Omega-hydroxylase-related fusion polypeptide variants with improved properties

Patent metadata

Field	Value
Publication number	US-2019002845-A1
Application number	US-201616063198-A
Country	US
Kind code	A1
Filing date	Dec 13, 2016
Priority date	Dec 15, 2015
Publication date	Jan 3, 2019
Grant date	—

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 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 12 and a mutation at each of amino acid positions
(a) 12, 27, 28, 119, 141, 157, 159, 231, 233, and 244 of SEQ ID NO: 38; (b) 12, 28, 119, 140, 157, 159, 233, 244, 254, and 407 of SEQ ID NO: 38; (c) 12, 27, 111, 119, 141, 157, 159, 231, 233, 244, and 254 of SEQ ID NO: 38; (d) 12, 28, 119, 140, 149, 157, 159, 231, 233, and 407 of SEQ ID NO: 38; (e) 12, 27, 28, 119, 140, 157, 159, 233, 244, and 407 of SEQ ID NO: 38; (f) 10, 11, 12, 28, 119, 141, 159, 231, 233, 244, and 407 of SEQ ID NO: 38; (g) 11, 12, 27, 28, 119, 141, 157, 159, 197, 231, 233, 244, 407, and 477 of SEQ ID NO: 38; (h) 11, 12, 28, 119, 141, 157, 159, 197, 231, 233, 244, and 407 of SEQ ID NO: 38; or (i) 11, 12, 27, 28, 119, 141, 149, 157, 159, 231, 233, and 407 of SEQ ID NO: 38; wherein said CYP153A-reductase hybrid fusion polypeptide variant catalyzes the conversion of a fatty acid to an omega-hydroxylated fatty acid.

2 . The CYP153A-reductase hybrid fusion polypeptide variant of claim 1 , wherein:
(a) the said mutations at amino acid positions 12, 27, 28, 119, 141, 157, 159, 231, 233, and 244 of SEQ ID NO: 38 are Q12W, R27L, Q28M, K119R, V141T, S157R, V159M, A231Y, S233L, and A244R, respectively; (b) the said mutations at amino acid positions 12, 28, 119, 140, 157, 159, 233, 244, 254, and 407 of SEQ ID NO: 38 are Q12W, Q28M, K119R, S140N, S157R, V159M, S233L, A244R, R254G, and N407G, respectively; (c) the said mutations at amino acid positions 12, 27, 111, 119, 141, 157, 159, 231, 233, 244, and 254 of SEQ ID NO: 38 are Q12W, R27L, F111A, K119R, V141T, S157R, V159M, A231Y, S233L, A244R, and R254G, respectively; (d) the said mutations at amino acid positions 12, 28, 119, 140, 149, 157, 159, 231, 233, and 407 of SEQ ID NO: 38 are Q12W, Q28M, K119R, S140N, P149G, S157R, V159M, A231Y, S233L, and N407G, respectively; (e) the said mutations at amino acid positions 12, 27, 28, 119, 140, 157, 159, 233, 244, and 407 of SEQ ID NO: 38 are Q12W, R27L, Q28M, K119R, S140N, S157R, V159M, S233L, A244R, and N407G, respectively; (f) the said mutations at amino acid positions 10, 11, 12, 28, 119, 141, 159, 231, 233, 244, and 407 of SEQ ID NO: 38 are D10Y, I11L, Q12W, Q28M, K119R, V141T, V159M, A231Y, S233L, A244R, and N407G, respectively; (g) the said mutations at amino acid positions 11, 12, 27, 28, 119, 141, 157, 159, 197, 231, 233, 244, 407, and 477 of SEQ ID NO: 38 are I11L, Q12W, R27L, Q28M, K119R, V141T, S157R, V159M, A197T, A231Y, S233L, A244R, N407G, and P477G, respectively; (h) the said mutations at amino acid positions 11, 12, 28, 119, 141, 157, 159, 197, 231, 233, 244, and 407 of SEQ ID NO: 38 are I11L, Q12W, Q28M, K119R, V141T, S157R, V159M, A197T, A231Y, S233L, A244R, and N407G, respectively; and (i) the said mutations at amino acid positions 11, 12, 27, 28, 119, 141, 149, 157, 159, 231, 233, and 407 of SEQ ID NO: 38 are I11L, Q12W, R27L, Q28M, K119R, V141T, P149G, S157R, V159M, A231Y, S233L, and N407G, respectively.

3 . The CYP153A-reductase hybrid fusion polypeptide variant of claim 2 , wherein:
(a) said CYP153A-reductase hybrid fusion polypeptide variant of (a) comprises the amino acid sequence of SEQ ID NO: 166; (b) said CYP153A-reductase hybrid fusion polypeptide variant of (b) comprises the amino acid sequence of SEQ ID NO: 168; (c) said CYP153A-reductase hybrid fusion polypeptide variant of (c) comprises the amino acid sequence of SEQ ID NO: 170; (d) said CYP153A-reductase hybrid fusion polypeptide variant of (d) comprises the amino acid sequence of SEQ ID NO: 172; (e) said CYP153A-reductase hybrid fusion polypeptide variant of (e) comprises the amino acid sequence of SEQ ID NO: 174; (f) said CYP153A-reductase hybrid fusion polypeptide variant of (f) comprises the amino acid sequence of SEQ ID NO: 176; (g) said CYP153A-reductase hybrid fusion polypeptide variant of (g) comprises the amino acid sequence of SEQ ID NO: 178; (h) said CYP153A-reductase hybrid fusion polypeptide variant of (h) comprises the amino acid sequence of SEQ ID NO: 180; and (i) said CYP153A-reductase hybrid fusion polypeptide variant of (i) comprises the amino acid sequence of SEQ ID NO: 182.

4 . A CYP153A-reductase hybrid fusion polypeptide variant selected from the group consisting of SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, and SEQ ID NO: 182.

5 . The CYP153A-reductase hybrid fusion polypeptide variant of any one of claims 1 to 4 , 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: 6 or SEQ ID NO: 38 in a corresponding host cell.

6 . The CYP153A-reductase hybrid fusion polypeptide variant of any one of claims 1 to 4 , wherein the CYP153A-reductase hybrid fusion polypeptide variant is a hybrid CYP153A-RedRhF fusion protein variant.

7 . A recombinant host cell expressing the CYP153A-reductase hybrid fusion polypeptide variant of any one of claims 1 to 4 .

8 . The recombinant host cell of claim 7 , further expressing a thioesterase polypeptide of EC 3.1.2.-, EC 3.1.1.5 or EC 3.1.2.14.

9 . The recombinant host cell of claim 8 , wherein the recombinant host cell produces a omega-hydroxylated fatty acid composition 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 an omega-hydroxylated fatty acid composition produced by a host cell expressing a corresponding CYP153A-reductase hybrid fusion polypeptide comprising SEQ ID NO: 38 or SEQ ID NO: 6, when cultured in medium containing a carbon source.

10 . A cell culture comprising the recombinant host cell of any one of claims 7 to 9 .

11 . A method of producing an omega-hydroxylated fatty acid, comprising:
(i) culturing the recombinant host cell of any one of claims 7 to 9 , or the cell culture according to claim 10 , in the presence of a carbon source; and (ii) harvesting an omega-hydroxylated fatty acid.

12 . A recombinant microorganism comprising a pathway engineered to express at least two nucleic acid sequences encoding a polypeptide comprising:
(i) a thioesterase of EC 3.1.2.-, EC 3.1.1.5, or EC 3.1.2.14; and (ii) a CYP153A-reductase hybrid fusion polypeptide variant of any one of claims 1 to 6 .

13 . The CYP153A-reductase hybrid fusion polypeptide variant of any one of claims 1 to 6 , or the recombinant host cell of any of claims 7 to 9 , or the cell culture according to claim 10 , or the method according to claim 11 , or the recombinant microorganism according to claim 12 , wherein said CYP153A-reductase hybrid fusion polypeptide variant is a self-sufficient CYP153A-RedRhF hybrid fusion polypeptide variant.

Assignees

• Reg Life Sciences Llc

Inventors

• Schirmer Andreas
• Zhu Baolong
• Popova Emanuela

Classifications

• C12N15/62

  DNA sequences coding for fusion proteins · CPC title

• C12N9/18

  Carboxylic ester hydrolases {(3.1.1)} · CPC title

• C12N9/0006 Primary

  acting on CH-OH groups as donors (1.1) · CPC title

• C12N15/81

  for yeasts · CPC title

• C12Y301/02

  Thioester hydrolases (3.1.2) · CPC title