Omega-hydroxylase-related fusion polypeptide variants with improved properties

Patent metadata

Field	Value
Publication number	US-10787648-B2
Application number	US-201616063198-A
Country	US
Kind code	B2
Filing date	Dec 13, 2016
Priority date	Dec 15, 2015
Publication date	Sep 29, 2020
Grant date	Sep 29, 2020

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 having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 38 and having at least one mutation at an amino acid position corresponding to amino acid position: 9, 10, 11, 12, 13, 14, 28, 61, 77, 119, 159, 231, 233, 327, 413, 703, 745, 747, 749, 757, 770, 771 or 784, wherein the CYP153A-reductase hybrid fusion polypeptide variant has 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 SE 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 the 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 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 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 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 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 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 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 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 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 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 1 , wherein the CYP153A-reductase hybrid fusion polypeptide variant comprises a mutation at amino acid position 12 and at each of amino acid positions:
(a) 27, 119, 140, 157, 159, 233, and 244 of SEQ ID NO: 38;
(b) 28, 61, 119, 231, 309, 413, and 480 of SEQ ID NO: 38;
(c) 119, 231, and 480 of SEQ ID NO: 38;
(d) 28, 77, 119, 141, 231 and 407 of SEQ ID NO: 38;
(e) 28, 61, 141, 231, and 407 of SEQ ID NO: 38;
(f) 28, 119, 231, and 244 of SEQ ID NO: 38;
(g) 28, 407, and 480 of SEQ ID NO: 38;
(h) 141, 231, 413, and 481 of SEQ ID NO: 38;
(i) 28, 111, 231, and 407 of SEQ ID NO: 38;
(j) 28, 61, 140, and 149 of SEQ ID NO: 38;
(k) 28, 77, 119, 159, 231, 254, 407, and 480 of SEQ ID NO: 38;
(l) 28, 254, 309, 407, and 451 of SEQ ID NO: 38;
(m) 28, 254, 309, 407, and 480 of SEQ ID NO: 38; or
(n) 28, 309, 407, 451, and 480 of SEQ ID NO: 38;
wherein the CYP153A-reductase hybrid fusion polypeptide variant catalyzes the conversion of a fatty acid to an omega-hydroxylated fatty acid.

4. The CYP153A-reductase hybrid fusion polypeptide variant of claim 3 , wherein:
(a) the mutations at amino acid positions 12, 27, 119, 140, 157, 159, 233, and 244 of SEQ ID NO: 38 are Q12W, R27L, K119R, S140N, S157R, V159M, S233L, and A244R, respectively;
(b) the mutations at amino acid positions 12, 28, 61, 119, 231, 309, 413, and 480 of SEQ ID NO: 38 are Q12R, Q28M, N61L, K119R, A231V, N309S, Y413R, and I480G, respectively;
(c) the mutations at amino acid positions 12, 119, 231, and 480 of SEQ ID NO: 38 are Q12R, K119R, A231V, and I480G, respectively;
(d) the mutations at amino acid positions 12, 28, 77, 119, 141, 231 and 407 of SEQ ID NO: 38 are Q12T, Q28M, R77Q, K119R, V141T, A231W and N407G, respectively;
(e) the mutations at amino acid positions 12, 28, 61, 141, 231, and 407 of SEQ ID NO: 38 are Q12R, Q28M, N61L, V141T, A231Y, and N407G, respectively;
(f) the mutations at amino acid positions 12, 28, 119, 231, and 244 of SEQ ID NO: 38 are Q12W, Q28M, K119R, A231Y, and A244R, respectively;
(g) the mutations at amino acid positions 12, 28, 407, and 480 of SEQ ID NO: 38 are Q12W, Q28T, N407G, and I480G, respectively;
(h) the mutations at amino acid positions 12, 141, 231, 413, and 481 of SEQ ID NO: 38 are Q12R, V141T, A231Y, Y413R, and G481I, respectively;
(i) the mutations at amino acid positions 12, 28, 111, 231, and 407 of SEQ ID NO: 38 are Q12T, Q28M, F111A, A231V, and N407G, respectively;
(j) the mutations at amino acid positions 12, 28, 61, 140, and 149 of SEQ ID NO: 38 are Q12T, Q28M, N61L, S140N, and P149R, respectively;
(k) the mutations at amino acid positions 12, 28, 77, 119, 159, 231, 254, 407, and 480 are Q12W, Q28T, R77Q, K119R, V159M, A231Y, R254G, N407G, and I480G, respectively;
(l) the mutations at amino acid positions 12, 28, 254, 309, 407, and 451 of SEQ ID NO: 38 are Q12W, Q28T, R254G, N309S, N407G, and V451M, respectively;
(m) the mutations at amino acid positions 12, 28, 254, 309, 407, and 480 of SEQ ID NO: 38 are Q12W, Q28T, R254G, N309S, N407G, and I480G, respectively; and
(n) the mutations at amino acid positions 12, 28, 309, 407, 451, and 480 of SEQ ID NO: 38 are Q12W, Q28T, N309S, N407G, V451M, and I480G, respectively.

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

7. The recombinant host cell of claim 6 , further expressing a thioesterase polypeptide of EC 3.1.2.-, EC 3.1.1.5 or EC 3.1.2.14.

8. The recombinant host cell of claim 7 , 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 cont

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