Omega-hydroxylase-related fusion polypeptides with improved properties

Patent metadata

Field	Value
Publication number	US-2017145390-A1
Application number	US-201515319272-A
Country	US
Kind code	A1
Filing date	Jun 16, 2015
Priority date	Jun 16, 2014
Publication date	May 25, 2017
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 ex pressing the omega-hydroxylase-related fusion polypeptides for the production of omega-hydroxylated fatty acid derivatives.

First claim

1 .- 47 . (canceled)

48 . The A CYP153A-reductase hybrid fusion polypeptide variant comprising at least 90% sequence identity to SEQ ID NO: 6 and having at least one mutation at an amino acid position selected from the group consisting of 796, 141, 27, 178, 309, 407, and 415.

49 . The CYP153A-reductase hybrid fusion polypeptide variant of claim 48 , wherein the CYP153A-reductase hybrid fusion polypeptide variant is a hybrid cyp153A-RedRhF fusion protein variant that catalyzes the conversion of a fatty acid to an omega-hydroxylated fatty acid.

50 . The CYP153A-reductase hybrid fusion polypeptide variant of claim 48 , wherein said mutation is selected from the group consisting of A796V, V141I, V141T, V141Q, V141G, V141M, V141L, R27L, R178N, N309R, N407A, and V415R.

51 . The CYP153A-reductase hybrid fusion polypeptide variant of claim 50 , 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 a titer of an omega-hydroxylated fatty acid produced by expression of a CYP153A-reductase hybrid fusion polypeptide of SEQ ID NO: 6.

52 . The CYP153A-reductase hybrid fusion polypeptide variant of claim 50 , selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22 SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 46.

53 . The CYP153A-reductase hybrid fusion polypeptide variant of claim 50 , having a mutation at:
(a) V141I and A231T, comprising SEQ ID NO: 32; (b) R27L, R82D, V141M, R178N and N407A, comprising SEQ ID NO: 34; (c) A796V, comprising SEQ ID NO: 38; (d) A796V, P666D and T516V, comprising SEQ ID NO: 40; (e) V141I, A231T and A796V, comprising SEQ ID NO: 42, (f) R27L, R82D, V141M, R178N, N407A and A796V, comprising SEQ ID NO: 44, or (g) V141T, A231T and A796V, comprising SEQ ID NO: 46.

54 . The CYP153A-reductase hybrid fusion polypeptide variant of claim 53 , wherein said CYP153A-reductase hybrid fusion polypeptide variant catalyzes the conversion of a fatty acid to an omega-hydroxylated fatty acid.

55 . A recombinant host cell expressing the CYP153A-reductase hybrid fusion polypeptide variant of claim 2 , further expressing a thioesterase polypeptide of EC 3.1.2.-, EC 3.1.1.5 or EC 3.1.2.14.

56 . The recombinant host cell of claim 55 , 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, when cultured in medium containing a carbon source.

57 . The recombinant host cell of claim 56 , wherein the omega-hydroxylated fatty acid is produced intracellularly or extracellularly.

58 . A cell culture comprising the recombinant host cell of claim 55 .

59 . The cell culture of claim 58 , wherein the omega-hydroxylated fatty acid comprises one or more of a C 12 , C 16 and a C 16:1 omega-hydroxylated fatty acid.

60 . The cell culture of claim 59 , wherein the omega-hydroxylated fatty acid comprises
(a) an unsaturated omega-hydroxylated C 16:1 fatty acid; or (b) a saturated omega-hydroxylated C 12 fatty acid.

61 . A method of producing an omega-hydroxylated fatty acid having an increase in titer, comprising: culturing the host cell of claim 8 with a carbon source; and harvesting an omega-hydroxylated fatty acid.

62 . The method of claim 61 , wherein said omega-hydroxylated fatty acid is a saturated omega-hydroxylated fatty acid, or an unsaturated omega-hydroxylated fatty acid.

63 . A recombinant microorganism for producing an omega-hydroxylated fatty acid derivative in vivo when grown in a fermentation broth in a presence of a carbon source from a renewable feedstock, wherein 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.-, EC 3.1.1.5, or EC 3.1.2.14; and a CYP153A-reductase hybrid fusion polypeptide variant.

64 . The recombinant microorganism of claim 48 , wherein said CYP153A-reductase hybrid fusion polypeptide variant is a self-sufficient CYP153A-RedRhF hybrid fusion protein variant.

65 . The recombinant microorganism of claim 48 , wherein said CYP153A-reductase hybrid fusion polypeptide variant has at least 90% sequence identity to any one of SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22 SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 46.

66 . A CYP153A-reductase hybrid fusion polypeptide variant comprising at least 90% sequence identity to SEQ ID NO: 38 and having at least one mutation at an amino acid position selected from the group consisting of 747, 12, 327, 14, 61, 28, 13, 771, 119, 10, 11, 28, 745, 9, 770, 413, 784, 749, 231, 233, 757 and 703.

67 . The CYP153A-reductase hybrid fusion polypeptide variant of claim 66 , wherein said mutation is selected from the group consisting of D747N, Q12W, Q12T, Q12R, P327D, R14F, N61L, Q28M, S13K, V771F, K119R, D10Y, I11L, Q28T, P745R, D9N, D9K, T770G, Y413R, M784I, E749L, A231Y, S233L, E757A, and L703G.

68 . The CYP153A-reductase hybrid fusion polypeptide variant of claim 67 , wherein said CYP153A-reductase hybrid fusion polypeptide variant catalyzes the conversion of a fatty acid to an omega-hydroxylated fatty acid.

Assignees

• Reg Life Sciences Llc

Inventors

• Zhu Baolong
• Schirmer Andreas W
• Chang Cindy

Classifications

• C12N9/0077 Primary

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

• C07K2319/00

  Fusion polypeptide · CPC title

• C12P7/6409

  Fatty acids · CPC title

• C12Y114/15003

  Alkane 1-monooxygenase (1.14.15.3) · CPC title

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title