Omega-hydroxylase-related fusion polypeptide variants with improved properties

Patent metadata

Field	Value
Publication number	US-11384341-B2
Application number	US-202017001015-A
Country	US
Kind code	B2
Filing date	Aug 24, 2020
Priority date	Dec 15, 2015
Publication date	Jul 12, 2022
Grant date	Jul 12, 2022

Abstract

The disclosure relates to omega-hydroxylase-related fusion polypeptides that result in improved omega-hydroxylated fatty acid derivative production when expressed in recombinant host cells. The disclosure further relates to microorganisms for expressing the omega-hydroxylase-related fusion polypeptides for the production of omega-hydroxylated fatty acid derivatives.

First claim

What is claimed is:

1. A cytochrome P450 CYP153A reductase (CYP153A-reductase) hybrid fusion polypeptide variant comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 38, wherein said CYP153A-reductase hybrid fusion polypeptide variant comprises a mutation at amino acid position 159.

2. The CYP153A-reductase hybrid fusion polypeptide variant of claim 1 , wherein expression of said CYP153A-reductase hybrid fusion polypeptide variant in a recombinant host cell results in a higher titer of an omega-hydroxylated fatty acid as compared to the titer of an omega-hydroxylated fatty acid produced by expression of the CYP153A-reductase hybrid fusion polypeptide of SEQ ID NO: 38 in a corresponding host cell.

3. The CYP153A-reductase hybrid fusion polypeptide variant of claim 1 , wherein the CYP153A-reductase hybrid fusion polypeptide variant is a hybrid CYP153A-RedRhF fusion protein variant.

4. The CYP153A-reductase hybrid fusion polypeptide variant of claim 1 , wherein the mutation is V159M.

5. A recombinant host cell expressing the CYP153A-reductase hybrid fusion polypeptide variant of claim 1 .

6. The recombinant host cell of claim 5 , further expressing a thioesterase polypeptide classified in EC 3.1.2.-.

7. The recombinant host cell of claim 6 , wherein the thioesterase polypeptide is classified in EC 3.1.1.5 or EC 3.1.2.14.

8. The recombinant host cell of claim 5 , wherein the recombinant host cell produces omega-hydroxylated fatty acids with a titer that is at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, or at least 30% greater than the titer of omega-hydroxylated fatty acids produced by a host cell expressing a corresponding CYP153A-reductase hybrid fusion polypeptide comprising SEQ ID NO: 38, when cultured in medium containing a carbon source.

9. A cell culture comprising the recombinant host cell of claim 5 .

10. A method of producing an omega-hydroxylated fatty acid, comprising:
(i) culturing the recombinant host cell of claim 5 in the presence of a carbon source; and
(ii) harvesting an omega-hydroxylated fatty acid.

11. A recombinant microorganism comprising a pathway engineered to express at least two nucleic acid sequences encoding a polypeptide comprising:
(i) a thioesterase classified in EC 3.1.2.-; and
(ii) a CYP153A-reductase hybrid fusion polypeptide variant of claim 1 .

Assignees

• Genomatica Inc

Inventors

• Schirmer Andreas
• Zhu Baolong
• Popova Emanuela

Classifications

• C12Y301/02

  Thioester hydrolases (3.1.2) · CPC title

• C12Y301/02014

  Oleoyl-[acyl-carrier-protein] hydrolase (3.1.2.14), i.e. ACP-thioesterase · CPC title

• C12Y301/01005

  Lysophospholipase (3.1.1.5) · CPC title

• C12Y106/02004

  NADPH-hemoprotein reductase (1.6.2.4), i.e. NADP-cytochrome P450-reductase · CPC title

• C12P7/6409

  Fatty acids · CPC title