Yeast strains with reduced fatty alcohol oxidase activity and method for the production of Ω-hydroxy fatty acids and dicarboxylic acids

• US 10,093,950 B2
• Filed: 12/10/2014
• Issued: 10/09/2018
• Est. Priority Date: 12/12/2013
• Status: Active Grant

• Current Assignee: Provivi, Inc.
• Original Assignee: Provivi, Inc.
• Inventors: Gatter, Michael, Matthaus, Falk, Barth, Gerold
• Primary Examiner(s): Fronda, Christian L
• Application Number:
  US15/103,191
• Publication Number:
  US 20160304913A1
• Time in Patent Office: 1,399 Days
• Field of Search: None
• CPC Class Codes: C12N 1/165 Yeast isolates
• 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 9/90 Isomerases (5.)
• CPC Class Codes: C12P 7/44 Polycarboxylic acids
• CPC Class Codes: C12P 7/6409 Fatty acids
• CPC Class Codes: C12P 7/6427 Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12R 2001/73 Candida lipolytica
• CPC Class Codes: C12Y 101/0302 Long-chain-alcohol oxidase (1.1.3.20)
• CPC Class Codes: C12Y 103/03006 Acyl-CoA oxidase (1.3.3.6)
• CPC Class Codes: C12Y 502/01008 Peptidylprolyl isomerase (5.2.1.8), i.e. cyclophilin

Abstract

The invention relates to various new yeast strains of the type Yarrowia lipolytica as well as relevant methods for the biocatalytic preparation of ω-hydroxy fatty acids or dicarboxylic acids with the aid of these strains, whereby the formation of ω-hydroxy fatty acids or dicarboxylic acids is advantageously increased.

Claims

1. A genetically engineered Yarrowia lipolytica strain with reduced fatty alcohol oxidase activity as compared to that of a wild type Yarrowia lipolytica strain,

wherein said genetically engineered Yarrowia lipolytica strain is engineered to eliminate the expression or activity of endogenous fatty alcohol oxidase encoded by the FAO1 YALI0B14014g gene, and

wherein the strain is further engineered to eliminate the expression or activity of each of the following endogenous acyl-CoA oxidases:

POX1, POX2, POX3, POX4, POX5, and POX6.

2. The genetically engineered Yarrowia lipolytica strain of claim 1, wherein the strain is further engineered to reduce or eliminate the expression or activity of one or more endogenous fatty alcohol dehydrogenases selected from the group consisting of FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, and ADH7.

3. The genetically engineered Yarrowia lipolytica strain of claim 2, wherein the strain is engineered to reduce or eliminate the expression or activity of FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, and ADH7.

4. The genetically engineered Yarrowia lipolytica strain of claim 1, wherein the strain is further engineered to increase the expression of one or more cytochrome P450 proteins selected from the group consisting of ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, ALK7, ALK8, ALK9, ALK10, ALK11, and ALK12.

5. The genetically engineered Yarrowia lipolytica strain of claim 1, wherein the strain is further engineered to increase the expression of the cytochrome P450 reductase protein CPR1.

6. The genetically engineered Yarrowia lipolytica strain of claim 1, wherein the strain is further engineered to reduce or eliminate the expression or activity of an endogenous phosphatidic acid dephosphohydrolase.

7. The genetically engineered Yarrowia lipolytica strain of claim 1, wherein the strain is further engineered to reduce or eliminate the expression or activity of an endogenous glycerol-3-phosphate-acyltransferase.

8. A method for the production of ω

-hydroxy fatty acids from a hydrophobic substrate, comprising;

(a) providing a genetically engineered Yarrowia lipolytica strain according to claim 1;

(b) cultivating the strain in a suitable cultivation medium; and

(c) contacting the strain with the hydrophobic substrate to form one or more ω

-hydroxy fatty acids.

9. The method of claim 8, further comprising step (d) of isolating the one or more (b-hydroxy fatty acids.

10. The method of claim 8, wherein the hydrophobic substrate is selected from the group consisting of n-alkanes having the general structure:

H₃C—

(CH₂)_n—

CH₃, alkenes having the general structure H₃C—

(C_nH_2n-2)—

CH₃, H₃C—

(C_nH_2n-4)—

CH₃ or H₃C—

(C_nH_2n-6)—

CH₃, fatty acids having the general structure;

HOOC—

(CH₂)_n—

CH₃, HOOC—

(C_nH_2n-2)—

CH₃, COOC—

(C_nH_2n-4)—

CH₃ or HOOC—

(C_nH_2n-6)—

CH₃, and fatty acid alkyl esters having the general structure ROOC—

(CH₂)_n—

CH₃, where n is in the range from 6 to 18.

11. The method of claim 8, wherein the hydrophobic substance is an n-alkane, an alkene or a fatty acid where n is in the range of 8 to 18.

12. The method of claim 8, wherein glucose is used as a carbon source.

13. The method of claim 8, wherein the pH value after step (c) of contacting is greater than 5.5.