Omega-hydroxylase-related fusion polypeptide variants with improved properties

Patent metadata

Field	Value
Publication number	US-12180515-B2
Application number	US-202217834681-A
Country	US
Kind code	B2
Filing date	Jun 7, 2022
Priority date	Dec 15, 2015
Publication date	Dec 31, 2024
Grant date	Dec 31, 2024

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:6, wherein:
the CYP153A-reductase hybrid fusion polypeptide variant comprises at least one mutation at one or more amino acid positions corresponding to positions 77, 159, 197, 451, or 643 of SEQ ID NO:6; and
the CYP153A-reductase hybrid fusion polypeptide variant catalyzes the conversion of a fatty acid or derivative thereof to an ω-hydroxy fatty acid or derivative thereof.

2. The CYP153A-reductase hybrid fusion polypeptide variant of claim 1 , comprising at least one mutation corresponding to R77Q, V159M, A197T, V451M, T516E, R643H, or P666K.

3. The CYP153A-reductase hybrid fusion polypeptide variant of claim 1 , further comprising one or more mutations at one or more amino acid positions corresponding to positions 6, 9, 10, 11, 12, 13, 14, 27, 28, 40, 41, 56, 61, 82, 111, 116, 119, 129, 131, 132, 134, 136, 138, 140, 141, 142, 144, 149, 153, 154, 157, 161, 162, 164, 168, 178, 204, 205, 228, 231, 233, 244, 254, 271, 273, 302, 304, 307, 308, 309, 327, 407, 413, 415, 419, 477, 480, 481, 516, 527, 544, 546, 557, 567, 591, 648, 649, 666, 696, 703, 706, 707, 708, 709, 710, 719, 720, 736, 741, 745, 747, 749, 757, 770, 771, 784, or 796 of SEQ ID NO:6.

4. The CYP153A-reductase hybrid fusion polypeptide variant of claim 3 , comprising one or more mutations corresponding to R6F, D9N, D9K, D10Y, I11C, I11L, Q12R, Q12T, Q12W, S13K, R14F, R27L, Q28M, Q28T, R40H, K41V, P56Q, N61L, R77Q, R82D, F111A, F116R, K119R, Q129R, I131L, L132T, D134G, P136T, P136C, G138F, S140N, V141I, V141Q, V141G, V141M, V141L, V141T, E142R, E142Q, F144R, P149G, P149R, D153G, V154G, V154A, S157R, S157V, V159M, G161A, G161P, V162C, A164N, L168V, R178N, A197T, G204V, R205L, M228R, A231T, A231V, A231W, A231Y, S233L, S233N, S233R, S233V, A244R, R254G, R258Y, E271F, E271D, P273M, T302M, L304W, G307A, G308W, N309R, N309S, P327D, N407A, N407G, Y413R, V415R, M419V, V451M, P477G, 1480G, G481I, T516V, T516E, T516G, D527E, D544N, P546G, E557R, E557W, E567S, E591Q, R643H, V648L, S649I, P666A, P666D, P666K, P666M, P666H, V696K, V696T, L703G, L706E, L706S, L706H, D707E, P708S, D709L, V710C, V710R, V710Q, R719W, D720V, A736V, N741G, P745K, P745R, D747N, E749L, E749M, E757A, T770G, V771F, M784I, or A796V.

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

6. The recombinant host cell of claim 5 , wherein:
the recombinant host cell produces an ω-hydroxy fatty acid or a derivative thereof; and
the ω-hydroxy fatty acid or derivative thereof is one or more of a saturated or unsaturated C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 ω-hydroxy fatty acid or derivative thereof.

7. The recombinant host cell of claim 6 , further expressing a thioesterase, an ester synthase, or a combination thereof.

8. The recombinant host cell of claim 7 , wherein the recombinant host cell produces an ω-hydroxy fatty acid, or an ω-hydroxy fatty acid derivative that is an ω-hydroxy fatty acid ester, or a combination thereof.

9. The recombinant host cell of claim 5 , further expressing:
(a) an alcohol dehydrogenase or an alcohol oxidase, wherein the recombinant host cell produces an ω-hydroxy fatty acid derivative that is an ω-oxo fatty acid, an ω-oxo fatty acid ester, or a combination thereof,
(b) an alcohol dehydrogenase or an alcohol oxidase, and an aldehyde dehydrogenase or an aldehyde oxidase, wherein the recombinant host cell produces an ω-hydroxy fatty acid derivative that is an α,ω-diacid, an ω-carboxy fatty acid ester, or a combination thereof,
(c) an alcohol dehydrogenase or an alcohol oxidase, an aldehyde dehydrogenase or an aldehyde oxidase, and an acyl-CoA ligase or an acyl-CoA transferase, wherein the recombinant host cell produces an ω-hydroxy fatty acid derivative that is an α,ω-diester;
(d) an alcohol dehydrogenase or an alcohol oxidase, and an aminotransferase or an amine dehydrogenase, wherein the recombinant host cell produces an ω-hydroxy fatty acid derivative that is an ω-amino fatty acid, an ω-amino fatty acid ester, or a combination thereof,
(e) an alcohol dehydrogenase and a carboxylic acid reductase, wherein the recombinant host cell produces an ω-hydroxy fatty acid derivative that is an α,ω-diol; or
(f) an acyl-ACP reductase and an alcohol dehydrogenase, wherein the recombinant host cell produces an ω-hydroxy fatty acid derivative that is an α,ω-diol.

10. The recombinant host cell of claim 9 , further expressing a thioesterase, or an ester synthase, or a combination thereof.

11. A recombinant host cell, expressing the CYP153A-reductase hybrid fusion polypeptide variant of claim 3 .

12. A cell culture, comprising the recombinant host cell of claim 5 .

13. A cell culture, comprising the recombinant host cell of claim 11 .

14. A method of producing an ω-hydroxy fatty acid or a derivative thereof, the method comprising:
(i) culturing the recombinant host cell of claim 5 in the presence of a carbon source; and
(ii) optionally harvesting the ω-hydroxy fatty acid or derivative thereof.

15. The method of claim 14 , wherein the ω-hydroxy fatty acid or derivative thereof is one or more of a saturated or unsaturated C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 ω-hydroxy fatty acid or derivative thereof.

16. The method of claim 14 , wherein the recombinant host cell produces an ω-hydroxy fatty acid or derivative thereof at 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 ω-hydroxy fatty acid or derivative thereof produced by a host cell comprising a CYP153A-reductase hybrid fusion polypeptide of SEQ ID NO:6.

17. The method of claim 15 , wherein the ω-hydroxy fatty acid or derivative thereof is an ω-hydroxy fatty acid, an ω-hydroxy fatty acid ester, an ω-oxo fatty acid, an ω-oxo fatty acid ester, an α,ω-diacid, an ω-carboxy fatty acid ester, an α,ω-diester, an ω-amino fatty acid, an ω-amino fatty acid ester, an α,ω-diol, or a combination thereof.

18. A method of producing an ω-hydroxy fatty acid or a derivative thereof, the method comprising:
(i) culturing the recombinant host cell of claim 11 the presence of a carbon source; and
(ii) optionally harvesting the ω-hydroxy fatty acid or derivative thereof.

19. The method of claim 18 , wherein the ω-hydroxy fatty acid or derivative thereof is one or more of a saturated or unsaturated C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 ω-hydroxy fatty acid or derivative thereof.

20. The method of claim 19 , wherein the ω-hydroxy fatty acid or derivative thereof is an ω-hydroxy fatty acid, an ω-hydroxy fatty acid ester, an ω-oxo fatty acid, an ω-oxo fatty acid ester, an α,ω-diacid, an ω-carboxy fatty acid ester, an α,ω-diester, an ω-amino fatty acid, an ω-amino fatty acid ester, an α,ω-diol, or a combination thereof.

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