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

• US 10,337,038 B2
• Filed: 07/18/2014
• Issued: 07/02/2019
• Est. Priority Date: 07/19/2013
• Status: Active Grant

• Current Assignee: Cargill Incorporated
• Original Assignee: Cargill Incorporated
• Inventors: Lynch, Michael, Louie, Michael, Copley, Shelley, Spindler, Eileen, Prather, Brittany, Lipscomb, Matthew, Lipscomb, Tanya, Liao, Hans, Hogsett, Dave, Evans, Ron
• Primary Examiner(s): Treptow, Nancy A
• Application Number:
  US14/904,862
• Publication Number:
  US 20160257975A1
• Time in Patent Office: 1,810 Days
• Field of Search: None
• 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: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 7/40 containing a carboxyl group including Peroxycarboxylic acids
• CPC Class Codes: C12P 7/52 Propionic acid; Butyric acids
• CPC Class Codes: C12P 7/62 Carboxylic acid esters
• CPC Class Codes: C12P 7/6409 Fatty acids
• CPC Class Codes: C12P 7/6436 Fatty acid esters
• CPC Class Codes: C12Y 203/0118 Beta-ketoacyl-acyl-carrier-protein synthase III (2.3.1.180)
• 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 organism comprising a heterologous nucleic acid sequence encoding a NphT7 polypeptide and a wax ester synthase,

wherein said microorganism produces a fatty acid or fatty acid-derived product having a carbon chain length of C4 or greater, and

wherein the NphT7 polypeptide comprises one or more amino acid substitutions at a position selected from the group consisting of Ser84, Val114, Gly288, Ile194, Gly318, Thr85, Gln90, Val196, Tyr144, Phe159, Ile147, and Phe217.

2. The genetically modified organism of claim 1 wherein the NphT7 polypeptide comprises one or more amino acid substitutions selected from the group consisting of a PDRP to HFLQ substitution for amino acids 86-89, F217A, F217E, F217G, F217I, F217L, F217M, F217P, F217S, F217T, F217V, F217W, G288S, G309S, I147A, I147C, I147D, I147E, I147F, I147G, I147H, I147K, I147L, I147M, I147N, I147P, I147Q, I147R, I147S, I147T, I147V, I147W, I147Y, V157F, V196G, and Y144L.

3. The genetically modified organism of claim 1 wherein the NphT7 polypeptide comprises two amino acid substitutions selected from the group consisting of I147T and F217V, I147T and Y144L, I147T and V196G, I147F and F217V, I147M and F217V, I147S and F217V, I147T and HFLQ, I147T and V157F, I147T and F217G, I147T and F217A, I147T and F217L, I147T and F217I, I147T and F217M, I147T and F217P, I147T and F217S, I147T and F217E, I147S and F217G, I147S and F217A, I147S and F217L, I147S and F217I, I147S and F217M, I147S and F217W, I147S and F217S, I147S and F217E, I147S and F217K, I147F and F217A, I147F and F217L, I147F and F217I, I147F and F217M, I147F and F217P, I147F and F217E, I147M and F217G, I147M and F217A, I147M and F217L, I147M and F217I, I147M and F217M, I147M and F217P, I147M and F217S, I147M and F217E, and I147M and F217K.

4. The genetically modified organism of claim 1 wherein the NphT7 polypeptide comprises three sets of amino acid substitutions selected from the group consisting of Y144L, I147T, and F217V;

I147T, F217V, and a PDRP to HFLQ substitution for amino acids 86-89;

I147T, V157F, and F217V; and

Y144L, I147T, and V157F.

5. The genetically modified organism of claim 1 wherein said microorganism is capable of producing a fatty ester.

6. The genetically modified organism of claim 1 further comprising a heterologous nucleic acid sequence encoding a termination enzyme that catalyzes production of the fatty acid-derived product selected from the group comprising a fatty alcohol, a fatty aldehyde, a fatty alkene, a fatty amide, a fatty ester, a fatty alkane, and a fatty diacid.

7. The genetically modified organism of claim 5 wherein the wax ester synthase is selected from the group comprising Maq1, Pcry1, Rjos1, and Abork1.

8. The genetically modified organism of claim 5 wherein the wax ester synthase comprises an amino acid sequence of at least 70% homology to any one of SEQ ID NO 289, SEQ ID NO 290, SEQ ID NO 291, and SEQ ID NO 292.

9. The genetically modified organism of claim 5 wherein the heterologous nucleic acid further encodes:

a) a ketoacyl-CoA reductase;

b) a hydroxyacyl-CoA dehydratase; and

c) an enoyl-CoA reductase.

10. The genetically modified organism of claim 9, wherein:

a) the ketoacyl-CoA reductase is selected from the group consisting of SEQ ID NO;

183 and SEQ ID NO;

137;

b) the hydroxyacyl-CoA dehydratase is selected from the group consisting of SEQ ID NO;

183, SEQ ID NO;

273, and SEQ ID NO;

274; and

c) the enoyl-CoA reductase is SEQ ID NO;

275.

11. The genetically modified organism of claim 1 wherein the organism is capable of using acetyl-CoA as a primer and malonyl-CoA as an extender molecule to produce a fatty acid or fatty acid derived product having a carbon chain length selected from 4, 6, 8, 10, 12, 14, 16, 18 and 20.