Microorganisms and methods for the production of fatty acids and fatty acid derived products

• US 10,465,213 B2
• Filed: 03/29/2016
• Issued: 11/05/2019
• Est. Priority Date: 08/10/2012
• Status: Active Grant

• Current Assignee: Cargill Incorporated
• Original Assignee: Cargill Incorporated
• Inventors: Liao, Hans, Spindler, Eileen, Warner, Joseph R., Louie, Michael, Ribble, Wendy, Prather, Brittany, Evans, Ron, Lipscomb, Tanya E. W., Lynch, Michael D.
• Primary Examiner(s): Mondesi, Robert B
• Assistant Examiner(s): Epstein, Todd M
• Application Number:
  US15/083,924
• Publication Number:
  US 20160340700A1
• Time in Patent Office: 1,316 Days
• Field of Search: None
• CPC Class Codes: C12N 1/205 Bacterial isolates
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/001 acting on the CH-CH group of donors (1.3)
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12P 7/04 acyclic
• CPC Class Codes: C12P 7/40 containing a carboxyl group including Peroxycarboxylic acids
• CPC Class Codes: C12P 7/6409 Fatty acids
• CPC Class Codes: C12R 2001/19 Escherichia coli
• CPC Class Codes: C12Y 101/01157 3-Hydroxybutyryl-CoA dehydrogenase (1.1.1.157)
• CPC Class Codes: C12Y 103/01009 Enoyl-[acyl-carrier-protein] reductase (NADH) (1.3.1.9)
• CPC Class Codes: C12Y 203/01194 Acetoacetyl-CoA synthase (2.3.1.194)

Abstract

This invention relates to metabolically engineered microorganism strains, such as bacterial strains, in which there is an increased utilization of malonyl-CoA for production of a fatty acid or fatty acid derived product, wherein the modified microorganism produces fatty acyl-CoA intermediates via a malonyl-CoA dependent but malonyl-ACP independent mechanism.

Claims

1. A genetically modified microorganism that produces a fatty acid or fatty acid derived product comprising (i) a genetic modification to overexpress a gene encoding an acetoacetyl-CoA synthase enzyme comprising Npht7, (ii) a deletion of a tesB gene and (iii) a genetic modification to overexpress a gene encoding AtTE thioesterase enzyme,

wherein:

the fatty acid or fatty acid derived product is produced via a growing acetyl-CoA or fatty acyl-CoA chain;

the acetyl-CoA or fatty acyl-CoA chain is extended with at least one malonyl-CoA molecule;

and

the fatty acid or fatty acid derived product is a mixture enriched for chain length C8 wherein the C8 chain comprises >

10% of the total fatty acid product on mass (g) basis.

2. The genetically modified microorganism of claim 1, wherein a butyryl-CoA intermediate is made from acetyl-CoA and malonyl-CoA.

3. The genetically modified microorganism of claim 1, wherein the microorganism comprises a genetic modification to convert an acyl-CoA into a fatty acid derived product, including an alcohol, aldehyde, alkene, alkane or diacid.

4. The genetically modified microorganism of claim 1, wherein the microorganism comprises a genetic modification to overexpress an enzyme selected from the group consisting of an enoyl-CoA reductase, a ketoacyl-CoA reductase and a 3-hydroxyacyl-CoA dehydratase.

5. The genetically modified microorganism of claim 4, wherein the microorganism comprises a genetic modification to overexpress an enoyl-CoA reductase that can utilize the cofactor NADH, NADPH or both NADH and NADPH, or a ketoacyl-CoA reductase that can utilize the cofactor NADH, NADPH or both NADH and NADPH.

6. The genetically modified microorganism of claim 4, wherein the enoyl-CoA reductase is E.C. No. 1.3.1.9, the ketoacyl-CoA reductase is E.C. No. 1.1.1.157, and the 3-hydroxyacyl-CoA dehydratase is E.C. No. 4.2.1.17.

7. The genetically modified microorganism of claim 4, wherein the enoyl-CoA reductase comprises a trans-2-enoyl-CoA reductase, the ketoacyl-CoA reductase comprises a 3-hydroxybutyryl-CoA dehydrogenase, and the 3-hydroxyacyl-CoA dehydratase comprises an enoyl-CoA hydratase.

8. The genetically modified microorganism of claim 1, wherein:

the microorganism produces a fatty acid and the microorganism comprises a genetic modification to overexpress a fatty acyl-CoA thioesterase, or a fatty acyl-CoA phosphotransferase and a fatty acid kinase;

the microorganism produces a fatty aldehyde and the microorganism comprises a genetic modification to overexpress a fatty acyl-CoA reductase;

the microorganism produces a fatty alcohol and the microorganism comprises a genetic modification to overexpress an alcohol-forming acyl-CoA reductase, a long-chain-fatty-acyl-CoA reductase and an alcohol dehydrogenase, or an aldehyde dehydrogenase and an alcohol dehydrogenase;

or

the microorganism produces a diacid and the microorganism comprises a genetic modification to overexpress one or more enzymes capable of catalyzing the conversion of a fatty acid to a diacid via omega or terminal oxidation.

9. The genetically modified microorganism of claim 1, wherein the microorganism is selected from bacteria, cyanobacteria, filamentous fungi and yeasts.