PRODUCTION OF FATTY ACID DERIVATIVES
Methods and compositions for producing fatty acid derivatives, for example, fatty esters, and commercial fuel compositions comprising fatty acid derivatives are described.
1 . A method of producing a fatty acid derivative, the method comprising culturing a host cell in the presence of a carbon source, wherein the host cell is genetically engineered to overexpress a gene encoding a thioesterase, a gene encoding an acyl-CoA synthase, and a gene encoding an ester synthase, wherein the gene encoding the thioesterase and the gene encoding the acyl-CoA synthase are integrated into the genomic DNA of the host cell.
2 . The method of claim 1 , further comprising isolating the fatty acid derivative.
3 . The method of claim 1 , further comprising culturing the host cell in the presence of an alcohol.
4 . The method of claim 1 , wherein the gene encoding a thioesterase is tesA, ‘tesA, fatB, fatB2, fatB3, fatA1, or fatA.
5 . The method of claim 1 , wherein the gene encoding an acyl-CoA synthase is fadD, fadK, BH3103, pfl-4354, EAV15023, fadD1, fadD2, RPC — 4074, fadDD, faa39, the gene that encodes the protein of GenBank Accession No. ZP — 01644875, or yhfL.
6 . The method of claim 1 , wherein the gene encoding an ester synthase is obtained from Acinetobacter sp., Alcanivorax borkumensis, Alcaligenes eutrophus, Mortierella alpina, Cryptococcus curvatus, Arabidopsis thaliana, Fundibacter jadensis, Pseudomonas aeruginosa, Rhodococcus opacus, Marinobacter hydrocarbonoclastics, Saccharomyces cerevisiae, Homo sapiens , or Simmondis chinensis.
7 . The method of claim 6 , wherein the gene encoding an ester synthase is wax/dgat, a gene encoding a wax synthase, a gene encoding a bifunctional ester synthase/acyl-CoA:diacylglycerol acyltransferase, ES9, ES8, DSM 8798, or AtfA2.
8 . The method of claim 1 , wherein the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis.
9 . The method of claim 8 , wherein the gene encoding the outer membrane protein receptor is a gene encoding an outer membrane ferrichrome transporter.
10 . The method of claim 8 , wherein the gene encoding the transcriptional regulator of fatty acid biosynthesis encodes a DNA transcriptional repressor.
11 . A method of producing a fatty acid derivative the method comprising culturing a host cell in the presence of a carbon source, wherein the host cell is genetically engineered to overexpress a gene encoding a thioesterase, a gene encoding an acyl-CoA synthase, and a gene encoding an ester synthase.
12 . The method of claim 11 , wherein the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of a gene encoding a pyruvate formate lyase, a lactate dehydrogenase, or both.
13 . The method of claim 1 , wherein the host cell is selected from the group consisting of a mammalian cell, plant cell, insect cell, yeast cell, fungus cell, filamentous fungi cell, cyanobacterial cell, and bacterial cell.
14 . The method of claim 1 , wherein the host cell is an E. coli cell.
15 . A fatty acid derivative produced by the method of claim 1 .
16 . A fatty ester produced by the method of claim 1 .
17 . A genetically engineered microorganism comprising at least one of a gene encoding a thioesterase, a gene encoding an acyl-CoA synthase, and a gene encoding an ester synthase, wherein the gene encoding the thioesterase and the gene encoding the acyl-CoA synthase are integrated into the genomic DNA of the microorganism, and wherein the microorganism produces an increased level of a fatty ester relative to a wild-type microorganism.
18 . A genetically engineered microorganism comprising an exogenous control sequence stably incorporated into the genomic DNA of the microorganism upstream of a gene encoding a thioesterase, and a gene encoding an acyl-CoA synthase, wherein the microorganism produces an increased level of a fatty ester relative to a wild-type microorganism.
19 . The genetically engineered microorganism of claim 18 , wherein the exogenous control sequence is a promoter.
20 . The genetically engineered microorganism of claim 19 , wherein the microorganism is genetically engineered to express, relative to a wild type microorganism, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis.
21 . The genetically engineered microorganism of claim 19 , wherein the microorganism is genetically engineered to express, relative to a wild type microorganism, a decreased level of at least one of a gene encoding a pyruvate formate lyase, and a gene encoding a lactate dehydrogenase.
22 . The genetically engineered microorganism of claim 20 , wherein the gene encoding the outer membrane protein receptor encodes a ferrichrome outer membrane transporter.
23 . The genetically engineered microorganism of claim 20 , wherein the gene encoding the transcriptional regulator encodes a DNA binding transcriptional repressor.
24 . The genetically engineered microorganism of claim 17 , selected from a Gram-negative or a Gram-positive bacterium.
25 . A method of producing a fatty acid derivative comprising culturing the genetically engineered microorganism of claim 17 , in the presence of an alcohol.
26 . A fatty acid derivative produced by the method of claim 25 .
27 . The fatty acid derivative of claim 26 , wherein the fatty acid derivative is a fatty ester.
28 . A biofuel composition comprising the fatty acid derivative of claim 26 .
29 . The biofuel composition of claim 28 , wherein the composition has a cloud point of about 5° C. or lower, of about 0° C. or lower, or of about −4° C. or lower.
30 . The biofuel composition of claim 28 , wherein the composition has a simulated distillation T90 of about 360° C. or lower, or of about 358° C. or lower.
31 . The biofuel composition of claim 28 , wherein the composition has a sulfur content of about 15 ppm or less, of about 12 ppm or less, or of about 10.7 ppm or less.
32 . The biofuel composition of claim 28 , wherein the composition has a Karl Fischer moisture content of about 0.1 wt. % or less, of about 0.08 wt. % or less, or of about 0.06 wt. % or less.
33 . The biofuel composition of claim 28 , wherein the composition has a total acid number of about 0.50 mg KOH/g or less, of about 0.20 mg KOH/g or less, or of about 0.15 mg KOH/g or less.
34 . The biofuel composition of claim 28 , wherein the composition has a combined calcium and magnesium content of about 5 ppm or less, of about 2 ppm or less, or of about 0.5 ppm or less.
35 . The biofuel composition of claim 28 , wherein the composition has a combined sodium and potassium content of about 5 ppm or less, of about 2.5 ppm or less, or of about 1.6 ppm or less.
36 . The biofuel composition of claim 28 , wherein the composition has a specific mass at 20° C. of about 850 to about 900 kg/m 3 , of about 860 to about 890 kg/m 3 , or of about 870 to about 880 kg/m 3 .
37 . The biofuel composition of claim 28 , wherein the composition has a kinematic viscosity at 40° C. of about 3 to about 6 mm 2 /s, of about 3.2 to about 5 mm 2 /s, or of about 3.5 to about 4 mm 2 /s.
38 . The biofuel composition of claim 28 , wherein the composition has a water content of about 500 mg/kg or less, of about 480 mg/kg or less, or of about 450 mg/kg or less.
39 . The biofuel composition of claim 28 , wherein the composition has a total contamination level of about 24 mg/kg or less, of about 20 mg/kg or less, of about 10 mg/kg or less, or of about 5 mg/kg or less.
40 . The biofuel composition of claim 28 , wherein the composition has a flash point of about 100° C. or higher, of about 110° C. or higher, or of about 120° C. or higher.
41 . The biofuel composition of claim 28 , wherein the composition has an ester content of about 96.5 wt. % or more, or of about 97 wt. % or more.
42 . The biofuel composition of claim 28 , wherein the composition has a carbon residue number of about 0.05 wt. % or less.
43 . The biofuel composition of claim 28 , wherein the composition has a sulfated ash level of 0.02 wt. % or less, or of 0.01 wt. % or less.
44 . The biofuel composition of claim 28 , wherein the composition has a phosphorus level of about 10 mg/kg or less, of about 5 mg/kg or less, or of about 1 mg/kg or less.
45 . The biofuel composition of claim 28 , wherein the composition has a copper corrosion score of 1.
46 . The biofuel composition of claim 28 , wherein the composition has a cold filter plugging point of about 19° C. or lower, of about 10° C. or lower, or of about 0° C. or lower.
47 . The biofuel composition of claim 28 , wherein the composition has a free glycerol level of about 0.02 wt. % or less.
48 . The biofuel composition of claim 28 , wherein the composition has a total glycerol level of about 0.25 wt. % or less, of about 0.15 wt. % or less, of about 0.10 wt. % or less, or of about 0.05 wt. % or less.
49 . The biofuel composition of claim 28 , wherein the composition has a monoacylglycerol level of about 0.02 wt. % or less.
50 . The biofuel composition of claim 28 , wherein the composition has a methanol or ethanol level of about 0.2 wt. % or less, of about 0.1 wt. % or less, or of about 0.02 wt. % or less.
51 . The biofuel composition of claim 28 , wherein the composition has an iodine number of about 65 g/100 g or less.
52 . The biofuel composition of claim 28 , wherein the composition has an oxidation stability at 110° C. of about 6 hours or longer, of about 9 hours or longer, or of about 11 hours or longer.