Cells and methods for producing fatty alcohols

Patent metadata

Field	Value
Publication number	US-9708630-B1
Application number	US-201414509408-A
Country	US
Kind code	B1
Filing date	Oct 8, 2014
Priority date	Oct 8, 2013
Publication date	Jul 18, 2017
Grant date	Jul 18, 2017

Abstract

Recombinant cells and methods for improved yield of fatty alcohols. The recombinant cells harbor a recombinant thioesterase gene, a recombinant acyl-CoA synthetase gene, and a recombinant acyl-CoA reductase gene. In addition, a gene product from one or more of an acyl-CoA dehydrogenase gene, an enoyl-CoA hydratase gene, a 3-hydroxyacyl-CoA dehydrogenase gene, and a 3-ketoacyl-CoA thiolase gene in the recombinant cells is functionally deleted. Culturing the recombinant cells produces fatty alcohols at high yields.

First claim

What is claimed is:

1. A recombinant cell for producing a fatty alcohol comprising a recombinant thioesterase gene, a recombinant acyl-CoA synthetase gene, and a recombinant acyl-CoA reductase gene, wherein the acyl-CoA reductase gene is configured to be present in the cell in exponential phase at a copy number of from 1 to 5 copies per copy of genomic DNA, wherein a gene in the cell selected from the group consisting of an acyl-CoA dehydrogenase gene, an enoyl-CoA hydratase gene, a 3-hydroxyacyl-CoA dehydrogenase gene, and a 3-ketoacyl-CoA thiolase gene is deleted, and wherein the recombinant cell is capable of producing a fatty alcohol.

2. The recombinant cell of claim 1 , wherein the acyl-CoA synthetase gene encodes a protein comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence at least 90% identical to SEQ ID NO: 12.

3. The recombinant cell of claim 1 , wherein the acyl-CoA synthetase gene is expressed in the recombinant cell at a level greater than 2-fold and less than 75-fold the endogenous expression level of a native acyl-CoA synthetase gene in the corresponding non-recombinant cell.

4. The recombinant cell of claim 1 , wherein the recombinant acyl-CoA reductase gene encodes an enzyme having both acyl-CoA reductase activity and aldehyde reductase activity.

5. The recombinant cell of claim 1 , further comprising a recombinant aldehyde reductase gene.

6. The recombinant cell of claim 1 , wherein the acyl-CoA reductase gene encodes a protein comprising the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence at least 90% identical to SEQ ID NO: 16.

7. The recombinant cell of claim 1 , wherein the acyl-CoA reductase gene and the acyl-CoA synthetase gene are included in the cell at a copy ratio of from about 5:1 (acyl-CoA reductase gene:acyl-CoA synthetase gene) to about 1:1 (acyl-CoA reductase gene:acyl-CoA synthetase gene).

8. The recombinant cell of claim 1 , wherein the relative level of expression of the recombinant acyl-CoA reductase gene as determined by quantitative PCR (qPCR) with respect to the level of expression of the recombinant acyl-CoA synthetase gene as determined by qPCR is the same as that obtained when the recombinant acyl-CoA reductase gene and the recombinant acyl-CoA synthetase gene are present in a copy ratio of about 5:1 (recombinant acyl-CoA reductase gene:recombinant acyl-CoA synthetase gene) to about 1:1 (recombinant acyl-CoA reductase gene:recombinant acyl-CoA synthetase gene) and the recombinant acyl-CoA reductase gene and the recombinant acyl-CoA synthetase gene each comprises the same promoter.

9. The recombinant cell of claim 1 , wherein the acyl-CoA dehydrogenase gene is deleted.

10. The recombinant cell of claim 1 , wherein the recombinant cell is E. coli and the gene fadE is deleted.

11. The recombinant cell of claim 1 , wherein a gene selected from the group consisting of the enoyl-CoA hydratase gene, the 3-hydroxyacyl-CoA dehydrogenase gene, and the 3-ketoacyl-CoA thiolase gene is deleted.

12. The recombinant cell of claim 1 , wherein the recombinant cell is E. coli and the genes fadA and fadI; fadB and fadJ; or fadA, fadI, fadB and fadJ are deleted.

13. The recombinant cell of claim 1 , wherein the thioesterase gene encodes a protein comprising the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence at least 90% identical to SEQ ID NO: 18.

14. The recombinant cell of claim 1 , wherein the recombinant cell is a microbial cell.

15. The recombinant cell of claim 1 , wherein the recombinant cell is a bacterial cell.

16. A method of producing a fatty alcohol comprising culturing the recombinant cell as recited in claim 1 under conditions effective to produce the fatty alcohol.

17. The method of claim 16 , comprising culturing the recombinant cell in a medium comprising a carbohydrate and no more than about 1 g L −1 dissolved, exogenous free fatty acid or salt thereof.

Assignees

• Wisconsin Alumni Res Found

Inventors

• Pfleger Brian F
• Youngquist J Tyler

Classifications

• C12Y102/0105

  Long-chain-fatty-acyl-CoA reductase (1.2.1.50) · CPC title

• C12N9/0008

  acting on the aldehyde or oxo group of donors (1.2) · CPC title

• C12Y602/01003

  Long-chain-fatty-acid-CoA ligase (6.2.1.3) · CPC title

• C12N9/93

  Ligases (6) · CPC title

• C12P7/04 Primary

  acyclic · CPC title