Production of fatty acids esters

Patent metadata

Field	Value
Publication number	US-9944959-B2
Application number	US-201514843525-A
Country	US
Kind code	B2
Filing date	Sep 2, 2015
Priority date	Sep 2, 2014
Publication date	Apr 17, 2018
Grant date	Apr 17, 2018

Abstract

A microbial cell is used for producing at least one fatty acid ester, wherein the cell is genetically modified to contain (i) at least one first genetic mutation that enables the cell to produce at least one fatty acid and/or acyl coenzyme A (CoA) thereof by increased enzymatic activity in the cell relative to the wild type cell of malonyl-CoA dependent and malonyl-ACP independent fatty acyl-CoA metabolic pathway, wherein the fatty acid contains at least 5 carbon atoms; and (ii) a second genetic mutation that increases the activity of at least one wax ester synthase in the cell relative to the wild type cell and the wax ester synthase has sequence identity of at least 50% to a polypeptide of SEQ ID NO: 1-8 and combinations thereof or to a functional fragment of any of the polypeptides for catalyzing the conversion of fatty acid and/or acyl coenzyme A thereof to the fatty acid ester.

First claim

The invention claimed is:

1. A microbial cell for producing at least one fatty acid ester, wherein the cell is genetically modified to comprise:
(i) a first genetic mutation that enables the cell to produce at least one fatty acid and/or acyl coenzyme A (CoA) thereof by increased enzymatic activity in the cell relative to a malonyl-CoA dependent and malonyl-ACP independent fatty acyl-CoA metabolic pathway of a wild type cell, wherein the fatty acid comprises at least 5 carbon atoms; and
(ii) a second genetic mutation that increases the activity of at least one wax ester synthase in the cell relative to the wild type cell and the wax ester synthase has sequence identity of at least 80% to a polypeptide set forth in SEQ ID NO: 1, 85% to a polypeptide as set forth in SEQ ID NO: 3, 85% to a polypeptide as set forth in SEQ ID NO: 4, 90% to a polypeptide as set forth in SEQ ID NO: 5, 95% to a polypeptide as set forth in SEQ ID NO: 7, or 98% to a polypeptide as set forth in SEQ ID NO: 8, and combinations thereof or to a functional fragment of any of the polypeptides for catalyzing the conversion of fatty acid and/or acyl coenzyme A thereof to the fatty acid ester.

2. The cell according to claim 1 , wherein the first genetic mutation results in an increase in the expression of at least one enzyme selected from the group consisting of acetoacetyl-CoA synthase, ketoacyl-CoA synthase (or elongase), ketoacyl-CoA thiolase, enoyl-CoA reductase, ketoacyl-CoA reductase and 3-hydroxyacyl-CoA dehydratase.

3. The cell according to claim 1 , further comprising a genetic mutation that results in a decrease in the expression of acetoacetyl-CoA thiolase relative to the wild type cell.

4. The cell according to claim 1 , wherein the cell comprises a third genetic mutation that reduces the fatty acid degradation capacity of the cell relative to the wild type cell.

5. The cell according to claim 4 , wherein the third genetic mutation results in a decrease in the expression of at least one enzyme selected from the group consisting of acyl-CoA dehydrogenase, 2,4-dienoyl-CoA reductase, enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase, each relative to the wild type cell.

6. The cell according to claim 1 , wherein the fatty acid is lauric acid, the acyl coenzyme A thereof is lauroyl coenzyme A and the fatty acid ester is selected from the group consisting of methyl laurate, ethyl laurate, propyl laurate, butyl laurate and pentyl laurate.

7. The cell according to claim 6 , wherein the cell comprises a first mutation that results in at least an increase in the expression of β-ketoacyl-ACP synthase III (fabH).

8. The cell according to claim 7 , wherein the β-ketoacyl-ACP synthase III (fabH) has sequence identity of
at least 85% to a polypeptide selected from the group consisting of SEQ ID NOs: 24-27 and combinations thereof.

9. The cell according to claim 6 , wherein the cell is further genetically modified to increase the expression of 3-hydroxyacyl coenzyme A dehydratase (3HCDh) and/or keto acyl-CoA reductase (KCR) relative to the wild type cell.

10. The cell according to claim 9 , wherein the 3HCDh is crotonase/enoyl-CoA hydratase (Crt) and the KCR is hydroxybutyric dehydrogenase (Hbd).

11. The cell according to claim 10 , wherein the Crt has sequence identity of at least 85% to a polypeptide as set forth in SEQ ID NO:28 and/or the Hbd has sequence identity of at least 85% to a polypeptide of SEQ ID NO:29.

12. The cell according to claim 1 , wherein the fatty acid ester is produced in the presence of at least one exogenous alcohol selected from the group consisting of methanol, ethanol, propanol, butanol and pentanol.

13. A method for producing methyl laurate, the method comprising:
contacting lauric acid and/or lauroyl coenzyme A with an isolated wax ester synthase that has sequence identity of at least 80% to a polypeptide set forth in SEQ ID NO: 1, 85% to a polypeptide as set forth in SEQ ID NO: 3, 85% to polypeptide as set forth in SEQ ID NO: 4, 90% to a polypeptide as set forth in SEQ ID No: 5, 95% to a polypeptide as set forth in SEQ ID NO: 7, or 98% to a polypeptide as set forth in SEQ In NO: 8.

14. A method for producing at least one fatty acid ester, the method comprising culturing a microbial cell which is genetically modified to comprise:
(i) a first genetic mutation that enables the cell to produce at least one fatty acid and/or acyl coenzyme A (CoA) thereof by increased enzymatic activity in the cell relative to a malonyl-CoA dependent and malonyl-ACP independent fatty acyl-CoA metabolic pathway of a wild type cell, wherein the fatty acid comprises at least 5 carbon atoms; and
(ii) a second genetic mutation that increases the activity of at least one wax ester synthase in the cell relative to the wild type cell and the wax ester synthase has sequence identity of at least 80% to a polypeptide set forth in SEQ ID NO: 1, 85% to a polypeptide as set forth in SEQ ID NO: 3, 85% to a polypeptide as set forth in SEQ ID NO: 4, 90% to a polypeptide as set forth in SEQ ID NO: 5, 95% to a polypeptide as set forth in SEQ ID NO: 7, or 98% to a polypeptide as set forth SEQ ID NO: 8, and combinations thereof or to a functional fragment of any of the polypeptides for catalyzing the conversion of fatty acid and/or acyl coenzyme A thereof to the fatty acid ester.

Assignees

• Cargill Inc

Inventors

• Grammann Katrin
• Wolter Jan
• Reinecke Liv
• Schaffer Steffen
• Spindler Eileen C
• Ribble Wendy K
• Prather Brittany L
• Poor Catherine B
• Lipscomb Tanya Warnecke
• Liao Hans H
• Hogsett Dave A
• Evans Ronald J

Classifications

• C12Y203/01075

  Long-chain-alcohol O-fatty-acyltransferase (2.3.1.75) · CPC title

• C12Y203/01041

  Beta-ketoacyl-acyl-carrier-protein synthase I (2.3.1.41) · CPC title

• C12N9/1029

  transferring groups other than amino-acyl groups (2.3.1) · CPC title

• C12P7/6436 Primary

  Fatty acid esters · CPC title

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title