YEAST STRAINS AND METHOD FOR THE PRODUCTION OF OMEGA-HYDROXY FATTY ACIDS AND DICARBOXYLIC ACIDS

• US 20160304913A1
• Filed: 12/10/2014
• Published: 10/20/2016
• Est. Priority Date: 12/12/2013
• Status: Active Grant

• Current Assignee: Provivi, Inc.
• Original Assignee: Technische Universitã¤T Dresden
• Inventors: GATTER, Michael, MATTHAUS, Falk, BARTH, Gerold
• Granted Patent: US 10,093,950 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 yeast strain of the type Y. lipolytica, exhibiting a reduction in the activities of the acyl-CoA oxidases, characterized in that in this additionally

a) the activities of relevant (fatty) alcohol dehydrogenases and/or

b) the activities of relevant (fatty) alcohol oxidases are reduced,

whereby an increased formation of ω

-hydroxy fatty acids according to the general structure;

HOOC—

(CH₂)_n—

OH, HOOC—

(C_nH_2n-2)—

CH₂OH, COOC—

(C_nH_2n-4)—

CH₂OH or HOOC—

(C_nH_2n-6)—

CH₂OH, where n is in the range from 6 to 16, takes place from a hydrophobic substrate.

2. The yeast strain according to claim 1, characterized in that in addition, the activities of individual or all cytochrome P450-gene products are increased.

3. The yeast strain according to claim 1 or 2, characterized in that in addition, the activity of the NADPH-cytochrome P450 reductase is increased.

4. The yeast strain according to any one of claims 1 to 3, characterized in that in addition, the activity of the phosphatidic acid dephosphohydrolase is reduced.

5. The yeast strain according to any one of claims 1 to 4, characterized in that in addition, the activity of the glycerol-3-phosphate-acyltransferase is reduced.

6. The yeast strain according to any one of claims 1 to 5, characterized in that the yeast strain exhibits a deletion of the genes for the acyl-CoA oxidases, the (fatty) alcohol dehydrogenases and/or the fatty (alcohol) oxidases.

7. Use of a yeast strain according to any one of claims 1 to 6 for the microbial production of ω

-hydroxy fatty acids according to the general structure;

HOOC—

(CH₂)_n—

OH, HOOC—

(C_nH_2n-2)—

CH₂OH, COOC—

(C_nH_2n-4)—

CH₂OH or HOOC—

(C_nH₂H_2n-6)—

CH₂OH, where n is in the range from 6 to 18, from a hydrophobic substrate.

10. The method according to claim 8 or 9, characterized in that the yeast cells of at least one yeast strain of the type Y. lipolytica are selected from a yeast strain according to claims 1 to 6.

17. The method according to any one of claims 8 to 10 or 11 to 16, characterized in that the hydrophobic substrate is selected from the group 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.

18. The method according to any one of the aforesaid claims 8 to 17, characterized in that in addition to the hydrophobic substrate, glucose is used as a carbon source.

19. The method according to any one of the aforesaid claims 8 to 18, characterized in that the glucose concentration in the course of the cultivation does not drop to 0 g l⁻

1.

20. The method according to any one of the aforesaid claims 8 to 19, characterized in that the nitrogen content in the culture medium after contact is less than 2 g l⁻

1.

21. The method according to any one of the aforesaid claims 8 to 20, characterized in that the pH value after contact is greater than 5.5.

22. The method according to any one of the aforesaid claims 8 to 21, characterized in that the hydrophobic substance is an n-alkane, an alkene or a fatty acid where n is in the range of 8 to 18.

8. Method for the biocatalytic preparation of ω

-hydroxy fatty acids from a hydrophobic substrate, which comprises the following steps;

d) Preparation of yeast cells of at least one yeast strain of the type Y. lipolytica, which exhibits

(iv) a reduction in the activities of the acyl-CoA oxidases,

(v) a reduced activity of relevant (fatty) alcohol dehydrogenases and

(vi) a reduced activity of relevant (fatty) alcohol oxidases

in a suitable cultivation medium,

e) contacting the yeast cells with the hydrophobic substrate, wherein the hydrophobic substrate is reacted with at least one of the enzymes of the yeast cells selected from the groups,

(iv) cytochrome P450-gene products,

(v) NADPH-cytochrome P450 reductase,

(vi) (fatty) aldehyde dehydrogenases

to form the reaction product ω

-hydroxy fatty acid,

f) isolation of the ω

-hydroxy fatty acid.

9. The method according to claim 8, characterized in that the reaction to form the reaction product ω

-hydroxy fatty acid additionally takes place with remaining (fatty) alcohol oxidases, (fatty) alcohol dehydrogenases.

11. Method for the biocatalytic preparation of dicarboxylic acids from a hydrophobic substrate, which comprises the following steps:

d) preparation of yeast cells of at least one yeast strain of the type Y. lipolytica, which exhibits

(iv) a reduction of the activities of the acyl-CoA oxidases,

(v) an enhanced activity of relevant (fatty) alcohol dehydrogenases and

(vi) an enhanced activity of relevant (fatty) alcohol oxidases

in a suitable cultivation medium,

e) contacting the yeast cells with the hydrophobic substrate, wherein the hydrophobic substrate is reacted with at least one of the enzymes of the yeast cells selected from the group,

(vi) of cytochrome P450-gene products,

(vii) of NADPH-cytochrome P450-reductase,

(viii) of (fatty) alcohol dehydrogenases according to a),

(ix) of (fatty) alcohol oxidases according to a) and

(x) (fatty) aldehyde dehydrogenases

to give the reaction product dicarboxylic acid,

f) isolation of the dicarboxylic acid.

12. The method according to claim 11, characterized in that in the yeast cells the activities of individual or all cytochrome P450-gene products are additionally increased.

13. The method according to claim 11 or 12, characterized in that in the yeast cells the activity of the NADPH-cytochrome P450-reductase is additionally increased.

14. The method according to any one of claims 11 to 13, characterized in that in the yeast cells the activities of relevant (fatty) alcohol dehydrogenases are additionally increased.

15. The method according to any one of claims 11 to 14, characterized in that in the yeast cells the activities of relevant (fatty) alcohol oxidases are additionally increased.

16. The method according to any one of claims 11 to 15, characterized in that in the yeast cells the activities of relevant (fatty) aldehyde dehydrogenases are additionally increased.