 mM phosphate (low phosphate medium), 1% (V/V) methanol (to produce fatty acid methyl esters) or ethanol (to produce fatty acid ethyl esters), chloramphenicol, and kanamycin as above, and l-ml aliquots dispensed into triplicate 16-mm glass tubes containing 64 pL of heptadecane or methyl tetradecanoate.
  • This synthetic medium functions as a limited phosphate medium that promotes the activity of a low phosphate inducible promoter such as the E.
  • the tubes are incubated at 30°C, 250 rpm for 4 hours. The incubation temperature is then raised to 37°C and incubation is continued for a further 20 hours. The entire culture is extracted with methyl tert-butyl ether and the extract analyzed for fatty acid esters by gas chromatography.
  • Seed 2 flasks contain 25-30 ml synthetic medium containing salts, glucose, NH 4 Cl, and is supplemented with vitamins, yeast extract, 35 pg/ml kanamycin, 20 pg/ml chloramphenicol, and 0-2% methanol. Seed 2 flasks are incubated at 32°C for 6-7 hours.
  • the ODeoo of a 1: 10 dilution of this culture is determined in order to inoculate a production flask to a final ODeoo of 0.01-0.025.
  • Production flasks contain 25 ml synthetic medium containing salts, glucose, NH 4 Cl, and supplemented with vitamins, yeast extract, 35 pg/ml kanamycin, 20 pg/ml chloramphenicol, 1.25-2.5 mM phosphate, 0-2% methanol, 3 - 6g/l glucose, and 15-40 g/l glycerol.
  • This synthetic medium functions as a limited phosphate medium that promotes the activity of a low phosphate inducible promoter such as the E.
  • in vitro assays are used to determine a specific activity of a 3-ketoacyl-CoA synthase for different chain length primers.
  • the assay can be used to determine the specific activity of a particular 3-ketoacyl-CoA synthase for a particular chain length primer.
  • the assay can be carried out to test for specific activity for a C6-C0A primer by reacting a C6-C0A primer and malonyl-CoA with a particular 3-ketoacyl-CoA synthase.
  • Magnesium chloride is added to the reaction mixture and complexes between the resulting 3- ketoC8-CoA and magnesium ion are formed.
  • the reaction progress can be monitored by detecting these 3-ketoC8-CoA and magnesium ion complexes at a UV absorbance of 303 nm.
  • These in vitro assays can be performed with cell lysates of cells expressing a 3-ketoacyl-CoA synthase and/or an isolated 3-ketoacyl-CoA synthase.
  • CoA synthase has specific activity against C6-C0A primers and that the assayed 3-ketoacyl-CoA synthase has 3-ketoC8-CoA synthase activity.
  • the specific activity for other chain length primers can be assayed in similar fashion by substituting a different chain length CoA primer.
  • a C2-CoA primer the specific activity for C2-CoA primers can be assayed and the 3-ketoC4-CoA synthase activity can be determined.
  • the specific activity for C4-CoA primers can be assayed and the 3-ketoC6-CoA synthase activity can be determined.
  • a C8-C0A primer the specific activity for C8-C0A primers can be assayed and the 3-ketoClO-CoA synthase activity can be determined.
  • the in vitro assay is carried out on isolated 3-ketoacyl-CoA synthase.
  • the 3-ketoacyl-CoA synthase can be isolated by methods known in the art.
  • the 3-ketoacyl-CoA synthase can be overexpressed as a fusion polypeptide with an affinity tag such as a poly-histidine tag and then purified by immobilized metal affinity chromatography.
  • the in vitro assay is then carried out on this isolated 3-ketoacyl-CoA synthase by preparing a reaction mixture of CoA primer (0.3 mM) and malonyl-CoA (0.3 mM), in a buffer of 5 mM MgCh and 50 mM Tris, pH 8.0.
  • the reaction is initiated by adding the isolated 3-ketoacyl-CoA synthase at various concentrations. As described above, the progress of the reaction can be monitored by detecting 3-ketoCX-CoA and magnesium ion complexes at a UV absorbance of 303 nm, where X represents the chain length of the resulting product.
  • the CoA primer is selected from C2-CoA, C4-CoA, C6-C0A, or C8-C0A.
  • the reactions can be carried out in 96-well microtiter plates.
  • sequence alignment was performed as a global protein sequence alignment with the Scoring Matrix BLOSUM62 and using Cbac (SEQ ID NO: 1) as the reference sequence.
  • Cbac SEQ ID NO: 1
  • FIGS. 1A, 1B, and 1C show graphical views of the sequence alignment. Each line corresponds to a 3-ketoacyl-CoA synthase enzyme as indicated by the designation. The amino acid position of the first residue of each row is indicated.
  • Each individual strain that was assayed was prepared using Recombinant Strain 1 and comprised a Type 1 A plasmid and a Type 2A plasmid.
  • Each Type 2A plasmid comprised the relevant 3-ketoacyl-CoA synthase enzyme with the PpstSIH promoter.
  • Each individual strain was cultured and assayed as described in the small scale fermentation method - 1 ml protocol described above. The results of the assays are as follows:
  • FIG. 2 shows the total amount of FAME produced in g/L per strain and the total amount of each of methyl hexanoate (C6), methyl octanoate (C8), methyl decanoate (C10), and methyl dodecanoate (C12) per strain.
  • FIG. 3 shows FAME production by each strain based on the chain length of the product as a percentage of the total FAME produced by that strain. As shown in the table above and in FIGS. 2 and 3, the Cbac strain has a higher titer for total FAME produced with a majority (72%) of the FAME produced as methyl octanoate (C8).
  • the Cbac strain also produced a very small amount of the total FAME produced as methyl decanoate (C10) (only 4%) or methyl dodecanoate (C12) (only 2%).
  • the higher production of FAME produced as methyl octanoate (C8) and the small production of FAME produced as methyl decanoate (C10) indicates that the Cbac strain has a higher specificity for production of methyl octanoate (C8) FAME.
  • the assay showed that all of the strains showed specificity for methyl octanoate (C8) because in each strain more methyl octanoate (C8) was produced than other chain lengths of FAME.
  • strains were able to produce methyl hexanoate (C6), methyl octanoate (C8), methyl decanoate (C10), and/or methyl dodecanoate (Cl 2) with the designated 3-ketoacyl-CoA synthase and without expressing a NphT7 or NphT7 mutant.
  • Mutants of the Cbac enzyme were generated by error-prone PCR. Briefly, the wild type gene corresponding to the Cbac enzyme was subjected to error-prone PCR using primers specific to the Cbac gene and EconoTaq DNA polymerase (Lucigen) with the thermocyling program: 98°C 2min, 30x[98°C 30s, 55°C 20s, 72°C lOOs], 72°C lOmin, 4°C hold. In addition, error-prone PCR reactions contained 50, 100, 150 or 200 mM MnCF. PCR fragments were purified with the DNA Clean and Concentrator kit (Zymo Research), digested with Dpnl at 37 °C for 1 h, and purified again.
  • the purified PCR fragments were inserted into Type 2A plasmids using 2x HiFi Assembly Master Mix, a two-fold molar excess of insert to plasmid, and incubation at 50°C for lh to generate Type 2A plasmids with Cbac mutants. These Cbac mutants were then assayed for their ability to generate 3-ketoC8-CoA from a C6-C0A substrate.
  • Cbac mutants with 3-ketoC8-CoA synthase activity were identified, including Cbac(V223A), Cbac(T46M), and Cbac(S256G). These assays show that some mutants of Cbac have improved 3-ketoC8-CoA synthase activity as compared to wild type Cbac.
  • Cbac(V223A), Cbac(T46M), and Cbac(S256G) mutants were each assayed for specific activity for either a C2-CoA primer, a C4-CoA primer, a C6-C0A primer, or a C8-C0A primer.
  • Wild type Cbac was also assayed for specific activity for either a C2-CoA primer, a C4- CoA primer, a C6-C0A primer, or a C8-C0A primer.
  • An in vitro assay was performed using the respective purified mutant Cbac enzyme and carried out as described in [0037].
  • FIG. 4 shows specific activity for either a C2-CoA primer, a C4-CoA primer, a
  • C6-C0A primer or a C8-C0A primer for each of the Cbac, Cbac(V223A), Cbac(T46M), and Cbac(S256G) mutant enzymes.
  • These in vitro assays show that the Cbac(V223A), Cbac(T46M), and Cbac(S256G) mutants have greater specific activity for certain CoA primers.
  • the Cbac(V223A), Cbac(T46M), and Cbac(S256G) mutants showed greater specific activity for C2-CoA primers, C4-CoA primers, and C6-C0A primers as compared to C8-C0A primers.
  • the wild type Cbac enzyme also showed greater specific activity for C2-CoA primers, C4-CoA primers, and C6-C0A primers as compared to C8-C0A primers.
  • the Cbac(V223A) mutant showed the greatest specific activity for C2-CoA primers, C4-CoA primers, and C6-C0A primers as compared to C8-C0A primers.
  • These in vitro activity assays also indicated that Cbac and the Cbac(V223A), Cbac(T46M), and Cbac(S256G) mutants likely possessed higher 3- ketoC8-CoA synthase activity and lower 3-ketoCl0-CoA synthase activity.
  • FAME fatty acid methyl esters
  • chain lengths e.g. C6, methyl hexanoate; C8, methyl octanoate; C10, methyl decanoate; and C12, methyl dodecanoate
  • Each individual strain that was assayed was prepared using Recombinant Strain 1 and comprised a Type 1A plasmid and a Type 2A plasmid.
  • Each Type 2A plasmid comprised wild type Cbac or a Cbac(V223A), Cbac(T46M), or a Cbac(S256G) mutant with the PpstSIH promoter.
  • Each individual strain was cultured and assayed as described in the small scale fermentation method -
  • FIGS. 5 and 6 show the results of the assays.
  • FIG. 5 shows the total amount of FAME produced in g/L per strain and the total amount of each of methyl hexanoate (C6), methyl octanoate (C8), methyl decanoate (C10), and methyl dodecanoate (C12) per strain.
  • FIG. 6 shows FAME production by each strain based on the chain length of the product as a percentage of the total FAME produced by that strain. As shown in the table above and in FIGS.
  • the Cbac(V223A) strain has a higher titer for total FAME produced with a majority (71%) of the FAME produced as methyl octanoate (C8) as compared to the wild type Cbac strain.
  • the Cbac(T46M) and Cbac(S256G) strains also produced higher titer for total FAME produced with a majority of the FAME produced as methyl octanoate (C8)(70% and 66% respectively).
  • the Cbac(V223A), Cbac(T46M), and Cbac(S256G) strains also produced a very small amount of the total FAME produced as methyl decanoate (C10) or methyl dodecanoate (C12).
  • the higher production of FAME produced as methyl octanoate (C8) and the small production of FAME produced as methyl decanoate (C10) indicates that the
  • Cbac(V223A), Cbac(T46M), and Cbac(S256G) strains have higher specificity for production of methyl octanoate (C8) FAME.
  • the assay showed that the Cbac(V223A), Cbac(T46M), and Cbac(S256G) strains showed specificity for methyl octanoate (C8) because in each strain more methyl octanoate (C8) was produced than other chain lengths of FAME.
  • strains were able to produce methyl hexanoate (C6), methyl octanoate (C8), methyl decanoate (C10), and/or methyl dodecanoate (C12) with a Cbac mutant and without expressing a NphT7 or NphT7 mutant.
  • Example 7 methyl hexanoate (C6), methyl octanoate (C8), methyl decanoate (C10), and/or methyl dodecanoate (C12) with a Cbac mutant and without expressing a NphT7 or NphT7 mutant.
  • Additional mutant enzymes were prepared using the Cbac(V223A) mutant.
  • Site- directed mutagenesis was used to generate Cbac(V223A, I246P), Cbac (V223A, I246L), and Cbac (V223A, S282T) double mutants.
  • Each of the Cbac(V223A, I246P), Cbac (V223A, I246L), and Cbac (V223A, S282T) double mutant strains was prepared using Recombinant Strain 1 and comprised a Type 1C plasmid and a Type 2A plasmid.
  • Each Type 2A plasmid comprised the relevant Cbac double mutant with a PpstSIH promoter.
  • Cbac(V223A, I246P), Cbac (V223A, I246L), and Cbac (V223A, S282T) mutants were each assayed for specific activity for either a C2-CoA primer, a C4-CoA primer, a C6-C0A primer, or a C8-C0A primer.
  • Cbac(V223A) was also assayed for specific activity for either a C2-CoA primer, a C4- CoA primer, a C6-C0A primer, or a C8-C0A primer.
  • An in vitro assay was performed using the respective purified mutant Cbac enzyme and carried out as described in [0037].
  • FIG. 7 shows specific activity for either a C2-CoA primer, a C4-CoA primer, a C6-C0A primer, or a C8-C0A primer for each of the Cbac(V223A), Cbac(V223A, I246P), Cbac (V223A, I246L), and Cbac (V223A, S282T) mutant enzymes.
  • Cbac(V223A, I246L) double mutant enzyme had greater specific activity for C2-CoA primers, C4-CoA primers, and C6-C0A primers as compared to the Cbac (V223A) mutant enzyme.
  • the Cbac(V223A, I246L) double mutant strain was assayed for the in vivo production of fatty acid methyl esters (FAME) in different recombinant strain backgrounds.
  • the wild type Cbac was also assayed in different recombinant strain backgrounds.
  • Each of the Cbac(V223A, I246L) double mutant strain and the wild type Cbac strain comprised a Type 2A plasmid. Strains were constructed as follows:
  • FIG. 8 shows the total amount of FAME produced in g/L per strain and the total amount of each of methyl hexanoate (C6), methyl octanoate (C8), methyl decanoate (C10), and methyl dodecanoate (C12) per strain.
  • a recombinant cell comprising:
  • heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-ketoacyl-CoA, wherein the polypeptide has SEQ ID NO: 2-9 or has at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 2-9,
  • the recombinant cell produces a fatty acid and/or a fatty acid chain product.
  • heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity
  • heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity.
  • heterologous gene encoding a polypeptide with enoyl-CoA reductase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17;
  • heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SE. ID NO: 11;
  • heterologous gene encoding a polypeptide with ester synthase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20; and/or
  • heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 24-26.
  • fatty acid chain product comprises one or more products selected from the group consisting of fatty acid ester, fatty alcohol, fatty acid amide, and fatty acid amine.
  • fatty acid ester comprises one or more esters selected from the group consisting of fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester, fatty acid butyl ester, fatty acid pentyl ester, and fatty acid hexyl ester.
  • amino acid residue 46 is not a threonine
  • amino acid residue 256 is not a serine
  • amino acid residue 246 is not a isoleucine
  • amino acid residue 282 is not a serine
  • amino acid residue 223 is an alanine
  • amino acid residue 46 is a methionine
  • amino acid residue 256 is a glycine
  • amino acid residue 246 is a proline or leucine
  • amino acid residue 282 is a threonine.
  • a cell culture comprising:
  • a recombinant cell comprising a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA, wherein the polypeptide has at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 2-10, wherein the recombinant cell produces a fatty acid and/or a fatty acid chain product;
  • heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity
  • heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity.
  • the heterologous gene encoding a polypeptide with enoyl-CoA reductase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17;
  • heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11;
  • heterologous gene encoding a polypeptide with ester synthase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20; and/or
  • heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 24-26.
  • fatty acid product comprises one or more of fatty acid ester, fatty alcohol, fatty acid amide, and fatty acid amine.
  • fatty acid ester comprises one or more of fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester, fatty acid butyl ester, fatty acid pentyl ester, and fatty acid hexyl ester.
  • a method of producing fatty acids and/or fatty acid chain products comprising culturing a recombinant cell in a culture medium, wherein:
  • the recombinant cell comprises a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA, wherein the polypeptide has SEQ ID NO: 2-10 or has at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 2-10; and
  • the recombinant cell is grown under conditions in which the heterologous gene is expressed.
  • heterologous gene encoding a polypeptide with enoyl-CoA reductase activity
  • heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity
  • heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity.
  • the heterologous gene encoding a polypeptide with enoyl-CoA reductase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17;
  • the heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11;
  • heterologous gene encoding a polypeptide with ester synthase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20; and/or
  • heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 24-26.
  • fatty acid chain product comprises one or more of fatty acid ester, fatty alcohol, fatty acid amide, and fatty acid amine.
  • fatty acid ester comprises one or more of fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester, fatty acid butyl ester, fatty acid pentyl ester, and fatty acid hexyl ester.
  • a method of producing fatty acid methyl ester comprising culturing a recombinant cell in a culture medium, wherein:
  • the recombinant cell comprises a heterologous gene encoding a polypeptide having SEQ ID NO: 10 or being at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 10; and
  • the recombinant cell is grown under conditions in which the heterologous gene is expressed.
  • polypeptide comprises at least one of the following features:
  • amino acid residue 223 is an alanine
  • amino acid residue 46 is a methionine
  • c) amino acid residue 256 is a glycine
  • amino acid residue 246 is a proline or leucine
  • e) amino acid residue 282 is a threonine.
  • heterologous gene encoding a polypeptide with enoyl-CoA reductase activity
  • heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity
  • heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity.
  • heterologous gene encoding a polypeptide with enoyl-CoA reductase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17;
  • heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11;
  • heterologous gene encoding a polypeptide with ester synthase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20; and/or
  • heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 24-26.

Abstract

A recombinant cell comprising a heterologous gene encoding a polypeptide with 3-ketoacyl-CoA synthase activity produces fatty acid or fatty acid chain product. The recombinant cell preferentially produces fatty acid or fatty acid chain product of C8 chain length. Methods for producing fatty acid or fatty acid chain product with the recombinant cell are also disclosed.

Description

METHODS AND GENETICALLY MODIFIED CELLS FOR PRODUCING C8 FATTY ACIDS AND FATTY ACID CHAIN PRODUCTS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No.
62/681,845, filed June 7, 2018, and entitled“METHODS AND GENETICALLY MODIFIED CELLS FOR PRODUCING C8 FATTY ACIDS AND FATTY ACID CHAIN PRODUCTS”, which application is hereby incorporated by reference herein in its entirety.
BACKGROUND
[0002] This disclosure relates to recombinant cells that produce fatty acids and fatty acid chain products. In some aspects, this disclosure relates to recombinant cells that produce C6- C10 fatty acids and C6-C10 fatty acid chain products such as C6-C10 fatty acid esters and C6- C10 fatty alcohols.
[0003] Fatty acids and fatty acid chain products have a number of important industrial uses. For instance, fatty acids and fatty acid chain products can be utilized as solvents for lacquers, paints, varnishes and other compositions; as plasticizers for organic resins, as fragrances and flavorings, as fuels for jet and other internal combustion engines, and as raw materials for making a variety of downstream products. Currently, fatty acids and fatty acid chain products are produced industrially from non-renewable fossil fuels or from plant oils such as palm and coconut oil. However, these conventional methods of producing fatty acids and fatty acid chain products have a number of drawbacks. A particular drawback is poor selectivity of a specific carbon chain length, and in particular, selectivity of C6-C10 chain lengths. The products of these conventional methods tend to be a mixture of compounds having a range of carbon chain lengths. Separating these mixtures of compounds can be difficult and can often lead to poor yields with much of the non-renewable fossil fuels or plant oils converted to lower- value byproducts that do not have the desired carbon chain lengths.
[0004] Some biological cells can naturally produce some types of fatty acid chain products. For example, almost all living cells produce triglycerides of fatty acids as well as other fatty acid esters. These triglycerides and other esters play important roles in the metabolism, cellular structure, and other biological processes of the cells, and can perform other useful functions such as storing energy. Therefore, some biological cells can potentially be used to produce fatty acid chain products industrially. Among other potential advantages, biological production of fatty acids and fatty acid products in some cases can rely on annually renewable carbon sources such as sugars, rather than on fossil fuels.
[0005] Biological cells tend to produce fatty acid groups with chain lengths of 12 carbon atoms or greater. Thus, naturally-occurring biological cells are a good source for C12 and higher fatty acids and their chain products. For example, triglycerides produced naturally by such cells can be hydrolyzed to produce C12 or higher fatty acids, which can in turn be converted to other derivatives such as esters or alcohols. On the other hand, few cells naturally produce fatty acids of C6 - C10 chain length in significant quantities.
[0006] Some biological cells can produce fatty acid chain products through a native metabolic pathway that starts with acetyl-CoA and malonyl-ACP. Acetyl-CoA condenses with malonyl-ACP with loss of carbon dioxide and CoA to produce 3-ketobutyryl-ACP. Subsequent enzymatic reactions convert the 3-ketobutyryl-ACP successively to 3-hydroxybutyryl-ACP, then to trans-2-butenoyl-ACP (with loss of water) and finally to butyryl-ACP. The butyryl-ACP can re-enter this reaction cycle in place of acetyl-CoA to produce hexanoyl-ACP. This cycle repeats itself, producing in each iteration a longer carbon atom chain by adding two carbon atoms at a time, until terminated by some other cellular process. However, this native metabolic pathway has low termination rates for C6-C10 chain lengths, resulting in low production of C6-C10 fatty acids and fatty acid chain products.
[0007] Some biological cells can be genetically modified to increase production of C6-
C10 chain lengths. These genetic modifications can include insertions of heterologous genes and modification and/or deletions of endogenous genes to increase production of C6-C10 chain lengths. Often, heterologous genes can be inserted to provide enzymes to catalyze specific reactions between chemical intermediates along a pathway that results in production of C6-C10 chain lengths. For example, Okamura et ak, in PNAS vol. 107, no. 25, pp. 11265-11270 (2010), reported that an enzyme produced by the nphT7 gene of a soil-isolated Streptomyces sp. strain can catalyze a single condensation of acetyl-CoA and malonyl-CoA to produce acetoacetyl- CoA. Additionally, U.S. Application No. 2014/0051136 disclosed that fatty acid chain products can be produced by a cell modified to include the nphT7 gene and additional heterologous genes. However, the amounts of C6 or longer chain fatty acid chain products produced were limited and selectivity of C6-C10 chain lengths was limited. WO 2015/010103 described mutants of the nphT7 gene that code for NphT7 enzymes that more efficiently catalyze the condensation of longer-chain acyl-CoA compounds with malonyl-CoA. Cells modified to include both the wild-type nphT7 gene and the nphT7 mutant gene produced greater relative quantities of longer fatty acids, but selectivity of C6-C10 chain lengths remained poor. C6-C10 fatty acid chain products in particular were made in only small amounts.
SUMMARY
[0008] Provided herein are nucleic acid sequences and proteins, as well as biological processes to produce fatty acids and/or fatty acid chain products with selectivity to C6-C10 chain lengths.
[0009] One aspect provides a recombinant cell comprising a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl- CoA, wherein the polypeptide has SEQ ID NO: 2-9 or has at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 2-9, wherein the recombinant cell produces a fatty acid and/or a fatty acid chain product. In one aspect, the polypeptide catalyzes one or more condensations selected from the group consisting of a) C2- CoA with malonyl-CoA to form 3-keto C4-CoA; b) C4-CoA with malonyl-CoA to form 3-keto C6-C0A; c) C6-C0A with malonyl-CoA to form 3-keto C8-C0A; d) C8-C0A with malonyl-CoA to form 3-keto ClO-CoA; and e) ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA. In another aspect, the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than C8-C0A with malonyl-CoA to form 3-keto ClO-CoA. In one aspect, the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8- CoA than ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA. In another aspect, the fatty acid and/or fatty acid product comprises a chain length of one or more of C6, C8, and/or C10.
In one aspect, the recombinant cell produces more fatty acid and/or fatty acid chain product of C8 chain length than other chain lengths. In another aspect, the total fatty acid and/or total fatty acid chain product comprises 35% or less of C10 chain length and/or the total fatty acid and/or total fatty acid chain product comprises 10% or less of C12 chain length. In another aspect, the total fatty acid and/or total fatty acid chain product comprises 40% or more of C8 chain length. In one aspect, the total fatty acid and/or total fatty acid chain product comprises 45% or less of C6 chain length.
[0010] In another aspect, the recombinant cell further comprises one or more of a heterologous gene encoding a polypeptide with enoyl-CoA reductase activity; a heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity; a heterologous gene encoding a polypeptide with ester synthase activity; and a heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity. In one aspect, the heterologous gene encoding a polypeptide with enoyl-CoA reductase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17; the heterologous gene encoding a polypeptide with bifunctional 3- hydroxyacyl-CoA dehydrogenase/dehydratase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11; the heterologous gene encoding a polypeptide with ester synthase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20; and/or the heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 24-26.
[0011] In one aspect, the polypeptide that catalyzes the condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA is under control of a constitutive promoter. In another aspect, the polypeptide that catalyzes the condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA is under control of an inducible promoter. In one aspect, the polypeptide that catalyzes the condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA is under control of an inducible promoter sensitive to lowering phosphate concentration. In another aspect, the polypeptide that catalyzes the condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA is under control of a PpstSIH promoter or a PphoE promoter. In one aspect, the fatty acid chain product comprises one or more products selected from the group consisting of fatty acid ester, fatty alcohol, fatty acid amide, and fatty acid amine. In another aspect, the fatty acid ester comprises one or more esters selected from the group consisting of fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester, fatty acid butyl ester, fatty acid pentyl ester, and fatty acid hexyl ester. In one aspect, the recombinant cell is a fungal cell, a bacterial cell, or a plant cell. In another aspect, the recombinant cell comprises an Escherichia coli species or Bacillus genus. In one aspect, the recombinant cell comprises a yeast cell.
[0012] One aspect provides a ketoacyl-CoA synthase having SEQ ID NO: 10 or being at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 10, wherein amino acid residue 223 is not a valine, amino acid residue 46 is not a threonine, amino acid residue 256 is not a serine, amino acid residue 246 is not a isoleucine, and/or amino acid residue 282 is not a serine. In one aspect, amino acid residue 223 is an alanine. In another aspect, amino acid residue 46 is a methionine. In one aspect, amino acid residue 256 is a glycine. In another aspect, amino acid residue 246 is a proline or leucine. In one aspect, amino acid residue 282 is a threonine. In one aspect, the ketoacyl-CoA synthase catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA. In another aspect, the ketoacyl-CoA synthase catalyzes one or more condensations selected from the group consisting of C2-CoA with malonyl-CoA to form 3-keto C4-CoA; C4-CoA with malonyl-CoA to form 3-keto C6-C0A; C6-C0A with malonyl-CoA to form 3-keto C8-C0A; C8-C0A with malonyl-CoA to form 3-keto ClO-CoA; and ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA. In another aspect, the ketoacyl-CoA synthase catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than C8-C0A with malonyl-CoA to form 3-keto ClO-CoA. In one aspect, the ketoacyl-CoA synthase catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
[0013] One aspect provides a ketoacyl-CoA synthase having SEQ ID NO: 10 or being at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 10, comprising at least one of the following features a) amino acid residue 223 is not a valine; b) amino acid residue 46 is not a threonine; c) amino acid residue 256 is not a serine; d) amino acid residue 246 is not a isoleucine; e) amino acid residue 282 is not a serine; f) amino acid residue 223 is an alanine; g) amino acid residue 46 is a methionine; h) amino acid residue 256 is a glycine; i) amino acid residue 246 is a proline or leucine; and j) amino acid residue 282 is a threonine.
[0014] One aspect provides a cell culture comprising a) a recombinant cell comprising a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA, wherein the polypeptide has at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 2-10, wherein the recombinant cell produces a fatty acid and/or a fatty acid chain product; and b) one or more fatty acids or fatty acid chain products produced by the recombinant cell, wherein at least one of the fatty acids or fatty acid chain products is present at a concentration of at least 0.1 g/L of the cell culture. In one aspect, the fatty acid and/or fatty acid product produced comprises a chain length of one or more of C6, C8, and/or C10. In another aspect, the cell culture comprises more fatty acid and/or fatty acid chain product of C8 chain length than other chain lengths. In one aspect, the total fatty acid and/or total fatty acid chain product comprises less than 30% C10. In another aspect, the recombinant cell further comprises one or more of a heterologous gene encoding a polypeptide with enoyl-CoA reductase activity; a heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity; a heterologous gene encoding a polypeptide with ester synthase activity; and a heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity. In another aspect, the heterologous gene encoding a polypeptide with enoyl-CoA reductase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17; the heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11; the heterologous gene encoding a polypeptide with ester synthase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20; and/or the heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 24-26. In one aspect, the fatty acid product comprises one or more of fatty acid ester, fatty alcohol, fatty acid amide, and fatty acid amine.
In another aspects, the fatty acid ester comprises one or more of fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester, fatty acid butyl ester, fatty acid pentyl ester, and fatty acid hexyl ester.
[0015] One aspect provides a method of producing fatty acids and/or fatty acid chain products comprising culturing a recombinant cell in a culture medium, wherein the recombinant cell comprises a heterologous gene encoding a polypeptide that catalyzes condensation of acyl- CoA with malonyl-CoA to form 3-keto acyl-CoA, wherein the polypeptide has SEQ ID NO: 2- 10 or has at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 2-10; and the recombinant cell is grown under conditions in which the heterologous gene is expressed. In one aspect, the polypeptide catalyzes one or more condensations selected from the group consisting of a) C2-CoA with malonyl-CoA to form 3- keto C4-CoA; b) C4-CoA with malonyl-CoA to form 3-keto C6-C0A; c) C6-C0A with malonyl- CoA to form 3-keto C8-C0A; d) C8-C0A with malonyl-CoA to form 3-keto ClO-CoA; and e) ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA. In another aspect, the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than C8- CoA with malonyl-CoA to form 3-keto ClO-CoA. In another aspect, the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA. In one aspect, the fatty acid and/or fatty acid product comprises a chain length of one or more of C6, C8, and/or C10. In another aspect, the recombinant cell produces more fatty acid and/or fatty acid chain product of C8 chain length than other chain lengths. In one aspect, the total fatty acid and/or total fatty acid chain product comprises 35% or less of C10 chain length. In another aspect, the total fatty acid and/or total fatty acid chain product comprises 41% or more of C8 chain length. In one aspect, the total fatty acid and/or total fatty acid chain product comprises 44% or less of C6 chain length.
[0016] In another aspect, the method further comprises one or more of a heterologous gene encoding a polypeptide with enoyl-CoA reductase activity; a heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity; a heterologous gene encoding a polypeptide with ester synthase activity; and a heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity. In one aspect, the heterologous gene encoding a polypeptide with enoyl-CoA reductase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17; the heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11; the heterologous gene encoding a polypeptide with ester synthase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20; and/or the heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 24-26. In another aspect, the fatty acid chain product comprises one or more of fatty acid ester, fatty alcohol, fatty acid amide, and fatty acid amine. In one aspect, the fatty acid ester comprises one or more of fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester, fatty acid butyl ester, fatty acid pentyl ester, and fatty acid hexyl ester. In another aspect, the recombinant cell is a fungal cell, a bacterial cell, or a plant cell. In one aspect, the recombinant cell comprises an Escherichia coli species or Bacillus genus. In another aspect, the recombinant cell comprises a yeast cell.
[0017] One aspect provides a method of producing fatty acid methyl ester comprising culturing a recombinant cell in a culture medium, wherein the recombinant cell comprises a heterologous gene encoding a polypeptide having SEQ ID NO: 10 or being at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 10; and the recombinant cell is grown under conditions in which the heterologous gene is expressed. In one aspect, the polypeptide comprises at least one of the following features a) amino acid residue 223 is an alanine; b) amino acid residue 46 is a methionine; c) amino acid residue 256 is a glycine; d) amino acid residue 246 is a proline or leucine; and/or e) amino acid residue 282 is a threonine. In one aspect, the polypeptide catalyzes one or more condensations selected from the group consisting of a) C2-CoA with malonyl-CoA to form 3-keto C4-CoA; b) C4-CoA with malonyl-CoA to form 3-keto C6-C0A; c) C6-C0A with malonyl-CoA to form 3-keto C8-C0A; d) C8-C0A with malonyl-CoA to form 3-keto ClO-CoA; and/or e) ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA. In another aspect, the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than C8-C0A with malonyl-CoA to form 3-keto C10- CoA. In one aspect, the polypeptide catalyzes condensation of more C6-C0A with malonyl- CoA to form 3-keto C8-C0A than ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA. In another aspect, the fatty acid methyl ester comprises a chain length of one or more of C6, C8, and/or C10. In one aspect, the recombinant cell produces more fatty acid methyl ester of C8 chain length than other chain lengths. In another aspect, the total fatty acid methyl ester comprises 35% or less of C10 chain length. In one aspect, the total fatty acid methyl ester comprises 41% or more of C8 chain length. In another aspect, the total fatty acid methyl ester comprises 44% or less of C6 chain length.
[0018] In one aspect, the recombinant cell further comprises one or more of a heterologous gene encoding a polypeptide with enoyl-CoA reductase activity; a heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity; a heterologous gene encoding a polypeptide with ester synthase activity; and a heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity. In another aspect, the heterologous gene encoding a polypeptide with enoyl-CoA reductase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17; the heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11; the heterologous gene encoding a polypeptide with ester synthase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20; and/or the heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 24-26. In one aspect, the recombinant cell is a fungal cell, a bacterial cell, or a plant cell. In another aspect, the recombinant cell comprises an Escherichia coli species or Bacillus genus. In one aspect, the recombinant cell comprises a yeast cell. BRIEF DESCRIPTION OF THE FIGURES
[0019] FIGS. 1A, 1B, and 1C show graphical views of a sequence alignment. Each line corresponds to a 3-ketoacyl-CoA synthase enzyme as indicated by the designation. The amino acid position of the first residue of each row is indicated.
[0020] FIG. 2 shows the total amount of FAME produced in g/L per strain and the total amount of each of methyl hexanoate (C6), methyl octanoate (C8), methyl decanoate (C10), and methyl dodecanoate (C12) per strain. The strains comprise 3-ketoacyl-CoA synthase enzyme from each of Clostridiales bacterium l_7_47_FAA, Clostridium clostridioforme, Clostridium bolteae 90A9, Clostridium saccharolyticum, Clostridium saccharolyticum, Clostridium clostridioforme 2_l_49FAA, Clostridium asparagiforme DSM 15981, Clostridium hathewayi, and Clostridium hathewayi WAL- 18680, respectively.
[0021] FIG. 3 shows FAME production by each strain based on the chain length of the product as a percentage of the total FAME produced by that strain. The strains comprise 3- ketoacyl-CoA synthase enzyme from each of Clostridiales bacterium l_7_47_FAA, Clostridium clostridioforme, Clostridium bolteae 90A9, Clostridium saccharolyticum, Clostridium saccharolyticum, Clostridium clostridioforme 2_l_49FAA, Clostridium asparagiforme DSM 15981, Clostridium hathewayi, and Clostridium hathewayi WAL-18680, respectively.
[0022] FIG. 4 shows specific activity for either a C2-CoA primer, a C4-CoA primer, a
C6-C0A primer, or a C8-C0A primer with malonyl-CoA for each of the Cbac, Cbac(V223A), Cbac(T46M), and Cbac(S256G) mutant enzymes.
[0023] FIG. 5 shows total amount of FAME produced in g/L per strain and the total amount of each of methyl hexanoate (C6), methyl octanoate (C8), methyl decanoate (C10), and methyl dodecanoate (C12) per strain. The strains comprise Cbac or a mutant Cbac(V223A), Cbac(T46M), or Cbac(S256G) enzyme.
[0024] FIG. 6 shows FAME production by each strain based on the chain length of the product as a percentage of the total FAME produced by that strain for each strain comprising Cbac, Cbac(V223A), Cbac(T46M), or Cbac(S256G) mutant enzyme.
[0025] FIG. 7 shows specific activity for either a C2-CoA primer, a C4-CoA primer, a
C6-C0A primer, or a C8-C0A primer with malonyl-CoA for each of the Cbac(V223A), Cbac(V223A, I246P), Cbac (V223A, I246L), and Cbac (V223A, S282T) mutant enzymes.
[0026] FIG. 8 shows total amount of FAME produced in g/L per strain and the total amount of each of methyl hexanoate (C6), methyl octanoate (C8), methyl decanoate (C10), and methyl dodecanoate (C12) per strain. The strains comprise either Cbac or Cbac(V223A, I246L) mutant, with or without NphT7, and with or without NphT7(LSVA).
DETAILED DESCRIPTION
[0027] Although some wild type biological cells can naturally produce some types of fatty acid chain products, these wild type biological cells tend to produce fatty acids and fatty acid chain products with chain lengths of C12 or higher. Few wild type biological cells naturally produce fatty acids and/or fatty acid chain products of C6-C10 chain length in significant quantities.
[0028] Applicants have shown that recombinant cells can be genetically engineered to express heterologous polypeptides for the biosynthesis of C6-C10 fatty acids and/or fatty acid chain products. These recombinant cells can be genetically modified to express identified enzymes that catalyze condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA (having 3-ketoacyl-CoA synthase activity) to produce C6-C10 fatty acids and/or fatty acid chain products. In particular, these identified enzymes having 3-ketoacyl-CoA synthase activity can catalyze the stepwise addition of malonyl-CoA to an acyl-CoA substrate to produce a C6-C10 fatty acid and/or fatty acid chain product. For example, an enzyme having 3-ketoacyl-CoA synthase activity can catalyze the addition of malonyl-CoA to a C2-CoA substrate to lead to a subsequent C4 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C4- CoA substrate to lead to a subsequent C6 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C6-C0A substrate to lead to a subsequent C8 fatty acid and/or fatty acid chain product, and/or the addition of malonyl-CoA to a C8-C0A substrate to lead to a subsequent C10 fatty acid and/or fatty acid chain product. In some aspects, an enzyme having 3-ketoacyl-CoA synthase activity can catalyze the addition of malonyl-CoA to a C2-CoA substrate (acetyl-CoA) to lead to a 3-keto C4-CoA, the addition of malonyl-CoA to a C4-CoA substrate to lead to a 3-keto C6-C0A, the addition of malonyl-CoA to a C6-C0A substrate to lead to a 3-keto C8-C0A, and/or the addition of malonyl-CoA to a C8-C0A substrate to lead to a 3-keto ClO-CoA.
[0029] In addition to enzymes having 3-ketoacyl-CoA synthase activity, the recombinant cells can be genetically modified to express other heterologous polypeptides important for the biosynthesis of C6-C10 fatty acids and/or fatty acid chain products. For example, the recombinant cells can be genetically modified to express one or more heterologous polypeptides capable of converting a 3-keto-CX-CoA substrate (e.g., 3-keto-C4-CoA, 3-keto-C6-CoA, 3- keto-C8-CoA, and/or 3-keto-ClO-CoA) to a 3-OH CX-CoA substrate (e.g., 3-OH-C4-CoA, 3- OH-C6-C0A, 3-OH-C8-CoA, and/or 3-OH-ClO-CoA) (where X represents the carbon chain length). The recombinant cells can be genetically modified to express one or more heterologous polypeptides capable of converting a 3-OH-CX-CoA substrate (e.g., 3-OH-C4-CoA, 3-OH-C6- CoA, 3-OH-C8-CoA, and/or 3-OH-ClO-CoA) to a trans-enoyl-CX-CoA substrate (e.g., enoyl- C4-CoA, enoyl-C6-CoA, enoyl-C8-CoA, and/or enoyl-ClO-CoA) (where X represents the carbon chain length). The recombinant cells can be genetically modified to express one or more heterologous polypeptides capable of converting a trans-enoyl-CX-CoA substrate (e.g., enoyl- C4-CoA, enoyl-C6-CoA, enoyl-C8-CoA, and/or enoyl-ClO-CoA) to a CX-CoA substrate (e.g., C4-CoA, C6-C0A, C8-C0A, and/or ClO-CoA) (where X represents the carbon chain length). Additionally, the recombinant cells can be genetically modified to express one or more heterologous polypeptides capable of converting a CX-CoA substrate (e.g., C4-CoA, C6-C0A, C8-C0A, and/or ClO-CoA) to a fatty acid ester.
[0030] As used herein, the singular forms“a,”“an,” and“the” include plural referents unless the context clearly dictates otherwise. For example, reference to“a nucleic acid” means one or more nucleic acids.
[0031] Methods well known to those skilled in the art can be used to construct genetic expression constructs and recombinant cells as described. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic DNA techniques, in vivo
recombination techniques, and polymerase chain reaction (PCR) techniques. For example, techniques as described in Green & Sambrook, 2012, MOLECULAR CLONING: A
LABORATORY MANUAL, Fourth Edition, Cold Spring Harbor Laboratory, New York;
Ausubel et al„ 1989, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, New York, and PCR Protocols: A Guide to Methods and Applications (Innis et ah, 1990, Academic Press, San Diego, CA) can be used to construct the genetic expression constructs and the recombinant cells described in this application.
[0032] The terms“polynucleotide,”“nucleotide,”“oligonucleotide,” and“nucleic acid” can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof.
[0033] Amino acid residues in all amino acid sequences described herein are ordered in the N-terminus to C-terminus direction. “Upstream” means in the direction toward the N- terminus, and“downstream” means toward the C-terminus direction. The“start” of an amino acid sequence is the first amino acid residue in the N-terminus direction. The first amino acid residue (amino acid residue 1) for any sequence or sub-sequence described herein is the amino acid residue at its N-terminus.
[0034] A“sub-sequence” is a sequence of amino acid residues contained within a larger amino acid sequence.
[0035] Polypeptides refer to polymeric chains of amino acids connected by peptide bonds. In some aspects, polypeptides can fold into unique three dimensional structures that allow the polypeptides to function as enzymes that catalyze distinct biochemical reactions. For example, a 3-ketoacyl-CoA synthase polypeptide catalyzes the condensation reaction of an acyl- CoA with malonyl-CoA to form a 3-ketoacyl-CoA
[0036] “Identity” is used herein to indicate the extent to which two (nucleotide or amino acid) sequences have the same residues at the same positions in side-by-side alignment of their sequences. The identity is expressed herein as a % identity as determined using BLAST (National Center for Biological Information (NCBI) Basic Local Alignment Search Tool) version 2.2.31 software (National Center for Biotechnology Information, U.S. National Library of Medicine, Bethesda, Maryland, USA), using default parameters unless indicated otherwise in this paragraph. Identity between amino acid sequences is determined using protein BLAST with the following parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 6; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: (Existence: 11, Extension: 1); Compositional adjustments: Conditional compositional score matrix adjustment; Filter: none selected; Mask: none selected. Nucleic acid % sequence identity between nucleic acid sequences is determined using standard nucleotide BLAST with the following default parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1, -2; Gap costs: Linear; Filter: Low complexity regions; Mask: Mask for lookup table only. A sequence having a % identity score of XX% (for example, 80%) to a reference sequence as determined in this manner is considered to be XX% identical to or, equivalently, have XX% sequence identity to, the reference sequence.
[0037] An amino acid residue of a sequence or sub-sequence under investigation
“aligns” to an amino acid residue of a reference sequence or sub-sequence when:
i) in the case of an entire sequence, the sequences are aligned using the BLAST version 2.2.31 software in the manner described above, and the amino acid residue of the sequence under investigation occupies the same position in the alignment as does the amino acid residue of the reference sequence;
ii) ii) in the case of a sub-sequence, the sequence containing the sub-sequence under investigation is aligned with the reference sequence, and the amino acid residue of the sub-sequence under investigation occupies the same position in the alignment as does the amino acid residue of the reference sub-sequence.
[0038] For example, the 3-ketoacyl-CoA synthase enzyme of wild type Clostridiales bacterium l_7_47_FAA (SEQ ID NO: 1) includes the following 9 amino acid residue sub sequence.
Figure imgf000015_0001
[0039] When the 3-ketoacyl-CoA synthase enzyme of wild type Clostridium
clostridioforme (SEQ ID NO: 2) is aligned with SEQ ID NO: 1 using the BLAST software as described, the following amino acid residues occupy the same positions in the alignment:
Figure imgf000015_0002
[0040] The phenylalanine (F) of the Clostridium clostridioforme sub-sequence
(corresponding to amino acid position 226 of the Clostridium clostridioforme full sequence) aligns to the phenylalanine at amino acid residue position 1 of the Clostridiales bacterium sub sequence and also to the phenylalanine at position 218 of the Clostridiales bacterium 3- ketoacyl-CoA synthase sequence. Also, each of alanine 227, valine 228, lysine 229, lysine 230, valine 231, proline 232, glutamine 233, and cystine 234 of the Clostridium clostridioforme full sequence align to each of alanine 2, valine 3, arginine 4, lysine 5, valine 6, proline 7, glutamic acid 8, and cystine 9 of the above Clostridiales bacterium sub-sequence, respectively. Likewise each of alanine 227, valine 228, lysine 229, lysine 230, valine 231, proline 232, glutamine 233, and cystine 234 of the Clostridium clostridioforme full sequence align to each of alanine 219, valine 220, arginine 221, lysine 222, valine 223, proline 224, glutamic acid 225, and cystine 226 of Clostridiales bacterium l_7_47_FAA full sequence, respectively.
[0041] A“recombinant cell” is a cell whose genetic material has been altered by a human using genetic engineering techniques including molecular cloning. Recombinant cells can comprise cells whose genetic material has been altered by the addition of genetic material from a cell of a different organism by genetic engineering. Recombinant cells can also comprise cells whose genetic material has been altered by the deletion of genetic material by genetic engineering. Recombinant cells can also comprise cells altered by the addition of extra copies of genetic material that is normally native to the recombinant cell by genetic engineering.
[0042] For purposes of this application, genetic material such as genes, promoters and terminators is“heterologous” if it is (i) non-native to the recombinant cell and/or (ii) is native to the recombinant cell, but is present at a location different than where that genetic material is present in the wild-type cell and/or (iii) is under the regulatory control of a non-native promoter and/or non-native terminator· Extra copies of native genetic material are considered as “heterologous” for purposes of this application, even if such extra copies are present at the same locus as that genetic material is present in the wild-type cell.
[0043] A polypeptide (such as a 3-ketoacyl-CoA synthase enzyme) is“heterologous” if it is non-native to a wild-type version of the recombinant cell, if it is native to the recombinant cell, but is expressed by a gene at a location different than where that gene is present in the wild- type version of the recombinant cell, if it is expressed by a gene that is under the regulatory control of a non-native promoter and/or non-native terminator, and/or if extra copies are present at the same locus where that gene is normally present in the wild-type version of the
recombinant cell.
[0044] A“3-ketoacyl-CoA synthase” is an enzyme that catalyzes the condensation reaction of an acyl-CoA with malonyl-CoA to form a 3-ketoacyl-CoA. One method for evaluating the ability of an enzyme to catalyze this reaction is by measuring the release of free CoA-SH using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), malonyl-CoA as the donor substrate, and a C2-C8 acyl-CoA as the primer substrate, in the presence of the enzyme. The formation of the corresponding 3-ketoacyl-CoA from any of these primer substrates indicates the enzyme is a 3-ketoacyl-CoA synthase. [0045] The ability of an enzyme to catalyze this condensation reaction can also be evaluated using C2-C8 acyl-CoA as the primer substrate and malonyl-CoA as the donor substrate in the presence of 5 mM Mg++ salt by measuring the increase in absorbance at 303 nm as a function of the increase in the formation of a Mg++-complex with the 3-ketoacyl-CoA product. 3-ketoacyl-CoA synthase will produce acetoacetyl-CoA from acetyl-CoA primer, 3- ketohexanoyl-CoA from butyryl-CoA primer, 3-ketooctanoyl-CoA from hexanoyl-CoA primer, and 3-ketodecanoyl-CoA from octanoyl-CoA primer.
[0046] EC numbers are established by the Nomenclature Committee of the International
Union of Biochemistry and Molecular Biology (NC-IUBMB) ( Enzyme Nomenclature
1992 [Academic Press, San Diego, California, ISBN 0-12-227164-5 (hardback), 0-12-227165-3 (paperback)] with Supplement 1 (1993), Supplement 2 (1994), Supplement 3 (1995),
Supplement 4 (1997) and Supplement 5 (in Eur. J. Biochem. 1994, 223, 1-5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur. J. Biochem. 1997, 250; 1-6, and Eur. J. Biochem. 1999, 264, 610-650). The EC numbers referenced herein are derived from the KEGG Ligand database, maintained by the Kyoto Encyclopedia of Genes and Genomics, sponsored in part by the University of Tokyo. Unless otherwise indicated, the EC numbers are as provided in the database as of April 2009.
[0047] A“CoA” or“CoA-SH”, as described here refers to Coenzyme A. A CoA substrate can refer to a straight chain carbon chain covalently linked to Coenzyme A. For example, C4-CoA refers to a C4 chain covalently linked to Coenzyme A via a thioester bond between the thiol of the Coenzyme A and a terminal carbon of the C4 chain.
[0048] A“fatty acid”, as described here, comprises a carboxylic acid with an aliphatic chain, the aliphatic chain having at least four carbon atoms.
[0049] A“fatty acid chain product” is a compound having a straight carbon chain formed in a series of one or more reactions at the site of the terminal carboxyl group of a fatty acid or thioester bond of a corresponding -CoA compound. A fatty acid chain product can comprise a straight carbon chain with a different end group such as, for example, an ester, an alcohol group, an amino group, an aldehyde group, a ketone, a methyl group, or an alkenyl group. Fatty acid chain product can include fatty acid ester, fatty alcohol, fatty acid amide, fatty acid imide, and fatty acid amine.
[0050] A“fatty acid ester” is an ester compound corresponding to the reaction product of a fatty acid and an alcohol (with loss of water). Fatty acid ester can also correspond to the reaction product of a fatty acyl CoA and an alcohol. Fatty acid ester can include fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester, fatty acid butyl ester, fatty acid pentyl ester, and fatty acid hexyl ester. Fatty acid methyl ester (FAME) is an ester compound corresponding to the reaction product of a fatty acid or a fatty acyl-CoA and methanol.
[0051] Chain lengths of fatty acids and fatty acid chain products are sometimes indicated herein by the shorthand“CX”, wherein X is a number designating the number of carbon atoms. The number of carbon atoms designated in each case represents the carbon length of the straight- chain compound (after removal of CoA or ACP coenzymes) formed by the recombinant cell through one or more iterations of the reaction cycle:
acyl-CoA (or acyl-ACP) + malonyl-CoA to form a 3-ketoacyl compound;
reduction of the 3-ketoacyl compounds to form a 3-hydroxyacyl compound;
dehydration of the 3-hydroxyacyl-CoA to form a 3-enoylacyl compound; and reduction of the 3-enoylacyl compound to the corresponding acyl compound.
[0052] Each iteration of this reaction cycle adds two carbon atoms to the starting acyl-
CoA or acyl-ACP. The number of carbon atoms does not include additional carbon atoms that may be added during the formation of any fatty acid chain products of the fatty acid, such as, for example, carbons included in an ester group following an esterification. Thus, hexanoic acid methyl ester is considered as a“C6” fatty acid ester compound, the carbon of the methyl ester group not being counted.
[0053] The“CX” designation can also apply or refer to fatty acid chain products. For example, C4 FAME refers to methyl butanoate, C6 FAME refers to methyl hexanoate, C8 FAME refers to methyl octanoate, C10 FAME refers to methyl decanoate, and C12 FAME refers to methyl dodecanoate. The“CX” designation can also be used for fatty acid
intermediates. For example, C2-CoA refers to acetyl-CoA, C4-CoA refers to butyryl-CoA, C6- CoA refers to hexanoyl-CoA, C8-C0A refers to octanoyl-CoA, ClO-CoA refers to decanoyl- CoA, and Cl2-CoA refers to dodecanoyl-CoA.
[0054] The term“engineered biosynthetic pathway” refers to a biosynthetic pathway that is genetically engineered into a recombinant cell and comprises the enzymes to carry out a sequence of steps to produce a desired product such as a fatty acid or fatty acid chain product.
In some aspects, one or more of the enzymes of the engineered biosynthetic pathway does not naturally occur in a wild type version of the recombinant cell. These enzymes can be introduced into the recombinant cell by using genetic engineering techniques to introduce one or more heterologous genes to overexpress the enzymes. In some aspects, one or more of the enzymes of the engineered biosynthetic pathway do not naturally occur in sufficient copy number in a wild- type version of the recombinant cell and additional copies of the enzymes must be introduced by using genetic engineering techniques to introduce one or more heterologous genes to overexpress the enzymes. In some cases, the engineered biosynthetic pathway also comprises genetic modifications to the recombinant cell to reduce or eliminate competing metabolic pathways and/or to reduce interfering activities such as degradation of desired products or necessary intermediates.
[0055] The term“endogenous” gene refers to a gene that is naturally found in a particular cell. The term“overexpress” is used to refer to the expression of a heterologous gene in a recombinant cell at levels higher than the level of gene expression in a wild type cell. The term“overexpress” can also refer to the expression of a polypeptide from a heterologous gene in a recombinant cell. In some aspects, an endogenous gene is deleted. The terms“deletion,” “deleted,”“knockout,” and“knocked out” can be used interchangeably to refer to an endogenous gene that has been engineered to no longer be expressed in a recombinant cell. In some aspects, a deleted/knocked out gene is an endogenous gene that is deleted to increase production of a desired product such as a fatty acid or fatty acid chain product.
[0056] The terms“variant” and“mutant” are used to describe a protein sequence that has been modified at one or more amino acids, compared to the wild type sequence of a particular protein. Mutations to the amino acid residues encoded by the wild-type genes are designated herein by the shorthand designation for the wild-type protein, followed in parentheses by a 3-, 4- or 5 character code consisting of a first letter designating the amino acid residue in the native enzyme, a 1-, 2- or 3-digit number indicating the position of that amino acid residue in the native enzyme, and a final letter designating the amino acid residue in that position in the mutated enzyme. The single-letter designations are IUPAC amino acid abbreviations as reported, for example, at Eur. J. Biochem. 138:9-37(1984). For example, the designation“Cbac(V223A)” indicates that a valine (V) at amino acid residue position 223 in the wild type 3-ketoacyl-CoA synthase enzyme from Clostridiales bacterium l_7_47_FAA (Cbac enzyme) has been replaced with an alanine (A). The designation“Cbac(V223X)” indicates that a valine (V) at amino acid residue position 223 in the wild type 3-ketoacyl-CoA synthase enzyme from Clostridiales bacterium l_7_47_FAA (Cbac enzyme) is replaced with an amino acid other than valine.
[0057] In some aspects, the recombinant cell is a prokaryotic cell. In some aspects, the recombinant cell is a eukaryotic cell.
[0058] In some aspects, the recombinant cell is a microorganism, and may be a single- celled microorganism. [0059] The recombinant cell may be a plant cell, including a cell from a plant within any of the Chlorophyta, Charophyta, Marchantiophyta, Anthocerotophyta, Bryophyta,
Lycopodiophyta, Pteridophyta, Cycadophyta, Ginkgophyta, Pinophyta, Gnetophyta or
Magnoliophyta plants. Such a plant cell may be, for example, a cell from a plant within any of the genera Arabidopsis, Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris, Populus, Saccharum, Salix, Simmondsia, and Zea.
[0060] The recombinant cell may be a fungi, microalgae, algae or red algae (heterokont) cell. The recombinant cell may be a yeast cell. A yeast or fungus cell may be an oleaginous yeast or fungus, and/or may be a Crabtree negative yeast or fungus.
[0061] The term“oleaginous fungi” refers to yeasts or filamentous fungi, which accumulate at least 10%, 12.5%, 15%, 17.5%, preferably at least 20% or even at least 25%
(w/w) of their biomass as lipid. They may even accumulate at least 30%, 40%, 50%, 60%, 70%, 80% (w/w) or more of their biomass as lipids. The biomass is usually measured as cell dry weight (CDW).
[0062] A“Crabtree -positive” organism is one that is capable of producing ethanol in the presence of oxygen, whereas a "Crabtree-negative" organism is not. A yeast cell having a Crabtree-negative phenotype is any yeast cell that does not exhibit the Crabtree effect. The term “Crabtree-negative” refers to both naturally occurring and genetically modified organisms. Briefly, the Crabtree effect is defined as the inhibition of oxygen consumption by a
microorganism when cultured under aerobic conditions due to the presence of a high
concentration of glucose (e.g., 10 g-glucose L-l). In other words, a yeast cell having a Crabtree positive phenotype continues to ferment irrespective of oxygen availability due to the presence of glucose, while a yeast cell having a Crabtree-negative phenotype does not exhibit glucose mediated inhibition of oxygen consumption. Crabtree-positive yeast produce an excess of alcohol rather than biomass production.
[0063] Examples of suitable yeast cells include, Pichia, Candida, Klebsiella, Hansenula,
Kluyveromyces, Trichosporon, Brettanomyces, Pachysolen, Issatchenkia, Yamadazyma
Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Debaryomyces, Cryptoococcus, Rhodotorula, Rhodosporidium, Lipomyces and Yarrowia. Examples of specific yeast cells include C. sonorensis, K. marxianus, K. thermotolerans, C. methane sorbosa, Saccharomyces bulderi (S. bulderi), I. orientalis, C. lambica, C. sorboxylosa, C. zemplinina, C. geochares, P. membranifaciens, Z. kombuchaensis, C. sorbosivorans, C. vanderwaltii, C. sorbophila, Z.
bisporus, Z. lentus, Saccharomyces bayanus (S. bayanus), D. caste llii, C, boidinii, C. etchellsii, K. lactis, P. jadinii, P. anomala, Saccharomyces cerevisiae ( S. cerevisiae ) Pichia galeiformis, Pichia sp. YB-4149 (NRRL designation ), Candida ethanolica, P. deserticola, P.
membranifaciens, P. fermentans, Rhodosporidium toruloide, Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporon pullas, T. cutaneum, Rhodotorula glutinous, R. garminis, Yarrowia lipolytica and Saccharomycopsis crataegensis ( S. crataegensis). Suitable strains of K. marxianus and C. sonorensis include those described in WO 00/71738 Al, WO 02/42471 A2, WO 03/049525 A2, WO 03/102152 A2 and WO
03/102201A2. Suitable strains of I. orientalis are ATCC strain 32196 and ATCC strain PTA- 6648.
[0064] In some aspects, the recombinant cell is a bacteria cell. The bacteria may be a gram-positive or gram-negative bacteria. It may be a cell within any of the Chlamydiae, green nonsulfur, actinobacteria, planctomycetes, spirochaetes, fusobacteria, cyanobacteria, thermophilic sulphate-reducer, acidobacteria or proteobacteria classifications of bacteria (Ciccarelli et al, Science 311 (5765): 1283-7 (2006).
[0065] Examples of suitable bacteria cells include, for example, those within any of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Streptomyces, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Bacteriophage, Brevibacterium, Acanthoceras, Acanthococcus, Acarvochloris, Achnanthes, Achnanthidiun, Actinastrum, Actinochloris, Actinocyclus,
Actinotaenium, Amphichrsis, Amphidiniunm, Amphikrikos, Amplhipleura, Amphiprora, Amphithrix, Amphora, Anabaena, Anabaenopsis, Aneumnastus, Ankistrodesmius, Ankyra, Anomoeoneis, Apatococcus, Aphanizomenon, Aphanocapsa, Aphanochaete, Aphanothece, Apiocystis, Apistonema, Arthrodesmus, Artherospira, Ascochloris, Asterionella, Asterococcus, Audouinella, Aularoseira, Bacillaria, Balbiania, Bambiusina, Bangia, Basichlamys,
Batrarhospermum, Binurlearia, Bitrichia, Blidingia, Botrdiopsis, Botrydium, Botryococcus, Botryosphaerella, Brachiomonas, Brachysira, Brachytrichia, Brebissonia, Bulbochaete, Bumnilleria, Buinilleriopsis, Caloneis, Calothrix, Campylodiscus, Capsosiphon, Carteria, Catena, Cavinula, Cenritractus, Centroniella, Ceratiunt, Chaetoceros, Chaetochloris,
Chaetomorpha, Chaetonella, Chaetonemna, Chaetopeltis, Chaetophora, Chaetosphaeridium, Chamaesiphion, Chara, Characiochloris, Characiopsis, Characium, Chorales, Chilomonas, Chlainomonas, Chlamydoblepharis, Chlamydocapsa, Chlamydomonas, Chlamydomonopsis, Chlamydomnyxa, Chlamydonephris, Chlorangiella, Chlorangiopsis, Chlorella, Chlorobotrys, Chlorobrachis, Chlorochytrium, Chloroccun, Chlorogloea, Chlorogloeopsis, Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema, Cholorphyta, Cholorosaccus, Cholorosarcina, Choricystis, Chromophyton, Chromulina, Chroococcidiopsis, Chrococcus, Chroodactylon, Chroomonas, Chroothece, Chrysamoeba, Chysapsis, Chrysidiastrum, Chrysocapsa,
Chrysocapsella, Chrysochaete, Chrysohromulina, Chrysococcus, Chrysocrinus,
Chrynsolepidomonas, Chrysolykos, Chrysonebula, Chrysophyta, Chrysopyxis, Chrysosaccus, Chrysophaerella, Chrysotephanosphaera, Clodophora, Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis, Coelastrella, Coelastrum, Coelosphaerium, Coenochloris,
Coenococcus, Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis,
Compsopogon, Conjugatophyta, Conoehaete, Coronastrum, Cosmarium, Cosmnioneis, Cosmocladium, Crateriportula, Craticula, Crinalium, Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta, Ctenophora, Cyanodictyon, Cyanonephron, Cyanophora,
Cyanophyta, Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella,
Cylindrocapsa, Cylindrocystis, Cylindrospermum, Cylindrotheca, Cymatopleura, Cymbella, Cymbeilonitzschia, Cystodinium Dactylococcopsis, Debarya, Denticula, Dermatochrysis, Dermorarpa, Dermocarpella, Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon, Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula,
Dichothrix, Dichtotomococcrus, Dicranochaete, Dictyochloris, Dictyococcus, Dictyosphaerium, Didymocystis, Didymogenes, Didymosphenia, Dilabifilum, Dimorphoccus, Dinobryon,
Dinocuccus, Diplochloris, Diploneis, Diplostauron, Distrionella, Docidium, Draparnaldia, Dunaliella, Dysmorphaocuccus, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema,
Enteromorpha, Entocladia, Entomoeis, Entophysalis, Ephichrysis, Epipyxis, Epithemia, Eremosphaura, Euastropsis, Euatstrum, Eucapsis, Eucocconeis, Eudorina, Euglena,
Euglenophyta, Eunotia, Eustigmatophyta, Eutreptia, Fallcia, Ficherella, Fragilaria,
Fragilariforma, Franceia, Frustulia, Curcilla, Geminella, Genicularia, Glaucocystis,
Glaucophyta, Glenodiniopsis, Glenodinium, Gloeomonas, Gloeoplax, Gloeothece, Geloeotila, Gloeotrichia, Gloiodictyon, Golenkinia, Golenkiniopsis, Gomontia, Gomphocymbella,
Gomphonema, Gomphosphaeria, Gonatozygon, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum, Granulochloris, Granulocystopsis, Groenbladia, Gymnodiunium, Gymnozyga, Gyrosignma, Haematocuccus, Hafniomonas, Hallassia, Hammatoidea, Hannaea, Hantzchia, Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Hemidinuim, Hemitonia, Heribaudiella, Heteromastix, Heterothrix, Hibberdia, Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema, Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium, Hyalotheca, Hydrianum, Hydrococcus, Hydrocoleum, Hydrocoryne, Hydrodictyon, Hydrosera, Hydrurus, Hyella, Hymenomonas, Isthmochloron, Johannesbaptistia, Juranyiella, Karayevia, Kathablepharis, Katodinium, Kaphyrion, Keratococcus, Kirchneriella, Klebsormidium, Kolbesia, Koliella, Komarekia, Korshikoviella, Kraskella, Lagerheimia, Lagynion,
Lamprothamnium, Lemanea, Lepocinclis, Leptosira, Lobococcus, Lobocystis, Lobomonas, Luticola, Lynbya, Malleochloris, Mallomonas, Mantoniella, Marssoniella, Martyana,
Mastigocloleus, Gastogloia, Melosira, Merismopedia, Mesostigma, Mesotaenium,
Micractinium, Micrasterias, Microchaete, Microcoleus, Microcystis, Microglena, Micromonas, Microspora, Microthamnion, Mischococcus, Monocrysis, Monodus, Monomastix,
Monoraphidium, Monostroma, Mougeotia, Mougeotiopsis, Myochloris, Myromecia,
Myxocarcina, Naegeliella, Nannochloris, Nautoccus, Navicula, Neglectella, Neidium,
Nephroclamys, Nephrocytium, Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia, Nodularia, Nostoc, Ochromonas, Oedogonium, Oligochaetophora, Onychonema, Oocadrium, Oocrystis, Opephora, Ophiocytium, Orthoseira, Oscillartoria, Oxyneis,
Pachycladella, Palmella, Palmodictyon, Pnadorina, Pannus, Paralia, Pascherina, Paulshulzia, Pediastrum, Pedinella, Pedinomonas, Pedinopera, Pelagodictyon, Penium, Peranema, Peridiniopsis, Peridinium, Peronia, Petroneis, Phacotus, Phacus, Phaeaster, Phaeodermatium, Phaeophyta, Phaeoshaera, Phaeothamnion, Phormidium, Phycopeltis, Phyllariochloris, Phyllocadium, Phyllomitas, Pinnilaria, Pitophora, Placoneis, Planctonema, Planktophaeria, Planothidium, Plectonema, Pleodorina, Pleurastrum, Pleurocapsa, Pleurocladia, Pleurodiscus, Pleurosigma, Pleurosira, Pleurotaenium, Pocillomanas, Podohedea, Polyblepharides, Polychaetophora, Polyedriella, Polyedriopsis, Polygoniochloris, Polyepidomanas, Polytaenia, Polytoma, Polytomella, Porphyridium, Posteriochromonas, Prasinochloris, Prasinocladus, Prasinophyta, Praisola, Prochlorphyta, Psammodictyon, Psammothidium, Pseudanabaena, Pseudenoclonium, Psuedocarteria, Pseudochate, Pseaudocharacium, Pseudococcomyxa, Pseudodictyosphaerium, Pseudokephyrion, Pseudoncobrysa, Pseudoquadrigula,
Pseudophaerocystis, Pseudostaurastrum, Pseudostraurosira, Pyrrophyta, Quadrichloris, Quadricoccus, Quadrigula, Radiocuccus, Radiobetalum, Raphidiopsis, Raphidocelis,
Raphidonema, Raphidophyta, Peimeria, Rhadorderma, Rhabomonas, Rhizoclonium,
Rhodomonas, Rhodiphyta, Rhoicosenia, Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus, Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix, Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia, Scytonema, Slenastrum,
Selenochloris, Sellaphora, Semiorbis, Siderocelis, Diderocystopsis, Dimonsenia, Siphononema, Sirocladium, Sirogonium, Skeletonema, Sorastrum, Spermatozopis, Sphaerellocystis, Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma, Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum, Spondylosium, Sporotetras, Spumella, Staurastrum, Stauerodesmus, Stauroneis, Staurosira, Staurrosiella, Stenopterobia, Stephanocostis,
Stephanodiscus, Stephanoporos, Stephanoshaera, Stichoccus, Stichogloea, Sigeoclonium, Stigonema, Stipitocuccus, Stokesiella, Stombomonas, Stylochrysalis, Stylodinium, Styloyxis, Stylosphaeridium, Surirella, Sykidion, Symploca, Synechococcus, Synechocystis, Synedra, Synochromonas, Synura, Tabellaria, Tabularia, Teilingia, Temnogametum, Tetmemorus, Tetrachlorella, Tetracyclus, Tetrademus, Tetraedriella, tetraedron, Tetraselmis, Tetraspora, Tetrastrum, Thalassiosira, Thanmiochaete, Thoakochloris, Thorea, Tolypella, Tolypothrix, Trachelomonas, Trachydiscus, Trebouxia, Trentepholia, Treubaria, Tribonema, Trichodesmium, Tricodiscus, Trochiscia, Tryblionella, Ulothrix, Uroglena, Uronema, Urosolenia, Urospora,
Uva, Vacuolaria, Vaucheria, Volvox, Volvulina, Westella, Woloszynskia, Xanthidium,
Xanthophyta, Exencoccus, Zygenema, Zygnemopsis, and Zygonium.
[0066] Specific examples of bacteria cells include Escherichia coli; Oligotropha carboxidovorans, Pseudomononas sp. Alcaligenes eutrophus ( Cupriavidus necator), Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis, Bacillus subtilis, Cupriavidus basilensis, Cupriavidus campinensis, Cupriavidus gilardi, Cupriavidus laharsis, Cupriavidus metallidurans, Cupriavidus oxalaticus, Cupriavidus pauculus, Cupriavidus pinatubonensis, Cupriavidus respiraculi, Cupriavidus taiwanensis, In some aspects, the bacterium is Nocardia sp. NRRL 5646, Nocardiafarcinica, Streptomyces griseus, Salinispora arenicola, or Clavibacter michiganenesis.
[0067] The recombinant cell may be a synthetic cell or a cell produced by a synthetic genome, as described in U.S. Patent Publication 2007/0264688, or 2007/0269862. The cell may be a CHO cell, a COS cell, a VERO cell, a BHK cell, a HeLa cell, a Cvl cell, an MDCK cell, a 293 cell, a 3T3 cell, or a PC 12 cell.
[0068] In some aspects, this application discloses a recombinant cell genetically modified to express one or more heterologous enzymes having 3-ketoacyl-CoA synthase activity. The heterologous enzymes having 3-ketoacyl-CoA synthase activity can comprise enzymes naturally found in Clostridiales bacterium l_7_47_FAA (SEQ ID NO: 1)
(Designation: Cbac), Clostridium clostridioforme (SEQ ID NO: 2) (Designation: Cclol), Clostridium bolteae 90A9 (SEQ ID NO: 3) (Designation: Cbol), Clostridium saccharolyticum (SEQ ID NO: 4) (Designation: Csacl), Clostridium saccharolyticum (SEQ ID NO: 5) (Designation: Csac2), Clostridium clostridioforme 2_l_49FAA (SEQ ID NO: 6) (Designation: Cclo2), Clostridium asparagiforme DSM 15981 (SEQ ID NO: 7) (Designation: Casp), Clostridium hathewayi (SEQ ID NO: 8) (Designation: Chat2), and/or Clostridium hathewayi WAL- 18680 (SEQ ID NO: 9) (Designation: Chatl).
[0069] In some aspects, a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA comprises the amino acid sequence of SEQ ID NO: 1 and corresponds to the polypeptide designated as Cbac. In other aspects, the heterologous enzyme having 3-ketoacyl-CoA synthase activity comprises a polypeptide having more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity to the amino acid sequence of SEQ ID NO: 1.
[0070] In some aspects, a recombinant cell genetically modified to express the polypeptide designated as Cbac produces fatty acids and/or fatty acid chain products. The recombinant cell expressing heterologous Cbac can also produce FAME. In other cases, the recombinant cell expressing heterologous Cbac can also produce a mixture of FAMEs with different carbon chain lengths. For example, the recombinant cell expressing heterologous Cbac can produce a FAME mixture comprising C6 FAME, C8 FAME, C10 FAME, and C12 FAME. In some cases, the recombinant cell expressing heterologous Cbac can also produce a FAME mixture comprising C6 FAME (methyl hexanoate), C8 FAME (methyl octanoate), C10 FAME (methyl decanoate), and C12 FAME (methyl dodecanoate) with C8 FAME (methyl octanoate) as the predominant species.
[0071] In some cases, the recombinant cell expressing heterologous Cbac can produce fatty acids and/or fatty acid chain products without the recombinant cell comprising an additional heterologous polypeptide with 3-ketoacyl-CoA synthase activity. The recombinant cell expressing heterologous Cbac can catalyze the addition of malonyl-CoA to a C2-CoA substrate to lead to a C4 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C4-CoA substrate to lead to a C6 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C6-C0A substrate to lead to a C8 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C8-C0A substrate to lead to a C 10 fatty acid and/or fatty acid chain product, and/or the addition of malonyl-CoA to a ClO-CoA substrate to lead to a C12 fatty acid and/or fatty acid chain product. In other cases, the recombinant cell expressing heterologous Cbac can also comprise one or more additional heterologous polypeptides with 3- ketoacyl-CoA synthase activity (e.g., a NphT7 polypeptide and/or a NphT7 polypeptide mutant as described below) to increase production of fatty acids and/or fatty acid chain products.
[0072] In some aspects, a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA comprises the amino acid sequence of SEQ ID NO: 2 and corresponds to the polypeptide designated as Cclol. In other aspects, the heterologous enzyme having 3-ketoacyl-CoA synthase activity comprises a polypeptide having more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity to the amino acid sequence of SEQ ID NO: 2.
[0073] In some aspects, a recombinant cell genetically modified to express the polypeptide designated as Cclol produces fatty acids and/or fatty acid chain products. The recombinant cell expressing heterologous Cclol can also produce FAME. In other cases, the recombinant cell expressing heterologous Cclol can also produce a mixture of FAMEs with different carbon chain lengths. For example, the recombinant cell expressing heterologous Cclol can produce a FAME mixture comprising C6 FAME, C8 FAME, C10 FAME, and C12 FAME. In some cases, the recombinant cell expressing heterologous Cclol can also produce a FAME mixture comprising C6 FAME (methyl hexanoate), C8 FAME (methyl octanoate), C10 FAME (methyl decanoate), and C12 FAME (methyl dodecanoate) with C8 FAME (methyl octanoate) as the predominant species.
[0074] In some cases, the recombinant cell expressing heterologous Cclol can produce fatty acids and/or fatty acid chain products without the recombinant cell comprising an additional heterologous polypeptide with 3-ketoacyl-CoA synthase activity. The recombinant cell expressing heterologous Cclol can catalyze the addition of malonyl-CoA to a C2-CoA substrate to lead to a C4 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C4-CoA substrate to lead to a C6 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C6-C0A substrate to lead to a C8 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C8-C0A substrate to lead to a C 10 fatty acid and/or fatty acid chain product, and/or the addition of malonyl-CoA to a ClO-CoA substrate to lead to a C12 fatty acid and/or fatty acid chain product. In other cases, the recombinant cell expressing heterologous Cclol can also comprise one or more additional heterologous polypeptides with 3- ketoacyl-CoA synthase activity (e.g., a NphT7 polypeptide and/or a NphT7 polypeptide mutant as described below) to increase production of fatty acids and/or fatty acid chain products. [0075] In some aspects, a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA comprises the amino acid sequence of SEQ ID NO: 3 and corresponds to the polypeptide designated as Cbol. In other aspects, the heterologous enzyme having 3-ketoacyl-CoA synthase activity comprises a polypeptide having more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity to the amino acid sequence of SEQ ID NO: 3.
[0076] In some aspects, a recombinant cell genetically modified to express the polypeptide designated as Cbol produces fatty acids and/or fatty acid chain products. The recombinant cell expressing heterologous Cbol can also produce FAME. In other cases, the recombinant cell expressing heterologous Cbol can also produce a mixture of FAMEs with different carbon chain lengths. For example, the recombinant cell expressing heterologous Cbol can produce a FAME mixture comprising C6 FAME, C8 FAME, C10 FAME, and C12 FAME. In some cases, the recombinant cell expressing heterologous Cbol can also produce a FAME mixture comprising C6 FAME (methyl hexanoate), C8 FAME (methyl octanoate), C10 FAME (methyl decanoate), and C12 FAME (methyl dodecanoate) with C8 FAME (methyl octanoate) as the predominant species.
[0077] In some cases, the recombinant cell expressing heterologous Cbol can produce fatty acids and/or fatty acid chain products without the recombinant cell comprising an additional heterologous polypeptide with 3-ketoacyl-CoA synthase activity. The recombinant cell expressing heterologous Cbol can catalyze the addition of malonyl-CoA to a C2-CoA substrate to lead to a C4 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C4-CoA substrate to lead to a C6 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C6-C0A substrate to lead to a C8 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C8-C0A substrate to lead to a C 10 fatty acid and/or fatty acid chain product, and/or the addition of malonyl-CoA to a ClO-CoA substrate to lead to a C12 fatty acid and/or fatty acid chain product. In other cases, the recombinant cell expressing
heterologous Cbol can also comprise one or more additional heterologous polypeptides with 3- ketoacyl-CoA synthase activity (e.g., a NphT7 polypeptide and/or a NphT7 polypeptide mutant as described below) to increase production of fatty acids and/or fatty acid chain products. [0078] In some aspects, a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA comprises the amino acid sequence of SEQ ID NO: 4 and corresponds to the polypeptide designated as Csacl. In other aspects, the heterologous enzyme having 3-ketoacyl-CoA synthase activity comprises a polypeptide having more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity to the amino acid sequence of SEQ ID NO: 4.
[0079] In some aspects, a recombinant cell genetically modified to express the polypeptide designated as Csacl produces fatty acids and/or fatty acid chain products. The recombinant cell expressing heterologous Csacl can also produce FAME. In other cases, the recombinant cell expressing heterologous Csacl can also produce a mixture of FAMEs with different carbon chain lengths. For example, the recombinant cell expressing heterologous Csacl can produce a FAME mixture comprising C6 FAME, C8 FAME, C10 FAME, and C12 FAME. In some cases, the recombinant cell expressing heterologous Csacl can also produce a FAME mixture comprising C6 FAME (methyl hexanoate), C8 FAME (methyl octanoate), C10 FAME (methyl decanoate), and C12 FAME (methyl dodecanoate) with C8 FAME (methyl octanoate) as the predominant species.
[0080] In some cases, the recombinant cell expressing heterologous Csacl can produce fatty acids and/or fatty acid chain products without the recombinant cell comprising an additional heterologous polypeptide with 3-ketoacyl-CoA synthase activity. The recombinant cell expressing heterologous Csacl can catalyze the addition of malonyl-CoA to a C2-CoA substrate to lead to a C4 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C4-CoA substrate to lead to a C6 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C6-C0A substrate to lead to a C8 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C8-C0A substrate to lead to a C 10 fatty acid and/or fatty acid chain product, and/or the addition of malonyl-CoA to a ClO-CoA substrate to lead to a C12 fatty acid and/or fatty acid chain product. In other cases, the recombinant cell expressing
heterologous Csacl can also comprise one or more additional heterologous polypeptides with 3- ketoacyl-CoA synthase activity (e.g., a NphT7 polypeptide and/or a NphT7 polypeptide mutant as described below) to increase production of fatty acids and/or fatty acid chain products. [0081] In some aspects, a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA comprises the amino acid sequence of SEQ ID NO: 5 and corresponds to the polypeptide designated as Csac2. In other aspects, the heterologous enzyme having 3-ketoacyl-CoA synthase activity comprises a polypeptide having more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity to the amino acid sequence of SEQ ID NO: 5.
[0082] In some aspects, a recombinant cell genetically modified to express the polypeptide designated as Csac2 produces fatty acids and/or fatty acid chain products. The recombinant cell expressing heterologous Csac2 can also produce FAME. In other cases, the recombinant cell expressing heterologous Csac2 can also produce a mixture of FAMEs with different carbon chain lengths. For example, the recombinant cell expressing heterologous Csac2 can produce a FAME mixture comprising C6 FAME, C8 FAME, C10 FAME, and C12 FAME. In some cases, the recombinant cell expressing heterologous Csac2 can also produce a FAME mixture comprising C6 FAME (methyl hexanoate), C8 FAME (methyl octanoate), C10 FAME (methyl decanoate), and C12 FAME (methyl dodecanoate) with C8 FAME (methyl octanoate) as the predominant species.
[0083] In some cases, the recombinant cell expressing heterologous Csac2 can produce fatty acids and/or fatty acid chain products without the recombinant cell comprising an additional heterologous polypeptide with 3-ketoacyl-CoA synthase activity. The recombinant cell expressing heterologous Csac2 can catalyze the addition of malonyl-CoA to a C2-CoA substrate to lead to a C4 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C4-CoA substrate to lead to a C6 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C6-C0A substrate to lead to a C8 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C8-C0A substrate to lead to a C 10 fatty acid and/or fatty acid chain product, and/or the addition of malonyl-CoA to a ClO-CoA substrate to lead to a C12 fatty acid and/or fatty acid chain product. In other cases, the recombinant cell expressing
heterologous Csac2 can also comprise one or more additional heterologous polypeptides with 3- ketoacyl-CoA synthase activity (e.g., a NphT7 polypeptide and/or a NphT7 polypeptide mutant as described below) to increase production of fatty acids and/or fatty acid chain products. [0084] In some aspects, a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA comprises the amino acid sequence of SEQ ID NO: 6 and corresponds to the polypeptide designated as Cclo2. In other aspects, the heterologous enzyme having 3-ketoacyl-CoA synthase activity comprises a polypeptide having more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity to the amino acid sequence of SEQ ID NO: 6.
[0085] In some aspects, a recombinant cell genetically modified to express the polypeptide designated as Cclo2 produces fatty acids and/or fatty acid chain products. The recombinant cell expressing heterologous Cclo2 can also produce FAME. In other cases, the recombinant cell expressing heterologous Cclo2 can also produce a mixture of FAMEs with different carbon chain lengths. For example, the recombinant cell expressing heterologous Cclo2 can produce a FAME mixture comprising C6 FAME, C8 FAME, C10 FAME, and C12 FAME. In some cases, the recombinant cell expressing heterologous Cclo2 can also produce a FAME mixture comprising C6 FAME (methyl hexanoate), C8 FAME (methyl octanoate), C10 FAME (methyl decanoate), and C12 FAME (methyl dodecanoate) with C8 FAME (methyl octanoate) as the predominant species.
[0086] In some cases, the recombinant cell expressing heterologous Cclo2 can produce fatty acids and/or fatty acid chain products without the recombinant cell comprising an additional heterologous polypeptide with 3-ketoacyl-CoA synthase activity. The recombinant cell expressing heterologous Cclo2 can catalyze the addition of malonyl-CoA to a C2-CoA substrate to lead to a C4 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C4-CoA substrate to lead to a C6 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C6-C0A substrate to lead to a C8 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C8-C0A substrate to lead to a C 10 fatty acid and/or fatty acid chain product, and/or the addition of malonyl-CoA to a ClO-CoA substrate to lead to a C12 fatty acid and/or fatty acid chain product. In other cases, the recombinant cell expressing
heterologous Cclo2 can also comprise one or more additional heterologous polypeptides with 3- ketoacyl-CoA synthase activity (e.g., a NphT7 polypeptide and/or a NphT7 polypeptide mutant as described below) to increase production of fatty acids and/or fatty acid chain products. [0087] In some aspects, a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA comprises the amino acid sequence of SEQ ID NO: 7 and corresponds to the polypeptide designated as Casp. In other aspects, the heterologous enzyme having 3-ketoacyl-CoA synthase activity comprises a polypeptide having more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity to the amino acid sequence of SEQ ID NO: 7.
[0088] In some aspects, a recombinant cell genetically modified to express the polypeptide designated as Casp produces fatty acids and/or fatty acid chain products. The recombinant cell expressing heterologous Casp can also produce FAME. In other cases, the recombinant cell expressing heterologous Casp can also produce a mixture of FAMEs with different carbon chain lengths. For example, the recombinant cell expressing heterologous Casp can produce a FAME mixture comprising C6 FAME, C8 FAME, C10 FAME, and C12 FAME. In some cases, the recombinant cell expressing heterologous Casp can also produce a FAME mixture comprising C6 FAME (methyl hexanoate), C8 FAME (methyl octanoate), C10 FAME (methyl decanoate), and C12 FAME (methyl dodecanoate) with C8 FAME (methyl octanoate) as the predominant species.
[0089] In some cases, the recombinant cell expressing heterologous Casp can produce fatty acids and/or fatty acid chain products without the recombinant cell comprising an additional heterologous polypeptide with 3-ketoacyl-CoA synthase activity. The recombinant cell expressing heterologous Casp can catalyze the addition of malonyl-CoA to a C2-CoA substrate to lead to a C4 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C4-CoA substrate to lead to a C6 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C6-C0A substrate to lead to a C8 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C8-C0A substrate to lead to a C 10 fatty acid and/or fatty acid chain product, and/or the addition of malonyl-CoA to a ClO-CoA substrate to lead to a C12 fatty acid and/or fatty acid chain product. In other cases, the recombinant cell expressing
heterologous Casp can also comprise one or more additional heterologous polypeptides with 3- ketoacyl-CoA synthase activity (e.g., a NphT7 polypeptide and/or a NphT7 polypeptide mutant as described below) to increase production of fatty acids and/or fatty acid chain products. [0090] In some aspects, a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA comprises the amino acid sequence of SEQ ID NO: 8 and corresponds to the polypeptide designated as Chat2. In other aspects, the heterologous enzyme having 3-ketoacyl-CoA synthase activity comprises a polypeptide having more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity to the amino acid sequence of SEQ ID NO: 8.
[0091] In some aspects, a recombinant cell genetically modified to express the polypeptide designated as Chat2 produces fatty acids and/or fatty acid chain products. The recombinant cell expressing heterologous Chat2 can also produce FAME. In other cases, the recombinant cell expressing heterologous Chat2 can also produce a mixture of FAMEs with different carbon chain lengths. For example, the recombinant cell expressing heterologous Chat2 can produce a FAME mixture comprising C6 FAME, C8 FAME, C10 FAME, and C12 FAME. In some cases, the recombinant cell expressing heterologous Chat2 can also produce a FAME mixture comprising C6 FAME (methyl hexanoate), C8 FAME (methyl octanoate), C10 FAME (methyl decanoate), and C12 FAME (methyl dodecanoate) with C8 FAME (methyl octanoate) as the predominant species.
[0092] In some cases, the recombinant cell expressing heterologous Chat2 can produce fatty acids and/or fatty acid chain products without the recombinant cell comprising an additional heterologous polypeptide with 3-ketoacyl-CoA synthase activity. The recombinant cell expressing heterologous Chat2 can catalyze the addition of malonyl-CoA to a C2-CoA substrate to lead to a C4 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C4-CoA substrate to lead to a C6 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C6-C0A substrate to lead to a C8 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C8-C0A substrate to lead to a C 10 fatty acid and/or fatty acid chain product, and/or the addition of malonyl-CoA to a ClO-CoA substrate to lead to a C12 fatty acid and/or fatty acid chain product. In other cases, the recombinant cell expressing
heterologous Chat2 can also comprise one or more additional heterologous polypeptides with 3- ketoacyl-CoA synthase activity (e.g., a NphT7 polypeptide and/or a NphT7 polypeptide mutant as described below) to increase production of fatty acids and/or fatty acid chain products. [0093] In some aspects, a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA comprises the amino acid sequence of SEQ ID NO 9 and corresponds to the polypeptide designated as Chatl. In other aspects, the heterologous enzyme having 3-ketoacyl-CoA synthase activity comprises a polypeptide having more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity to the amino acid sequence of SEQ ID NO: 9.
[0094] In some aspects, a recombinant cell genetically modified to express the polypeptide designated as Chatl produces fatty acids and/or fatty acid chain products. The recombinant cell expressing heterologous Chatl can also produce FAME. In other cases, the recombinant cell expressing heterologous Chatl can also produce a mixture of FAMEs with different carbon chain lengths. For example, the recombinant cell expressing heterologous Chatl can produce a FAME mixture comprising C6 FAME, C8 FAME, C10 FAME, and C12 FAME. In some cases, the recombinant cell expressing heterologous Chatl can also produce a FAME mixture comprising C6 FAME (methyl hexanoate), C8 FAME (methyl octanoate), C10 FAME (methyl decanoate), and C12 FAME (methyl dodecanoate) with C8 FAME (methyl octanoate) as the predominant species.
[0095] In some cases, the recombinant cell expressing heterologous Chatl can produce fatty acids and/or fatty acid chain products without the recombinant cell comprising an additional heterologous polypeptide with 3-ketoacyl-CoA synthase activity. The recombinant cell expressing heterologous Chatl can catalyze the addition of malonyl-CoA to a C2-CoA substrate to lead to a C4 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C4-CoA substrate to lead to a C6 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C6-C0A substrate to lead to a C8 fatty acid and/or fatty acid chain product, the addition of malonyl-CoA to a C8-C0A substrate to lead to a C 10 fatty acid and/or fatty acid chain product, and/or the addition of malonyl-CoA to a ClO-CoA substrate to lead to a C12 fatty acid and/or fatty acid chain product. In other cases, the recombinant cell expressing
heterologous Chatl can also comprise one or more additional heterologous polypeptides with 3- ketoacyl-CoA synthase activity (e.g., a NphT7 polypeptide and/or a NphT7 polypeptide mutant as described below) to increase production of fatty acids and/or fatty acid chain products. [0096] In some aspects, a Cbac variant with the identified amino acid substitution of
V223A can comprise an enzyme with increased specific activity for C2-CoA, C4-CoA, C6-C0A, and C8-C0A substrates when compared to the respective polypeptide with unaltered sequence.
In some cases, the Cbac variant with the identified amino acid substitution of V223A can comprise more than twice the specific activity for C2-CoA, C4-CoA, and C6-C0A substrates when compared to the respective polypeptide with unaltered sequence. In other cases, the Cbac variant with the identified amino acid substitution of V223A can comprise a similar specific activity for C8-C0A substrates when compared to the respective polypeptide with unaltered sequence. A Cbac variant with the identified amino acid substitution of T46M can comprise a similar or slightly reduced specific activity for C2-CoA, C4-CoA, C6-C0A, and C8-C0A substrates when compared to the respective polypeptide with unaltered sequence. A Cbac variant with the identified amino acid substitution of S256G can comprise an increased or similar specific activity for C2-CoA, C4-CoA, C6-C0A, and C8-C0A substrates when compared to the respective polypeptide with unaltered sequence.
[0097] In some aspects, a recombinant cell with a Cbac(V223A) variant can comprise an increased total FAME production of a mixture of C6-FAME (methyl hexanoate), C8-FAME (methyl octanoate), C10-FAME (methyl decanoate), and C12-FAME (methyl dodecanoate) when compared to a recombinant cell with the respective polypeptide with unaltered sequence.
In some cases, the recombinant cell with the Cbac(V223A) variant can comprise an increased total FAME production when compared to the recombinant cell with respective polypeptide with unaltered sequence. In some cases, the recombinant cell with the Cbac(V223A) variant can comprise an increased total FAME production of 40% or more when compared to the recombinant cell with the respective polypeptide with unaltered sequence. In some cases, the recombinant cell with the Cbac(T46M) variant can comprise an increased total FAME production when compared to the recombinant cell with the respective polypeptide with unaltered sequence. In some cases, the recombinant cell with the Cbac(T46M) variant can comprise an increased total FAME production of 20% or more when compared to the recombinant cell with the respective polypeptide with unaltered sequence. In some cases, the recombinant cell with the Cbac(S256G) variant can comprise an increased total FAME production when compared to the recombinant cell with the respective polypeptide with unaltered sequence. In some cases, the recombinant cell with the Cbac(S256G) variant can comprise an increased total FAME production of 9% or more when compared to the
recombinant cell with the respective polypeptide with unaltered sequence. [0098] In some aspects, a recombinant cell with a Cbac(V223A) variant, a Cbac(T46M) variant, or a Cbac(S256G) variant can comprise a similar ratio of FAME production by carbon chain length when compared to the recombinant cell with the Cbac polypeptide with unaltered sequence. For example, in each case, C8-FAME (methyl octanoate) comprises 60%, 65%, 70%, or more of the total FAME produced. Likewise, C6-FAME (methyl hexanoate) comprises 20%, 25%, 30%, or less of the total FAME produced. Similarly, C10-FAME (methyl decanoate) comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or less of the total FAME produced. Also, C12-FAME (methyl dodecanoate) comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or less of the total FAME produced.
[0099] In some aspects, a Cbac variant with more than one identified amino acid substitution can comprise an altered specific activity for C2-CoA, C4-CoA, C6-C0A, and C8- CoA substrates when compared to the respective polypeptide with unaltered sequence. In some cases, a Cbac(V223A, I246P) variant can comprise similar specific activity for C2-CoA, less specific activity for C4-CoA, less specific activity for C6-C0A substrates, and less specific activity for C8-C0A substrates when compared to the respective polypeptide with unaltered sequence. In other cases, a Cbac(V223A, I246L) variant can comprise increased specific activity for C2-CoA, increased specific activity for C4-CoA, increased specific activity for C6- CoA substrates, and similar specific activity for C8-C0A substrates when compared to the respective polypeptide with unaltered sequence. In some instances, a Cbac(V223A, I246L) variant can comprise 30% or greater specific activity for C2-CoA, 30% or greater specific activity for C4-CoA, 100% or greater specific activity for C6-C0A substrates, and similar specificity for C8-C0A substrates when compared to the respective polypeptide with unaltered sequence. In some cases, a Cbac(V223A, S282T) variant can comprise similar specific activity for C2-CoA, similar specific activity for C4-CoA, increased specific activity for C6-C0A substrates, and similar specific activity for C8-C0A substrates when compared to the respective polypeptide with unaltered sequence. In some cases, a Cbac(V223A, I246L, S282T) variant can comprise reduced specific activity for C2-CoA, reduced specific activity for C4-CoA, reduced specific activity for C6-C0A substrates, and reduced specific activity for C8-C0A substrates when compared to the respective polypeptide with unaltered sequence.
[0100] In some aspects, the recombinant cell can comprise heterologous 3-ketoacyl-CoA synthase enzymes having complementary acyl-CoA chain length specificities. For example, a heterologous NphT7 comprising high specific activity for C2-CoA substrates can be combined with another heterologous 3-ketoacyl-CoA synthase enzyme. Likewise, a heterologous NphT7 LSVA variant (see below) comprising high specific activity for C4-CoA substrates can be combined with another heterologous 3-ketoacyl-CoA synthase enzyme. In other words, heterologous enzymes with certain high specific activities (e.g., for C2-CoA substrates and/or C4-CoA substrates) can be paired with heterologous enzymes having other high specific activities (e.g., for C6-C0A substrates and/or C8-C0A substrates).
[0101] In some aspects, a recombinant cell comprising heterologous Cbac and heterologous NphT7 comprises increased total FAME production compared to a recombinant cell comprising heterologous Cbac and no heterologous NphT7 or heterologous NphT7 LSVA variant. In some cases, a recombinant cell comprising heterologous Cbac and heterologous NphT7 comprises 35% or more total FAME production compared to a recombinant cell comprising heterologous Cbac and no heterologous NphT7 or heterologous NphT7 LSVA variant. A recombinant cell comprising heterologous Cbac and heterologous NphT7 LSVA comprises similar total FAME production compared to a recombinant cell comprising heterologous Cbac and no heterologous NphT7 or heterologous NphT7 LSVA variant. A recombinant cell comprising heterologous Cbac, heterologous NphT7, and heterologous NphT7 LSVA variant comprises increased total FAME production compared to a recombinant cell comprising heterologous Cbac and no heterologous NphT7 or heterologous NphT7 LSVA variant. In some cases, a recombinant cell comprising heterologous Cbac, heterologous NphT7, and heterologous NphT7 LSVA variant comprises 65% or more total FAME production compared to a recombinant cell comprising heterologous Cbac and no heterologous NphT7 or heterologous NphT7 LSVA variant.
[0102] In some aspects, a recombinant cell comprising heterologous Cbac(V223A,
I246L) variant and heterologous NphT7 comprises increased total FAME production compared to a recombinant cell comprising heterologous Cbac(V223A, I246L) variant and no
heterologous NphT7 or heterologous NphT7 LSVA variant. In some cases, a recombinant cell comprising heterologous Cbac(V223A, I246L) variant and heterologous NphT7 comprises 65% or more total FAME production compared to a recombinant cell comprising heterologous Cbac(V223A, I246L) variant and no heterologous NphT7 or heterologous NphT7 LSVA variant. A recombinant cell comprising heterologous Cbac(V223A, I246L) variant and heterologous NphT7 LSVA variant comprises increased total FAME production compared to a recombinant cell comprising heterologous Cbac(V223A, I246L) variant and no heterologous NphT7 or heterologous NphT7 LSVA. In some cases, a recombinant cell comprising heterologous Cbac(V223A, I246L) variant and heterologous NphT7 LSVA variant comprises 25% or more total FAME production compared to a recombinant cell comprising heterologous Cbac(V223A, I246L) variant and no heterologous NphT7 or heterologous NphT7 LSVA variant. A recombinant cell comprising heterologous Cbac(V223A, I246L) variant, heterologous NphT7, and heterologous NphT7 LSVA variant comprises increased total FAME production compared to a recombinant cell comprising heterologous Cbac(V223A, I246L) variant and no
heterologous NphT7 or heterologous NphT7 LSVA variant. In some cases, a recombinant cell comprising heterologous Cbac(V223A, I246L) variant, heterologous NphT7, and heterologous NphT7 LSVA variant comprises 70% or more total FAME production compared to a recombinant cell comprising heterologous Cbac(V223A, I246L) variant and no heterologous NphT7 or heterologous NphT7 LSVA variant.
[0103] In some aspects, this application discloses a recombinant cell with an engineered biosynthetic pathway comprising one or more heterologous enzymes that convert chemical precursors and/or substrates into desired chemical products. In some cases, the engineered biosynthetic pathway comprises heterologous enzymes to synthesize the desired chemical product. In some instances, individual heterologous enzymes work in a stepwise fashion to convert a precursor into the desired chemical product. The engineered biosynthetic pathway can comprise one or more identified heterologous enzymes having 3-ketoacyl-CoA synthase activity to produce fatty acids and fatty acid chain products of C6-C10 chain length. In other words, the engineered biosynthetic pathway can comprise one or more of Cbac, Cclol, Cbol, Csacl,
Csac2, Cclo2, Casp, Chat2, Chatl, and their respective variants to produce fatty acids and fatty acid chain products of C6-C10 chain length. The engineered biosynthetic pathway can also comprise additional heterologous enzyme(s) that work in combination with one or more of Cbac, Cclol, Cbol, Csacl, Csac2, Cclo2, Casp, Chat2, Chatl, and their respective variants to produce fatty acids and fatty acid chain products of C6-C10 chain length.
[0104] In some aspects, the recombinant cell comprises one or more of Cbac, Cclol,
Cbol, Csacl, Csac2, Cclo2, Casp, Chat2, Chatl, and their respective variants to catalyze the reaction of acyl-CoA with malonyl-CoA to produce fatty acids and fatty acid chain products of C6-C10 chain length. However, the reaction of acyl-CoA with malonyl-CoA produces a 3- ketoacyl-CoA compound that must be reduced to the corresponding acyl compound before it can condense with another molecule of malonyl-CoA to extend the chain. The reduction takes place in three steps, the first being the reduction of the 3-ketoacyl group to the corresponding 3- hydroxyacyl group. The second reaction is a dehydration to the corresponding trans-2-enoylacyl compound, which is reduced in a third step to the corresponding acyl-CoA. The first reaction step is enzymatically catalyzed by a keto-CoA reductase (KCR) enzyme (EC 1.1.1.35). The second step is enzymatically catalyzed by a 3-hydroxy-acyl-CoA dehydratase (3HDh) enzyme (EC 4.2.1.17). Some bifunctional enzymes catalyze both of the first and second step reactions (EC 1.1.1.35 and EC 4.2.1.55). The third reaction step is enzymatically catalyzed by an enoyl- CoA reductase (ECR) enzyme (EC 1.1.1.32).
[0105] Accordingly, the engineered biosynthetic pathway preferably further comprises at least one of (1) a heterologous KCR gene that encodes for a KCR enzyme; (2) a heterologous 3HDh gene that encodes for a 3HDh enzyme; (3) a heterologous gene that encodes for a bifunctional enzyme that catalyzes both of the first and second reaction steps (EC 1.1.1.35 and 4.1.2.55) and (4) a heterologous ECR gene that encodes for an ECR enzyme. Preferably, the recombinant cell contains at least (1), (2) and (4) or at least (3) and (4). In each case, the gene preferably is under the control of promoter and/or terminator sequences active in the
recombinant cell.
[0106] The KCR enzyme may be, for example, one encoded by a P. aeruginosa fadB gene and/or having an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 11, one encoded by a P. aeruginosa fadG gene and/or having an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 12, one encoded by a C. beijerinckii hbd gene and/or having an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 11, and others as described in WO 2015/010103.
[0107] The 3HDh enzyme may be, for example, one encoded by a C. acetobutylicum crt
(short-chain-enoyl-CoA hydratase) gene and/or having an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 14, one encoded by a P. putida ech (enoyl-CoA hydratase/aldolase) gene and/or having an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 15, and others as described in WO 2015/010103.
[0108] Suitable bifunctional enzymes that catalyze both the first and second reactions steps include, for example, one encoded by an E. colifadB gene and/or having an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 16; one encoded by an R. novegicus ech2 gene, and others as described in WO 2015/010103.
[0109] Suitable ECR enzymes include, for example, one encoded by a T denticola ter gene and/or having an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 17. [0110] The recombinant cell can further include at least one heterologous 3-ketobutyryl-
CoA synthase gene, different from the modified 3-ketoacyl-CoA synthases described above, which encodes for a 3-ketobutyryl-CoA synthase. The heterologous 3-ketobutyryl-CoA synthase gene may encode for a 3-ketobutyryl-CoA synthase enzyme that is at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to any of those identified as SEQ ID NO: 1-120 of WO 2015/10103.
[0111] In some aspects, the heterologous 3-ketobutyryl-CoA synthase gene is a
Streptomyces Sp CL190 gene and/or a gene that encodes for an NphT7 enzyme that is at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 18.
[0112] In some aspects, the recombinant cell includes at least one gene that encodes for a modified NphT7 enzyme as described in WO 2015/10103. The modified NphT7 enzyme comprises an amino acid sequence having at least 70% but less than 100% to SEQ ID NO: 18. The modified NphT7 enzyme may have, for example, one or more amino acid substitutions selected from the group consisting of H100L, I147T, F217V, Y144L, V157F, G309S, G288S, a PDRP to HFLQ substitution for amino acid residues 86-89, 1147F, I147M, I147Q, I147S, I147C, 1147E, I147N, I147W, I147D, I147R, I147P, I147L, V196G, I147G, I147H, I147K, I147V,
1147 A, I147Y, F217G, F217A, F217L, F217I, F217M, F217T, F217P, F217S, F217E, F217L, F217V, F217W, S323A and S323V, and any combination of any two or more thereof.
[0113] In some aspects, the modified NphT7 enzyme comprises at least one amino acid substitution selected from the group consisting of I147V, I147S, I147T, and at least one additional amino acid substitution selected from H100L, F217V, S323A and S323V. In some aspects, the modified NphT7 enzyme corresponds to SEQ ID NO: 19. In some aspects, the modified NphT7 enzyme comprises an I147V, I147S or I147T amino acid substitution and an S323A amino acid substitution (corresponding to SEQ ID NO: 19 in which amino acid 100 is H, amino acid 147 is V, S or T, amino acid 217 is F and amino acid 323 is A). In some aspects, the modified NphT7 enzyme comprises an H100L substitution, an I147V, I147S or I147T amino acid substitution, an F217V substitution and an S323A amino acid substitution (corresponding to SEQ ID NO: 19 in which amino acid residue 100 is L, amino acid residue 147 is V, S or T, amino acid residue 217 is V and amino acid residue 323 is A).
[0114] In some aspects, the recombinant cell includes both of (1) a Streptomyces Sp
CL190 nphT7 gene and/or a gene that encodes for an NphT7 enzyme that is at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 18 and (2) a modified NphT7 enzyme having one or more amino acid substitutions selected from the group consisting of H100L, I147T, F217V, Y144L, V157F, G309S, G288S, a PDRP to HFLQ substitution for amino acid residues 86-89, 1147F, I147M, I147Q, I147S, I147C, 1147E, I147N, I147W, I147D, I147R, I147P, I147L, V196G, I147G, I147H, I147K, I147V, I147A, I147Y, F217G, F217A, F217L, F217I, F217M, F217T, F217P, F217S, F217E, F217L, F217V, F217W, S323A and S323V, and any combination of any two or more thereof. In preferred embodiments, the recombinant cell includes a gene that encodes for an enzyme having SEQ ID NO: 18 and another gene that encodes for an enzyme having SEQ ID NO: 19. In especially preferred embodiments, the recombinant cell includes a gene that encodes for an enzyme having SEQ ID NO: 18 and another gene that encodes for an enzyme having SEQ ID NO: 19 in which amino acid residue 100 is H or L, amino acid residue 147 is S, T or V, amino acid residue 271 is F or V and amino acid residue 323 is A.
[0115] The recombinant cell can produce one or more enzymes that terminate the acyl elongation cycle and produce a product having the desired chain length. Such a termination enzyme may or may not be heterologous. The selection of termination enzyme may depend on whether the desired product is a fatty acid or a derivative thereof such as a fatty alcohol, a fatty aldehyde, a fatty alkene, a fatty amide, a fatty ester or a fatty alkane.
[0116] The recombinant cell in some aspects includes a heterologous thioesterase gene that encodes for a thioesterase such as an acyl-CoA esterase, in which case the product will be a fatty acid. Suitable thioesterases include those described in Table 11 of WO 2015/101013.
[0117] In some aspects the recombinant cell includes a gene that encodes for an ester synthase, in which case the product typically is a fatty acid ester. Suitable ester synthases have amino acid sequences at least 80%, at least 90%, at least 95%, at least 99% or at least 100% identical to any of the Marinobacter aquacolei Maql enzyme (SEQ ID NO: 289 of WO 2015/10103), the Psychrobacter cryohaloentis Pcryl enzyme (SEQ ID NO: 290 of WO
2015/10103), the Rhodococcus jostii Rjosl enzyme (SEQ ID NO: 291 of WO 2015/10103), the, Alcanivorax borkumensis strain SK2 Aborkl enzyme (SEQ ID NO: 292 of WO 2015/10103) and the Hahella chejuensis hche gene (SEQ ID NO: 20). The ester synthase may have an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99% or at least 100% identical to the Hahella chejuensis Hche ester synthase (SEQ ID NO: 20).
[0118] The recombinant cell may also include one or more genes that encode for one or more of a fatty acyl-CoA reductase (alcohol or aldehyde forming), a fatty aldehyde reductase, an acyl-ACP reductase, an acyl-CoA:ACP acyltransferase, an acyl-CoA hydrolase, a carboxylic acid reductase, an aldehyde dehydrogenase and/or an acyl-ACP reductase. [0119] The recombinant cell also may include (A) one or more genes that encode for a carboxyl transferase subunit a enzyme, (EC 6.3.1.2) such as an E. coli accA enzyme or an enzyme that is at least 80%, at least 90%, at least 95% or at least 99% identical thereto; (B) one or more genes that encode for a biotin carboxyl carrier protein, (EC 6.4.1.2) such as an E. coli accB enzyme or an enzyme that is at least 80%, at least 90%, at least 95% or at least 99% identical thereto; (C) one or more genes that encode for a biotin carboxylase subunit enzyme,
(EC 6.3.4.14) such as an E. coli accC enzyme or an enzyme that is at least 80%, at least 90%, at least 95% or at least 99% identical thereto; (D) a carboxyl transferase subunit b (EC 6.4.1.2), such as an E. coli accD enzyme or an enzyme that is at least 80%, at least 90%, at least 95% or at least 99% identical thereto, (E) a fused E. coli accD subunit and accA subunit enzyme (SEQ ID NO: 26); or a combination of any two or more thereof. In some aspects, all of (A) - (E) are present.
[0120] In some aspects, the recombinant cell described here further comprises one or more additional genetic modifications to reduce or eliminate the expression of certain endogenous enzymes in the recombinant cell. Reducing or eliminating the expression of these certain endogenous enzymes in the recombinant cell can increase the production of desired products such as fatty acids and/or fatty acid chain products. These reduced or eliminated endogenous enzymes include one or more of the following enzymes:
Methylglyoxal synthase (EC 4.2.3.3), for example that encoded by the E. coli mgsA gene.
Lactate dehydrogenase (EC 1.1.1.27), for example that encoded by the E. coli IdhA gene.
Phosphotransacetylase (EC 2.3.1.8), for example that encoded by the E. coli pta gene.
Acetate kinase (EC 2.7.2.1), for example that encoded by E. coli ackA gene.
Acyl-CoA synthase (EC 6.2.1.3), for example that encoded by the E. colifadD gene.
Pyruvate formate lyase (EC 2.3.1.54), for example that encoded by the E.coli pflB gene.
Pyruvate oxidase (EC 1.2.2.2), for example that encoded by the E. coli poxB gene.
Fused acetaldehyde-CoA dehydrogense (EC 1.2.1.10), for example that encoded by the E. coli adhE gene.
Trigger factor (EC 5.2.1.8), for example that encoded by the E. coli tig gene.
Restriction endonuclease (EC 3.1.21.3), for example that encoded by the E. coli hsdr514 gene.
The atoDAEB operon.
Acyl-CoA thioesterase (EC 3.1.2.-), for example that encoded by the E. coli tesB or yciA gene. Acyl-coenzyme A dehydrogenase (EC 1.3.8.7), for example that encoded by the E. coli fadE gene.
3-ketoacyl-CoA thiolase (EC 2.3.1.16), for example that encoded by the E. colifadA gene.
L-ribulokinase (EC2.7.1.16), for example that encoded by the E. coli araB gene.
L-ribulose-5-phosphate-4-epimerase (EC 5.1.3.4), for example that encoded by the E. coli araD gene.
Beta-D-galactosidase (EC 3.2.1.23), for example that encoded by the E.coli lacZ gene.
Lambda phage lysogen.
Rhamnulose-l -phosphate aldolase (EC 4.1.2.19), for example that encoded by the E. coli rhaD gene.
Rhamnulokinase (EC 2.7.1.5), for example that encoded by the E. coli rhaB gene.
F mating factor.
Truncated RNase PH (EC2.7.7.56) Rph-1 gene.
[0121] Other genetic modifications may be present in the recombinant cell, including any of those described in WO 2015/10103.
[0122] Any heterologous gene may be operatively linked to a promoter and/or terminator sequence that is functional in the recombinant strain. The promoter may be an inducible promoter that functions only under certain conditions. For example, a low phosphate inducible promoter such as the promoter of the wild-type E. coli phoE gene ( PphoE ), is a useful promoter for the 3-ketoacyl synthase gene. Such a promoter is active in a low phosphate environment. Accordingly, a recombinant cell in which the 3-ketoacyl synthase gene is under the control of an E. coli phoE promoter or another low phosphate inducible promoter may be cultivated in a fermentation medium containing no more than 25 mM phosphate, especially no more than 20 mM, no more than 2 mM, no more than lmM, no more than 0.5 mM, or no more than 0.25 mM phosphate. In some aspects, the promoter that is a low phosphate inducible promoter is the promoter for the pstS gene ( PpstS ). This promoter may be constructed to include a binding site for Integration Host Factor and is thus designated PpstSIH (Lyzen et ak, Plasmid 60:125 (2008)).
[0123] Any heterologous gene may be integrated into the genome of the recombinant strain and/or present in one or more plasmids. If integrated into the genome, the heterologous gene may be inserted at a targeted or random location. Transformation methods such as electroporation and chemical methods (including calcium chloride and/or lithium acetate methods) known in the art are suitable. Examples of suitable transformation methods are described, for example, in Molecular Cloning: A Laboratory Manual, 4th Ed. Spring Harbor Press 2012. In general, no special transformation methods are necessary to produce the recombinant cells.
[0124] Deletions and/or disruptions of native genes can be performed by transformation methods, by mutagenesis and/or by forced evolution methods. In mutagenesis methods, cells are exposed to ultraviolet radiation or a mutagenic substance, under conditions sufficient to achieve a high kill rate (60-99.9%, preferably 90-99.9%) of the cells. Surviving cells are then plated and selected or screened for cells having the deleted or disrupted metabolic activity. Disruption or deletion of the desired native gene(s) can be confirmed through PCR or Southern analysis methods.
[0125] The recombinant cells described herein are used to produce compounds having a straight-chain alkyl group. The recombinant cells are grown under conditions such that they produce such compounds, and the compounds are recovered.
[0126] When the recombinant cell is a plant cell, the plant can be grown and the compound having the straight-chain alkyl group can be recovered from the plant or any portion thereof, such as roots, stems, leaves, flowers, seeds, seed pods and the like, in which the compound accumulates during the growth of the plant.
[0127] Single-cell and other microcells can be used in a culturing process to produce such compounds.
[0128] Culturing is performed generally by forming a culture medium that includes at least one carbon source that is capable of being metabolized by the recombinant cell to produce the product compounds and nutrients as may be required by the specific recombinant cell. The nutrients may include, for example, at least one nitrogen source such as yeast extract, peptone, tryptone, soy flour, com steep liquor, or casein, at least one phosphorus source, one or more vitamins such as biotin, vitamin B12 and derivatives of vitamin B 12, thiamin, pantothenate, one or more trace metals and the like. The fermentation medium may also contain additional materials such as anti-foam agents, biocides, buffers and the like.
[0129] In some cases, such as the production of fatty acid esters, the culture medium may also include a reagent that reacts with the straight-chain compound to produce the desired product. In the specific case of fatty acid esters, for example the culture medium preferably contains an alkanol such as methanol, ethanol or a C3-C8 alkanol. The alkanol reacts to produce the corresponding ester. A native or heterologous ester synthase, or other appropriate enzyme, may be expressed by the recombinant cell to catalyze such a reaction. [0130] Generally, the culture medium is inoculated with the recombinant cell, and the inoculum is cultured in the medium so that the cell density increases to a cell density suitable for production. The culture medium is then maintained at conditions sufficient for the recombinant cells to produce the desired product.
[0131] Suitable culture conditions will of course depend on the requirements of the particular recombinant strain. The temperature of the culture medium may be, for example from 20°C to 70°C, with a temperature of 25 to 40°C being preferred for most recombinant cells.
[0132] The pH of the culture medium may be, for example, from 2.0 to 10.0, from 3.0 to
9.0 or from 6.0 to pH 8.5.
[0133] It is contemplated that the described aspects may be practiced using either batch, fed-batch or continuous processes and that any known mode of bio-production would be suitable.
[0134] The culturing may be performed under aerobic, microaerobic, or anaerobic conditions, as required or can be tolerated by the particular recombinant cell. Generally, no special culturing equipment is needed to perform the fermentation. The equipment may include, for example, a tank suitable for holding the recombinant cell and the culture medium; a line for discharging contents from the culture tank to an extraction and/or separation vessel; and an extraction and/or separation vessel suitable for removal of the chemical product from cell culture waste.
[0135] The carbon source is one or more carbon-containing compounds that can be metabolized by the recombinant cell as a source of carbon. Examples of suitable carbon sources include sugars such as glucose, sucrose, fructose, lactose, C-5 sugars such as xylose and arabinose, glycerol and polysaccharides such as starch and cellulose. Other suitable carbon sources include fermentable sugars as may be obtained from cellulosic and lignocellulosic biomass through processes of pretreatment and saccharification, as described, for example, in U.S. Patent Publication No. 2007/0031918A1, hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Other suitable carbon sources include high-fructose com syrup, cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Still other suitable carbon sources include carbon dioxide, carbon monoxide, methanol, methylamine and glucosamine.
[0136] The culturing process may be continued until a titer of the desired product reaches at least 0.01, at least 0.05, at least 0.1, at least 0.25, at least 0.5 or at least 1 g per liter of culture medium (g/L). The fermentation process may be continued until the titer reaches, for example, up to 40, up to 45, up to 50, up to 80, up to 100, or up to 120 g/L. The specific productivity may be, for example, from 0.01 and 0.60 grams of the desired product per gram of cells on a dry weight basis per hour (g chemical product/g DCW-hr). The volumetric productivity achieved may be at least 0.005 g of the desired product per liter per hour (g/L-hr), at least 0.01 g/L-hr, at least 0.1 g/L-hr or at least 0.5 g/L-hr, and may be up to, for example, 10 g/L-hr, up to 5 g/L-hr or up to 1 g/L-hr. [0212] In some aspects, specific productivity as measured over a 24-hour fermentation (culture) period may be greater than about 0.01, 0.05, 0.10, 0.20, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 or 12.0 grams of chemical product per gram DCW of cells (based on the final DCW at the end of the 24-hour period).
SEQUENCES
SEQ ID NO: 1 Clostridiales bacterium 1_7_47_FAA
MTTRI IGTGS YVPEQIVTNN DLAQIVETND EWIRSRTGIG ERRIATTEST SYMAANAAMR 60 ALEQSGVKPE EIDLILLGTS SPDYCFPNGA CEVQGMIGAV NAACYDISAA CTGFVYALNT 120 AHAFISSGIY KTALVIGSDV LSKLIDWTDR GTCVLFGDGA GAVWKADET GILGINMHSD 180 GTKGNVLTCG SRTNGNFLLG KKPELGYMTM DGQEVFKFAV RKVPECIKQV LDDAGVAAAE 240 VRYFVIHQAN YRIIESIAKR LKVSVDCFPV NMEHYGNTSG ASVPLLLDEI NRKGMLESGD 300 KIVFSGFGAG LTWGATLLEW 320
SEQ ID NO: 2 Clostridium clostridioforme
MDSGWVRRMT TRIVGTGSYV PEQIVTNDDL AKIVETNDEW IRSRTGIGAR RIATSESTSY 60 MAAEASIKAL ENAGVKPEEI DLILLATSSP DYCFPNGACE VQGRIGAVNA ACFDLSVACT 120 GFVYALNTAH AFISSGIYKT ALVIGADVLS KLIDWTDRGT CVLFGDGAGA VWKADETGI 180 LGMNMHSDGT KGGVLTCGSR TNGNFLMGKK PELGYMTMDG QEVFKFAVKK VPQCIMEVLD 240 DTGVKAEDVR YFAMHQANYR IIESIAKRLK VNVDRFPVNM EHYGNTSGAS VPLLLDEMNR 300 KGMLSAGDKV VLSGFGAGLT WGAALLEW 328
SEQ ID NO: 3 Clostridium bolteae 90A9
MDSGWVRRMT TRIVGTGSYV PEQIVTNDDL AKIVETNDEW IRSRTGIGAR RIATSESTSY 60 MAAEASIKAL ENAGVKPEEI DLILLATSSP DYCFPNGACE VQGRIGAVNA ACFDLSVACT 120 GFVYALNTAH AFISSGIYKT ALVIGADVLS KLIDWTDRGT CVLFGDGAGA VWKADETGI 180 LGMNMHSDGT KGGVLTCGSR TNGNFLLGKK PELGYMTMDG QEVFKFAVKK VPQCITEVLE 240 DTGVKAEDVR YFAIHQANYR IIESIAKRLK VNVDRFPVNM EHYGNTSGAS VPLLLDEMNR 300 KGMLSAGDKV VLSGFGAGLT WGAALLEW 328
SEQ ID NO: 4 Clostridium saccharolyticum
MTARQAPGGG GCHKQTEKRE SDMTTRI IGI GSYVPDTWT NKDLMEFLDT DDAWIRERTG 60 ICERRVSKEM GTCALAVEAA KRAVSDAGID PKEIDLIVLA TSSGDRAFPA AAMDVQAAIG 120 AVNAVGFDIT AACSGFIFGL HIAHSFFMSG IYKTALIVGA EALSKWDWT DRGTCILFGD 180 GAGAAWRAS EDGGI IKTLM GSDGTKGWTL ECQARNLGNC LNGVKPELGF MKMDGKEVFK 240 FAVRKVPEIV EDILKEAEMT PEEIKYFVLH QANYRILEAA SRRLKIPMEK IPVNIDRYGN 300 TSAASIPILL DEMKQEGKLK RGDKLVLAGF GSGMTWGATL LEW 343
SEQ ID NO: 5 Clostridium saccharolyticum
MGEFMTTRI I GTGSAVPEQV VTNEDLARLV DTSDEWIRTR TGIKERRIAS AESGTSDLAI 60 QAAKEALNHA GVSAEELDI I ILATSSADCC FPSGACEVQA AIGALHAAAF DISAACSGFV 120 YALNTVHGFF KAGIYQTGLV IGADTLSKLI DWNDRSTCVL FGDGAGAAW RAEEKGILHT 180 VMGADGTRGK VLECGGRTTG NFLTGKKPEL GYMTMDGQEV FKFAAKTVPE SIKRWEESG 240
TAMEEIKYFI LHQANYRIFE SIAKRLKIPM EKFPTNLDRY GNTSGATIPI LLDEMNREGK 300
FQRGDKIVLA GFGAGLTWGA TLLEW 325
SEQ ID NO: 6 Clostridium clostridioforme 2_1_49FAA
MLMDSGWVRR MTTRIVGTGS YVPEQIVTND DLAKIVETND EWIRSRTGIG ARRIATNEGT 60
SYMAAEASVN ALENAGVKPD EIDLILLATS SPDYCFPNGA CEVQDRIGAI NAACFDISAA 120
CTGFVYALNT AHAFISSGIY KAALVIGADV LSKLIDWTDR GTCVLFGDGA GAVWKADET 180
GILGMNMHSD GTKGGVLTCG SRTNGNFLLG KKPELGYMTM DGQEVFKFAV KKVPQCITEV 240
LEDTGVKAED VRYFAIHQAN YRIIESIAKR LKVNVDRFPV NMEHYGNTSG ASVPILLDEL 300
NRKGMLSAGD KWLSGFGAG LTWGAALLEW 330
SEQ ID NO: 7 Clostridium asparagiforme DSM 15981
MTTRIVGTGA YVPEQIATND DLARIVETND EWIRSRTGIG ERRIATTETN SYMAAQAAKQ 60
ALDQAGIAPE DVDLILLATS SPDYCFPNGA CEIQEQIGAV NAAGYDISAA CTGFVFALNT 120
AHAFIQAGIY RTALVIGSDV LSKLLDWTDR GTCVLFGDGA GAVWQAADR GVIGVKMHSD 180
GTKGGVLTCG ARTNGNFLLG KKPELGYMTM DGQEVFKFAV KKVPEI IKEL LEENRTSLEE 240
IRYFVLHQAN YRI IESVAKR LKADISKFPA NMEHYGNTSG GSIPLLLDEM NRKGMLAPGD 300
KIVLSGFGAG LTWGATLVEW 320
SEQ ID NO: 8 Clostridium hathewayi
MGEIMTTRI I GTGSAVPKQV VTNDDLAKIV DTSDEWIRPR TGIRERRIAA AESGTTDLAA 60
EAARMAIEQS GIKPEELDI I VLATSSGDCC FPNGACEVQA AVGAVNAVAF DISAACSGFV 120
FALNTVHSFL SAGIYRTGLV IGADTLSKLV DWNDRSTCVL FGDGAGAAW RAEDTGVIGL 180
TMGADGTKGD VLKCGGRTTG NFLTGKKPEL GYMSMDGQEV FRFAVKTVPE AIKKTLAGSG 240
TELEEIKYFI LHQSNYRISE SIAKRLKLPM DKFPANLERY GNTSGASVPI LLDELNREGK 300
LKPGDKLLLA GFGAGLTWGT TLLEW 325
SEQ ID NO: 9 Clostridium hathewayi WAL-18680
MTTKI IGTGS YVPERWSND DLAKWETSD EWIQSRTGIR ERRIADCEGT SELAAKAAAA 60
AMENAGIEAS EIDIIILATS SPDNCFPNGA CEVQAAIGAE NAVAFDISAA CTGFVYALNT 120
MHAFLKSGLY QTGLVIGADV MSKLTDWTDR GTCVLFGDGA GAAWRAEES GIVRMVMKAD 180
GKKSHVLTCK ARTSENFSTG KTPELGYTAM DGQEVFKFAV KKVPECVRQV LEESGTDIEE 240
IKYFVMHQAN YRIFESIAKR LKVSMERIPM NMDRYGNTSG ASVPIMLDEL NREGKLQPGD 300
KLILSGFGAG LTWGATLIEW 320
SEQ ID NO: 10 Artificial Sequence - Cbac variant
Residue 46: Thr or Met
Residue 223: Val or Ala
Residue 256: Ser or Gly
Residue 282: Ser or Thr
MTTRI IGTGS YVPEQIVTNN DLAQIVETND EWIRSRTGIG ERRIAXTEST SYMAANAAMR 60
ALEQSGVKPE EIDLILLGTS SPDYCFPNGA CEVQGMIGAV NAACYDISAA CTGFVYALNT 120
AHAFISSGIY KTALVIGSDV LSKLIDWTDR GTCVLFGDGA GAVWKADET GILGINMHSD 180
GTKGNVLTCG SRTNGNFLLG KKPELGYMTM DGQEVFKFAV RKXPECIKQV LDDAGVAAAE 240
VRYFVXHQAN YRI IEXIAKR LKVSVDCFPV NMEHYGNTSG AXVPLLLDEI NRKGMLESGD 300
KIVFSGFGAG LTWGATLLEW 320
SEQ ID NO: 11 Pseudomonas aeruginosa
MSLQGKVALV TGASRGIGQA IALELGRLGA WIGTATSAS GAEKIAETLK ANGVEGAGLV 60 LDVSSDESVA ATLEHIQQHL GQPLIWNNA GITRDNLLVR MKDDEWFDW NTNLNSLYRL 120 SKAVLRGMTK ARWGRI INIG SWGAMGNAG QTNYAAAKAG LEGFTRALAR EVGSRAITVN 180 AVAPGFIDTD MTRELPEAQR EALLGQIPLG RLGQAEEIAK WGFLASDGA AYVTGATVPV 240 NGGMYMS 247 SEQ ID NO: 12 Pseudomonas aeruginosa
MSLQGKVALV TGASRGIGQA IALELGRLGA WIGTATSAS GAEKIAETLK ANGVEGAGLV 60 LDVSSDESVA ATLEHIQQHL GQPLIWNNA GITRDNLLVR MKDDEWFDW NTNLNSLYRL 120 SKAVLRGMTK ARWGRI INIG SWGAMGNAG QTNYAAAKAG LEGFTRALAR EVGSRAITVN 180 AVAPGFIDTD MTRELPEAQR EALLAQIPLG RLGQAEEIAK WGFLASDGA AYVTGATVPV 240 NGGMYMS 247
SEQ ID NO: 13 Clostridium bei jerinckii
MKKIFVLGAG TMGAGIVQAF AQKGCEVIVR DIKEEFVDRG IAGITKGLEK QVAKGKMSEE 60 DKEAILSRIS GTTDMKLAAD CDLWEAAIE NMKIKKEIFA ELDGICKPEA ILASNTSSLS 120 ITEVASATKR PDKVIGMHFF NPAPVMKLVE I IKGIATSQE TFDAVKELSV AIGKEPVEVA 180 EAPGFWNGI LIPMINEASF ILQEGIASVE DIDTAMKYGA NHPMGPLALG DLIGLDVCLA 240 IMDVLFTETG DNKYRASSIL RKYVRAGWLG RKSGKGFYDY SK 282
SEQ ID NO: 14 Clostridium acetobutylicum
MELNNVILEK EGKVAWTIN RPKALNALNS DTLKEMDYVI GEIENDSEVL AVILTGAGEK 60 SFVAGADISE MKEMNTIEGR KFGILGNKVF RRLELLEKPV IAAVNGFALG GGCEIAMSCD 120 IRIASSNARF GQPEVGLGIT PGFGGTQRLS RLVGMGMAKQ LIFTAQNIKA DEALRIGLVN 180 KWEPSELMN TAKEIANKIV SNAPVAVKLS KQAINRGMQC DIDTALAFES EAFGECFSTE 240 DQKDAMTAFI EKRKIEGFKN R 261
SEQ ID NO: 15 Pseudomonas putida
MSDTEVPVLA EVRNRVGHLA LNRPVGLNAL TLQMIRITWR QLHAWESDPE IVAWLRANG 60 EKAFCAGGDI RSLYDSYQAG DDLHHVFLEE KYSLDQYIHG YPKPIVALMD GFVLGGGMGL 120 VQGTALRWT ERVKMGMPET SIGYFPDVGG SYFLPRLPGE LGLYLGITGI QIRAADALYA 180 RLADWCLPSE RISEFDRRLD QISWGYAPRE ILAGLLSSLA SNRLLGAELK SLHPAIDEHF 240 TQPDLSAIRA SLQAERRPEY QDWAEQTVEL LNNRSPLAMS ATLKLLRLGR TLSLANCFEL 300 ELHLERQWFA KGDLIEGVRA LLIDKDKTPR WNPPTLEQLD TNRVNEFFDG FQPAT 355
SEQ ID NO: 16 Escherichia coli
MVYKGDTLYL DWLEDGIAEL VFDAPGSVNK LDTATVASLG EAIGVLEQQS DLKGLLLRSN 60 KAAFIVGADI TEFLSLFLVP EEQLSQWLHF ANSVFNRLED LPVPTIAAVN GYALGGGCEC 120 VLATDYRLAT PDLRIGLPET KLGIMPGFGG SVRMPRMLGA DSALEI IAAG KDVGADQALK 180 IGLVDGWKA EKLVEGAKAV LRQAINGDLD WKAKRQPKLE PLKLSKIEAT MSFTIAKGMV 240 AQTAGKHYPA PITAVKTIEA AARFGREEAL NLENKSFVPL AHTNEARALV GIFLNDQYVK 300 GKAKKLTKDV ETPKQAAVLG AGIMGGGIAY QSAWKGVPW MKDINDKSLT LGMTEAAKLL 360 NKQLERGKID GLKLAGVIST IHPTLDYAGF DRVDIWEAV VENPKVKKAV LAETEQKVRQ 420 DTVLASNTST IPISELANAL ERPENFCGMH FFNPVHRMPL VEIIRGEKSS DETIAKWAW 480 ASKMGKTPIV VNDCPGFFVN RVLFPYFAGF SQLLRDGADF RKIDKVMEKQ FGWPMGPAYL 540 LDWGIDTAH HAQAVMAAGF PQRMQKDYRD AIDALFDANR FGQKNGLGFW RYKEDSKGKP 600 KKEEDAAVED LLAEVSQPKR DFSEEEI IAR MMIPMVNEW RCLEEGI IAT PAEADMALVY 660 GLGFPPFHGG AFRWLDTLGS AKYLDMAQQY QHLGPLYEVP EGLRNKARHN EPYYPPVEPA 720 RPVGDLKTA 729
SEQ ID NO: 17 Treponema denticola
MIVKPMVRNN ICLNAHPQGC KKGVEDQIEY TKKRITAEVK AGAKAPKNVL VLGCSNGYGL 60 ASRITAAFGY GAATIGVSFE KAGSETKYGT PGWYNNLAFD EAAKREGLYS VTIDGDAFSD 120 EIKAQVIEEA KKKGIKFDLI VYSLASPVRT DPDTGIMHKS VLKPFGKTFT GKTVDPFTGE 180 LKEISAEPAN DEEAAATVKV MGGEDWEVGS NS 212
SEQ ID NO: 18 Streptomyces sp . CL190
MTDVRFRI IG TGAYVPERIV SNDEVGAPAG VDDDWITRKT GIRQRRWAAD DQATSDLATA 60 AGRAALKAAG ITPEQLTVIA VATSTPDRPQ PPTAAYVQHH LGATGTAAFD VNAVCSGTVF 120 ALSSVAGTLV YRGGYALVIG ADLYSRILNP ADRKTWLFG DGAGAMVLGP TSTGTGPIVR 180 RVALHTFGGL TDLIRVPAGG SRQPLDTDGL DAGLQYFAMD GREVRRFVTE HLPQLIKGFL 240 HEAGVDAADI SHFVPHQANG VMLDEVFGEL HLPRATMHRT VETYGNTGAA SIPITMDAAV 300 RAGSFRPGEL VLLAGFGGGM AASFALIEW 329
SEQ ID NO: 19 Artificial Sequence NphT7 variant
RESIDUE 99: His or Leu
RESIDUE 147 Ser, Thr, or Val
RESIDUE 217 Phe or Val
RESIDUE 323 Ser, Val or Ala
MTDVRFRI IG TGAYVPERIV SNDEVGAPAG VDDDWITRKT GIRQRRWAAD DQATSDLATA 60 AGRAALKAAG ITPEQLTVIA VATSTPDRPQ PPTAAYVQXL LGATGTAAFD VNAVCSGTVF 120 ALSSVAGTLV YRGGYALVIG ADLYSRXLNP ADRKTWLFG DGAGAMVLGP TSTGTGPIVR 180 RVALHTFGGL TDLIRVPAGG SRQPLDTDGL DAGLQYXAMD GREVRRFVTE HLPQLIKGFL 240 HEAGVDAADI SHFVPHQANG VMLDEVFGEL HLPRATMHRT VETYGNTGAA SIPITMDAAV 300 RAGSFRPGEL VLLAGFGGGM AAXFALIEW 329
SEQ ID NO: 20 Hahella chejuensis
MTPLSPVDQI FLWLEKRQQP MHVGGLHIFS FPDDADAKYM TELAQQLRAY ATPQAPFNRR 60 LRQRWGRYYW DTDAQFDLEH HFRHEALPKP GRIRELLAHV SAEHSNLMDR ERPMWECHLI 120 EGIRGRRFAV YYKAHHCMLD GVAAMRMCVK SYSFDPTATE MPPIWAISKD VTPARETQAP 180 AAGDLVHSLS QLVEGAGRQL ATVPTLIREL GKNLLKARDD SDAGLIFRAP PSILNQRITG 240 SRRFAAQSYA LERFKAIGKA FQATVNDWL AVCGSALRNY LLSRQALPDQ PLIAMAPMSI 300 RQDDSDSGNQ IAMILANLGT HIADPVRRLE LTQASARESK ERFRQMTPEE AVNYTALTLA 360 PSGLNLLTGL APKWQAFNW ISNVPGPNKP LYWNGARLEG MYPVSIPVDY AALNITLVSY 420 RDQLEFGFTA CRRTLPSMQR LLDYIEQGIA ELEKAAGV 458
SEQ ID NO: 21 Escherichia coli
MGFLSGKRIL VTGVASKLSI AYGIAQAMHR EGAELAFTYQ NDKLKGRVEE FAAQLGSDIV 60 LQCDVAEDAS IDTMFAELGK VWPKFDGFVH SIGFAPGDQL DGDYVNAVTR EGFKIAHDIS 120 SYSFVAMAKA CRSMLNPGSA LLTLSYLGAE RAIPNYNVMG LAKASLEANV RYMANAMGPE 180 GVRVNAISAG PIRTLAASGI KDFRKMLAHC EAVTPIRRTV TIEDVGNSAA FLCSDLSAGI 240 FGEWHVDGG FSIAAMNELE LK 262
SEQ ID NO: 22 Escherichia coli
aaatcagact gaagacttta tctctctgtc ataaaactgt catattcctt acatataact 60 gtcacctgtt tgtcctattt tgcttctcgt agccaacaaa caatgcttta tgaatcctcc 120 c 121
SEQ ID NO: 23 Escherichia coli
gatcttgata tcaaacgaac gttttagcag gactgtcgtc ggttgccaac catctgcgag 60 caaagcatgg cgttttgttg cgcgggatca gcaagcctag cggcagttgt ttacgctttt 120 attacagatt taataaatta ccacatttta agaatattat taatctgtaa tatatcttta 180 acaatctcag gttaaaaact ttcctgtttt caacg 215
SEQ ID NO: 24 Escherichia coli
MDIRKIKKLI ELVEESGISE LEISEGEESV RISRAAPAAS FPVMQQAYAA PMMQQPAQSN 60 AAAPATVPSM EAPAAAEISG HIVRSPMVGT FYRTPSPDAK AFIEVGQKVN VGDTLCIVEA 120 MKMMNQIEAD KSGTVKAILV ESGQPVEFDE PLWIE 156
SEQ ID NO: 25 Escherichia coli
MLDKIVIANR GEIALRILRA CKELGIKTVA VHSSADRDLK HVLLADETVC IGPAPSVKSY 60 LNIPAI ISAA EITGAVAIHP GYGFLSENAN FAEQVERSGF IFIGPKAETI RLMGDKVSAI 120 AAMKKAGVPC VPGSDGPLGD DMDKNRAIAK RIGYPVIIKA SGGGGGRGMR WRGDAELAQ 180 SISMTRAEAK AAFSNDMVYM EKYLENPRHV EIQVLADGQG NAIYLAERDC SMQRRHQKW 240 EEAPAPGITP ELRRYIGERC AKACVDIGYR GAGTFEFLFE NGEFYFIEMN TRIQVEHPVT 300 EMITGVDLIK EQLRIAAGQP LSIKQEEVHV RGHAVECRIN AEDPNTFLPS PGKITRFHAP 360 GGFGVRWESH IYAGYTVPPY YDSMIGKLIC YGENRDVAIA RMKNALQELI IDGIKTNVDL 420 QIRIMNDENF QHGGTNIHYL EKKLGLQEK 449
SEQ ID NO: 26 Escheri :hia coli
MSWIERIKSN ITPTRKASIP EGVWTKCDSC GQVLYRAELE RNLEVCPKCD HHMRMTARNR 60 LHSLLDEGSL VELGSELEPK DVLKFRDSKK YKDRLASAQK ETGEKDALW MKGTLYGMPV 120 VAAAFEFAFM GGSMGSWGA RFVRAVEQAL EDNCPLICFS ASGGARMQEA LMSLMQMAKT 180 SAALAKMQER GLPYISVLTD PTMGGVSASF AMLGDLNIAE PKALIGFAGP RVIEQTVREK 240 LPPGFQRSEF LIEKGAIDMI VRRPEMRLKL ASILAKLMNL PAPNPEAPRE GVWPPVPDQ 300 EPEALSGGGG SGGGGSGGGG SGGGGSAAAS LNFLDFEQPI AELEAKIDSL TAVSRQDEKL 360 DINIDEEVHR LREKSVELTR KIFADLGAWQ IAQLARHPQR PYTLDYVRLA FDEFDELAGD 420 RAYADDKAIV GGIARLDGRP VMI IGHQKGR ETKEKIRRNF GMPAPEGYRK ALRLMQMAER 480 FKMPIITFID TPGAYPGVGA EERGQSEAIA RNLREMSRLG VPWCTVIGE GGSGGALAIG 540 VGDKVNMLQY STYSVISPEG CASILWKSAD KAPLAAEAMG I IAPRLKELK LIDS I IPEPL 600 GGAHRNPEAM AASLKAQLLA DLADLDVLST EDLKNRRYQR LMSYGYA 647
SEQ ID NO: 27 Escherichia coli
ggtttgaata aatgacaaaa agcaaagcct ttgtgccgat gaatctctat actgtttcac 60 a 61
SEQ ID NO: 28 Escherichia coli
gagttaacca cgcggcttgc caacggggtc tgaatcgctt tttttgtata taatgcgtgt 60
EXAMPLES
[0137] The following examples are provided to illustrate the disclosure, but are not intended to limit the scope thereof. All parts and percentages are by weight unless otherwise indicated.
Example 1.
[0138] The following recombinant E. coli strains were used in the following examples as indicated.
[0139] Recombinant Strain 1 is a mutant of the E. coli strain designated BW25113, available from the E. coli Genetic Strain Center (CGSC#7636; Dept of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut), having the following additional genetic modifications:
Figure imgf000049_0001
Figure imgf000050_0001
[0140] Recombinant Strain 2 is a mutant of the BW25113 E. coli strain with the following genetic modifications:
Figure imgf000050_0002
[0141] Recombinant Strain 3 is a mutant of the BW25113 E. coli strain with the following genetic modifications:
Figure imgf000050_0003
[0142] The following recombinant plasmids were used in the following examples as indicated.
[0143] Type 1 plasmids are pACYC plasmids containing the pl5a origin of replication and a chloramphenicol resistance marker:
[0144] Type 1A: this plasmid includes an E. coli bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase (fadB) gene (SEQ ID NO: 11) and a T denticola enoyl-CoA (ter) gene (SEQ ID NO: 17) cassette, all under a native E. coli pstSIH promoter (SEQ ID NO: 22) and a native E. coli terminator. This plasmid also contains a Hahella chejuensis ester synthase gene (SEQ ID NO: 20) fused to a DNA sequence encoding a protein fragment containing 6 histidine residues and a protease recognition site under an E. coli phoE promoter (SEQ ID NO: 23).
[0145] Type 1B: this plasmid includes a mutated Streptomyces sp. nphT7 gene encoding for a 3-ketoacyl-CoA synthase having H100L, I147S, F217V and S323A mutations (the “LSVA” NphT7 mutant, SEQ ID NO: 19), an E. coli bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase (fadB) (SEQ ID NO: 11) gene and a T. denticola enoyl-CoA (ter) gene (SEQ ID NO: 17) cassette, all under a native E. coli pstSIH promoter (SEQ ID NO: 22) and a native E. coli terminator. This plasmid also contains a Hahella chejuensis ester synthase gene (SEQ ID NO: 20) fused to a DNA sequence encoding a protein fragment containing 6 histidine residues and a protease recognition site under an E. coli phoE promoter (SEQ ID NO: 23), and an ACC (acetyl-CoA carboxylase) cassette including fused E. coli accD and accA genes (SEQ ID NO: 26) with a E. coli tpiA promoter (SEQ ID NO: 27) and a cassette including the E. coli accB (SEQ ID NO: 24) and E. coli accC genes (SEQ ID NO: 25) under an E. coli rpiA promoter (SEQ ID NO: 28).
[0146] Type 1C: this plasmid includes an E. coli bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase (fadB) gene (SEQ ID NO: 11) and a T. denticola enoyl-CoA (ter) gene (SEQ ID NO: 17) cassette, all under a native E. coli pstSIH promoter (SEQ ID NO: 22) and a native E. coli terminator. This plasmid also contains a Hahella chejuensis ester synthase gene (SEQ ID NO: 20) fused to a DNA sequence encoding a protein fragment containing 6 histidine residues and a protease recognition site under an E. coli phoE promoter (SEQ ID NO: 23), and an ACC (acetyl-CoA carboxylase) cassette including fused E. coli accD&nd accA genes (SEQ ID NO: 26) with a E. coli tpiA promoter (SEQ ID NO: 27) and a cassette including the E. coli accB (SEQ ID NO: 24) and E. coli accC genes (SEQ ID NO: 25) under an E. coli rpiA promoter (SEQ ID NO: 28). [0147] Type 2 plasmids are pET Plasmids containing a ColEl origin of replication and a kanamycin resistance marker:
[0148] Type 2A: this plasmid includes the 3-ketoacyl-CoA synthase gene to be evaluated
(fused to a DNA sequence that encodes an N-terminal protein fragment containing 6 histidine residues and a protease recognition site unless indicated otherwise) under an E. coli promoter and an E. coli terminator. The E. coli promoter is either the promoter for the native pstS gene (PpstSIH promoter) or that for the native phoE gene (PphoE promoter), both of which are induced in response to lowering phosphate concentrations as phosphate is incorporated into cell biomass during growth, or to medium with low phosphate concentrations.
[0149] 3-ketoacyl-CoA synthase genes are synthesized based on published polypeptide sequence information for various wild type enzymes. Site-specific mutants of the synthesized 3- ketoacyl-CoA synthase genes are generated by oligonucleotide-directed mutagenesis. The sources of the wild- type genes, the short-hand designations used herein for each of them, and the amino acid sequence of the native enzyme produced by the wild-type genes are as follows:
Figure imgf000052_0001
*GenInfo Identifier (GI) refers to sequence identification numbers used by the GenBank genetic sequence database at the National Center for Biotechnology Information, U.S. National Library of Medicine, Bethesda, Maryland, USA
[0150] Mutations to the amino acid residues encoded by the wild- type genes are designated herein by the shorthand designation for the wild-type enzyme, followed in parentheses by a 3-, 4- or 5 character code consisting of a first letter designating the amino acid residue in the native enzyme, a 1-, 2- or 3-digit number indicating the position of that amino acid residue in the native enzyme, and a final letter designating the amino acid residue in that position in the mutated enzyme. The single-letter designations are IUPAC amino acid abbreviations as reported, for example, at Eur. J. Biochem. 138:9-37(1984). For example, the designation“Cbac(V223A)” indicates that a valine (V) at amino acid residue position 147 in the wild type Clostridiales bacterium l_7_47_FAA enzyme has been replaced with an alanine (A).
[0151] Production of recombinant E. coli strains:
[0152] Recombinant E. coli strains are prepared using standard electroporation methods.
In each case, one of the above described recombinant strains (e.g. Recombinant Strain 1, Recombinant Strain 2, or Recombinant Strain 3) is transformed with a“Type 1” plasmid (e.g. Type 1A, Type 1B, or Type 1C) and an above-described“Type 2” plasmid (e.g. Type 2A). For each Type 2 plasmid, the 3-ketoacyl-CoA synthase and the promoter for the 3-ketoacyl-CoA synthase gene are as indicated in the specific examples below.
[0153] Small scale fermentation method - 1 ml protocol
[0154] A culture of synthetic medium containing salts, glucose, NH4Cl, and
supplemented with vitamins, yeast extract, 35 pg/ml kanamycin and 20 pg/ml chloramphenicol is inoculated with the strain to be tested and grown overnight at 30°C. The ODeoo of a 1: 10 dilution of this culture is determined, and a volume of the original culture corresponding to 8 OD units is centrifuged and the supernatant discarded. The pelleted cells are resuspended thoroughly in 4 mL of fresh medium containing, 30 g/L glucose, 0.158 mM phosphate (low phosphate medium), 1% (V/V) methanol (to produce fatty acid methyl esters) or ethanol (to produce fatty acid ethyl esters), chloramphenicol, and kanamycin as above, and l-ml aliquots dispensed into triplicate 16-mm glass tubes containing 64 pL of heptadecane or methyl tetradecanoate. This synthetic medium functions as a limited phosphate medium that promotes the activity of a low phosphate inducible promoter such as the E. coli phoE promoter (SEQ ID NO: 23) or E. coli pstSIH promoter (SEQ ID NO: 22). The tubes are incubated at 30°C, 250 rpm for 4 hours. The incubation temperature is then raised to 37°C and incubation is continued for a further 20 hours. The entire culture is extracted with methyl tert-butyl ether and the extract analyzed for fatty acid esters by gas chromatography.
[0155] Small scale fermentation method - shake flask protocol
[0156] A culture of synthetic medium containing salts, glucose, NH4Cl, and
supplemented with vitamins, yeast extract, 35 pg/ml kanamycin and 20 pg/ml chloramphenicol is inoculated with the strain to be tested and grown overnight at 32°C. The ODeoo of a 1: 10 dilution of this culture is determined in order to inoculate a Seed 2 flask to a final ODeoo of 0.3. Seed 2 flasks contain 25-30 ml synthetic medium containing salts, glucose, NH4Cl, and is supplemented with vitamins, yeast extract, 35 pg/ml kanamycin, 20 pg/ml chloramphenicol, and 0-2% methanol. Seed 2 flasks are incubated at 32°C for 6-7 hours.
[0157] The ODeoo of a 1: 10 dilution of this culture is determined in order to inoculate a production flask to a final ODeoo of 0.01-0.025. Production flasks contain 25 ml synthetic medium containing salts, glucose, NH4Cl, and supplemented with vitamins, yeast extract, 35 pg/ml kanamycin, 20 pg/ml chloramphenicol, 1.25-2.5 mM phosphate, 0-2% methanol, 3 - 6g/l glucose, and 15-40 g/l glycerol. This synthetic medium functions as a limited phosphate medium that promotes the activity of a low phosphate inducible promoter such as the E. coli phoE promoter (SEQ ID NO: 23) or E. coli pstSIH promoter (SEQ ID NO: 22). Production flasks are incubated at 32°C. When phosphate is depleted the following additions are made: 2 ml methyl tetradecanoate, 1 ml 12.5% Polysorbate 80 (sold under the trademark name Tween 80), 1-1.25 ml 50% glycerol. Methanol (0-0.5 ml) is added at the beginning of the production assay or right after phosphate depletion. Additional methanol can be added after phosphate depletion to compensate for methanol evaporation. Flasks are incubated at 35 °C for a further 24 hours. Samples are taken at 24 hours and extracted with 0.1 % HC1 in MTBE (Methyl tert butyl ether). The extracts are analyzed for fatty acid esters by gas chromatography.
[0158] In vitro assay for specific activity for different chain length primers
[0159] In vitro assays are used to determine a specific activity of a 3-ketoacyl-CoA synthase for different chain length primers. In some aspects, the assay can be used to determine the specific activity of a particular 3-ketoacyl-CoA synthase for a particular chain length primer. For example, the assay can be carried out to test for specific activity for a C6-C0A primer by reacting a C6-C0A primer and malonyl-CoA with a particular 3-ketoacyl-CoA synthase.
Magnesium chloride is added to the reaction mixture and complexes between the resulting 3- ketoC8-CoA and magnesium ion are formed. The reaction progress can be monitored by detecting these 3-ketoC8-CoA and magnesium ion complexes at a UV absorbance of 303 nm. These in vitro assays can be performed with cell lysates of cells expressing a 3-ketoacyl-CoA synthase and/or an isolated 3-ketoacyl-CoA synthase.
[0160] The presence of 3-ketoC8-CoA products indicates that the assayed 3-ketoacyl-
CoA synthase has specific activity against C6-C0A primers and that the assayed 3-ketoacyl-CoA synthase has 3-ketoC8-CoA synthase activity. The specific activity for other chain length primers can be assayed in similar fashion by substituting a different chain length CoA primer. For example, by substituting a C2-CoA primer, the specific activity for C2-CoA primers can be assayed and the 3-ketoC4-CoA synthase activity can be determined. By substituting a C4-CoA primer, the specific activity for C4-CoA primers can be assayed and the 3-ketoC6-CoA synthase activity can be determined. By substituting a C8-C0A primer, the specific activity for C8-C0A primers can be assayed and the 3-ketoClO-CoA synthase activity can be determined.
[0161] In some aspects, the in vitro assay is carried out on isolated 3-ketoacyl-CoA synthase. The 3-ketoacyl-CoA synthase can be isolated by methods known in the art. For example, the 3-ketoacyl-CoA synthase can be overexpressed as a fusion polypeptide with an affinity tag such as a poly-histidine tag and then purified by immobilized metal affinity chromatography. The in vitro assay is then carried out on this isolated 3-ketoacyl-CoA synthase by preparing a reaction mixture of CoA primer (0.3 mM) and malonyl-CoA (0.3 mM), in a buffer of 5 mM MgCh and 50 mM Tris, pH 8.0. The reaction is initiated by adding the isolated 3-ketoacyl-CoA synthase at various concentrations. As described above, the progress of the reaction can be monitored by detecting 3-ketoCX-CoA and magnesium ion complexes at a UV absorbance of 303 nm, where X represents the chain length of the resulting product. The CoA primer is selected from C2-CoA, C4-CoA, C6-C0A, or C8-C0A. The reactions can be carried out in 96-well microtiter plates.
Example 2.
[0162] An amino acid sequence alignment of the 3-ketoacyl-CoA synthase enzymes,
Cbac (SEQ ID NO: 1), Cclol (SEQ ID NO: 2), Cbol (SEQ ID NO: 3), Csacl (SEQ ID NO: 4), Csac2 (SEQ ID NO: 5), Cclo2 (SEQ ID NO: 6), Casp (SEQ ID NO: 7), Chat2 (SEQ ID NO: 8), and Chatl (SEQ ID NO: 9), was carried out with the Clone Manager software package
(Scientific & Educational Software, Denver, Colorado). The sequence alignment was performed as a global protein sequence alignment with the Scoring Matrix BLOSUM62 and using Cbac (SEQ ID NO: 1) as the reference sequence. The results of the sequence alignment are as follows:
Figure imgf000055_0001
Figure imgf000056_0001
[0163] FIGS. 1A, 1B, and 1C show graphical views of the sequence alignment. Each line corresponds to a 3-ketoacyl-CoA synthase enzyme as indicated by the designation. The amino acid position of the first residue of each row is indicated.
Example 3.
[0164] Individual strains containing a single heterologous 3-ketoacyl-CoA synthase enzyme (e.g., Cbac (SEQ ID NO: 1), Cclol (SEQ ID NO: 2), Cbol (SEQ ID NO: 3), Csacl (SEQ ID NO: 4), Csac2 (SEQ ID NO: 5), Cclo2 (SEQ ID NO: 6), Casp (SEQ ID NO: 7), Chat2 (SEQ ID NO: 8), and Chatl (SEQ ID NO: 9) were assayed for the production of fatty acid methyl esters (FAME). The production of FAMEs of different chain lengths (e.g. C6, methyl hexanoate; C8, methyl octanoate; C10, methyl decanoate; and C12, methyl dodecanoate) was assayed. Each individual strain that was assayed was prepared using Recombinant Strain 1 and comprised a Type 1 A plasmid and a Type 2A plasmid. Each Type 2A plasmid comprised the relevant 3-ketoacyl-CoA synthase enzyme with the PpstSIH promoter. Each individual strain was cultured and assayed as described in the small scale fermentation method - 1 ml protocol described above. The results of the assays are as follows:
Figure imgf000056_0002
[0165] The results of the assays are also shown in FIGS. 2 and 3. FIG. 2 shows the total amount of FAME produced in g/L per strain and the total amount of each of methyl hexanoate (C6), methyl octanoate (C8), methyl decanoate (C10), and methyl dodecanoate (C12) per strain. FIG. 3 shows FAME production by each strain based on the chain length of the product as a percentage of the total FAME produced by that strain. As shown in the table above and in FIGS. 2 and 3, the Cbac strain has a higher titer for total FAME produced with a majority (72%) of the FAME produced as methyl octanoate (C8). The Cbac strain also produced a very small amount of the total FAME produced as methyl decanoate (C10) (only 4%) or methyl dodecanoate (C12) (only 2%). The higher production of FAME produced as methyl octanoate (C8) and the small production of FAME produced as methyl decanoate (C10) indicates that the Cbac strain has a higher specificity for production of methyl octanoate (C8) FAME. The assay showed that all of the strains showed specificity for methyl octanoate (C8) because in each strain more methyl octanoate (C8) was produced than other chain lengths of FAME. Also, the strains were able to produce methyl hexanoate (C6), methyl octanoate (C8), methyl decanoate (C10), and/or methyl dodecanoate (Cl 2) with the designated 3-ketoacyl-CoA synthase and without expressing a NphT7 or NphT7 mutant.
Example 4.
[0166] Mutants of the Cbac enzyme were generated by error-prone PCR. Briefly, the wild type gene corresponding to the Cbac enzyme was subjected to error-prone PCR using primers specific to the Cbac gene and EconoTaq DNA polymerase (Lucigen) with the thermocyling program: 98°C 2min, 30x[98°C 30s, 55°C 20s, 72°C lOOs], 72°C lOmin, 4°C hold. In addition, error-prone PCR reactions contained 50, 100, 150 or 200 mM MnCF. PCR fragments were purified with the DNA Clean and Concentrator kit (Zymo Research), digested with Dpnl at 37 °C for 1 h, and purified again. The purified PCR fragments were inserted into Type 2A plasmids using 2x HiFi Assembly Master Mix, a two-fold molar excess of insert to plasmid, and incubation at 50°C for lh to generate Type 2A plasmids with Cbac mutants. These Cbac mutants were then assayed for their ability to generate 3-ketoC8-CoA from a C6-C0A substrate.
[0167] Cells containing Cbac mutants were cultured, lysed with lysozyme, centrifuged, and the clarified lysate was transferred to individual wells of a microtiter plate. The in vitro assay is then carried out by adding a reaction mixture of CoA primer (0.3 mM), malonyl-CoA (0.3 mM), FabG enzyme and NADPH (0.5 mM) in a buffer of 50 mM Tris, pH 8.0, to each individual well. The reactions are incubated at room temperature for 20 minutes and then stopped by the addition of acetic acid. The 3-hydroxy C8-C0A product is detected by LC-UV. Several Cbac mutants with 3-ketoC8-CoA synthase activity were identified, including Cbac(V223A), Cbac(T46M), and Cbac(S256G). These assays show that some mutants of Cbac have improved 3-ketoC8-CoA synthase activity as compared to wild type Cbac.
Example 5.
[0168] Several mutants of Cbac were assayed for their specific activity for specific CoA primers. The Cbac(V223A), Cbac(T46M), and Cbac(S256G) mutants were each assayed for specific activity for either a C2-CoA primer, a C4-CoA primer, a C6-C0A primer, or a C8-C0A primer. Wild type Cbac was also assayed for specific activity for either a C2-CoA primer, a C4- CoA primer, a C6-C0A primer, or a C8-C0A primer. An in vitro assay was performed using the respective purified mutant Cbac enzyme and carried out as described in [0037].
[0169] The results of the assays are as follows:
Figure imgf000058_0001
[0170] FIG. 4 shows specific activity for either a C2-CoA primer, a C4-CoA primer, a
C6-C0A primer, or a C8-C0A primer for each of the Cbac, Cbac(V223A), Cbac(T46M), and Cbac(S256G) mutant enzymes. These in vitro assays show that the Cbac(V223A), Cbac(T46M), and Cbac(S256G) mutants have greater specific activity for certain CoA primers. For instance, the Cbac(V223A), Cbac(T46M), and Cbac(S256G) mutants showed greater specific activity for C2-CoA primers, C4-CoA primers, and C6-C0A primers as compared to C8-C0A primers. The wild type Cbac enzyme also showed greater specific activity for C2-CoA primers, C4-CoA primers, and C6-C0A primers as compared to C8-C0A primers. The Cbac(V223A) mutant showed the greatest specific activity for C2-CoA primers, C4-CoA primers, and C6-C0A primers as compared to C8-C0A primers. These in vitro activity assays also indicated that Cbac and the Cbac(V223A), Cbac(T46M), and Cbac(S256G) mutants likely possessed higher 3- ketoC8-CoA synthase activity and lower 3-ketoCl0-CoA synthase activity.
Example 6.
[0171] The wild type Cbac enzyme and the Cbac(V223A), Cbac(T46M), and
Cbac(S256G) mutants were assayed for the production of fatty acid methyl esters (FAME). The production of FAMEs of different chain lengths (e.g. C6, methyl hexanoate; C8, methyl octanoate; C10, methyl decanoate; and C12, methyl dodecanoate) was assayed. Each individual strain that was assayed was prepared using Recombinant Strain 1 and comprised a Type 1A plasmid and a Type 2A plasmid. Each Type 2A plasmid comprised wild type Cbac or a Cbac(V223A), Cbac(T46M), or a Cbac(S256G) mutant with the PpstSIH promoter. Each individual strain was cultured and assayed as described in the small scale fermentation method -
1 ml protocol described above. The results of the assays are as follows:
Figure imgf000059_0001
[0172] The results of the assays are also shown in FIGS. 5 and 6. FIG. 5 shows the total amount of FAME produced in g/L per strain and the total amount of each of methyl hexanoate (C6), methyl octanoate (C8), methyl decanoate (C10), and methyl dodecanoate (C12) per strain. FIG. 6 shows FAME production by each strain based on the chain length of the product as a percentage of the total FAME produced by that strain. As shown in the table above and in FIGS. 5 and 6, the Cbac(V223A) strain has a higher titer for total FAME produced with a majority (71%) of the FAME produced as methyl octanoate (C8) as compared to the wild type Cbac strain. Likewise, the Cbac(T46M) and Cbac(S256G) strains also produced higher titer for total FAME produced with a majority of the FAME produced as methyl octanoate (C8)(70% and 66% respectively). The Cbac(V223A), Cbac(T46M), and Cbac(S256G) strains also produced a very small amount of the total FAME produced as methyl decanoate (C10) or methyl dodecanoate (C12). The higher production of FAME produced as methyl octanoate (C8) and the small production of FAME produced as methyl decanoate (C10) indicates that the
Cbac(V223A), Cbac(T46M), and Cbac(S256G) strains have higher specificity for production of methyl octanoate (C8) FAME. The assay showed that the Cbac(V223A), Cbac(T46M), and Cbac(S256G) strains showed specificity for methyl octanoate (C8) because in each strain more methyl octanoate (C8) was produced than other chain lengths of FAME. Also, the strains were able to produce methyl hexanoate (C6), methyl octanoate (C8), methyl decanoate (C10), and/or methyl dodecanoate (C12) with a Cbac mutant and without expressing a NphT7 or NphT7 mutant. Example 7.
[0173] Additional mutant enzymes were prepared using the Cbac(V223A) mutant. Site- directed mutagenesis was used to generate Cbac(V223A, I246P), Cbac (V223A, I246L), and Cbac (V223A, S282T) double mutants. Each of the Cbac(V223A, I246P), Cbac (V223A, I246L), and Cbac (V223A, S282T) double mutant strains was prepared using Recombinant Strain 1 and comprised a Type 1C plasmid and a Type 2A plasmid. Each Type 2A plasmid comprised the relevant Cbac double mutant with a PpstSIH promoter. The Cbac(V223A, I246P), Cbac (V223A, I246L), and Cbac (V223A, S282T) mutants were each assayed for specific activity for either a C2-CoA primer, a C4-CoA primer, a C6-C0A primer, or a C8-C0A primer. Cbac(V223A) was also assayed for specific activity for either a C2-CoA primer, a C4- CoA primer, a C6-C0A primer, or a C8-C0A primer. An in vitro assay was performed using the respective purified mutant Cbac enzyme and carried out as described in [0037].
[0174] The results of the assays are as follows:
Figure imgf000060_0001
[0175] The results of the assays are also shown in FIG. 7. FIG. 7 shows specific activity for either a C2-CoA primer, a C4-CoA primer, a C6-C0A primer, or a C8-C0A primer for each of the Cbac(V223A), Cbac(V223A, I246P), Cbac (V223A, I246L), and Cbac (V223A, S282T) mutant enzymes. These in vitro assays showed that the Cbac(V223A, I246L) double mutant enzyme had greater specific activity for C2-CoA primers, C4-CoA primers, and C6-C0A primers as compared to the Cbac (V223A) mutant enzyme. These in vitro activity assays also indicated that the Cbac(V223A, I246L) double mutant enzyme likely possessed higher 3-keto C8-C0A synthase activity and lower 3-ketoCl0-CoA synthase activity. Example 8.
[0176] The Cbac(V223A, I246L) double mutant strain was assayed for the in vivo production of fatty acid methyl esters (FAME) in different recombinant strain backgrounds. The wild type Cbac was also assayed in different recombinant strain backgrounds. Each of the Cbac(V223A, I246L) double mutant strain and the wild type Cbac strain comprised a Type 2A plasmid. Strains were constructed as follows:
Figure imgf000061_0001
[0177] Each individual strain was cultured and assayed as described in the Small scale fermentation method - shake flask protocol described above. The results of the assays are as follows:
Figure imgf000062_0001
(-) = absent in genotype
(+) = present in genotype
[0178] The results of the assays are also shown in FIG. 8. FIG. 8 shows the total amount of FAME produced in g/L per strain and the total amount of each of methyl hexanoate (C6), methyl octanoate (C8), methyl decanoate (C10), and methyl dodecanoate (C12) per strain.
These in vivo assays showed that the Cbac(V223A, I246L) double mutant strain generated higher titer of FAME than the wild type strain notwithstanding whether nphT7 and/or nphT7 LSVA was present.
EMBODIMENTS
1. A recombinant cell comprising:
a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-ketoacyl-CoA, wherein the polypeptide has SEQ ID NO: 2-9 or has at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 2-9,
wherein the recombinant cell produces a fatty acid and/or a fatty acid chain product.
2. The recombinant cell of embodiment 1, wherein the polypeptide catalyzes one or more condensations selected from the group consisting of:
a) C2-CoA with malonyl-CoA to form 3-keto C4-CoA;
b) C4-CoA with malonyl-CoA to form 3-keto C6-C0A;
c) C6-C0A with malonyl-CoA to form 3-keto C8-C0A;
d) C8-C0A with malonyl-CoA to form 3-keto ClO-CoA; and
e) ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
3. The recombinant cell of any one of embodiments 1-2, wherein the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than C8-C0A with malonyl-CoA to form 3-keto ClO-CoA.
4. The recombinant cell of any one of embodiments 1-2, wherein the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
5. The recombinant cell of any one of embodiments 1-4, wherein the fatty acid and/or fatty acid product comprises a chain length of one or more of C6, C8, and/or C10.
6. The recombinant cell of any of embodiments 1-5, wherein the recombinant cell produces more fatty acid and/or fatty acid chain product of C8 chain length than other chain lengths.
7. The recombinant cell of any one of embodiments 1-6, wherein the total fatty acid and/or total fatty acid chain product comprises 35% or less of C10 chain length and/or the total fatty acid and/or total fatty acid chain product comprises 10% or less of C12 chain length. 8. The recombinant cell of any one of embodiments 1-7, wherein the total fatty acid and/or total fatty acid chain product comprises 40% or more of C8 chain length.
9. The recombinant cell of any one of embodiments 1-8, wherein the total fatty acid and/or total fatty acid chain product comprises 45% or less of C6 chain length.
10. The recombinant cell of any one of embodiments 1-9, further comprising one or more of: a heterologous gene encoding a polypeptide with enoyl-CoA reductase activity;
a heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity;
a heterologous gene encoding a polypeptide with ester synthase activity; and
a heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity.
11. The recombinant cell of embodiment 10, wherein:
the heterologous gene encoding a polypeptide with enoyl-CoA reductase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17;
the heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SE. ID NO: 11;
the heterologous gene encoding a polypeptide with ester synthase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20; and/or
the heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 24-26.
12. The recombinant cell of any one of embodiments 1-10, wherein the polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA is under control of a constitutive promoter.
13. The recombinant cell of any one of embodiment 1-11, wherein the polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA is under control of an inducible promoter. 14. The recombinant cell of embodiment 13, wherein the polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA is under control of an inducible promoter sensitive to lowering phosphate concentration.
15. The recombinant cell of embodiment 14, wherein the polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA is under control of a PpstSIH promoter or a PphoE promoter.
16. The recombinant cell of any one of embodiments 1-15, wherein the fatty acid chain product comprises one or more products selected from the group consisting of fatty acid ester, fatty alcohol, fatty acid amide, and fatty acid amine.
17. The recombinant cell of embodiment 16, wherein the fatty acid ester comprises one or more esters selected from the group consisting of fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester, fatty acid butyl ester, fatty acid pentyl ester, and fatty acid hexyl ester.
18. The recombinant cell of any one of embodiments 1-17, wherein the recombinant cell is a fungal cell, a bacterial cell, or a plant cell.
19. The recombinant cell of embodiment 18, wherein the recombinant cell comprises an Escherichia coli species or Bacillus genus.
20. The recombinant cell of embodiment 18, wherein the recombinant cell comprises a yeast cell.
21. A ketoacyl-CoA synthase having SEQ ID NO: 10 or being at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 10, wherein amino acid residue 223 is not a valine, amino acid residue 46 is not a threonine, amino acid residue 256 is not a serine, amino acid residue 246 is not a isoleucine, and/or amino acid residue 282 is not a serine.
22. The ketoacyl-CoA synthase of embodiment 21, wherein amino acid residue 223 is an alanine.
23. The ketoacyl-CoA synthase of embodiment 21, wherein amino acid residue 46 is a methionine. 24. The ketoacyl-CoA synthase of embodiment 21, wherein amino acid residue 256 is a glycine.
25. The ketoacyl-CoA synthase of embodiment 21, wherein amino acid residue 246 is a proline or leucine.
26. The ketoacyl-CoA synthase of embodiment 21, wherein amino acid residue 282 is a threonine.
27. The ketoacyl-CoA synthase of embodiment 21, wherein the ketoacyl-CoA synthase catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA.
28. The ketoacyl-CoA synthase of embodiment 21, wherein the ketoacyl-CoA synthase catalyzes one or more condensations selected from the group consisting of:
C2-CoA with malonyl-CoA to form 3-keto C4-CoA;
C4-CoA with malonyl-CoA to form 3-keto C6-C0A;
C6-C0A with malonyl-CoA to form 3-keto C8-C0A;
C8-C0A with malonyl-CoA to form 3-keto ClO-CoA; and
ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
29. The ketoacyl-CoA synthase of any one of embodiments 21-28, wherein the ketoacyl-CoA synthase catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than C8-C0A with malonyl-CoA to form 3-keto ClO-CoA.
30. The ketoacyl-CoA synthase of any one of embodiments 21-29, wherein the ketoacyl-CoA synthase catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
31. A ketoacyl-CoA synthase having SEQ ID NO: 10 or being at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 10, comprising at least one of the following features: a) amino acid residue 223 is not a valine;
b) amino acid residue 46 is not a threonine;
c) amino acid residue 256 is not a serine;
d) amino acid residue 246 is not a isoleucine;
e) amino acid residue 282 is not a serine;
f) amino acid residue 223 is an alanine; g) amino acid residue 46 is a methionine;
h) amino acid residue 256 is a glycine;
i) amino acid residue 246 is a proline or leucine; and
j) amino acid residue 282 is a threonine.
32. A cell culture comprising:
a) a recombinant cell comprising a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA, wherein the polypeptide has at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 2-10, wherein the recombinant cell produces a fatty acid and/or a fatty acid chain product; and
b) one or more fatty acids or fatty acid chain products produced by the recombinant cell, wherein at least one of the fatty acids or fatty acid chain products is present at a concentration of at least 0.1 g/L of the cell culture.
33. The cell culture of embodiment 32, wherein the fatty acid and/or fatty acid product produced comprises a chain length of one or more of C6, C8, and/or C10.
34. The cell culture of embodiment 33, wherein the cell culture comprises more fatty acid and/or fatty acid chain product of C8 chain length than other chain lengths.
35. The cell culture of any one of embodiments 33-34, wherein total fatty acid and/or total fatty acid chain product comprises less than 30% C10.
36. The cell culture of any one of embodiments 32-35, wherein the recombinant cell further comprises one or more of: a heterologous gene encoding a polypeptide with enoyl-CoA reductase activity;
a heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity;
a heterologous gene encoding a polypeptide with ester synthase activity; and
a heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity.
37. The cell culture of embodiment 36, wherein: the heterologous gene encoding a polypeptide with enoyl-CoA reductase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17;
the heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11;
the heterologous gene encoding a polypeptide with ester synthase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20; and/or
the heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 24-26.
38. The cell culture of any one of embodiments 32-37, wherein the fatty acid product comprises one or more of fatty acid ester, fatty alcohol, fatty acid amide, and fatty acid amine.
39. The cell culture of embodiment 38, wherein the fatty acid ester comprises one or more of fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester, fatty acid butyl ester, fatty acid pentyl ester, and fatty acid hexyl ester.
40. A method of producing fatty acids and/or fatty acid chain products comprising culturing a recombinant cell in a culture medium, wherein:
the recombinant cell comprises a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA, wherein the polypeptide has SEQ ID NO: 2-10 or has at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 2-10; and
the recombinant cell is grown under conditions in which the heterologous gene is expressed.
41. The method of embodiment 40, wherein the polypeptide catalyzes one or more condensations selected from the group consisting of:
a) C2-CoA with malonyl-CoA to form 3-keto C4-CoA;
b) C4-CoA with malonyl-CoA to form 3-keto C6-C0A;
c) C6-C0A with malonyl-CoA to form 3-keto C8-C0A;
d) C8-C0A with malonyl-CoA to form 3-keto ClO-CoA; and e) ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
42. The method of any one of embodiments 40-41, wherein the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than C8-C0A with malonyl-CoA to form 3-keto ClO-CoA.
43. The method of any one of embodiments 40-42, wherein the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
44. The method of any one of embodiments 40-43, wherein the fatty acid and/or fatty acid product comprises a chain length of one or more of C6, C8, and/or C10.
45. The method of any of embodiments 40-44, wherein the recombinant cell produces more fatty acid and/or fatty acid chain product of C8 chain length than other chain lengths.
46. The method of any one of embodiments 40-45, wherein the total fatty acid and/or total fatty acid chain product comprises 35% or less of C10 chain length.
47. The method of any one of embodiments 40-46, wherein the total fatty acid and/or total fatty acid chain product comprises 41% or more of C8 chain length.
48. The method of any one of embodiments 40-47, wherein the total fatty acid and/or total fatty acid chain product comprises 44% or less of C6 chain length.
49. The method of any one of embodiments 40-48, further comprising one or more of:
a heterologous gene encoding a polypeptide with enoyl-CoA reductase activity;
a heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity;
a heterologous gene encoding a polypeptide with ester synthase activity; and
a heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity.
50. The method of embodiment 49, wherein:
the heterologous gene encoding a polypeptide with enoyl-CoA reductase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17; the heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11;
the heterologous gene encoding a polypeptide with ester synthase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20; and/or
the heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 24-26.
51. The method of any one of embodiments 40-50, wherein the fatty acid chain product comprises one or more of fatty acid ester, fatty alcohol, fatty acid amide, and fatty acid amine.
52. The method of embodiment 51, wherein the fatty acid ester comprises one or more of fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester, fatty acid butyl ester, fatty acid pentyl ester, and fatty acid hexyl ester.
53. The method of any one of embodiments 50-52, wherein the recombinant cell is a fungal cell, a bacterial cell, or a plant cell.
54. The method of embodiment 53, wherein the recombinant cell comprises an Escherichia coli species or Bacillus genus.
55. The method of embodiment 53, wherein the recombinant cell comprises a yeast cell.
56. A method of producing fatty acid methyl ester comprising culturing a recombinant cell in a culture medium, wherein:
the recombinant cell comprises a heterologous gene encoding a polypeptide having SEQ ID NO: 10 or being at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 10; and
the recombinant cell is grown under conditions in which the heterologous gene is expressed.
57. The method of embodiment 56, wherein the polypeptide comprises at least one of the following features:
a) amino acid residue 223 is an alanine; b) amino acid residue 46 is a methionine;
c) amino acid residue 256 is a glycine;
d) amino acid residue 246 is a proline or leucine; and
e) amino acid residue 282 is a threonine.
58. The method of any one of embodiment 56-57, wherein the polypeptide catalyzes one or more condensations selected from the group consisting of:
a) C2-CoA with malonyl-CoA to form 3-keto C4-CoA;
b) C4-CoA with malonyl-CoA to form 3-keto C6-C0A;
c) C6-C0A with malonyl-CoA to form 3-keto C8-C0A;
d) C8-C0A with malonyl-CoA to form 3-keto ClO-CoA; and
e) ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
59. The method of any one of embodiments 56-58, wherein the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than C8-C0A with malonyl-CoA to form 3-keto ClO-CoA.
60. The method of any one of embodiments 56-59, wherein the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
61. The method of any one of embodiments 56-60, wherein the fatty acid methyl ester comprises a chain length of one or more of C6, C8, and/or C10.
62. The method of any of embodiments 56-61, wherein the recombinant cell produces more fatty acid methyl ester of C8 chain length than other chain lengths.
63. The method of any one of embodiments 56-62, wherein the total fatty acid methyl ester comprises 35% or less of C10 chain length.
64. The method of any one of embodiments 56-63, wherein the total fatty acid methyl ester comprises 41% or more of C8 chain length.
65. The method of any one of embodiments 56-64, wherein the total fatty acid methyl ester comprises 44% or less of C6 chain length. 66. The method of any one of embodiments 56-65, wherein the recombinant cell further comprises one or more of:
a heterologous gene encoding a polypeptide with enoyl-CoA reductase activity;
a heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity;
a heterologous gene encoding a polypeptide with ester synthase activity; and
a heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity.
67. The method of embodiment 66, wherein:
the heterologous gene encoding a polypeptide with enoyl-CoA reductase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17;
the heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11;
the heterologous gene encoding a polypeptide with ester synthase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20; and/or
the heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 24-26.
68. The method of any one of embodiments 56-67, wherein the recombinant cell is a fungal cell, a bacterial cell, or a plant cell.
69. The method of embodiment 68, wherein the recombinant cell comprises an Escherichia coli species or Bacillus genus.
70. The method of embodiment 68, wherein the recombinant cell comprises a yeast cell.

Claims

1. A recombinant cell comprising:
a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA, wherein the polypeptide has SEQ ID NO: 2-9 or has at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 2-9,
wherein the recombinant cell produces a fatty acid and/or a fatty acid chain product.
2. The recombinant cell of claim 1, wherein the polypeptide catalyzes one or more condensations selected from the group consisting of:
a) C2-CoA with malonyl-CoA to form 3-keto C4-CoA;
b) C4-CoA with malonyl-CoA to form 3-keto C6-C0A;
c) C6-C0A with malonyl-CoA to form 3-keto C8-C0A;
d) C8-C0A with malonyl-CoA to form 3-keto ClO-CoA; and
e) ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
3. The recombinant cell of any one of claims 1-2, wherein the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than C8-C0A with malonyl-CoA to form 3-keto ClO-CoA.
4. The recombinant cell of any one of claims 1-2, wherein the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
5. The recombinant cell of any one of claims 1-4, wherein the fatty acid and/or fatty acid product comprises a chain length of one or more of C6, C8, and/or C10.
6. The recombinant cell of any of claims 1-5, wherein the recombinant cell produces more fatty acid and/or fatty acid chain product of C8 chain length than other chain lengths.
7. The recombinant cell of any one of claims 1-6, wherein the total fatty acid and/or total fatty acid chain product comprises 35% or less of C10 chain length and/or the total fatty acid and/or total fatty acid chain product comprises 10% or less of C12 chain length.
8. The recombinant cell of any one of claims 1-7, wherein the total fatty acid and/or total fatty acid chain product comprises 40% or more of C8 chain length
9. The recombinant cell of any one of claims 1-8, wherein the total fatty acid and/or total fatty acid chain product comprises 45% or less of C6 chain length
10. The recombinant cell of any one of claims 1-9, further comprising one or more of:
a heterologous gene encoding a polypeptide with enoyl-CoA reductase activity;
a heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity;
a heterologous gene encoding a polypeptide with ester synthase activity; and
a heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity.
11. The recombinant cell of claim 10, wherein:
the heterologous gene encoding a polypeptide with enoyl-CoA reductase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17;
the heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11;
the heterologous gene encoding a polypeptide with ester synthase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20; and/or
the heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 24-26.
12. The recombinant cell of any one of claims 1-10, wherein the polypeptide that catalyzes the condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA is under control of a constitutive promoter.
13. The recombinant cell of any one of claim 1-11, wherein the polypeptide that catalyzes the condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA is under control of an inducible promoter
14. The recombinant cell of claim 13, wherein the polypeptide that catalyzes the condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA is under control of an inducible promoter sensitive to lowering phosphate concentration
15. The recombinant cell of claim 14, wherein the polypeptide that catalyzes the condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA is under control of a PpstSIH promoter or a PphoE promoter.
16. The recombinant cell of any one of claims 1-15, wherein the fatty acid chain product comprises one or more products selected from the group consisting of fatty acid ester, fatty alcohol, fatty acid amide, and fatty acid amine.
17. The recombinant cell of claim 16, wherein the fatty acid ester comprises one or more esters selected from the group consisting of fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester, fatty acid butyl ester, fatty acid pentyl ester, and fatty acid hexyl ester.
18. The recombinant cell of any one of claims 1-17, wherein the recombinant cell is a fungal cell, a bacterial cell, or a plant cell.
19. The recombinant cell of claim 18, wherein the recombinant cell comprises an Escherichia coli species or Bacillus genus.
20. The recombinant cell of claim 18, wherein the recombinant cell comprises a yeast cell.
21. A ketoacyl-CoA synthase having SEQ ID NO: 10 or being at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 10, wherein amino acid residue 223 is not a valine, amino acid residue 46 is not a threonine, amino acid residue 256 is not a serine, amino acid residue 246 is not a isoleucine, and/or amino acid residue 282 is not a serine.
22. The ketoacyl-CoA synthase of claim 21, wherein amino acid residue 223 is an alanine.
23. The ketoacyl-CoA synthase of claim 21, wherein amino acid residue 46 is a methionine.
24. The ketoacyl-CoA synthase of claim 21, wherein amino acid residue 256 is a glycine.
25. The ketoacyl-CoA synthase of claim 21, wherein amino acid residue 246 is a proline or leucine.
26. The ketoacyl-CoA synthase of claim 21, wherein amino acid residue 282 is a threonine.
27. The ketoacyl-CoA synthase of claim 21, wherein the ketoacyl-CoA synthase catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA.
28. The ketoacyl-CoA synthase of claim 21, wherein the ketoacyl-CoA synthase catalyzes one or more condensations selected from the group consisting of:
C2-CoA with malonyl-CoA to form 3-keto C4-CoA;
C4-CoA with malonyl-CoA to form 3-keto C6-C0A;
C6-C0A with malonyl-CoA to form 3-keto C8-C0A;
C8-C0A with malonyl-CoA to form 3-keto ClO-CoA; and
ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
29. The ketoacyl-CoA synthase of any one of claims 21-28, wherein the ketoacyl-CoA synthase catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than C8-C0A with malonyl-CoA to form 3-keto ClO-CoA.
30. The ketoacyl-CoA synthase of any one of claims 21-29, wherein the ketoacyl-CoA synthase catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
31. A ketoacyl-CoA synthase having SEQ ID NO: 10 or being at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 10, comprising at least one of the following features: a) amino acid residue 223 is not a valine;
b) amino acid residue 46 is not a threonine;
c) amino acid residue 256 is not a serine;
d) amino acid residue 246 is not a isoleucine;
e) amino acid residue 282 is not a serine;
f) amino acid residue 223 is an alanine;
g) amino acid residue 46 is a methionine;
h) amino acid residue 256 is a glycine;
i) amino acid residue 246 is a proline or leucine; and
j) amino acid residue 282 is a threonine.
32. A cell culture comprising:
a) a recombinant cell comprising a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA, wherein the polypeptide has at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 2-10, wherein the recombinant cell produces a fatty acid and/or a fatty acid chain product; and
b) one or more fatty acids or fatty acid chain products produced by the recombinant cell, wherein at least one of the fatty acids or fatty acid chain products is present at a concentration of at least 0.1 g/L of the cell culture.
33. The cell culture of claim 32, wherein the fatty acid and/or fatty acid product produced comprises a chain length of one or more of C6, C8, and/or C10.
34. The cell culture of claim 33, wherein the cell culture comprises more fatty acid and/or fatty acid chain product of C8 chain length than other chain lengths.
35. The cell culture of any one of claims 33-34, wherein total fatty acid and/or total fatty acid chain product comprises less than 30% C10.
36. The cell culture of any one of claims 32-35, wherein the recombinant cell further comprises one or more of:
a heterologous gene encoding a polypeptide with enoyl-CoA reductase activity;
a heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity;
a heterologous gene encoding a polypeptide with ester synthase activity; and
a heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity.
37. The cell culture of claim 36, wherein:
the heterologous gene encoding a polypeptide with enoyl-CoA reductase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17;
the heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11;
the heterologous gene encoding a polypeptide with ester synthase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20; and/or
the heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 24-26.
38. The cell culture of any one of claims 32-37, wherein the fatty acid product comprises one or more of fatty acid ester, fatty alcohol, fatty acid amide, and fatty acid amine.
39. The cell culture of claim 38, wherein the fatty acid ester comprises one or more of fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester, fatty acid butyl ester, fatty acid pentyl ester, and fatty acid hexyl ester.
40. A method of producing fatty acids and/or fatty acid chain products comprising culturing a recombinant cell in a culture medium, wherein:
the recombinant cell comprises a heterologous gene encoding a polypeptide that catalyzes condensation of acyl-CoA with malonyl-CoA to form 3-keto acyl-CoA, wherein the polypeptide has SEQ ID NO: 2-10 or has at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 2-10; and
the recombinant cell is grown under conditions in which the heterologous gene is expressed.
41. The method of claim 40, wherein the polypeptide catalyzes one or more condensations selected from the group consisting of:
a) C2-CoA with malonyl-CoA to form 3-keto C4-CoA;
b) C4-CoA with malonyl-CoA to form 3-keto C6-C0A;
c) C6-C0A with malonyl-CoA to form 3-keto C8-C0A;
d) C8-C0A with malonyl-CoA to form 3-keto ClO-CoA; and
e) ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
42. The method of any one of claims 40-41, wherein the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than C8-C0A with malonyl-CoA to form 3-keto ClO-CoA.
43. The method of any one of claims 40-42, wherein the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
44. The method of any one of claims 40-43, wherein the fatty acid and/or fatty acid product comprises a chain length of one or more of C6, C8, and/or C10.
45. The method of any of claims 40-44, wherein the recombinant cell produces more fatty acid and/or fatty acid chain product of C8 chain length than other chain lengths.
46. The method of any one of claims 40-45, wherein the total fatty acid and/or total fatty acid chain product comprises 35% or less of C10 chain length.
47. The method of any one of claims 40-46, wherein the total fatty acid and/or total fatty acid chain product comprises 41% or more of C8 chain length.
48. The method of any one of claims 40-47, wherein the total fatty acid and/or total fatty acid chain product comprises 44% or less of C6 chain length.
49. The method of any one of claims 40-48, further comprising one or more of:
a heterologous gene encoding a polypeptide with enoyl-CoA reductase activity;
a heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity;
a heterologous gene encoding a polypeptide with ester synthase activity; and
a heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity.
50. The method of claim 49, wherein:
the heterologous gene encoding a polypeptide with enoyl-CoA reductase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17;
the heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11;
the heterologous gene encoding a polypeptide with ester synthase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20; and/or
the heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 24-26.
51. The method of any one of claims 40-50, wherein the fatty acid chain product comprises one or more of fatty acid ester, fatty alcohol, fatty acid amide, and fatty acid amine.
52. The method of claim 51, wherein the fatty acid ester comprises one or more of fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester, fatty acid butyl ester, fatty acid pentyl ester, and fatty acid hexyl ester.
53. The method of any one of claims 50-52, wherein the recombinant cell is a fungal cell, a bacterial cell, or a plant cell.
54. The method of claim 53, wherein the recombinant cell comprises an Escherichia coli species or Bacillus genus.
55. The method of claim 53, wherein the recombinant cell comprises a yeast cell.
56. A method of producing fatty acid methyl ester comprising culturing a recombinant cell in a culture medium, wherein:
the recombinant cell comprises a heterologous gene encoding a polypeptide having SEQ ID NO: 10 or being at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 10; and
the recombinant cell is grown under conditions in which the heterologous gene is expressed.
57. The method of claim 56, wherein the polypeptide comprises at least one of the following features:
a) amino acid residue 223 is an alanine;
b) amino acid residue 46 is a methionine;
c) amino acid residue 256 is a glycine;
d) amino acid residue 246 is a proline or leucine; and/or
e) amino acid residue 282 is a threonine.
58. The method of any one of claim 56-57, wherein the polypeptide catalyzes one or more condensations selected from the group consisting of:
a) C2-CoA with malonyl-CoA to form 3-keto C4-CoA;
b) C4-CoA with malonyl-CoA to form 3-keto C6-C0A;
c) C6-C0A with malonyl-CoA to form 3-keto C8-C0A; d) C8-C0A with malonyl-CoA to form 3-keto ClO-CoA; and/or
e) ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
59. The method of any one of claims 56-58, wherein the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than C8-C0A with malonyl-CoA to form 3-keto ClO-CoA.
60. The method of any one of claims 56-59, wherein the polypeptide catalyzes condensation of more C6-C0A with malonyl-CoA to form 3-keto C8-C0A than ClO-CoA with malonyl-CoA to form 3-keto Cl2-CoA.
61. The method of any one of claims 56-60, wherein the fatty acid methyl ester comprises a chain length of one or more of C6, C8, and/or C10.
62. The method of any of claims 56-61 , wherein the recombinant cell produces more fatty acid methyl ester of C8 chain length than other chain lengths.
63. The method of any one of claims 56-62, wherein the total fatty acid methyl ester comprises 35% or less of C10 chain length.
64. The method of any one of claims 56-63, wherein the total fatty acid methyl ester comprises 41% or more of C8 chain length.
65. The method of any one of claims 56-64, wherein the total fatty acid methyl ester comprises 44% or less of C6 chain length.
66. The method of any one of claims 56-65, wherein the recombinant cell further comprises one or more of:
a heterologous gene encoding a polypeptide with enoyl-CoA reductase activity;
a heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity;
a heterologous gene encoding a polypeptide with ester synthase activity; and
a heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity.
67. The method of claim 66, wherein:
the heterologous gene encoding a polypeptide with enoyl-CoA reductase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17;
the heterologous gene encoding a polypeptide with bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11;
the heterologous gene encoding a polypeptide with ester synthase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20; and/or
the heterologous gene encoding a polypeptide with acetyl-CoA carboxylase activity comprises at least 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 24-26.
68. The method of any one of claims 56-67, wherein the recombinant cell is a fungal cell, a bacterial cell, or a plant cell.
69. The method of claim 68, wherein the recombinant cell comprises a Escherichia coli species or Bacillus genus.
70. The method of claim 68, wherein the recombinant cell comprises a yeast cell.
PCT/US2019/035757 2018-06-07 2019-06-06 Methods and genetically modified cells for producing c8 fatty acids and fatty acid chain products WO2019236819A1 (en)

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