Production of fatty acids esters

• US 10,494,654 B2
• Filed: 03/12/2018
• Issued: 12/03/2019
• Est. Priority Date: 09/02/2014
• Status: Active Grant

• Current Assignee: Cargill, Inc. (Agricultural Services Division)
• Original Assignee: Cargill, Inc. (Agricultural Services Division)
• Inventors: Grammann, Katrin, Wolter, Jan, Reinecke, Liv, Schaffer, Steffen, Spindler, Eileen E., Ribble, Wendy K., Robinson, Brittany L., Poor, Catherine B., Lipscomb, Tanya Warnecke, Liao, Hans H., Hogsett, David A., Evans, Ronald J.
• Primary Examiner(s): Noakes, Suzanne M
• Assistant Examiner(s): Lee, Jae W
• Application Number:
  US15/918,806
• Publication Number:
  US 20180273990A1
• Time in Patent Office: 631 Days
• Field of Search: None
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12P 7/6436 Fatty acid esters
• CPC Class Codes: C12P 7/649 Biodiesel, i.e. fatty acid alkyl esters
• CPC Class Codes: C12Y 203/01041 Beta-ketoacyl-acyl-carrier-protein synthase I (2.3.1.41)
• CPC Class Codes: C12Y 203/01075 Long-chain-alcohol O-fatty-acyltransferase (2.3.1.75)
• CPC Class Codes: H05K 999/99 dummy group
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

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.

Claims

1. A microbial cell for producing at least one fatty acid ester, wherein the cell comprises:

(i) a first genetic mutation that enables the cell to produce at least one fatty acyl coenzyme A (CoA) intermediate by 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 relative to a wild type cell; 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, wherein the wax ester synthase has sequence identity of at least 80% to a polypeptide set forth in SEQ ID NO;

1, at least 80% to a polypeptide set forth in SEQ ID NO;

2, at least 85% to a polypeptide as set forth in SEQ ID NO;

3, at least 85% to a polypeptide as set forth in SEQ ID NO;

4, at least 90% to a polypeptide as set forth in SEQ ID NO;

5, at least 95% to a polypeptide as set forth in SEQ ID NO;

7, or at least 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 the fatty acyl coenzyme A intermediate to a fatty acid ester.

2. The cell according to claim 1, wherein the at least one fatty acyl coenzyme A (CoA) intermediate comprises a C4 to C10 acyl-CoA substrate.

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 cell is genetically modified to increase the expression of β

-ketoacyl-ACP synthase III (fabH) relative to the wild type cell.

7. The cell according to claim 6, 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.

8. The cell according to claim 1, wherein the cell is 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.

9. The cell according to claim 8, wherein the 3HCDh is crotonase/enoyl-CoA hydratase (Crt) and the KCR is hydroxybutyric dehydrogenase (Hbd).

10. The cell according to claim 9, 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.

11. The cell according to claim 1, wherein the fatty acid ester is a fatty acid methyl ester (FAME) and/or a fatty acid ethyl ester (FAEE).

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. The cell according to claim 1, wherein the cell is a yeast cell, a fungi cell, or a bacteria cell.

14. The cell according to claim 13, wherein the cell is a selected from the group consisting of Pseudomonas putida, Escherichia coli, and Saccharomyces cerevisiae.

15. A method for producing fatty acid ester, the method comprising:

contacting at least one fatty acyl coenzyme A (CoA) intermediate with an isolated wax ester synthase that has sequence identity of at least 80% to a polypeptide set forth in SEQ ID NO;

1, at least 85% to a polypeptide as set forth in SEQ ID NO;

3, at least 85% to a polypeptide as set forth in SEQ ID NO;

4, at least 90% to a polypeptide as set forth in SEQ ID NO;

5, at least 95% to a polypeptide as set forth in SEQ ID NO;

7, or at least 98% to a polypeptide as set forth in SEQ ID NO;

8.

16. A method for producing at least one fatty acid ester, the method comprising culturing a microbial cell which comprises:

(i) a first genetic mutation that enables the cell to produce at least one fatty acyl coenzyme A (CoA) intermediate by 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 relative to a wild type cell; 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, wherein the wax ester synthase has sequence identity of at least 80% to a polypeptide set forth in SEQ ID NO;

1, at least 80% to a polypeptide set forth in SEQ ID NO;

2, at least 85% to a polypeptide as set forth in SEQ ID NO;

3, at least 85% to a polypeptide as set forth in SEQ ID NO;

4, at least 90% to a polypeptide as set forth in SEQ ID NO;

5, at least 95% to a polypeptide as set forth in SEQ ID NO;

7, or at least 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 the fatty acyl coenzyme A intermediate to a fatty acid ester.

17. The cell according to claim 16, wherein the fatty acid ester is a fatty acid methyl ester (FAME) and/or a fatty acid ethyl ester (FAEE).

18. The cell according to claim 16, 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.