GENETICALLY MODIFIED CELLS THAT PRODUCE C6-C10 FATTY ACID DERIVATIVES

Abstract

Genes encoding mutant 3-ketoacyl-CoA synthases are introduced into host cells. Certain of the mutants enhance the production of shorter-chain fatty acids and derivatives by the cell than do the wild-type (unmutated) enzymes. In other cases, the chain length is not significantly affected, but productivity is enhanced. In specific cases, both a shift toward lower chain length and higher productivity is seen. Cells producing the mutant 3-ketoacyl-CoA synthases are especially suitable for producing C6-C10 fatty acids and derivatives.

Claims

1. A ketoacyl-CoA synthase having SEQ ID NO:119 or being at least 80% identical to SEQ ID NO:119, wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 296 of SEQ ID NO: 119 is alanine, valine, isoleucine, or leucine.

2. The ketoacyl-CoA synthase of claim 1, comprising at least one of the following features i) to xliii): i) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 18 of SEQ ID NO:119 is alanine; ii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 22 of SEQ ID NO:119 is methionine; iii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 30 of SEQ ID NO:119 is alanine; iv) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 38 of SEQ ID NO:119 is valine; v) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 39 of SEQ ID NO:119 is valine; vi) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 51 of SEQ ID NO:119 is alanine, cysteine, aspartic acid, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine; vii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 54 of SEQ ID NO:119 is valine; viii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 69 of SEQ ID NO:119 is valine; ix) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 83 of SEQ ID NO:119 is asparagine; x) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 94 of SEQ ID NO:119 is threonine, leucine, glutamic acid, or alanine; xi) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 111 of SEQ ID NO:119 is cysteine; xii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 116 of SEQ ID NO:119 is glycine; xiii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 127 of SEQ ID NO:119 is a threonine; xiv) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 130 of SEQ ID NO:119 is glycine; xv) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 152 of SEQ ID NO:119 is cysteine, leucine, methionine or threonine; xvi) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 154 of SEQ ID NO:119 is glycine; xvii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 178 of SEQ ID NO:119 is leucine or threonine; xviii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 190 of SEQ ID NO:119 is phenylalanine or tyrosine; xix) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 210 of SEQ ID NO:119 is valine; xx) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 223 of SEQ ID NO:119 is histidine; xxi) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 231 of SEQ ID NO:119 is isoleucine; xxii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 232 of SEQ ID NO:119 is valine; xxiii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 236 of SEQ ID NO:119 is leucine or methionine; xxiv) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 241 of SEQ ID NO:119 is proline; xxv) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 268 of SEQ ID NO:119 is alanine; xxvi) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 271 of SEQ ID NO:119 is isoleucine; xxvii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 274 of SEQ ID NO:119 is glutamic acid; xxviii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 278 of SEQ ID NO:119 is arginine, glutamic acid, aspartic acid, glutamine; xxix) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 280 of SEQ ID NO:119 is isoleucine; xxx) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 282 of SEQ ID NO:119 is glycine; xxxi) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 184 of SEQ ID NO:119 is isoleucine, leucine, methionine or valine; xxxii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 302 of SEQ ID NO:119 is threonine; xxxiii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 312 of SEQ ID NO:119 is aspartic acid; xxxiv) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 313 of SEQ ID NO:119 is glutamic acid or methionine; xxxv) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 315 of SEQ ID NO:119 is lysine; xxxvi) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 317 of SEQ ID NO:119 is alanine; xxxvii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 322 of SEQ ID NO:119 is glycine; xxxviii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 328 of SEQ ID NO:119 is glycine; xxxix) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 329 of SEQ ID NO:119 is glycine; xl) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 344 of SEQ ID NO:119 is glutamic acid, aspartic acid or asparagine; xli) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 356 of SEQ ID NO:119 is glycine or serine; xlii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 368 of SEQ ID NO:119 is arginine; and xliii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 370 of SEQ ID NO:119 is threonine.

3. The ketoacyl-CoA synthase of claim 1, wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 296 of SEQ ID NO:119 is alanine.

4.-9. (canceled)

10. The 3-ketoacyl-CoA synthase of claim 1, wherein the amino acid sequence is at least 80% identical to SEQ ID NO:73.

11.-30. (canceled)

31. A method for making one or more compounds having a straight-chain alkyl group, the method comprising culturing a genetically modified cell in a fermentation medium and recovering the compound(s) having a straight-chain alkyl group from the fermentation medium; wherein the genetically modified cell comprises a heterologous nucleic acid sequence encoding the ketoacyl-CoA synthase of claim 1.

32. (canceled)

33. A method for making one or more compounds having a straight-chain alkyl group, the method comprising culturing a genetically modified cell in a fermentation medium and recovering the compound(s) having a straight-chain alkyl group from the fermentation medium; wherein the genetically modified cell comprises a heterologous nucleic acid sequence encoding a ketoacyl-CoA synthase having SEQ ID NO:119 or being at least 80% identical to SEQ ID NO:119, wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 184 of SEQ ID NO:119 is isoleucine, leucine, methionine, or valine.

34. The method of claim 33, wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 184 of SEQ ID NO:119 is isoleucine.

35. The method of claim 33, wherein the ketoacyl-CoA synthase further comprising at least one of the following features: the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 236 of SEQ ID NO:119 is leucine; the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 268 of SEQ ID NO:119 is alanine; the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 296 of SEQ ID NO:119 is alanine; the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 328 of SEQ ID NO:119 is glycine; the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 317 of SEQ ID NO:119 is alanine; and combinations thereof.

36. The method of claim 31, wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 317 of SEQ ID NO:119 is alanine.

37. The method of claim 31, wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 236 of SEQ ID NO:119 is leucine.

38. The method of claim 31, wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 268 of SEQ ID NO:119 is alanine.

39. The method of claim 31, wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 296 of SEQ ID NO: 119 is alanine.

40. The method of claim 31, wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 328 of SEQ ID NO: 119 is glycine.

41. The method of claim 31, wherein the amino acid sequence is SEQ ID NO:135.

42. A ketoacyl-CoA synthase having SEQ ID NO: 42 or being at least 80% identical to SEQ ID NO: 42, wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 186 of SEQ ID NO: 42 is isoleucine, leucine, methionine, cysteine, valine, glutamine, phenylalanine, aspartate, asparagine, or tyrosine.

43. The ketoacyl-CoA synthase of claim 42, wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid reside 186 of SEQ ID NO: 42 is isoleucine.

44. The ketoacyl-CoA synthase of claim 42, wherein the ketoacyl-CoA synthase is at least 90% identical to SEQ ID NO: 43.

45. The ketoacyl-CoA synthase of claim 42, comprising at least one of the following features i) to iv): i) an amino acid residue of the ketoacyl-CoA synthase aligns with amino acid residue 241 of SEQ ID NO: 42 is glutamate, leucine, phenylalanine, tyrosine, methionine, or aspartate; ii) an amino acid residue of the ketoacyl-CoA synthase aligns with amino acid residue 239 of SEQ ID NO: 42 is aspartate, asparagine, or glutamine; iii) an amino acid residue of the ketoacyl-CoA synthase aligns with amino acid residue 246 of SEQ ID NO: 42 is arginine or lysine; or iv) an amino acid residue of the ketoacyl-CoA synthase aligns with amino acid residue 243 of SEQ ID NO: 42 is glutamate.

46. A genetically modified cell comprising a heterologous nucleic acid sequence encoding the ketoacyl-CoA synthase of claim 42.

47. A method for making one or more compounds having a straight-chain alkyl group, the method comprising culturing the genetically modified cell of claim 46 in a fermentation medium and recovering the compound(s) having a straight-chain alkyl group from the fermentation medium.

Description

EXAMPLES

[0159] The following host E. coli strains are used in the following examples as indicated.

[0160] Host 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, Conn.), having the following additional genetic modifications:

TABLE-US-00004 Designation Description ΔldhA::frt Deletion of native lactate dehydrogenase ΔpflB::frt Deletion of native pyruvate formate lyase ΔmgsA::frt Deletion of methylglyoxal synthase ΔpoxB::frt Deletion of pyruvate oxidase Δpta-ack::frt Deletion of phosphotransacetylase and acetate kinase Δtig:frt Deletion of trigger factor protein ΔatoDAEB::frt Deletion to disrupt short-chain poly-(R)-3-hydroxybuty- rate synthesis ΔfadD:frt Deletion of native acyl-CoA synthase ΔtesB::frt Deletion of native thioesterase ΔyciA::frt Deletion of native thioesterase fabI(ts)_zeo Insertion of E. coli fabI gene encoding a protein having SEQ ID NO 85, except the serine at position 241 replaced by phenylalanine (S241F modification) and a zeomyin resistance marker at the 3′ end and deletion of the wildtype fabI gene.

[0161] Host Strain 2 is a mutant of the BW 25113 E. coli strain with the following additional genetic modifications:

TABLE-US-00005 Designation Description ΔldhA::frt Deletion of native lactate dehydrogenase ΔpflB::frt Deletion of native pyruvate formate lyase ΔmgsA::frt Deletion of methylglyoxal synthase ΔpoxB::frt Deletion of pyruvate oxidase Δpta-ack::frt Deletion of phosphotransacetylase and acetate kinase Δtig:frt Deletion of trigger factor protein ΔatoDAEB::frt Deletion to disrupt short-chain poly-(R)-3-hydroxybuty- rate synthesis ΔfadD::frt Deletion of native acyl-CoA synthase ΔtesB::frt Deletion of native thioesterase ΔyciA::frt Deletion of native thioesterase Δadhe Deletion of native aldehyde-alcohol dehydrogenase fabI(ts) Insertion of modified E. coli fabI gene encoding an enzyme having SEQ ID NO. 85, with the serine at position 241 replaced by phenylalanine (S241F modification), and deletion of the wildtype fabI gene P.sub.pstsIH-nphT7- Insertion of a gene encoding for Streptomyces Sp. CL190 ter-TT-loxP acetoacetyl-CoA synthase (NphT7, SEQ. ID. NO. 83) and a gene encoding for Treponema denticola enoyl-CoA reductase (ter, SEQ. ID. NO. 84) under control of E. coli pstsIH promoter and an E. coli terminator at locus of native adhE gene

[0162] Host Strain 3 is a mutant of the BW 25113 E. coli strain with the following genetic modifications:

TABLE-US-00006 Designation Description ΔldhA::frt Deletion of native lactate dehydrogenase ΔpflB::frt Deletion of native pyruvate formate lyase ΔmgsA::frt Deletion of methylglyoxal synthase ΔpoxB::frt Deletion of pyruvate oxidase Δpta-ack::frt Deletion of phosphotransacetylase and acetate kinase Δtig::frt Deletion of trigger factor protein ΔatoDAEB::frt Deletion to disrupt short-chain poly-(R)-3-hydroxybuty- rate synthesis ΔfadD::frt Deletion of native acyl-CoA synthase ΔtesB::frt Deletion of native thioesterase ΔyciA::frt Deletion of native thioesterase Δadhe Deletion of native aldehyde-alcohol dehydrogenase P.sub.pstsIH-nphT7- Insertion of a gene encoding for Streptomyces Sp. CL190 ter-TT-loxP acetoacetyl-CoA synthase (NphT7, SEQ. ID. NO. 83) and a gene encoding for Treponema denticola enoyl-CoA reductase (ter, SEQ. ID. NO. 84) under control of E. coli pstsIH promoter and an E. coli terminator at locus of native adhE gene

[0163] Production of 3-ketoacyl-CoA synthase genes and mutants 3-ketoacyl-CoA synthase genes are synthesized based on published sequence information for various wild type 3-ketoacyl-CoA synthase genes. Site-specific mutants of the synthesized 3-ketoacyl-CoA synthase genes are generated by oligonucleotide-directed mutagenesis. The sources of the wild-type genes and 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:

TABLE-US-00007 Enzyme Encoded Source Species Designation (wild-type strain) Acinetobacter schindleri CIP Asch SEQ ID NO. 8 107287 Acinetobacter schindleri Asch-2 SEQ ID NO. 86 NIPH 900 Acinetobacter johnsonii Ajoh-2 SEQ ID NO. 35 SH046 Acinetobacter lwoffii SH145 Alwo SEQ ID NO. 88 Acinetobacter sp. NIPH 713 ANIP71 SEQ ID NO. 89 Pseudomonas stutzeri ATCC Pstu SEQ ID NO. 90 17588 Alishewanella agri BL06 Aagr SEQ ID NO. 91

[0164] In each case, the 3-ketoacyl-CoA synthase gene is fused to a DNA sequence encoding a protein fragment containing 6 histidine residues and a protease recognization site, and incorporated into a pET plasmid having a ColE1 origin of replication and a kanamycin resistance marker.

[0165] Mutations to the amino acid residues encoded by the wild-type genes are designated herein by the shorthand designation for the wild-type strain, followed in parenthesis 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 “Asch(T184I)” indicates that a threonine (T) at amino acid residue position 184 in the wild type Acinetobacter schindleri CIP 107287 enzyme has been replaced with an isoleucine (I).

[0166] Production of multiply-mutated 3-ketoacyl-CoA synthase genes. Multi-mutated genes are prepared from wild-type or mutated (parent) 3-ketoacyl-CoA synthase genes using error-prone PCR as the mutagenic method. Error-prone PCR of the 3-ketoacyl-CoA synthase gene is carried out using primers having SEQ ID NO. 94 and SEQ ID NO. 95 and EconoTaq DNA polymerase (Lucigen) with the thermocyling program: 94° C. 2 min, 30×[94° C. 20 s, 55° C. 20 s, 72° C. 72 s], 72° C. 10 min, 4° C. hold. In addition, error-prone PCR reactions contain 50, 100, 150 or 200 μM MnCl.sub.2. PCR fragments are purified with the DNA Clean and Concentrator kit (Zymo Research), digested with DpnI at 37° C. for 1 h, and purified again. The plasmid and insert are assembled using 2×HiFi Assembly Master Mix, a two-fold molar excess of insert to plasmid and incubation at 50° C. for 1 h.

[0167] The amino acid sequences of the gene produced by multiply-mutated genes are designated by a shorthand as described above, with the mutations listed sequentially. For example, “Asch(T184I,S328V)” indicates that a threonine (T) at amino acid residue position 184 in the wild type Acinetobacter schindleri CIP 107287 enzyme has been replaced with an isoleucine (I) and a serine at position 328 has been replaced with valine.

Production of Mutant E. coli Strains

[0168] Mutant E. coli strains are prepared using standard electroporation methods. In each case, the host strain is transformed with a “Type 1” plasmid and a “Type 2” plasmid as described below.

[0169] Type 1 plasmids are pACYC plasmids containing the p15a origin of replication and chloramphenicol resistance marker. The Type 1 plasmids used in the following examples are:

[0170] Type 1A: 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. 82), an E. coli bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase (fadB) gene and a T. denticola enoyl-CoA (ter) gene cassette, all under a native E. coli pstsIH promoter and a native E. coli terminator. This plasmid also contains a Hahella chejuensis ester synthase gene fused to a DNA sequence encoding a protein fragment containing 6 histidine residues and a protease recognition site under an E. coli phoE promoter, and an ACC (acetyl-CoA carboxylase) cassette including fused E. coli accA and accD genes with a E. coli tpiA promoter and a cassette including the E. coli accB and E. coli accC genes under an E. coli rpiA promoter.

[0171] Type 1B: this plasmid includes a mutated Streptomyces sp. gene encoding for an nphT7 enzyme having 11475 and F217V modifications (the “SV” NphT7 mutant, SEQ ID NO. 96) and a T. denticola enoyl-CoA (ter) gene cassette, under a native E. coli pstsIH promoter and a native E. coli terminator. The plasmid further contains an E. coli bifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase (fadB) gene under a native E. coli pstsIH promoter; and a Hahella chejuensis ester synthase gene fused to a DNA sequence encoding a protein fragment containing 6 histidine residues and a protease recognition site under an E. coli phoE promoter.

[0172] Plasmid type 2A includes a ColE1 origin of replication, a kanamycin resistance marker and 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), the latter of which is a low phosphate inducible type. The 3-ketoacyl-CoA synthase and the promoter for the 3-ketoacyl-CoA synthase gene are as indicated in the specific examples below.

[0173] Plasmid 2B includes a ColE1 origin of replication, a kanamycin resistance marker and the 3-ketoacyl-CoA synthase gene to be evaluated (in some cases without the His-tag) under the PpstsIH (SEQ ID NO. 118) or PphoE promoter (SEQ ID NO. 117) and an E. coli terminator. The 3-ketoacyl-CoA synthase and the promoter for its gene are as indicated in the specific examples below.

Small-Scale Fermentation Method

[0174] A culture of synthetic medium containing salts, glucose, NH.sub.4Cl, and supplemented with vitamins, yeast extract, 35 μg/ml kanamycin and 20 μg/ml chloramphenicol is inoculated with the strain to be tested and grown overnight at 30° C. The OD.sub.600 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 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 1-ml aliquots dispensed into triplicate 16-mm glass tubes containing 64 μL of heptadecane or methyl tetradecanoate. This represents a limited phosphate medium that promotes the activity of a low phosphate inducible promoter such as the E. coli PphoE promoter (SEQ ID NO. 117). 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.

Small-Scale Fermentation Method Used for Examples 80-116

[0175] A culture of synthetic medium containing salts, glucose, NH.sub.4Cl, and supplemented with vitamins, yeast extract, 35 μg/ml kanamycin and 20 μg/ml chloramphenicol is inoculated with the strain to be tested and grown overnight at 32° C. The OD.sub.600 of a 1:10 dilution of this culture is determined in order to inoculate a Seed 2 flask to a final OD.sub.600 of 0.3. Seed 2 flasks contain 25-30 ml synthetic medium containing salts, glucose, NH.sub.4Cl, and supplemented with vitamins, yeast extract, 35 μg/ml kanamycin and 20 μg/ml chloramphenicol, 0-2% methanol. Seed 2 flasks are incubated at 32° C. for 6-7 hours.

[0176] The OD.sub.600 of a 1:10 dilution of this culture is determined in order to inoculate a production flask to a final OD.sub.600 of 0.01-0.025. Production flasks contain 25 ml synthetic medium containing salts, glucose, NH.sub.4Cl, and supplemented with vitamins, yeast extract, 35 μg/ml kanamycin and 20 μg/ml chloramphenicol, 1.25-2.5 mM phosphate, 0-2% methanol, 3-6 g/l glucose, 15-40 g/l glycerol. This represents a limited phosphate medium that promotes the activity of a low phosphate inducible promoter such as the E. coli PphoE promoter (SEQ ID NO. 117). Production flasks are incubated at 32° C. When phosphate is depleted the following additions are made: 2 ml methyl myristate, 1 ml 12.5% 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 at 24 hours are taken and extracted with 0.1% HCL in MTBE (Methyl tert butyl ether) and the extracts are analyzed for fatty acid esters by gas chromatography.

Small-Scale Fermentation Method Used for Examples 117-148

[0177] A shallow 96-well plate of synthetic medium containing salts, glucose, NH.sub.4Cl, and supplemented with vitamins, yeast extract, 35 μg/ml kanamycin and 20 μg/ml chloramphenicol is inoculated with the strain to be tested and grown overnight at 30° C. The culture from the shallow 96-well plate is used to inoculate a deep well production plate with 2-3 μl per well. Each well of the production plate contains 400 μl synthetic medium containing salts, glucose, NH.sub.4Cl, and supplemented with vitamins, 35 μg/ml kanamycin and 20 μg/ml chloramphenicol, 1.25 mM phosphate, 2% methanol, 3 g/l glucose, 30 g/l glycerol. This represents a limited phosphate medium that promotes the activity of a low phosphate inducible promoter such as the E. coli PphoE promoter (SEQ ID NO. 117). Production plates are incubated at 32° C. When phosphate is depleted, 26 μl methyl myristate is added to each well. Plates are incubated at 35° C. for a further 20 hours. Samples at 20 hours are taken and diluted with acetonitrile and analyzed for fatty acid esters by gas chromatography.

Examples 1-15 and Comparative Samples A-C—Asch(T184I) Variants

[0178] Mutant E. coli strain Examples 1 and 2 contain a modified Asch gene encoding a 3-ketoacyl-CoA synthase Asch(T184I) (SEQ ID NO. 9). Comparative Samples A and B contain the wild-type gene (encoding for SEQ ID NO. 8). Details of strain construction are as follows:

TABLE-US-00008 Host Plasmid 1 NphT7 Plasmid 2 3-ketoacyl-CoA synthase Designation Strain Type mutant Type Promoter Mutant Ex. 1 1 1B SV 2A PpstsIH Asch(T184I) Comp. A* 1 1B SV 2A PpstsIH wt Asch Ex. 2 2 1A LSVA 2A PphoE Asch(T184I) Comp. B* 2 1A LSVA 2A PphoE wt Asch *Not an example of this invention.

[0179] Each of Examples 1 and 2 and Comparative Samples are cultured to produce fatty acid methyl esters using the small-scale method described above. Total fatty acid methyl ester (FAME) and amounts of C6, C8 and C10 fatty acid esters produced are as indicated in the following table. Selectivities to C8 and C10 fatty acid esters are indicated under the respective “%” columns; these are calculated as the titer of the C8 or C10 fatty acid ester over the total fatty acid ester production. Amounts of higher- and lower-carbon number fatty acid esters are not shown separately, but are included in the total FAME production value indicated.

TABLE-US-00009 Total FAME C6 C8 C10 3-ketoacyl-CoA production production production production Designation synthase (g/L) g/L) g/L % g/L % Ex. 1 Asch(T184I) 1.17 0.21 0.75 64.1 0.22 18.5 Comp. A* Wt** Asch 1.43 0.15 0.01 <1 1.27 88.5 Ex. 2 Asch(T184I) 1.21 0.35 0.68 56.2 0.14 11.4 Comp. B* Wt** Asch 2.13 0.27 0.07 3.1 1.75 82.1 *Not an example of this invention. **“Wt” in this and subsequent tables indicates the wildtype enzyme.

[0180] The wildtype Asch enzyme produces about 82% C10 fatty acid methyl esters, with minimal amounts of the C8 esters. By changing the threonine at position 184 to isoleucine, production is shifted from almost exclusively C10 fatty acid esters to mainly C8 fatty acid esters, with some increased selectivity toward C6 fatty acid esters also being seen. The mutant strain Examples 1 and 2 are useful for producing a mixture of fatty acid esters enriched in the C8 esters.

[0181] Mutant E. coli strain Examples 3-5 similarly contain a modified Asch gene encoding a mutated 3-ketoacyl-CoA synthase (Asch(T184L), Asch(T184M) or Asch(T184V)). Comparative Sample C again contains the wild-type gene. Details of strain construction are as follows:

TABLE-US-00010 Host Plasmid 1 NphT7 Plasmid 2 3-ketoacyl-CoA synthase Designation Strain Type mutant Type Promoter Mutant Ex. 3 1 1B SV 2A PpstsIH Asch(T184L) Ex. 4 1 1B SV 2A PpstsIH Asch(T184M) Ex. 5 1 1B SV 2A PpstsIH Asch(T184V) Comp. C* 1 1B SV 2A PpstsIH wt Asch *Not an example of this invention.

[0182] Each of Examples 3-5 and Comparative Sample C are cultivated to produce fatty acid ethyl esters using the small-scale method described above. Total fatty acid ethyl ester (FAEE) and amounts of C6, C8 and C10 fatty acid esters produced are as indicated in the following table, as are selectivities to C8 and C10 fatty acid esters. Amounts of higher- and lower-carbon number fatty acid esters are not shown separately, but are included in the total FAEE production value indicated.

TABLE-US-00011 Total FAEE C6 C8 C10 3-ketoacyl-CoA production production production production Designation synthase (g/L) g/L) g/L % g/L % Ex. 3 Asch(T184L) 0.88 0.30 0.22 24.4 0.37 41.6 Ex. 4 Asch(T184M) 0.75 0.51 0.23 30.1 0.01 1.7 Ex. 5 Asch(T184V) 1.05 0.19 0.51 48.2 0.36 33.9 Comp. C wt Asch 1.22 0.13 0.01 0.7 1.09 88.7 *Not an example of this invention.

[0183] As seen above with Comparative Samples A and B, the wildtype Asch enzyme leads to mainly C10 fatty acid ester production. The T184L, T184M and T184V variations all shift production from C10 fatty acid esters to mainly C6 and C8 fatty acid esters. The T184M variation in Example 4 reduces C10 fatty acid ester production to less than 2%. Selectivity to C8 fatty acid esters is almost 50% for the T184V variation.

[0184] Mutant E. coli strains Examples 6-15 similarly contain a mutated Asch gene encoding a mutated 3-ketoacyl-CoA synthase with multiple mutations as indicated in the following table. Details of strain construction are as follows:

TABLE-US-00012 Host Plasmid 1 NphT7 Plasmid 2 3-ketoacyl-CoA synthase Designation Strain Type mutant Type Promoter Mutant Ex. 6 1 1A LSVA 2A PpstsIH Asch(N152T, T184I) Ex. 7 1 1A LSVA 2A PpstsIH Asch(N152L, T184I) Ex. 8 1 1A LSVA 2A PpstsIH Asch(N152M, T184I) Ex. 9 1 1A LSVA 2A PpstsIH Asch(N152C, T184I) Ex. 10 1 1A LSVA 2A PpstsIH Asch(A69V, T184I, S328G) Ex. 11 1 1A LSVA 2A PpstsIH Asch(G111C, T184I, S328G) Ex. 12 1 1A LSVA 2A PpstsIH Asch(D39V, T184I, S328G) Ex. 13 1 1A LSVA 2A PpstsIH Asch(D39V, G111C, T184I, S328G) Ex. 14 2 1A LSVA 2A PpstsIH Asch(T184I, V268A, V296A, S328G) Ex. 15 2 1A LSVA 2A PpstsIH Asch(T184I, V268A, K278R, S328G)

[0185] Each of Examples 6-15 are cultivated to produce fatty acid methyl esters using the small-scale method described above. Total fatty acid methyl ester (FAME) and amounts of C6, C8 and C10 fatty acid esters produced are as indicated in the following table, as are selectivities to C8 and and C10 fatty acid esters. Amounts of higher- and lower-carbon number fatty acid esters are not shown separately, but are included in the total FAME production value indicated.

TABLE-US-00013 Total FAME C6 C8 C10 3-ketoacyl-CoA production production production production Designation synthase (g/L) g/L) g/L % g/L % Ex. 6 Asch(N152T, T184I) 0.91 0.11 0.53 58.2 0.27 29.3 Ex. 7 Asch(N152L, T184I) 0.41 0.12 0.23 57.3 0.05 12.2 Ex. 8 Asch(N152M, T184I) 1.01 0.12 0.57 56.3 0.33 32.2 Ex. 9 Asch(N152C, T184I) 0.69 0.06 0.39 56.8 0.20 28.3 Ex. 10 Asch(A69V, T184I, 0.67 0.04 0.53 79.7 0.05 7.5 S328G) Ex. 11 Asch(G111C, T184I, 0.58 0.05 0.45 78.1 0.03 5.7 S328G) Ex. 12 Asch(D39V, T184I, 0.61 0.04 0.48 79.6 0.04 7.0 S328G) Ex. 13 Asch(D39V, G111C, 0.60 0.08 0.44 74.3 0.03 4.7 T184I, S328G) Ex. 14 Asch(T184I, V268A, 1.033 0.04 0.84 79.8 0.12 13.2 V296A, S328G) Ex. 15 Asch(T184I, V268A, 1.375 0.01 0.96 67.2 0.37 29.4 K278R, S328G)

[0186] As this data shows, the multiply-mutated enzymes that contain the T184 mutations all shift production away from C10 fatty acid esters to mainly C8 fatty acid esters. Mutated enzymes having the S328G variation are especially selective toward C8 fatty acid esters in this evaluation, with selectivities approaching 80%.

Examples 16-18 and Comparative Sample D—Asch-2 Variants

[0187] Mutant E. coli strain Examples 16-18 contain a modified Asch-2 gene encoding a 3-ketoacyl-CoA synthase (Asch-2(T184M), Asch-2(T184V) and Asch-2(T184I), SEQ ID NOs. 22-24, respectively). Comparative Sample D contains the wild-type gene (encoding for SEQ ID NO. 86). Details of strain construction are as follows:

TABLE-US-00014 Host Plasmid 1 NphT7 Plasmid 2 3-ketoacyl-CoA synthase Designation Strain Type mutant Type Promoter Mutant Comp. D* Host 1 1 A LSVA 2A PpstsIH wt Asch-2 Ex. 16 Host 1 1 A LSVA 2A PpstsIH Asch-2 (T184M) Ex. 17 Host 1 1 A LSVA 2A PpstsIH Asch-2 (T184V) Ex. 18 Host 1 1 A LSVA 2A PpstsIH Asch-2 (T184I)

[0188] Each of Examples 16-18 and Comparative Sample D is cultivated to produce fatty acid methyl esters using the small-scale method described above. Total fatty acid methyl ester (FAME) and amounts of C6, C8 and C10 fatty acid esters produced are as indicated in the following table, as are selectivities to C8 and C10 fatty acid esters. Amounts of higher- and lower-carbon number fatty acid esters are not shown separately, but are included in the total FAEE production value indicated.

TABLE-US-00015 Total FAME C6 C8 C10 3-ketoacyl-CoA production production production production Designation synthase (g/L) g/L) g/L % g/L % Comp. D* wt Asch-2 2.75 0.03 0 0 2.66 96.7 Ex. 16 Asch-2 (T184M) 0.82 0.30 0.36 44.0 0.11 13.8 Ex. 17 Asch-2 (T184V) 1.66 0.07 0.57 34.1 0.97 58.7 Ex. 18 Asch-2 (T184I) 2.29 0.05 0.72 31.2 1.47 64.2 *Not an example of this invention.

[0189] The wildtype Asch-2 gene produces C10 fatty acid esters almost exclusivily. The T184 variations all shift production from C10 fatty acid esters towards C6 and C8 fatty acid esters. Selectivity to C8 fatty acid esters is increased from zero to about 30-45% with these modifications to the Asch-2 gene.

Examples 19-22 and Comparative Sample E—Alwo Variants

[0190] Mutant E. coli strain Examples 19-22 contain a modified Alwo gene encoding a mutated 3-ketoacyl-CoA synthase (Alwo(T184L), Alwo(T184M), Alwo(T184V) and Alwo(T184I), SEQ ID NOs. 26-29, respectively). Comparative Sample E contains the wild-type gene (encoding for SEQ ID NO. 88). Details of strain construction are as follows:

TABLE-US-00016 Host Plasmid 1 NphT7 Plasmid 2 3-ketoacyl-CoA synthase Designation Strain Type mutant Type Promoter Mutant Comp. E* 1 1B SV 2A PpstsIH wt Alwo Ex. 19 1 1B SV 2A PpstsIH Alwo(T184L) Ex. 20 1 1B SV 2A PpstsIH Alwo(T184M) Ex. 21 1 1B SV 2A PpstsIH Alwo(T184V) Ex. 22 1 1B SV 2A PpstsIH Alwo(T184I)

[0191] Each of Examples 19-22 and Comparative Sample E are cultivated to produce fatty acid ethyl esters using the small-scale method described above. Total fatty acid ethyl ester (FAEE) and amounts of C6, C8 and C10 fatty acid esters produced are as indicated in the following table, as are selectivities to C8 and C10 fatty acid esters. Amounts of higher- and lower-carbon number fatty acid esters are not shown separately, but are included in the total FAEE production value indicated.

TABLE-US-00017 Total FAEE C6 C8 C10 3-ketoacyl-CoA production production production production Designation synthase (g/L) g/L) g/L % g/L % Comp. E* wt Alwo 0.95 0.05 0.00 0 0.89 93.7 Ex. 19 Alwo(T184L) 0.73 0.31 0.20 27.7 0.22 29.6 Ex. 20 Alwo(T184M) 0.69 0.46 0.22 31.2 0.01 1.42 Ex. 21 Alwo(T184V) 1.27 0.11 0.40 31.2 0.76 60.0 Ex. 22 Alwo(T184I) 0.85 0.19 0.53 61.8 0.13 15.5 *Not an example of this invention.

[0192] The effect of the T184 variations in the Alwo enzyme is similar to those seen in the Asch and Asch-2 mutations. Whereas the wildtype Alwo enzyme produces over 90% C10 fatty acid esters and no C8 fatty acid esters, the T184L, M, V and I variations all shift production toward C6 and C8 fatty acid esters. The T184M and T184I variations are particularly effective in this regard, with C10 fatty acid ester selectivity being reduced to below 20% in each of those cases and C8 fatty acid ester selectivity exceeding 60% in the T184I case.

Examples 23-26 and Comparative Sample F—Ajoh-2 Variants

[0193] Mutant E. coli strain Examples 23-26 contain a modified Ajoh-2 gene encoding a 3-ketoacyl-CoA synthase (Ajoh-2(T184L), Ajoh-2(T184M), Ajoh-2(T184V) and Ajoh-2(T184I), SEQ ID NOs. 36-39, respectively). Comparative Sample F contains the wild-type gene (encoding for SEQ ID NO. 35). Details of strain construction are as follows:

TABLE-US-00018 Host Plasmid 1 NphT7 Plasmid 2 3-ketoacyl-CoA synthase Designation Strain Type mutant Type Promoter Mutant Comp. F* 1 1A LSVA 2A PpstsIH wt Ajoh-2 Ex. 23 1 1A LSVA 2A PpstsIH Ajoh-2 (T184L) Ex. 24 1 1A LSVA 2A PpstsIH Ajoh-2 (T184M) Ex. 25 1 1A LSVA 2A PpstsIH Ajoh-2 (T184V) Ex. 26 1 1A LSVA 2A PpstsIH Ajoh-2 (T184I)

[0194] Each of Examples 23-26 and Comparative Sample F are cultivated to produce fatty acid methyl esters using the small-scale method described above. Total fatty acid methyl ester (FAME) and amounts of C6, C8 and C10 fatty acid esters produced are as indicated in the following table, as are selectivities to C8 and C10 fatty acid esters. Amounts of higher- and lower-carbon number fatty acid esters are not shown separately, but are included in the total FAEE production value indicated.

TABLE-US-00019 Total FAME C6 C8 C10 3-ketoacyl-CoA production production production production Designation synthase (g/L) g/L) g/L % g/L % Comp. F* wt Ajoh-2 2.65 0.09 0.00 0.0 2.50 96.0 Ex. 23 Ajoh-2 (T184L) 0.90 0.10 0.17 19.3 0.57 63.9 Ex. 24 Ajoh-2 (T184M) 0.60 0.28 0.23 38.4 0.04 6.6 Ex. 25 Ajoh-2 (T184V) 2.38 0.15 0.59 24.9 1.58 66.3 Ex. 26 Ajoh-2 (T184I) 0.97 0.12 0.39 39.9 0.42 43.4 *Not an example of the invention.

[0195] The same general pattern is seen with the Ajoh-2 gene modifications. The high selectivity of the wildtype gene to C10 fatty acid ester production is shifted toward C6 and C8 fatty acid ester production. The T184M and T184I variations are particularly effective in this regard. The T184V variation results in a large increase of overall productivity compared to the T184L, M and I variations.

Examples 27-29 and Comparative Sample G—ANIP71 Variants

[0196] Mutant E. coli strain Examples 27-29 contain a modified ANIP71 gene encoding a 3-ketoacyl-CoA synthase (ANIP71(T184M), ANIP71(T184V) and ANIP71(T184I), SEQ ID NOs. 32, 34 and 31, respectively). Comparative Sample G contains the wild-type gene (encoding for SEQ ID NO. 89). Details of strain construction are as follows:

TABLE-US-00020 Host Plasmid 1 NphT7 Plasmid 2 3-ketoacyl-CoA synthase Designation Strain Type mutant Type Promoter Mutant Comp. G* 1 1A LSVA 2A PpstsIH wt ANIP71 Ex. 27 1 1A LSVA 2A PpstsIH ANIP71 (T184M) Ex. 28 1 1A LSVA 2A PpstsIH ANIP71 (T184V) Ex. 29 1 1A LSVA 2A PpstsIH ANIP71 (T184I)

[0197] Each of Examples 27-29 and Comparative Sample G are cultivated to produce fatty acid methyl esters using the small-scale method described above. Total fatty acid methyl ester (FAME) and amounts of C6, C8 and C10 fatty acid esters produced are as indicated in the following table, as are selectivities to C8 and C10 fatty acid esters. Amounts of higher- and lower-carbon number fatty acid esters are not shown separately, but are included in the total FAME production value indicated.

TABLE-US-00021 Total FAME C6 C8 C10 3-ketoacyl-CoA production production production production Designation synthase (g/L) g/L) g/L % g/L % Comp. G* wt ANIP71 2.71 0.04 0 0 2.61 96.5 Ex. 27 ANIP71 (T184M) 0.49 0.27 0.15 30.1 0.03 6.1 Ex. 28 ANIP71 (T184V) 2.46 0.10 0.58 23.4 1.72 70.0 Ex. 29 ANIP71 (T184I) 0.21 0.04 0.10 47.8 0.03 14.2 *Not an example of the invention.

[0198] As before, the T184 variations all shift production from C10 fatty acid esters towards C8 fatty acid esters, with the T184M and T184I variations being particularly effective. The T184V variation results in a large increase of overall productivity compared with the T184M and T184I variations. The T184I variation exhibits the highest selectivity to C8 fatty acid esters.

[0199] Examples 30-31 and Comparative Sample H—Pstu Variants

[0200] Mutant E. coli strain Examples 30-31 contain a modified Pstu gene encoding a 3-ketoacyl-CoA synthase (Pstu(C186M) and Pstu(C186I), respectively, SEQ ID NO. 40, with position 186 being M and I, respectively). Comparative Sample H contains the wild-type gene (encoding for SEQ ID NO. 90). Details of strain construction are as follows:

TABLE-US-00022 Host Plasmid 1 NphT7 Plasmid 2 3-ketoacyl-CoA synthase Designation Strain Type mutant Type Promoter Mutant Comp. H* 1 1A LSVA 2A PpstsIH wt Pstu Ex. 30 1 1A LSVA 2A PpstsIH Pstu (C186M) Ex. 31 1 1A LSVA 2A PpstsIH Pstu(C186I)

[0201] Each of Examples 30-31 and Comparative Sample H are cultivated to produce fatty acid methyl esters using the small-scale method described above. Total fatty acid methyl ester (FAME) and amounts of C6, C8 and C10 fatty acid esters produced are as indicated in the following table, as are selectivities to C8 and C10 fatty acid esters. Amounts of higher- and lower-carbon number fatty acid esters are not shown separately, but are included in the total FAME production value indicated.

TABLE-US-00023 Total FAME C6 C8 C10 3-ketoacyl-CoA production production production production Designation synthase (g/L) g/L) g/L % g/L % Comp. H* wt Pstu 2.19 0.03 0 0 2.08 95.1 Ex. 30 Pstu (C186M) 0.53 0.22 0.24 44.5 0.03 5.3 Ex. 31 Pstu(C186I) 0.14 0.02 0.08 54.6 0 0 *Not an example of the invention.

[0202] The wildtype Pstu enzyme produces C10 fatty acid esters almost exclusively. The C186M and I variations both shift production towards C6 and C8 fatty acid esters, with selectivity toward C8 fatty acid esters being about 40-60%.

Examples 32-61 and Comparative Sample I—Aagr Variants

[0203] Mutant E. coli strains Examples 32-61 contain a modified Aagr gene encoding a 3-ketoacyl-CoA synthase, as indicated in the following tables. Comparative Sample I contains the wild-type gene (encoding for SEQ ID NO. 91). Details of strain construction are as follows:

TABLE-US-00024 Host Plasmid 1 NphT7 Plasmid 2 3-ketoacyl-CoA synthase Designation Strain Type mutant Type Promoter Mutant Comp. I* 1 1B SV 1A PpstsIH wt Aagr Ex. 32 1 1B SV 1A PpstsIH Aagr(A186I) Ex. 33 1 1B SV 1A PpstsIH Aagr(A186M) Ex. 34 1 1B SV 1A PpstsIH Aagr(A186L) Ex. 35 1 1B SV 1A PpstsIH Aagr(A186T) Ex. 36 1 1B SV 1A PpstsIH Aagr(A186C) Ex. 37 1 1B SV 1A PpstsIH Aagr(A186V) Ex. 38 1 1B SV 1A PpstsIH Aagr(A186Q) Ex. 39 1 1B SV 1A PpstsIH Aagr(A186F) Ex. 40 1 1B SV 1A PpstsIH Aagr(A186D) Ex. 41 1 1B SV 1A PpstsIH Aagr(A186N) Ex. 42 1 1B SV 1A PpstsIH Aagr(A186Y) Ex. 43 1 1B SV 1A PpstsIH Aagr(A186I, I241D) Ex. 44 1 1B SV 1A PpstsIH Aagr(A186I, I241E) Ex. 45 1 1B SV 1A PpstsIH Aagr(A186I, I241D, H246R) Ex. 46 1 1B SV 1A PpstsIH Aagr(A186I, I241E, H246R) Ex. 47 1 1B SV IA PpstsIH Aagr(A186I, C239N, H246R) Ex. 48 1 1B SV 1A PpstsIH Aagr(A186I, C239N, I241F) Ex. 49 1 1B SV 1A PpstsIH Aagr(A186I, C239N, I241Y) Ex. 50 1 1B SV 1A PpstsIH Aagr(A186I, C239D) Ex. 51 1 1B SV 1A PpstsIH Aagr(A186I, I241L) Ex. 52 1 1B SV 1A PpstsIH Aagr(A186I, I241F) Ex. 53 1 1B SV 1A PpstsIH Aagr(A1861, I241Y) Ex. 54 1 1B SV 1A PpstsIH Aagr(A186I, H246R) Ex. 55 1 1B SV 1A PpstsIH Aagr(A186I, H246K) Ex. 56 1 1B SV 1A PpstsIH Aagr(A186I, C239N) Ex. 57 1 1B SV 1A PpstsIH Aagr(A186I, C239Q) Ex. 58 1 1B SV 1A PpstsIH Aagr(A186I, I241M) Ex. 59 1 1B SV 1A PpstsIH Aagr(A186I, I241D, H246K) Ex. 60 1 1B SV 1A PpstsIH Aagr(A186I, I241Y, H246R) Ex. 61 1 1B SV 1A PpstsIH Aagr(A186I, I241E, H246K)

[0204] Each of Examples 32, 35-42, 44 and 50-61 and Comparative Sample I are cultivated to produce fatty acid ethyl esters in the small-scale method described above. Total fatty acid ethyl ester (FAEE) and amounts of C8 and C10 fatty acid esters produced are as indicated below in the following table, as are selectivities to C8, C10 and C12 fatty acid esters. Amounts of higher- and lower-carbon number fatty acid esters are not shown separately, but are included in the total FAEE production value indicated. “Small” under the “C12 Production” column means that most FAEE production not specifically accounted for in the table is the C6 ethyl ester, with less than 5% C12 fatty acid esters being produced.

TABLE-US-00025 Total FAEE C8 C10 C12 3-ketoacyl-CoA production production production production, Designation synthase (g/L) g/L % g/L % % Comp. I* wt Aagr 0.84 0.01 1.2 0.57 67.9 18.0 Ex. 32 Aagr(A186I) 1.32 0.40 22.7 0.62 47.0 0.4 Ex. 35 Aagr(I186T) 1.16 0.02 1.9 0.81 69.8 11.0 Ex. 36 Aagr(A186C) 0.96 0.01 1.5 0.64 67.0 13.4 Ex. 37 Aagr(A186V) 1.43 0.13 8.9 1.08 75.4 1.2 Ex. 38 Aagr(A186Q) 0.73 0.13 18.3 0.15 21.0 0.5 Ex. 39 Aagr(A186F) 0.97 0.09 9.0 0 0.4 0.3 Ex. 40 Aagr(A186D) 0.55 0.01 2.1 0.40 72.5 7.8 Ex. 41 Aagr(A186N) 0.99 0.02 1.7 0.66 66.7 11.5 Ex. 42 Aagr(A186Y) 0.62 0.07 10.8 0 0 0 Ex. 44 Aagr(A186I, I241E) 1.08 0.22 20.7 0.60 55.5 Small Ex. 50 Aagr(A186I, C239D) 0.20 0.05 26.1 0.02 12.4 Small Ex. 51 Aagr(A186I, I241L) 1.27 0.28 22.3 0.62 48.5 Small Ex. 52 Aagr(A186I, I241F) 1.27 0.24 18.6 0.67 58.1 Small Ex. 53 Aagr(A186I, I241Y) 1.34 0.25 18.5 0.81 60.4 Small Ex. 54 Aagr(A186I, H246R) 1.22 0.36 29.5 0.60 49.0 Small Ex. 55 Aagr(A186I, H246K) 0.93 0.26 28.5 0.35 38.1 Small Ex. 56 Aagr(A186I, C239N) 1.32 0.21 15.8 0.80 60.9 Small Ex. 57 Aagr(A186I, C239Q) 0.11 0.03 28.5 0.02 14.3 Small Ex. 58 Aagr(A186I, I241M) 1.17 0.20 17.2 0.70 60.2 Small Ex. 59 Aagr(A186I, I241D, 0.36 0.13 36.7 0.10 26.1 Small H246K) Ex. 60 Aagr(A186I, I241Y, 1.18 0.33 27.6 0.68 57.2 Small H246R) Ex. 61 Aagr(A186I, I243E, 0.42 0.14 32.7 0.09 22.5 Small H246K) *Not an example of the invention.

[0205] The wildtype Aagr enzyme produces 18% C12 fatty acid esters in this evaluation. The replacement of the wildtype Aagr reduces C12 fatty acid ester production, in most cases in favor of higher selectivity toward C8 and/or C10 fatty acid esters.

[0206] Each of Examples 32-34 and 43-49 are cultivated to produce fatty acid methyl esters using the small-scale method described above. Total fatty acid methyl ester (FAME) and amounts of C8 and C10 fatty acid esters produced are as indicated in the following table, as are selectivities to C8, C10 and C12 fatty acid esters. Amounts of higher- and lower-carbon number fatty acid esters are not shown separately, but are included in the total FAME production value indicated. “Small” under the “C12 Production” column means that most FAME production not accounted for in the table is the C6 methyl ester. with less than 5% C12 fatty acid esters being produced.

TABLE-US-00026 Total FAME C8 C10 C12 3-ketoacyl-CoA production production production production, Designation synthase (g/L) g/L % g/L % % Ex. 32 Aagr(A186I) 1.46 0.20 14.0 1.06 72.8 0.5 Ex. 33 Aagr(A186M) 1.07 0.26 23.9 0.1 9.33 Small Ex. 34 Aagr(A186L) 0.41 0.04 9.9 0.3 73.4 Small Ex. 43 Aagr(A186I, I241D) 0.96 0.15 15.5 0.74 77.2 Small Ex. 44 Aagr(A186I, I241E) 0.99 0.13 12.8 0.81 81.6 Small Ex. 45 Aagr(A186I, I241D, 0.74 0.18 24.5 0.51 68.8 Small H246R) Ex. 46 Aagr(A186I, I241E, 0.42 0.14 32.2 0.24 57.4 Small H246R) Ex. 47 Aagr(A186I, C239N, 0.09 0.04 48.8 0.04 41.7 Small H246R) Ex. 48 Aagr(A186I, C239N, 1.04 0.9 8.5 0.90 86.5 Small I241F) Ex. 49 Aagr(A186I, C239N, 1.17 0.10 8.7 1.02 86.6 Small I241Y)

[0207] As the data in the foregoing two table shows, the wild-type Aagr enzyme produces a significant fraction (18%) of C12 fatty acid esters. The A186I modification, by itself or accompanied by additional modifications, reduces C12 fatty acid ester production in favor of C8 and/or C10 fatty acid esters, and in some cases also in favor of C6 fatty acid esters, both in FAEE and FAME production.

Examples 62-71 and Comparative Samples J and K—Asch Variants

[0208] Mutant E. coli strains Examples 62-71 contain a modified Asch gene encoding a 3-ketoacyl-CoA synthase as indicated in the following tables. Comparative Samples J and K contain the wild-type gene (encoding for SEQ ID NO. 8). In Examples 62-64 and Comparative Sample J only, the mutant Asch gene is fused to a DNA sequence encoding a protein fragment containing 6 histidine residues and a protease recognition site. Details of strain construction are as follows:

TABLE-US-00027 Host Plasmid 1 NphT7 Plasmid 2 3-ketoacyl-CoA synthase Designation Strain Type mutant Type Promoter Mutant Comp. J. 2 1A LSVA 2A PpstsIH Wt Asch Ex. 62 2 1A LSVA 2A PpstsIH Asch(K278R) Ex. 63 2 1A LSVA 2A PpstsIH Asch(V296A) Ex. 64 2 1A LSVA 2A PpstsIH Asch(K278R, V296A) Comp. K. 2 1A LSVA 2B PphoE Wt Asch Ex. 65 2 1A LSVA 2B PphoE Asch(V317A) Ex. 66 2 1A LSVA 2B PphoE Asch(K278R, V317A) Ex. 67 2 1A LSVA 2B PphoE Asch(V296A, V317A) Ex. 68 2 1A LSVA 2B PphoE Asch(M271I) Ex. 69 2 1A LSVA 2B PphoE Asch(M271I, V296A) Ex. 70 2 1A LSVA 2B PphoE Asch(M178L) Ex. 71 2 1A LSVA 2B PphoE Asch(M178L, V296A)

[0209] Each of 62-71 and Comparative Samples J and K are cultivated to produce fatty acid methyl esters using the small-scale method described above. Total fatty acid methyl ester (FAME) and amounts of C6, C8 and C10 fatty acid esters produced are as indicated in the following table. Amounts of higher- and lower-carbon number fatty acid esters are not shown separately, but are included in the total FAME production value indicated.

TABLE-US-00028 Total FAME C6 3-ketoacyl-CoA production production C8 C10 Designation synthase (g/L) g/L) production production Comp. J Wt Asch 2.27 0.03 2.19 Ex. 62 Asch(K278R) 2.77 0.03 2.66 Ex. 63 Asch(V296A) 3.08 0.04 2.96 Ex. 64 Asch(K278R, V296A) 3.06 0.05 2.93 Comp. K. Wt Asch 4.18 0.23 0.03 3.87 Ex. 65 Asch(V317A) 4.66 0.29 0.04 4.27 Ex. 66 Asch(K278R, V317A) 4.33 0.29 0.04 3.95 Ex. 67 Asch(V296A, V317A) 4.89 0.26 0.03 4.55 Ex. 68 Asch(M271I) 4.38 0.27 0.04 4.01 Ex. 69 Asch(M271I, V296A) 4.40 0.27 0.02 4.06 Ex. 70 Asch(M178L) 4.63 0.37 0.04 4.17 Ex. 71 Asch(M178L, V296A) 4.64 0.40 0.03 4.16

[0210] As this data shows, the K278R, the V296A, the V317A, the M271I and M178L mutations all result in an increase in total productivity of the cell, compared to the wild-type Asch enzyme. Productivity is improved in these examples by as much as 36%. The generally higher productivity of Comp. K and Examples 65-71 as compared to Comp. J and Examples 62-64 is believed to be attributable to the combination of having the 3-ketoacyl synthase under the control of a low phosphate inducible promoter together with the selection of a low phosphate medium.

Examples 72-74—Asch(T184I) Variants

[0211] Mutant E. coli strains Examples 72-74 similarly contain a mutated Asch gene encoding a mutated 3-ketoacyl-CoA synthase with multiple mutations as indicated in the following table. Details of strain construction are as follows:

TABLE-US-00029 Host Plasmid 1 NphT7 Plasmid 2 3-ketoacyl-CoA synthase Designation Strain Type mutant Type Promoter Mutant Ex. 72 2 1A LSVA 2A PpstsIH Asch(T184I, V268A, V296A, S328G) Ex. 73 2 1A LSVA 2A PpstsIH Asch(T184I, V268A, V296A, V317A, S328G) Ex. 74 2 1A LSVA 2A PpstsIH Asch (V30A, T184I, V268A, V296A, V317A, S328G)

[0212] Each of examples 72-74 are cultivated to produce fatty acid methyl esters using the small-scale method described above. Total fatty acid methyl ester (FAME) and amounts of C6, C8 and C10 fatty acid esters produced are as indicated in the following table, as are selectivities to C8 and and C10 fatty acid esters. Amounts of higher- and lower-carbon number fatty acid esters are not shown separately, but are included in the total FAME production value indicated.

TABLE-US-00030 Total FAME C6 C8 C10 3-ketoacyl-CoA production production production production Designation synthase (g/L) g/L) g/L % g/L % Ex. 72 Asch (T184I, V268A, 1.03 0.04 0.84 79.8 0.12 13.2 V296A, S328G) Ex. 73 Asch (T184I, V268A, 1.51 0.00 0.93 62 0.51 34 V296A, V317A, S328G) Ex. 74 Asch (V30A, T184I, 1.47 0.05 1.17 80 0.19 11 V268A, V296A, V317A, S328G)

Examples 75-77

[0213] E. coli mutants are prepared by transforming E. coli strain BW25113 with a “Type 1” plasmid and a “Type 2” plasmid, using electroporation methods described before. Details of strain construction are as follows:

TABLE-US-00031 Example Plasmid 1 NphT7 Plasmid 2 3-ketoacyl-CoA synthase No. Type mutant Type Promoter Mutant Ex. 75 1B SV 2A PpstsIH Asch T184I Ex. 76 1A LSVA 2A PphoE Asch T184I Ex. 77 1A LSVA 2A PpstsIH Asch T184I

[0214] All of Examples 75-77 exhibit good selectivities toward C6-C10 fatty acid esters, when evaluated using the small-scale fermentation method.

Example 78

[0215] The Saccharomyces cereuisiae strain IMX581 (Mans, R., H. M. van Rossum, et al. (2015). CRISPR/Cas9: a molecular tool for simultaneous introduction of multiple genetic modifications in Saccharomyces cereuisiae. FEMS Yeast Res 15(2)) has Cas9 nuclease integrated in its chromosome such that it can be used as the host strain for manipulating the genome using CRISPR technology (US20140068797 A1). The guide RNA (gRNA) is expressed from either pMEL or pROS series of plasmids. The genes of the non-native fatty acid and fatty acid ester pathway are integrated in the chromosome of IMX581 using this technology. The gRNA sequences are designed using Yeastriction online tool (Robert Mans, Harmen M. van Rossum, Melanie Wijsman, Antoon Backx, Niels G. A. Kuijpers, Marcel van den Broek, Pascale Daran-Lapujade, Jack T. Pronk, Antonius J. A. van Maris, Jean-Marc G. Daran (2015) CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cereuisiae. FEMS Yeast Research 16). The gRNA sequence is introduced into pMEL plasmid using complementary primers that have 50 bp of homology and are PAGE-purified. The primers are dissolved in distilled water to a final concentration of 100 μM, the primers are mixed in 1:1 molar ratio, and the mixture is heated to 95° C. for 5 min and annealed by cooling to room temperature. The primers are mixed with pMEL10 as template and the mixture is amplified using Q5 High Fidelity 2× Master Mix (New England BioLabs (Ipswich, Mass.). The PCR product is digested with DpnI for 30 minutes and the PCR product purified on an agarose gel. The protocol for simultaneous integration and deletion is described in Mans et al (supra). Using the protocol, genes that encode for the proteins listed in the table below are integrated into loci in the S. cereuisiae chromosome as listed below. The terminators and promoters that are used to express the genes are also listed in the table.

TABLE-US-00032 Enzyme Gene Encoded Target locus Promoter Terminator NphT7 SEQ ID NO. PDC1 gene Native PDC1 Native PDC1 83 NphT7 (LVSA) SEQ ID NO. CIT3 gene Native TDH3 Native ADH1 variant 82 Mutant 3- SEQ ID NO. ADH1 gene Native TEF1 Native ADH1 ketoacyl-CoA 11 synthase 3-ketoacyl-CoA SEQ ID. NO. GDH1 gene Native PGK1 Native CYC1 reductase 98 3-hydroxyacyl- SEQ ID NO. GAL1 gene Native GPD1 Native ADH1 CoA reductase 99 Enoyl-CoA SEQ ID NO. GAL10 gene Native PGK1 Native CYC1 reductase 84 Ester synthase SEQ ID NO. GPD1 gene Native GPD1 Native GPD1 104

[0216] The engineered yeast is grown in 250 mL shake flasks at 30° C. in 25 mL of synthetic defined medium supplemented with 10 g/L of glucose as carbon source. The flasks are shaken at 200 rpm for 24 h. Fatty acid or fatty acid methyl ester acid is measured in the supernatant.

Example 79

[0217] The oleaginous yeast Yarrowia lipolytica strain LGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol. 82(1):43-9 (2002)). CRISPR/Cas9: a molecular tool for simultaneous introduction of multiple genetic modifications in Y. lipolytica. The host is engineered with Cas9 nuclease integrated in its chromosome such that it can be used as the host strain for manipulating the genome using CRISPR technology (US20140068797 A1). The guide RNA (gRNA) is expressed from either pMEL or pROS series of plasmids. The genes of the non-native fatty acid and fatty acid ester pathway are integrated in the chromosome of Y. lipolytica using this technology. The gRNA sequences are designed using Yeastriction online tool (Robert Mans, Harmen M. van Rossum, Melanie Wijsman, Antoon Backx, Niels G. A. Kuijpers, Marcel van den Broek, Pascale Daran-Lapujade, Jack T. Pronk, Antonius J. A. van Maris, Jean-Marc G. Daran (2015) CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cereuisiae. FEMS Yeast Research 16). The gRNA sequence is introduced into pMEL plasmid using complementary primers that have 50 bp of homology and are PAGE-purified. The primers are dissolved in distilled water to a final concentration of 100 μM, the primers are mixed in 1:1 molar ratio, and the mixture is heated to 95° C. for 5 min and annealed by cooling to room temperature. The primers are mixed with pMEL10 as template and the mixture is amplified using Q5 High Fidelity 2× Master Mix (New England BioLabs (Ipswich, Mass.). The PCR product is digested with DpnI for 30 minutes and the PCR product purified on an agarose gel. The protocol for simultaneous integration and deletion is described in Mans et al (supra). Using the protocol, genes that encode for the proteins listed in the table below are integrated into the loci in the Y. lipolytica chromosome. Examples of the terminator and promoters that are used to express the genes are also listed in the table.

TABLE-US-00033 Enzyme Gene Encoded Target locus Promoter Terminator NphT7 SEQ ID NO. PDC1 gene Native PDC1 Native PDC1 83 NphT7 (LVSA) SEQ ID NO. XPR2 gene Native TDH3 Native ADH1 variant 82 Mutant 3- SEQ ID NO. ADH1 gene Native TEF1 Native ADH1 ketoacyl-CoA 11 synthase 3-ketoacyl-CoA SEQ ID. NO. GDH1 gene NativePGK1 Native CYC1 reductase 98 3-hydroxyacyl- SEQ ID NO. GAL1 gene Native GPD1 Native ADH1 CoA reductase 99 Enoyl-CoA SEQ ID NO. GAL10 gene Native PGK1 Native CYC1 reductase 84 Ester synthase SEQ ID NO. GPD1 gene Native GPD1 Native GPD1 104

[0218] The engineered yeast is grown in 250 mL shake flasks at 30° C. in 25 mL of synthetic defined media supplemented with 10 g/L of glucose as carbon source. The flasks are shaken at 200 rpm for 24 h. Fatty acid or fatty acid methyl ester acid is measured in the supernatant.

Examples 80-96—Asch(T184I) Variants.SUB.[GC1]

[0219] The mutant 3-ketoacyl-CoA synthase having SEQ ID NO. 20 is selected as a promising candidate for further improvement through additional mutations. Inventive Control A is produced by introducing mutant 3-ketoacyl-CoA synthase having SEQ ID NO. 20 into E. coli host strain 3. This inventive example serves as a basis for comparison for the additional mutant Examples 80-96. Mutant E. coli strains Examples 80-96 contain a mutated Asch gene encoding a mutated 3-ketoacyl-CoA synthase having the same mutations as SEQ ID NO. 20 together with one or more additional mutations. All mutations differing from the wild-type 3-ketoacyl-CoA synthase (SEQ ID NO. 8) are indicated in the following table. Details of strain construction are as follows:

TABLE-US-00034 Host NphT7 3-ketoacyl-CoA synthase Designation Strain mutant Promoter Mutant Inventive 3 LSVA PphoE Asch (T184I, V268A, V296A, S328G), SEQ Control A ID NO. 20 Ex. 80 3 LSVA PphoE Asch(T184I, V268A, V296A, K313E, S328G, A370T), SEQ ID NO. 121 Ex. 81 3 LSVA PphoE Asch(T18A, T184I, V268A, V296A, S328G), SEQ ID NO. 122 Ex. 82 3 LSVA PphoE Asch(T184I, V268A, V296A, S328G, S329G), SEQ ID NO. 123 Ex. 83 3 LSVA PphoE Asch(I127T, T184I, V268A, V296A, S328G), SEQ ID NO. 124 Ex. 84 3 LSVA PphoE Asch(T184I, V268A, K274E, V296A, S328G), SEQ ID NO. 125 Ex 85 3 LSVA PphoE Asch(, T184I, N231I, V268A, V296A, , S328G), SEQ ID NO. 204 Ex. 86 3 LSVA PphoE Asch(A38V, M178T, T184I, V268A, V296A, A312D, S328G), SEQ ID NO. 126 Ex. 87 3 LSVA PphoE Asch(D116G, T184I, F190Y, L241P, V268A, V296A, S328G), SEQ ID NO. 127 Ex. 88 3 LSVA PphoE Asch(I94T, T184I, V268A, V296A, S328G), SEQ ID NO. 128 Ex. 89 3 LSVA PphoE Asch(L22M, T184I, V268A, V296A, K313M, S328G), SEQ ID NO. 129 Ex. 90 3 LSVA PphoE Asch(T184I, V268A, V296A, K313E, S328G), SEQ ID NO. 130 Ex. 91 3 LSVA PphoE Asch(T184I, V268A, V296A, S328G, A370T), SEQ ID NO. 131 Ex. 92 3 LSVA PphoE Asch(T18A, T184I, V268A, V296A, K313E, S328G, A370T), SEQ ID NO. 132 Ex. 93 3 LSVA PphoE Asch(T184I, V268A, V296A, K313E, S328G, S329G, A370T), SEQ ID NO. 133 Ex. 94 3 LSVA PphoE Asch(T184I, F236L, V268A, V296A, K313E, V317A, S328G), SEQ ID NO. 134 Ex. 95 3 LSVA PphoE Asch(T184I, F236M, V268A, V296A, V317A, S328G), SEQ ID NO. 135 Ex. 96 3 LSVA PphoE Asch(T184I, I232V, V268A, V296A, S328G), SEQ ID NO. 136

[0220] Each of examples 80-96 are cultivated to produce fatty acid methyl esters using the shake flask method described above. Amounts of C8 and C10 fatty acid esters are measured. The ability of each example to increase production of C8 and/or C10 fatty esters is recorded as indicated. The ability of each example to increase specificity for C8 and/or C10 is recorded as indicated.

TABLE-US-00035 Increase C8 Increase Increase C8 FAME total FAME FAME Specificity relative to relative to relative to Inventive Inventive Inventive Designation 3-ketoacyl-CoA synthase Control A Control A * Control A ** Ex. 80 Asch(T184I, V268A, V296A, + ++ − K313E, S328G, A370T) Ex. 81 Asch(T18A, T184I, V268A, V296A, S328G) + ++ + Ex. 82 Asch(T184I, V268A, V296A, ++ ++ − S328G, S329G) Ex. 83 Asch (I127T, T184I, A268A, V296A, S328G) +++ +++ ◯ Ex. 84 Asch(T184I, V268A, K274E, V296A, S328G) ++ ++ ◯ Ex. 85 Asch(, T184I, N231I, V268A, V296A, ,S328G) +++ ++ ◯ Ex. 86 Asch(A38V, M178T, T184I, V268A, V296A, + + ◯ A312D, S328G) Ex. 87 Asch(D116G, T184I, F190Y, L241P, V268A, + + ◯ V296A, S328G) Ex. 88 Asch(I94T, T184I, V268A, V296A, S328) ++ ++ + Ex. 89 Asch(L22M, T184I, V268A, V296A, K313M, ++ ++ +++ S328G) Ex. 90 Asch(T184I, V268A, V296A, K313E, S328G) + + + Ex. 91 Asch(T184I, V268A, V296A, S328G, A370T) ++ ++ − Ex. 92 Asch(T18A, T184I, V268A, V296A, K313E, + + − S328G, A370T) Ex. 93 Asch(T184I, V268A, V296A, K313E, S328G, + + ++ S329G, A370T) Ex. 94 Asch(T184I, F236L, V268A, V296A, K313E, + + +++ V317A, S328G) Ex. 95 Asch(T184I, F236M, V268A, V296A, V317A, + S328G) Ex. 96 Asch(T184I, I232V, V268A, V296A, S328G) +++ +++ − * (+) = Increase over control strain ** (+) = Increase over control strain; (−) = No change over control strain; (◯) = Decrease over control strain

Examples 97-99—Asch(T184I) Variants.SUB.[GC2]

[0221] A mutant 3-ketoacyl-CoA synthase having SEQ ID NO. 20, except that the valines appearing at amino acids 30 and 317 each are replaced with alanine, is selected as a candidate for further improvement through additional mutations. Inventive Control B is produced by introducing this mutant 3-ketoacyl-CoA synthase into E. coli host strain 2. This inventive example serves as a basis for comparison for the additional mutants Examples 97-99. Mutant E. coli strains Examples 97-99 contain a mutated Asch gene encoding a mutated 3-ketoacyl-CoA synthase having the same mutations as that of Inventive Control B together with one or more additional mutations. All mutations differing from the wild-type 3-ketoacyl-CoA synthase (SEQ ID NO. 8) are indicated in the following table. Details of strain construction are as follows:

TABLE-US-00036 Host NphT7 3-ketoacyl-CoA synthase Designation Strain mutant Promoter Mutant Inventive 2 LSVA PpstsIH Asch(V30A, T184I, V268A, V296A, Control B V317A, S328G) Ex. 97 2 LSVA PpstsIH Asch(V30A, T184I, V268A, E282G, V296A, V317A, S328G), SEQ ID NO. 137 Ex. 98 2 LSVA PpstsIH Asch(V30A, T184I, V268A, V296A, V317A, D322G, S328G), SEQ ID NO. 138 Ex. 99 2 LSVA PpstsIH Asch(V30A, T184I, E210V, V268A, V296A, V317A, S328G), SEQ ID NO. 139

[0222] Each of examples 97-99 are cultivated to produce fatty acid methyl esters using the shake flask method described above. Amounts of C8 and C10 fatty acid esters are measured. The ability of each example to increase production of C8 and/or C10 fatty esters is recorded as indicated. The ability of each example to increase specificity for C8 and/or C10 is recorded as indicated.

TABLE-US-00037 Increase C8 Increase Increase C8 FAME total FAME FAME Specificity relative to relative to relative to Inventive Inventive Inventive Designation 3-ketoacyl-CoA synthase Control B Control B * Control B** Ex. 97 Asch(V30A, T184I, V268A, E282G, +++ ++ ◯ V296A, V317A, S328G) Ex. 98 Asch(V30A, T184I, V268A, V296A, +++ +++ − V317A, D322GS328G) Ex. 99 Asch(V30A, T184I, E210V, V268A, + + − V296A, V317A, S328G) * (+) = Increase over control strain **(+) = Increase over control strain; (−) = No change over control strain; (◯) = Decrease over control strain

Examples 100-116—Asch(T184I) Variants.SUB.[GC3]

[0223] A mutant 3-ketoacyl-CoA synthase corresponding to SEQ ID NO. 8 with six specific mutations as indicated in the following table, is selected as a candidate for further improvement through additional mutations. Inventive Control C is produced by introducing this mutant 3-ketoacyl-CoA synthase into E. coli host strain 3. This inventive example serves as a basis for comparison for the additional mutants Examples 100-116. Mutant E. coli strains Examples 100-116 contain a mutated Asch gene encoding a mutated 3-ketoacyl-CoA synthase having the same mutations as that of Inventive Control C together with one or more additional mutations. All mutations differing from the wild-type 3-ketoacyl-CoA synthase (SEQ ID NO. 8) are indicated in the following table. Details of strain construction are as follows:

TABLE-US-00038 Host NphT7 3-ketoacyl-CoA synthase Designation Strain mutant Promoter Mutant Inventive 3 LSVA PpstsIH Asch (V30A, T184I, V268A, V296A, V317A, Control C V328G) Ex. 100 3 LSVA PpstsIH Asch(V30A, T184I, V268A, V296A, E315K, V317A, S328G, H368R), SEQ ID NO. 140 Ex. 101 3 LSVA PpstsIH Asch(V30A, A54V, T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 141 Ex. 102 3 LSVA PpstsIH Asch(V30A, T184I, V268A, V296A, I302T, V317A, S328G, H368R), SEQ ID NO. 142 Ex. 103 3 LSVA PpstsIH Asch(V30A, A54V, T184I, V268A, V296A, I302T V3I7A, S328G, H368R), SEQ ID NO. 143 Ex. 104 3 LSVA PpstsIH Asch(V30A, T184I, V268A, M271I, V296A, V317A, S328G, H368R), SEQ ID NO. 144 Ex. 105 3 LSVA PpstsIH Asch(V30A, T184I, V268A, V296A, V317A, S328G, A356G, H368R), SEQ ID NO. 145 Ex. 106 3 LSVA PpstsIH Asch(V30A, T184I, V268A, V296A, V317A, S328G, A356S, H368R), SEQ ID NO. 146 Ex. 107 3 LSVA PpstsIH Asch(V30A, A154G, T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 147 Ex. 108 3 LSVA PpstsIH Asch(V30A, T184I, I232V, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 148 Ex. 109 3 LSVA PpstsIH Asch(V30A, A54V, A154G, T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 149 Ex. 110 3 LSVA PpstsIH Asch(V30A, A154G, T184I, V268A, M271I, V296A, I302T, V317A, S328G, H368R), SEQ ID NO. 150 Ex. 111 3 LSVA PpstsIH Asch(V30A, A54V, A154G, T184I, V268A, M271I, V296A, I302T, V317A, S328G, H368R), SEQ ID NO. 151 Ex. 112 3 LSVA PpstsIH Asch(V30A, A54V, T184I, V268A, M271I, V296A, I302T, V317A, S328G, H368R), SEQ ID NO. 152 Ex. 113 3 LSVA PpstsIH Asch(V30A, A54V, T184I, V268A, M271I, V296A, V317A, S328G, H368R), SEQ ID NO. 153 Ex. 114 3 LSVA PpstsIH Asch(V30A, T184I, V268A,M271I, V296A, I302T, V317A, S328G, H368R), SEQ ID NO. 154 Ex. 115 3 LSVA PpstsIH Asch(V30A, A54V, A151G, T184I, V268A, M271I, V296A, V317A, S328G, H368R), SEQ ID NO. 155 Ex. 116 3 LSVA PpstsIH Asch(V30A, A154G, T184I, V268A, M271I, V296A, V317A, S328G, H368R), SEQ ID NO. 156

[0224] Each of examples 100-116 are cultivated to produce fatty acid methyl esters using the shake flask method described above. Amounts of C8 and C10 fatty acid esters are measured. The ability of each example to increase production of C8 and/or C10 fatty esters is recorded as indicated.

TABLE-US-00039 Increase C8 Increase Increase C8 FAME total FAME FAME Specificity relative to relative to relative to Inventive Inventive Inventive Designation 3-ketoacyl-CoA synthase Control C Control C* Control C** Ex. 100 Asch(V30A, T184I, V268A, V296A, ++ + ◯ E315K, V317A, S328G, H368R) Ex. 101 Asch(V30A, A54V, T184I, V268A, ++ ++ + V296A, V317A, S328G,H368R) Ex. 102 Asch(V30A, T184I, V268A, V296A, + + + I302T, V317A, S328G, H368R) Ex. 103 Asch(V30A, A54V, T184I, V268A, ++ ++ + V296A, I302TV317A, S328G, H368R) Ex. 104 Asch(V30A, T184I, V268A, M271I ++ + + V296A, V317A, S328G, H368R) Ex. 105 Asch(V30A, T184I, V268A, V296A, +++ +++ + V317A, S328G, A356G, H368R) Ex. 106 Asch(V30A, T184I, V268A, V296A, + + + V317A, S328G, A356S, H368R) Ex. 107 Asch(V30A, A154G, T184I, V268A, ++ +++ + V296A, V317A, S328G, H368R) Ex. 108 Asch(V30A, T184I, I232V, V268A, +++ ++ + V296A, V317A, S328G, H368R) Increase C8 Increase Increase C8 FAME total FAME FAME Specificity relative to relative to relative to Designation 3-ketoacyl-CoA synthase Ex. 107 Ex. 107* Ex. 107** Ex. 109 Asch(V30A, A54V, A154G, T184I, + + ◯ V268A, V296A, V317A, S328G, H368R) Ex. 110 Asch(V30A, A154G, T184I, V268A, M271I, + + − V296A, I302T, V317A, S328G, H368R) Ex. 111 Asch(V30A, A54V, A154G, T184I, +++ +++ ◯ V268A, M271I, V296A, I302T, V317A, S328G, H368R) Increase C8 Increase Increase C8 FAME total FAME FAME Specificity relative to relative to relative to Designation 3-ketoacyl-CoA synthase Ex. 104 Ex. 104* Ex. 104** Ex. 112 Asch(V30A, A54V, T184I, V268A, +++ +++ + M271I, V296A, I302T, V317A, S328G, H368R) Ex. 113 Asch(V30A, A54V, T184I, V268A, + + + M271I, V296A, V317A, S328G, H368R) Ex. 114 Asch(V30A, T184I, V268A, M271I, V296A, +++ +++ + I302T, V317A, S328G, H368R) Ex. 115 Asch(V30A, A54V, A154G, T184I, + + + V268A, M271I, V296A, V317A, S328G, H368R) Ex. 116 Asch(V30A, A154G, T184I, V268A, + ++ + M271IV296A, V317A, S328G, H368R) *(+) = Increase over control strain **(+) = Increase over control strain; (−) = No change over control strain; (◯) = Decrease over control strain

Examples 117-133—Asch(G51) Variants

[0225] Mutant E. coli strains Examples 117-133 similarly contain a mutated Asch gene encoding a mutated 3-ketoacyl-CoA synthase with multiple mutations as indicated in the following table. Details of strain construction are as follows:

TABLE-US-00040 Host NphT7 3-ketoacyl-CoA synthase Designation Strain mutant Promoter Mutant Ex. 117 3 LSVA PphoE Asch(G51A, T184I, V268A, V296A, S328G), SEQ ID NO. 172 Ex. 118 3 LSVA PphoE Asch(G51C, T184I, V268A, V296A, S328G), SEQ ID NO. 173 Ex. 119 3 LSVA PphoE Asch(G51D, T184I, V268A, V296A, S328G), SEQ ID NO. 174 Ex. 120 3 LSVA PphoE Asch(G51H, T184I, V268A, V296A, S328G), SEQ ID NO. 175 Ex. 121 3 LSVA PphoE Asch(G51I, T184I, V268A, V296A, S328G), SEQ ID NO. 176 Ex. 122 3 LSVA PphoE Asch(G51K, T184I, V268A, V296A, S328G), SEQ ID NO. 177 Ex. 123 3 LSVA PphoE Asch(G51L, T184I, V268A, V296A, S328G), SEQ ID NO. 178 Ex. 124 3 LSVA PphoE Asch(G51M, T184I, V268A, V296A, S328G), SEQ ID NO. 179 Ex. 125 3 LSVA PphoE Asch(G51N, T184I, V268A, V296A, S328G), SEQ ID NO. 180 Ex. 126 3 LSVA PphoE Asch(G51P, T184I, V268A, V296A, S328G), SEQ ID NO. 181 Ex. 127 3 LSVA PphoE Asch(G51Q, T184I, V268A, V296A, S328G), SEQ ID NO. 182 Ex. 128 3 LSVA PphoE Asch(G51R, T184I, V268A, V296A, S328G), SEQ ID NO. 183 Ex. 129 3 LSVA PphoE Asch(G51S, T184I, V268A, V296A, S328G), SEQ ID NO. 184 Ex. 130 3 LSVA PphoE Asch(G51T, T184I, V268A, V296A, S328G), SEQ ID NO. 185 Ex. 131 3 LSVA PphoE Asch(G51V, T184I, V268A, V296A, S328G), SEQ ID NO. 186 Ex. 132 3 LSVA PphoE Asch(G51W, T184I, V268A, V296A, S328G), SEQ ID NO. 187 Ex. 133 3 LSVA PphoE Asch(G51Y, T184I, V268A, V296A, S328G), SEQ ID NO. 188

[0226] Each of examples 117-133 are cultivated to produce fatty acid methyl esters using the shake flask method described above. Total fatty acid methyl ester (FAME) and amounts of C8 and C10 fatty acid esters produced are as indicated in the following table, as are relative percentages of C8 and and C10 fatty acids. Amounts of higher- and lower-carbon number fatty acid esters are not shown separately, but are included in the total FAME production value indicated.

TABLE-US-00041 Total FAME production C8 FAME relative to g/L relative 3-ketoacyl-CoA Inventive to Inventive Designation synthase Control A Control A Ex. 117 Asch(G51A, + + T184I, V268A, V296A, S328G), SEQ ID NO. 172 Ex. 118 Asch(G51C, + + T184I, V268A, V296A, S328G), SEQ ID NO. 173 Ex. 119 Asch (G51D, +++ ++ T184I, V268A, V296A, S328G), SEQ ID NO. 174 Ex. 120 Asch(G51H, ++ ++ T184I, V268A, V296A, S328G), SEQ ID NO. 175 Ex. 121 Asch(G51I, + + T184I, V268A, V296A, S328G), SEQ ID NO. 176 Ex. 122 Asch (G51K, +++ +++ T184I, V268A, V296A, S328G), SEQ ID NO. 177 Ex. 123 Asch(G51L, +++ +++ T184I, V268A, V296A, S328G), SEQ ID NO. 178 Ex. 124 Asch(G51M, +++ +++ T184I, V268A, V296A, S328G), SEQ ID NO. 179 Ex. 125 Asch(G51N, +++ +++ T184I, V268A, V296A, S328G), SEQ ID NO. 180 Ex. 126 Asch(G51P, +++ +++ T184I, V268A, V296A, S328G), SEQ ID NO. 181 Ex. 127 Asch (G51Q, +++ +++ T184I, V268A, V296A, S328G), SEQ ID NO. 182 Ex. 128 Asch(G51R, + + T184I, V268A, V296A, S328G), SEQ ID NO. 183 Ex. 129 Asch(G51S, ++ ++ T184I, V268A, V296A, S328G), SEQ ID NO. 184 Ex. 130 Asch(G51T, + ++ T184I, V268A, V296A, S328G), SEQ ID NO. 185 Ex. 131 Asch(G51V, + ++ T184I, V268A, V296A, S328G), SEQ ID NO. 186 Ex. 133 Asch(G51W, ++ ++ T184I, V268A, V296A, S328G), SEQ ID NO. 187 Ex. 133 Asch(G51Y, +++ ++ T184I, V268A, V296A, S328G), SEQ ID NO. 188

Examples 134-148—Asch(G51) Variants

[0227] A mutant 3-ketoacyl-CoA synthase corresponding to SEQ ID NO. 8 with seven specific mutations as indicated in the following table, is selected as a candidate for further improvement through additional mutations. Inventive Control D is produced by introducing this mutant 3-ketoacyl-CoA synthase into E. coli host strain 3. This inventive example serves as a basis for comparison for the additional mutants Examples 134-148. Mutant E. coli strains Examples 134-148 contain a mutated Asch gene encoding a mutated 3-ketoacyl-CoA synthase having the same mutations as that of Inventive Control D together with one or more additional mutations. All mutations differing from the wild-type 3-ketoacyl-CoA synthase (SEQ ID NO. 8) are indicated in the following table. Details of strain construction are as follows:

TABLE-US-00042 Host NphT7 3-ketoacyl-CoA synthase Designation Strain mutant Promoter Mutant Inventive 3 LSVA PpstsIH Asch (V30A, T184I, V268A, V296A, V317A, Control D S238G, H368R) Ex. 134 3 LSVA PpstsIH Asch(V30A, G51C, T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 189 Ex. 135 3 LSVA PpstsIH Asch(V30A, G51D, T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 190 Ex. 136 3 LSVA PpstsIH Asch(V30A, G51E, T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 191 Ex. 137 3 LSVA PpstsIH Asch(V30A, G51F, T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 192 Ex. 138 3 LSVA PpstsIH Asch(V30A, G51H, T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 193 Ex. 139 3 LSVA PpstsIH Asch(V30A, G51I, T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 194 Ex. 140 3 LSVA PpstsIH Asch(V30A, G51K, T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 195 Ex. 141 3 LSVA PpstsIH Asch(V30A, G51M, T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 196 Ex. 142 3 LSVA PpstsIH Asch(V30A, G51N, T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 197 Ex. 143 3 LSVA PpstsIH Asch(V30A, G51P, T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 198 Ex. 144 3 LSVA PpstsIH Asch(V30A, G51R, T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 199 Ex. 145 3 LSVA PpstsIH Asch(V30A, G51S, T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 200 Ex. 146 3 LSVA PpstsIH Asch(V30A, G51T, T184L V268A V296A, V317A, S328G, H368R), SEQ ID NO. 201 Ex. 147 3 LSVA PpstsIH Asch(V30A, G51W, T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 202 Ex. 148 3 LSVA PpstsIH Asch(V30A, G51Y, T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 203

[0228] Each of examples 134-148 are cultivated to produce fatty acid methyl esters using the shake flask method described above. Total fatty acid methyl ester (FAME) and amounts of C8 and C10 fatty acid esters produced are as indicated in the following table, as are relative percentages of C8 and and C10 fatty acids. Amounts of higher- and lower-carbon number fatty acid esters are not shown separately, but are included in the total FAME production value indicated.

TABLE-US-00043 Total FAME production C8 FAME relative to g/L relative Inventive to Inventive Designation 3-ketoacyl-CoA synthase Control D Control D Ex. 134 Asch(V30A, G51C, + +++ T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 189 Ex. 135 Asch(V30A, G51D, +++ ++ T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 190 Ex. 136 Asch(V30A, G51E, +++ ++ T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 191 Ex. 137 Asch(V30A, G51F, +++ ++ T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 192 Ex. 138 Asch(V30A, G51H, + + T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 193 Ex. 139 Asch(V30A, G51I, + +++ T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 194 Ex. 140 Asch(V30A, G51K, +++ +++ T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 195 Ex. 141 Asch(V30A, G51M, ++ ++ T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 196 Ex. 142 Asch(V30A, G51N, +++ ++ T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 197 Ex. 143 Asch(V30A, G51P, + + T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 198 Ex. 144 Asch(V30A, G51R, + + T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 199 Ex. 145 Asch(V30A, G51S, + ++ T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 200 Ex. 146 Asch(V30A, G51T, + +++ T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 201 Ex. 147 Asch(V30A, G51W, ++ ++ T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 202 Ex. 148 Asch(V30A, G51Y, + + T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 203

EMBODIMENTS

[0229] 1. A 3-ketoacyl-CoA synthase having an amino acid sequence characterized in including at least one of a) a sub-sequence at least 80% identical to SEQ ID NO. 1, provided that an amino acid residue that aligns to amino acid residue 8 of SEQ ID NO. 1 is leucine, valine, isoleucine or methionine and amino acid residue 2 is leucine or methionine; b) a sub-sequence at least 80% identical to SEQ ID NO. 2, provided that an amino acid residue that aligns to amino acid residue 6 of SEQ ID NO. 2 is isoleucine or methionine and c) a sub-sequence at least 80% identical to SEQ ID NO. 3, provided that an amino acid residue that aligns with amino acid residue 6 of SEQ ID NO. 3 is isoleucine, methionine, threonine, cysteine, valine, glutamine, phenylalanine, aspartic acid, asparagine or tyrosine.
2. A 3-ketoacyl-CoA synthase having an amino acid sequence characterized in including at least one of a) SEQ ID NO. 1, b) SEQ ID NO. 2 and c) SEQ ID NO. 3.
3. The 3-ketoacyl-CoA synthase of embodiment 2 wherein the amino acid sequence includes SEQ ID NO. 1.
4. The 3-ketoacyl-CoA synthase of any of embodiments 1-3 wherein the amino acid sequence further includes at least one of a) SEQ ID NO. 4 or SEQ ID NO. 161, b) SEQ ID NO. 5 and c) SEQ ID NO. 6 or SEQ ID NO. 162.
5. The 3-ketoacyl-CoA synthase of embodiment 4 wherein the amino acid sequence includes SEQ ID NO. 44.
6. The 3-ketoacyl-CoA synthase of embodiment 5 wherein the amino acid sequence includes a sub-sequence at least 85% identical to SEQ ID NO. 45, provided that an amino acid residue of the 3-ketoacyl-CoA synthase that aligns to amino acid residue 35 of SEQ ID NO. 5 is leucine, valine, isoleucine or methionine.
7. The 3-ketoacyl-CoA synthase of embodiment 5 wherein the amino acid sequence includes SEQ ID NO. 45.
8. The 3-ketoacyl-CoA synthase of any of embodiments 4-7 wherein the amino acid sequence includes SEQ ID NO. 4 or SEQ ID NO. 161, SEQ ID NO. 5 and SEQ ID NO. 6 or SEQ ID NO. 162.
9. The 3-ketoacyl-CoA synthase of embodiment 7 wherein the amino acid sequence further includes SEQ ID NO. 46.
10. The 3-ketoacyl-CoA synthase of embodiment 8 wherein the amino acid sequence includes SEQ ID NO. 47.
11. The 3-ketoacyl-CoA synthase of any of embodiments 1-10 wherein the amino acid sequence includes SEQ ID NO. 48.
12. The 3-ketoacyl-CoA synthase of embodiment 3 wherein the amino acid sequence is at least 80% identical to SEQ ID NO. 49.
13. The 3-ketoacyl-CoA synthase of embodiment 3 wherein the amino acid sequence has SEQ ID NO. 49.
14. The 3-ketoacyl-CoA synthase of any of embodiments 1-13 wherein the amino acid sequence is at least 50% identical to SEQ ID NO. 8.
15. The 3-ketoacyl-CoA synthase of embodiment 14 wherein the amino acid sequence is at least 80% identical to SEQ ID NO. 8.
16. A 3-ketoacyl-CoA synthase having an amino acid sequence is selected from the group consisting of SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 92, SEQ ID NO. 93, any one of SEQ ID NOs 121-157 or any one of SEQ ID NOs. 172-204.
17. The 3-ketoacyl-CoA synthase of embodiment 2 wherein the amino acid sequence includes SEQ ID NO. 2.
18. The 3-ketoacyl-CoA synthase of embodiment 17 wherein the amino acid sequence further includes at least one of a) SEQ ID NO. 4 or SEQ ID NO. 161, b) SEQ ID NO. 5 and c) SEQ ID NO. 6 or SEQ ID NO. 162.
19. The 3-ketoacyl-CoA synthase of embodiment 18 wherein the amino acid sequence is at least 50% identical to SEQ ID NO. 40.
20. The 3-ketoacyl-CoA synthase of embodiment 19 wherein the amino acid sequence is at least 80% identical to SEQ ID NO. 40.
21. The 3-ketoacyl-CoA synthase of embodiment 18 which has SEQ. ID NO. 40.
22. The 3-ketoacyl-CoA synthase of embodiment 2 wherein the amino acid sequence includes SEQ ID NO. 3.
23. The 3-ketoacyl-CoA synthase of embodiment 22 wherein the amino acid sequence further includes a) SEQ ID NO. 4 or SEQ ID NO. 161, b) SEQ ID NO. 5 and c) SEQ ID NO. 6 or SEQ ID NO. 162.
24. The 3-ketoacyl-CoA synthase of embodiment 22 wherein the amino acid sequence includes SEQ ID NO. 41.
25. The 3-ketoacyl-CoA synthase of embodiment 22 wherein the amino acid sequence is at least 50% identical to SEQ ID NO. 42 or 43.
26. The 3-ketoacyl-CoA synthase of embodiment 22 wherein the amino acid sequence at least 80% identical to SEQ ID NO. 42 or 43.
27. The 3-ketoacyl-CoA synthase of embodiment 22 wherein the amino acid sequence is SEQ. ID NO. 42 or 43.
28. A 3-ketoacyl-CoA synthase having an amino acid sequence characterized in including SEQ ID. NO. 50.
29. The 3-ketoacyl-CoA synthase of embodiment 28 wherein the amino acid sequence includes SEQ ID NO. 51.
30. The 3-ketoacyl-CoA synthase of embodiment 28 wherein the amino acid sequence includes SEQ ID NO. 52.
31. The 3-ketoacyl-CoA synthase of embodiment 28 wherein the amino acid sequence includes SEQ ID NO. 53.
32. The 3-ketoacyl-CoA synthase of any of embodiments 23-31 wherein the amino acid sequence further includes SEQ ID NO. 46.
33. The 3-ketoacyl-CoA synthase of embodiment 28 wherein the amino acid sequence includes SEQ ID NO. 54.
34. The 3-ketoacyl-CoA synthase of any of embodiments 28-33 wherein the amino acid sequence further includes SEQ ID NO. 48.
35. The 3-ketoacyl-CoA synthase of embodiment 28 wherein the amino acid sequence includes SEQ ID NO. 55.
36. A 3-ketoacyl-CoA synthase having an amino acid sequence characterized in including SEQ ID. NO. 56.
37. The 3-ketoacyl-CoA synthase of embodiment 36 wherein the amino acid sequence includes SEQ ID NO. 57.
38. The 3-ketoacyl-CoA synthase of embodiment 36 or 37 wherein the amino acid sequence further includes SEQ ID NO. 51. 39. The 3-ketoacyl-CoA synthase of embodiment 36 or 37 wherein the amino acid sequence further includes SEQ ID NO. 52.
40. The 3-ketoacyl-CoA synthase of embodiment 36 or 37 wherein the amino acid sequence further includes SEQ ID NO. 53.
41. The 3-ketoacyl-CoA synthase of embodiment 36 wherein the amino acid sequence includes SEQ ID NO. 58.
42. The 3-ketoacyl-CoA synthase of any of embodiments 36-41 wherein the amino acid sequence further includes SEQ ID NO. 48.
43. The 3-ketoacyl-CoA synthase of embodiment 36 wherein the amino acid sequence includes SEQ ID NO. 59.
44. A 3-ketoacyl-CoA synthase having an amino acid sequence characterized in including SEQ ID. NO. 60.
45. The 3-ketoacyl-CoA synthase of embodiment 44 wherein the amino acid sequence includes SEQ ID. NO. 61.
46. The 3-ketoacyl-CoA synthase of embodiment 44 wherein the amino acid sequence includes SEQ ID. NO. 62.
47. The 3-ketoacyl-CoA synthase of embodiment 44 wherein the amino acid sequence includes SEQ ID. NO. 63.
48. The 3-ketoacyl-CoA synthase of any of embodiments 44-46 wherein the amino acid includes SEQ ID. NO. 46.
49. The 3-ketoacyl-CoA synthase of any of embodiments 44-48 wherein the amino acid sequence includes SEQ ID. NO. 48.
50. The 3-ketoacyl-CoA synthase of embodiment 44 wherein the amino acid sequence includes SEQ ID. NO. 64.
51. A 3-ketoacyl-CoA synthase having an acid sequence characterized in including SEQ ID. NO. 65.
52. The 3-ketoacyl-CoA synthase of embodiment 51 wherein the amino acid sequence includes SEQ ID. NO. 51.
53. The 3-ketoacyl-CoA synthase of any of embodiments 51 or 52 wherein the amino acid sequence includes SEQ ID. NO. 66.
54. The 3-ketoacyl-CoA synthase of any of embodiments 51-53 wherein the amino acid sequence includes SEQ ID. NO. 48.
55. The 3-ketoacyl-CoA synthase of any of embodiments 51-54 wherein the amino acid sequence includes at least one of a) SEQ ID. NO. 4 or SEQ ID NO. 161 and b) SEQ ID NO. 5.
56. The 3-ketoacyl-CoA synthase of any of embodiments 51-55 wherein the amino acid sequence includes SEQ ID. NO. 53.
57. The 3-ketoacyl-CoA synthase of embodiment 51 wherein the amino acid sequence includes SEQ ID. NO. 67.
58. The 3-ketoacyl-CoA synthase of embodiment 51 wherein the amino acid sequence includes SEQ ID. NO. 68.
59. A 3-ketoacyl-CoA synthase comprising a heterologous nucleic acid sequence encoding a 3-ketoacyl-CoA synthase having an amino sequence characterized in including SEQ ID. NO. 69.
60. The 3-ketoacyl-CoA synthase of embodiment 59 wherein the amino acid sequence further includes SEQ ID. NO. 51.
61. The 3-ketoacyl-CoA synthase of embodiment 59 or 60 wherein the amino acid sequence includes SEQ ID NO. 52.
62. The 3-ketoacyl-CoA synthase of embodiment 59 or 69 wherein the amino acid sequence includes SEQ ID NO. 53.
63. The 3-ketoacyl-CoA synthase of any of embodiments 56-62 wherein the amino acid sequence includes at least one of a) SEQ ID NO. 5 and b) SEQ ID NO. 6 or SEQ ID NO. 162.
64. The 3-ketoacyl-CoA synthase of any of embodiments 59-63 wherein the amino acid sequence includes SEQ ID NO. 46.
65. The 3-ketoacyl-CoA synthase of embodiment 59 or 60 wherein the amino acid sequence includes SEQ ID NO. 70.
66. The 3-ketoacyl-CoA synthase of any of embodiments 59-65 wherein the amino acid sequence includes SEQ ID NO. 48.
67. The 3-ketoacyl-CoA synthase of embodiment 59 wherein the amino acid sequence includes ID NO. 71.
68. A ketoacyl-CoA synthase having SEQ ID NO. 110 or being at least 80% identical to SEQ ID NO. 110, comprising in each case at least one of features i)-xiii):

[0230] i) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 30 of SEQ ID NO. 110 is alanine;

[0231] ii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 39 of SEQ ID NO. 110 is valine;

[0232] iii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 69 of SEQ ID NO. 110 is valine;

[0233] iv) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 111 of SEQ ID NO. 110 is cysteine;

[0234] v) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 152 of SEQ ID NO. 110 is cysteine, leucine, methionine or threonine;

[0235] vi) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 178 of SEQ ID NO. 110 is leucine;

[0236] vii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 184 of SEQ ID NO. 110 is isoleucine, leucine, methionine or valine;

[0237] viii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 268 of SEQ ID NO. 110 is alanine;

[0238] ix) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 271 of SEQ ID NO. 110 is isoleucine;

[0239] x) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 278 of SEQ ID NO. 110 is arginine;

[0240] xi) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 296 of SEQ ID NO. 110 is alanine;

[0241] xii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 317 of SEQ ID NO. 110 is alanine;

[0242] xiii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 328 of SEQ ID NO. 110 is glycine.

69. A ketoacyl-CoA synthase having SEQ ID NO. 119 or being at least 80% identical to SEQ ID NO. 119, comprising in each case at least one of features i) to xliii):

[0243] i) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 18 of SEQ ID NO. 119 is alanine;

[0244] ii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 22 of SEQ ID NO. 119 is methionine;

[0245] iii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 30 of SEQ ID NO. 119 is alanine;

[0246] iv) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 38 of SEQ ID NO. 119 is valine;

[0247] v) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 39 of SEQ ID NO. 119 is valine;

[0248] vi) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 51 of SEQ ID NO. 119 is alanine, cysteine, aspartic acid, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine;

[0249] vii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 54 of SEQ ID NO. 119 is valine;

[0250] viii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 69 of SEQ ID NO. 119 is valine;

[0251] ix) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 83 of SEQ ID NO. 119 is asparagine;

[0252] x) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 94 of SEQ ID NO. 119 is threonine, leucine, glutamic acid, or alanine;

[0253] xi) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 111 of SEQ ID NO. 119 is cysteine;

[0254] xii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 116 of SEQ ID NO. 119 is glycine;

[0255] xiii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 127 of SEQ ID NO. 119 is a threonine;

[0256] xiv) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 130 of SEQ ID NO. 119 is glycine;

[0257] xv) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 152 of SEQ ID NO. 119 is cysteine, leucine, methionine or threonine;

[0258] xvi) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 154 of SEQ ID NO. 119 is glycine;

[0259] xvii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 178 of SEQ ID NO. 119 is leucine or threonine;

[0260] xviii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 190 of SEQ ID NO. 119 is phenylalanine or tyrosine;

[0261] xix) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 210 of SEQ ID NO. 119 is valine;

[0262] xx) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 223 of SEQ ID NO. 119 is histidine;

[0263] xxi) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 231 of SEQ ID NO. 119 is isoleucine;

[0264] xxii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 232 of SEQ ID NO. 119 is valine;

[0265] xxiii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 236 of SEQ ID NO. 119 is leucine or methionine;

[0266] xxiv) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 241 of SEQ ID NO. 119 is proline;

[0267] xxv) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 268 of SEQ ID NO. 119 is alanine;

[0268] xxvi) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 271 of SEQ ID NO. 119 is isoleucine;

[0269] xxvii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 274 of SEQ ID NO. 119 is glutamic acid;

[0270] xxviii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 278 of SEQ ID NO. 119 is arginine, glutamic acid, aspartic acid, glutamine;

[0271] xxix) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 280 of SEQ ID NO. 119 is isoleucine;

[0272] xxx) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 282 of SEQ ID NO. 119 is glycine;

[0273] xxxi) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 296 of SEQ ID NO. 119 is alanine;

[0274] xxxii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 302 of SEQ ID NO. 119 is threonine;

[0275] xxxiii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 312 of SEQ ID NO. 119 is aspartic acid;

[0276] xxxiv) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 313 of SEQ ID NO. 119 is glutamic acid or methionine;

[0277] xxxv) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 315 of SEQ ID NO. 119 is lysine;

[0278] xxxvi) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 317 of SEQ ID NO. 119 is alanine;

[0279] xxxvii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 322 of SEQ ID NO. 119 is glycine;

[0280] xxxviii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 328 of SEQ ID NO. 119 is glycine;

[0281] xxxix) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 329 of SEQ ID NO. 119 is glycine;

[0282] xl) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 344 of SEQ ID NO. 119 is glutamic acid, aspartic acid or asparagine;

[0283] xli) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 356 of SEQ ID NO. 119 is glycine or serine;

[0284] xlii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 368 of SEQ ID NO. 119 is arginine; and

[0285] xliii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 370 of SEQ ID NO. 119 is threonine.

70. The ketoacyl-CoA synthase of embodiment 69 wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 184 of SEQ ID NO. 119 is isoleucine, leucine, methionine or valine.
71. The ketoacyl-CoA synthase of embodiment 69 wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 184 of SEQ ID NO. 119 is isoleucine.
72. .sub.[GC4]The ketoacyl-CoA synthase of any of embodiments 69-71 wherein:

[0286] the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 268 of SEQ ID NO. 119 is alanine;

[0287] the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 296 of SEQ ID NO. 119 is alanine; and

[0288] the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 328 of SEQ ID NO. 119 is glycine.

73. The ketoacyl-CoA synthase of any of embodiments 69-72 wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 317 of SEQ ID NO. 119 is alanine.
74. The ketoacyl-CoA synthase of any of embodiments 70-73 wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 30 of SEQ ID NO. 119 is alanine.
75. The ketoacyl-CoA synthase of any of embodiments 70-74 wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 368 of SEQ ID NO. 119 is arginine.
76. The ketoacyl-CoA synthase of any of embodiments 69-75 wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 51 of SEQ ID NO. 119 is alanine, cysteine, aspartic acid, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine.
77. The ketoacyl-CoA synthase of any of embodiments 69-76 wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 51 of SEQ ID NO. 119 is alanine.
78. The 3-ketoacyl-CoA synthase having an amino acid sequence selected from the group consisting of any of SEQ ID NOs. 121-157.
79. A ketoacyl-CoA synthase having SEQ ID NO. 110 or being at least 80% identical to SEQ ID NO. 110, comprising in each case at least one of features i)-xiii):

[0289] i) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 30 of SEQ ID NO. 110 is alanine;

[0290] ii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 39 of SEQ ID NO. 110 is valine;

[0291] iii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 69 of SEQ ID NO. 110 is valine;

[0292] iv) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 111 of SEQ ID NO. 110 is cysteine;

[0293] v) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 152 of SEQ ID NO. 110 is cysteine, leucine, methionine or threonine;

[0294] vi) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 178 of SEQ ID NO. 110 is leucine;

[0295] vii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 184 of SEQ ID NO. 110 is isoleucine, leucine, methionine or valine;

[0296] viii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 268 of SEQ ID NO. 110 is alanine;

[0297] ix) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 271 of SEQ ID NO. 110 is isoleucine;

[0298] x) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 278 of SEQ ID NO. 110 is arginine;

[0299] xi) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 296 of SEQ ID NO. 110 is alanine;

[0300] xii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 317 of SEQ ID NO. 110 is alanine;

[0301] xiii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 328 of SEQ ID NO. 110 is glycine.

80. The ketoacyl-CoA synthase of embodiment 79 wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 184 of SEQ ID NO. 120 is isoleucine, leucine, methionine or valine.
81. The ketoacyl-CoA synthase of embodiment 79 wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 184 of SEQ ID NO. 120 is isoleucine.
82. .sub.[GC5]The ketoacyl-CoA synthase of any of embodiments 79-81 wherein:

[0302] the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 268 of SEQ ID NO. 120 is alanine;

[0303] the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 296 of SEQ ID NO. 120 is alanine; and

[0304] the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 328 of SEQ ID NO. 120 is glycine.

83. The ketoacyl-CoA synthase of any of embodiments 79-82 wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 317 of SEQ ID NO. 120 is alanine.
84. The ketoacyl-CoA synthase of any of embodiments 80-83 wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 30 of SEQ ID NO. 120 is alanine.
85. The ketoacyl-CoA synthase of any of embodiments 80-84 wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 368 of SEQ ID NO. 120 is arginine.
86. A ketoacyl-CoA synthase having SEQ ID NO. 110 or being at least 80% identical to SEQ ID NO. 110, comprising in each case at least one of features i)-xiii):

[0305] i) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 30 of SEQ ID NO. 110 is alanine;

[0306] ii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 39 of SEQ ID NO. 110 is valine;

[0307] iii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 69 of SEQ ID NO. 110 is valine;

[0308] iv) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 111 of SEQ ID NO. 110 is cysteine;

[0309] v) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 152 of SEQ ID NO. 110 is cysteine, leucine, methionine or threonine;

[0310] vi) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 178 of SEQ ID NO. 110 is leucine;

[0311] vii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 184 of SEQ ID NO. 110 is isoleucine, leucine, methionine or valine;

[0312] viii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 268 of SEQ ID NO. 110 is alanine;

[0313] ix) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 271 of SEQ ID NO. 110 is isoleucine;

[0314] x) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 278 of SEQ ID NO. 110 is arginine;

[0315] xi) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 296 of SEQ ID NO. 110 is alanine;

[0316] xii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 317 of SEQ ID NO. 110 is alanine;

[0317] xiii) an amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 328 of SEQ ID NO. 110 is glycine.

87. The ketoacyl-CoA synthase of embodiment 86 wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 184 of SEQ ID NO. 110 is isoleucine, leucine, methionine or valine.
88. The ketoacyl-CoA synthase of embodiment 86 wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 184 of SEQ ID NO. 110 is isoleucine.
89. .sub.[GC6]The ketoacyl-CoA synthase of any of embodiments 86-88 wherein:

[0318] the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 268 of SEQ ID NO. 110 is alanine;

[0319] the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 296 of SEQ ID NO. 110 is alanine; and

[0320] the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 328 of SEQ ID NO. 110 is glycine.

90. The ketoacyl-CoA synthase of any of embodiments 86-89 wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 317 of SEQ ID NO. 110 is alanine.
91. The ketoacyl-CoA synthase of any of embodiments 87-90 wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 30 of SEQ ID NO. 110 is alanine.
92. The ketoacyl-CoA synthase of any of embodiments 87-91 wherein the amino acid residue of the ketoacyl-CoA synthase that aligns with amino acid residue 368 of SEQ ID NO. 110 is arginine.
93. A 3-ketoacyl-CoA synthase having an acid sequence characterized in including SEQ ID. NO. 170.
94. A 3-ketoacyl-CoA synthase gene encoding for a 3-ketoacyl-CoA synthase of any of embodiments 1-93.
95. A genetically modified cell comprising a heterologous nucleic acid sequence encoding a 3-ketoacyl-CoA synthase of any of embodiments 1-93.
96. A genetically modified cell comprising a 3-ketoacyl-CoA synthase gene of embodiment 94.
97. The genetically modified cell of embodiment 95 or 96 wherein the 3-ketoacyl-CoA synthase is malonyl-CoA-dependent.
98. The genetically modified cell of any of embodiment 95-97 which further comprises a heterologous nucleic acid sequence that encodes a malonyl-CoA-dependent 3-ketohexanoyl-CoA synthase.
99. The genetically modified cell of embodiment 98 wherein the heterologous nucleic acid sequence that encodes a malonyl-CoA-dependent 3-ketohexanoyl-CoA synthase includes SEQ ID. NO. 82.
100. The genetically modified cell of embodiment 98 wherein the heterologous nucleic acid sequence that encodes a malonyl-CoA-dependent 3-ketohexanoyl-CoA synthase includes SEQ ID. NO. 82 in which amino acid residue 100 is leucine, amino acid residue 147 is serine, threonine or phenylalanine, amino acid residue 217 is valine and amino acid residue 323 is valine.
101. The genetically modified cell of any of embodiments 95-100 which further comprises a heterologous nucleic acid sequence that encodes a malonyl-CoA-dependent 3-ketobutyryl-CoA synthase.
102. The genetically modified cell of embodiment 101 wherein the heterologous nucleic acid sequence that encodes a malonyl-CoA-dependent 3-ketobutyryl-CoA synthase includes SEQ ID. NO. 83.
103. The genetically modified cell of any of embodiments 95-102 further comprising a heterologous nucleic acid sequence that encodes a 3-ketoacyl-CoA reductase.
104. The genetically modified cell of embodiment 103 wherein the 3-ketoacyl-CoA reductase has or is at least 80% identical to any one of SEQ. ID. NO. 103, SEQ. ID. NO. 102 or SEQ. ID. NO. 101.
105. The genetically modified cell of any of embodiments 95-104 further comprising a heterologous nucleic acid sequence that encodes a 3-hydroxyacyl-CoA dehydratase.
106. The genetically modified cell of any of embodiments 95-105 further comprising a heterologous nucleic acid sequence that encodes for an enzyme that reduces a 3-ketoacyl-CoA to form a corresponding 3-hydroxyacyl-CoA and dehydrates the 3-ketoacyl-CoA to form a corresponding 2-trans-enoyl-CoA.
107. The genetically modified cell of embodiment 106 wherein the heterologous nucleic acid sequence that encodes for an enzyme that reduces a 3-ketoacyl-CoA to form a corresponding 3-hydroxyacyl-CoA and dehydrates the 3-hydroxyacyl-CoA to form a corresponding 2-trans-enoyl-CoA has or is at least 80% identical to SEQ. ID. NO. 98.
108. The genetically modified cell of any of embodiments 95-107 further comprising a heterologous nucleic acid sequence that encodes an enoyl-CoA reductase.
109. The genetically modified cell of any of embodiments 95-108 further comprising a heterologous nucleic acid sequence that encodes an ester synthase.
110. The genetically modified cell of any of embodiments 95-109 further comprising a deletion of a native LDH gene.
111. The genetically modified cell of any of embodiments 95-110 further comprising a deletion of a native pyruvate formate lyase gene.
112. The genetically modified cell of any of embodiments 95-111 further comprising deletion of a native methylglyoxal synthase gene.
113. The genetically modified cell of any of embodiments 95-112 further comprising a deletion of a native phosphotransacetylase gene.
114. The genetically modified cell of any of embodiments 95-113 further comprising a deletion of a native thioesterase gene.
115. The genetically modified cell of any of embodiments 95-114 further comprising a deletion of a native adhE gene.
116. The genetically modified cell of any of embodiments 95-115 further comprising a deletion of a native atoDAEB operon.
117. The genetically modified cell of any of embodiments 95-116 further comprising deletion of a native fadD gene.
118. The genetically modified cell of any of embodiments 95-111 further comprising a deletion of a native phosphotransacetylase gene.
119. The genetically modified cell of any of embodiments 95-118 further comprising a heterologous nucleic acid sequence that encodes for any of a fatty acyl-CoA reductase, a fatty aldehyde reductase, an acyl-ACP reductase, an acyl-CoA:ACP acyltransferase, a thioesterase, an acyl-CoA hydrolase, a carboxylic acid reductase, a CoA hydrolase, an aldehyde dehydrogenase, a carboxylic acid reductase and an acyl-ACP reductase.
120. The genetically modified cell of any of embodiments 95-119 which is a bacteria.
121. The genetically modified cell of embodiment 120 wherein the bacteria is E. coli.
122. A process for making one or more compounds having a straight-chain alkyl group, comprising culturing the genetically modified cell of any of embodiments 95-121 in a fermentation medium and recovering the compound(s) having a straight-chain alkyl group from the fermentation medium.
123. The process of embodiment 122 wherein at least 40% by weight of the compound(s) having a straight-chain alkyl group have 6-10 carbon atoms in the straight-chain alkyl group.
124. The process of embodiment 122 wherein at least 60% by weight of the compound(s) having a straight-chain alkyl group have 6-10 carbon atoms in the straight-chain alkyl group.
125. The process of any of embodiments 122-124 wherein the compound(s) having a straight-chain alkyl group are fatty alcohols.
126. The process of any of embodiments 122-124 wherein the compound(s) having a straight-chain alkyl group are fatty amides.
127. The process of any of embodiments 122-124 wherein the compound(s) having a straight-chain alkyl group are fatty diacids or fatty diacid esters.
128. The process of any of embodiments 122-124 wherein the compound(s) having a straight-chain alkyl group are fatty acids.
129. The process of any of embodiments 122-124 wherein the compound(s) having a straight-chain alkyl group fatty acid esters.
130. The process of embodiment 129 wherein the fatty acid esters are methyl and/or ethyl esters.
131. The process of embodiment 130 wherein the fermentation medium includes methanol and/or ethanol.
132. The process of any of embodiments 122-131 wherein at least 60% by weight of the compound(s) having a straight-chain alkyl group have 8 carbon atoms in the straight-chain alkyl group.
133. The process of any of embodiments 122-131 wherein at least 80% by weight of the compound(s) having a straight-chain alkyl group have 8 carbon atoms in the straight-chain alkyl group.
134. The process of any of embodiments 122-131 wherein at least 90% by weight of the compound(s) having a straight-chain alkyl group have 8 carbon atoms in the straight-chain alkyl group.
135. The process of any of embodiments 122-131 wherein at least 60% by weight of the compound(s) having a straight-chain alkyl group have 10 carbon atoms in the straight-chain alkyl group.
136. The process of any of embodiments 122-131 wherein at least 80% by weight of the compound(s) having a straight-chain alkyl group have 10 carbon atoms in the straight-chain alkyl group.
137. The process of any of embodiments 122-131 wherein at least 90% by weight of the compound(s) having a straight-chain alkyl group have 8 carbon atoms in the straight-chain alkyl group.
138. The process of any of embodiments 122-131 wherein at least 95% by weight of the compound(s) having a straight-chain alkyl group have 8 carbon atoms in the straight-chain alkyl group.
139. The process of any of embodiments 122-138 wherein at least 0.05 grams of the one or more compound(s) having a straight-chain alkyl group are produced per liter of fermentation broth per hour.
140. The process of any of embodiments 122-138 wherein at least 0.1 grams of the one or more compound(s) having a straight-chain alkyl group are produced per liter of fermentation broth per hour.
141. The process of any of embodiments 122-138 wherein at least 0.25 grams of the one or more compound(s) having a straight-chain alkyl group are produced per liter of fermentation broth per hour.
142. Use of a genetically modified cell of any of embodiments 95-121 to produce one or more compounds having a straight-chain alkyl group, wherein at least 60% by weight of the one or more compounds has 6 to 10 carbon atoms in the straight-chain alkyl group.
145. Use of a genetically modified cell of any of embodiments 95-121 to produce one or more compounds having a straight-chain alkyl group, wherein at least 80% by weight of the one or more compounds has 8 to 10 carbon atoms in the straight-chain alkyl group.
146. A cell that produces a 3-ketoacyl-CoA synthase of any of embodiments 1-93.