Methods of producing omega-hydroxylated fatty acid derivatives

Patent metadata

Field	Value
Publication number	US-2016130616-A1
Application number	US-201414897285-A
Country	US
Kind code	A1
Filing date	Jun 16, 2014
Priority date	Jun 14, 2013
Publication date	May 12, 2016
Grant date	—

Abstract

The disclosure relates to omega-hydroxylated fatty acid derivatives and methods of producing them. Herein, the disclosure encompasses a novel and environmentally friendly production method that provides omega-hydroxylated fatty acid derivatives at high purity and yield. Further encompassed are recombinant microorganisms that produce omega-hydroxylated fatty acid derivatives through selective fermentation.

First claim

We claim:

1 . A recombinant microorganism for producing an ω-hydroxy fatty acid derivative in vivo when grown in a fermentation broth in a presence of a carbon source from a renewable feedstock, said microorganism comprising a pathway engineered to express at least two nucleic acid sequences encoding a polypeptide comprising:
(a) a thioesterase of EC 3.1.2.-, 3.1.1.5, or 3.1.2.14; or an ester synthase of EC 2.3.1.75 or EC 2.3.1.20; and
(b) a modified ω-hydroxylase of EC 1.14.15.3.

2 . The recombinant microorganism of claim 1 , wherein said modified ω-hydroxylase has a modified cytochrome P450 monoxogyenase enzymatic activity and efficiently catalyzes the ω-position of hydrocarbon chains in vivo.

3 . The recombinant microorganism of claim 2 , wherein said modified ω-hydroxylase is a CYP153A-reductase hybrid fusion polypeptide.

4 . The recombinant microorganism of claim 3 , wherein said CYP153A-reductase hybrid fusion polypeptide is a self-sufficient CYP153A-RedRhF hybrid fusion protein.

5 . The recombinant microorganism of claim 1 , wherein said modified ω-hydroxylase is a ω-hydroxylase hybrid fusion protein variant of EC 1.14.15.3.

6 . The recombinant microorganism of claim 5 , wherein said ω-hydroxylase hybrid fusion protein variant has at least 90% sequence identity to SEQ ID NO: 6 and has one or more mutations selected from the group consisting of V141I, V141T, V141Q, V141G, V141M, V141L, R27L, R82D, R178N, A231T, N309R, N407A, V415R, T516V, P666A, P666D and A796V.

7 . The recombinant microorganism of claim 1 , wherein the recombinant microorganism produces ω-hydroxy fatty acid derivatives selected from the group consisting of ω-hydroxy fatty acids and ω-hydroxy fatty acid methyl esters.

8 . The recombinant microorganism of claim 1 , wherein the recombinant microorganism is engineered to further express a nucleic acid sequence encoding a polypeptide comprising an alcohol dehydrogenase of EC 1.1.1.1/2 or an alcohol oxidase of EC 1.1.3.13 or EC 1.1.3.20.

9 . The recombinant microorganism of claim 8 , wherein the recombinant microorganism produces ω-hydroxy fatty acid derivatives selected from the group consisting of ω-oxo fatty acids and ω-oxo fatty acid methyl esters.

10 . The recombinant microorganism of claim 8 , wherein the recombinant microorganism is engineered to further express a nucleic acid sequence encoding a polypeptide comprising an aldehyde dehydrogenase of EC 1.2.1.3/4/5 or an aldehyde oxidase of EC 1.2.3.1.

11 . The recombinant microorganism of claim 10 , wherein the recombinant microorganism produces ω-hydroxy fatty acid derivatives that are α,ω-diacids or ω-carboxy fatty acid methyl esters.

12 . The recombinant microorganism of claim 10 , wherein the recombinant microorganism is engineered to further express a nucleic acid sequence encoding a polypeptide comprising an acyl-CoA ligase of EC 6.2.1.3 or an acyl-CoA transferase of EC 2.8.3.6.

13 . The recombinant microorganism of claim 12 , wherein the recombinant microorganism produces ω-hydroxy fatty acid derivatives that are α,ω-diesters.

14 . The recombinant microorganism of claim 8 , wherein the recombinant microorganism is engineered to further express a nucleic acid sequence encoding a polypeptide comprising an amino transferase of EC 2.6.1 or an amine dehydrogenases of EC 1.4.9, EC 1.4.98 or EC 1.4.99.

15 . The recombinant microorganism of claim 14 , wherein the recombinant microorganism produces ω-hydroxy fatty acid derivatives that are selected from the group consisting of ω-amino fatty acids and ω-amino fatty acid methyl esters.

16 . The recombinant microorganism of claim 1 , wherein the recombinant microorganism is engineered to further express a nucleic acid sequence encoding a polypeptide comprising an alcohol dehydrogenase of EC 1.1.-.- and a carboxylic acid reductase of 1.2.99.

17 . The recombinant microorganism of claim 16 , wherein ω-hydroxy fatty acid derivative is a α,ω-diol.

18 . A cell culture comprising the microorganism according to any one of claims 1 - 17 .

19 . The cell culture of claim 18 , wherein said cell culture produces ω-hydroxy fatty acid derivatives selected from the group consisting of, ω-hydroxy free fatty acids; ω-hydroxy fatty acid methyl esters; ω-oxo fatty acids; ω-oxo fatty acid methyl esters; α,ω-diacids; α,ω-diols; ω-amino fatty acids; and ω-amino fatty acid methyl esters.

20 . A method of producing a ω-hydroxy fatty acid derivative comprising:
(a) providing a recombinant microorganism in a fermentation broth, said microorganism comprising a pathway engineered to express at least two nucleic acid sequences encoding a polypeptide comprising a thioesterase of EC 3.1.2.-, 3.1.1.5, or 3.1.2.14; or an ester synthase of EC 2.3.1.75 or EC 2.3.1.20; and
a modified ω-hydroxylase of EC 1.14.15.3;

(b) adding a renewable feedstock containing a carbon source to said fermentation broth; and
(c) isolating a ω-hydroxy fatty acid derivative from said fermentation broth.

21 . The method of claim 20 , wherein said modified ω-hydroxylase is a CYP153A-reductase hybrid fusion polypeptide.

22 . The method of claim 21 , wherein said CYP153A-reductase hybrid fusion polypeptide is a self-sufficient CYP153A-RedRhF hybrid fusion protein.

23 . The method of claim 20 , wherein said modified ω-hydroxylase is a ω-hydroxylase hybrid fusion protein variant of EC 1.14.15.3.

24 . The method of claim 23 , wherein said ω-hydroxylase hybrid fusion protein variant has at least 90% sequence identity to SEQ ID NO: 6 and has one or more mutations selected from the group consisting of V141I, V141T, V141Q, V141G, V141M, V141L, R27L, R82D, R178N, A231T, N309R, N407A, V415R, T516V, P666A, P666D and A796V.

25 . The method of claim 20 , wherein said ω-hydroxy fatty acid derivative is an ω-hydroxy free fatty acid or an ω-hydroxy fatty acid methyl ester.

26 . The method of claim 20 , wherein the recombinant microorganism is engineered to further express a nucleic acid sequence encoding a polypeptide comprising an alcohol dehydrogenase of EC 1.1.1.1/2 or an alcohol oxidase of EC 1.1.3.13 or EC 1.1.3.20.

27 . The method of claim 26 , wherein said ω-hydroxy fatty acid derivative is an ω-oxo fatty acid or ω-oxo fatty acid methyl ester.

28 . The method of claim 26 , wherein the recombinant microorganism is engineered to further express a nucleic acid sequence encoding a polypeptide comprising an aldehyde dehydrogenase of EC 1.2.1.3/4/5 or an aldehyde oxidase of EC 1.2.3.1.

29 . The method of claim 28 , wherein said ω-hydroxy fatty acid derivative is an α,ω-diacid or an α,ω-fatty acid di-methyl ester.

30 . The method of claim 26 , wherein the recombinant microorganism is engineered to further express a nucleic acid sequence encoding a polypeptide comprising an amino transferase of EC 2.6.1 or an amine dehydrogenases of EC 1.4.9, EC 1.4.98 or EC 1.4.99.

31 . The method of claim 30 , wherein said ω-hydroxy fatty acid derivative is an ω-amino fatty acid or ω-amino fatty acid methyl ester.

32 . The method of claim 20 , wherein the recombinant microorganism is engineered to further express a nucleic acid encoding a polypeptide comprising a carboxylic acid reductase of EC 1.2.99.6 or and an alcohol dehydrogenase of EC 1.1.-.-.

33 . The method of clai

Assignees

• Reg Life Sciences Llc

Inventors

• Schirmer Andreas W
• Wang Haibo
• Del Cardayre Stephen B
• Hu Zhihao
• Hom Louis G
• Zhu Baolong
• Chang Cindy
• Popova Emanuela E

Classifications

• C12N9/0077

  with a reduced iron-sulfur protein as one donor (1.14.15) · CPC title

• C12Y102/01042

  Hexadecanal dehydrogenase (acylating) (1.2.1.42), i.e. fatty acyl-CoA reductase · CPC title

• C12Y114/15003

  Alkane 1-monooxygenase (1.14.15.3) · CPC title

• C12Y101/00

  Oxidoreductases acting on the CH-OH group of donors (1.1) · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title