Novel car enzymes and improved production of fatty alcohols

Patent metadata

Field	Value
Publication number	US-2018135028-A1
Application number	US-201715695205-A
Country	US
Kind code	A1
Filing date	Sep 5, 2017
Priority date	Apr 2, 2012
Publication date	May 17, 2018
Grant date	—

Abstract

The disclosure relates to variant carboxylic acid reductase (CAR) enzymes for the improved production of fatty alcohols in recombinant host cells.

First claim

1 .- 57 . (canceled)

58 . A variant carboxylic acid reductase (CAR) polypeptide comprising an amino acid sequence having at least 85% sequence identity to the wild-type CAR polypeptide of SEQ ID NO: 7, wherein said variant CAR polypeptide is genetically engineered to have at least one mutation at an amino acid position selected from the group consisting of S3R, D18R, D18L, D18T, D18P, E20V, E20S, E20R, S22R, S22N, S22G, L80R, R87G, R87E, V191S, F288R, F288S, F288G, Q473L, Q473W, Q473Y, Q473I, Q473H, A535S, D750A, R827C, R827A, I870L, R873S, V926A, V926E, S927K, S927G, M930K, M930R and L1128W, wherein expression of said variant CAR polypeptide in a recombinant host cell results in a fatty alcohol composition, a fatty aldehyde composition or a composition that is a mixture of fatty aldehydes and fatty alcohols wherein the fatty alcohols and fatty aldehydes are produced at a higher titer than is produced by a recombinant host cell expressing the wild type CAR polypeptide of SEQ ID NO:7.

59 . The variant CAR polypeptide of claim 58 , wherein said variant polypeptide comprises:
(a) mutation A535S; (b) mutations E20R, F288G, Q473I and A535S; (c) mutations E20R, F288G, Q473H, A535S, R827A and S927G; (d) mutations E20R, S22R, F288G, Q473H, A535S, R827A and S927G (e) mutations S3R, E20R, S22R, F288G, Q473H, A535S, R873S, S927G, M930R and L1128W; (f) mutations E20R, S22R, F288G, Q473H, A535S, R873S, S927G, M930R and LI 128W. (g) mutations D18R, E20R, S22R, F288G, Q473I, A535S, S927G, M930K and L1128W; (h) mutations E20R, S22R, F288G, Q473I, A535S, R827C, V926E, S927K and M930R; (i) mutations D18R, E20R, 288G, Q473I, A535S, R827C, V926E, M930K and LI 128W; (j) mutations E20R, S22R, F288G, Q473H, A535S, R827C, V926A, S927K and M930R; (k) mutations E20R, S22R, F288G, Q473H, A535S and R827C; (l) mutations E20R, S22R, F288G, Q473I, A535S, R827C and M930R; (m) mutations E20R, S22R, F288G, Q473I, A535S, I870L, S927G and M930R; (n) mutations E20R, S22R, F288G, Q473I, A535S, I870L and S927G; (o) mutations D18R, E20R, S22R, F288G, Q473I, A535S, R827C, I870L, V926A and S927G; (p) mutations E20R, S22R, F288G, Q473H, A535S, R827C, I870L and L1128W; (q) mutations D18R, E20R, S22R, F288G, Q473H, A535S, R827C, I870L, S927G and L1128W; (r) mutations E20R, S22R, F288G, Q473I, A535S, R827C, I870L, S927G and L1128W; (s) mutations E20R, S22R, F288G, Q473I, A535S, R827C, I870L, S927G, M930K and L1128W; (t) mutations E20R, S22R, F288G, Q473H, A535S, I870L, S927G and M930K; (u) mutations E20R, F288G, Q473I, A535S, I870L, M930K; (v) mutations E20R, S22R, F288G, Q473H, A535S, S927G, M930K and L1128W; (w) mutations D18R, E20R, S22R, F288G, Q473I, A535S, S927G and L1128W; (x) mutations E20R, S22R, F288G, Q473I, A535S, R827C, I870L and S927G; (y) mutations D18R, E20R, S22R, F288G, Q473I, A535S, R827C, I870L, S927G and L1128W; (z) mutations D18R, E20R, S22R, F288G, Q473I, A535S, S927G, M930R and L1128W; (xx) mutations E20R, S22R, F288G, Q473H, A535S, V926E, S927G and M930R; or (zz) mutations E20R, S22R, F288G, Q473H, A535S, R827C, I870L, V926A and L1128W.

60 . A recombinant host cell comprising a polynucleotide sequence encoding a variant carboxylic acid reductase (CAR) polypeptide having at least 85% sequence identity to the wild-type CAR polypeptide of SEQ ID NO: 7 and having at least one mutation at an amino acid position selected from the group consisting of S3R, D18R, D18L, D18T, D18P, E20V, E20S, E20R, S22R, S22N, S22G, L80R, R87G, R87E, V191S, F288R, F288S, F288G, Q473L, Q473W, Q473Y, Q473I, Q473H, A535S, D750A, R827C, R827A, I870L, R873S, V926A, V926E, S927K, S927G, M930K, M930R and L1128W, wherein the recombinant host cell produces a fatty alcohol composition, a fatty aldehyde composition or a composition that is a mixture of fatty aldehydes and fatty alcohols wherein the fatty alcohols and fatty aldehydes are produced at a higher titer or yield than is produced by a host cell expressing the wild type CAR polypeptide of SEQ ID NO: 7 when cultured in a medium containing a carbon source under conditions effective to express said variant CAR polypeptide.

61 . The recombinant host cell of claim 60 , further comprising a polynucleotide encoding a thioesterase polypeptide.

62 . The recombinant host cell of claim 61 , further comprising a polynucleotide encoding
(i) a FabB polypeptide and a FadR polypeptide, or (ii) a fatty aldehyde reductase.

63 . The recombinant host cell of claim 60 , wherein the titer or yield is at least 3 times greater than the titer or the yield of a host cell expressing the corresponding wild type CAR polypeptide when cultured under the same conditions as the recombinant host cell.

64 . The recombinant host cell of claim 63 , wherein the titer is in a range that is between 30 g/L to 250 g/L, or between 90 g/L to 120 g/L.

65 . The recombinant host cell of claim 64 , wherein the yield is in a range that is between 10% to 40%.

66 . A cell culture comprising the recombinant host cell of claim 60 .

67 . The cell culture of claim 66 wherein the cell culture has productivity that is at least 3 times greater than the productivity of a cell culture that expresses the corresponding wild type CAR polypeptide.

68 . The cell culture of claim 67 , wherein said productivity ranges from 0.7 mg/L/hr to 3 g/L/hr.

69 . The cell culture of claim 66 , wherein the fatty alcohol composition, fatty aldehyde composition or composition that is a mixture of fatty aldehydes and fatty alcohols collects in an organic phase extracellularly.

70 . The cell culture of claim 69 , wherein the fatty alcohol composition, fatty aldehyde composition or composition that is a mixture of fatty aldehydes and fatty alcohols comprises one or more of a C6, C8, C10, C12, C13, C14, C15, C16, C17, or C18 fatty alcohol, fatty aldehyde or combination thereof.

71 . The cell culture of claim 69 , wherein the fatty alcohol composition, fatty aldehyde composition or composition that is a mixture of fatty aldehydes and fatty alcohols comprises a C10:1, C12:1, C14:1, C16:1, or a C18:1 unsaturated fatty alcohol or a C10:1, C12:1, C14:1, C16:1, or a C18:1 unsaturated fatty aldehyde or a combination thereof.

72 . The cell culture of claim 69 , wherein the fatty alcohol composition, fatty aldehyde composition or composition that is a mixture of fatty aldehydes and fatty alcohols comprises C12 and C14 fatty alcohols and/or C12 and C14 fatty aldehydes.

73 . The cell culture of claim 72 , wherein the fatty alcohol composition, fatty aldehyde composition or composition that is a mixture of fatty aldehydes and fatty alcohols comprises C12 and C14 fatty alcohols and/or C12 and C14 fatty aldehydes at a ratio of about 3:1.

74 . The cell culture of claim 69 , wherein the fatty alcohol composition comprises unsaturated fatty alcohols, the fatty aldehyde composition comprises unsaturated fatty aldehydes and the composition that is a mixture of fatty aldehydes and fatty alcohols comprises unsaturated fatty alcohols and unsaturated fatty aldehydes.

75 . The cell culture of claim 74 , wherein the unsaturated fatty alcohols and unsaturated fatty aldehydes have a double bond at position 7 in the carbon chain between C7 and C8 from the reduced end of the fatty alcohol or fatty aldehyde.

76 . The cell culture of claim 69 , wherein the fatty alcohol composition comprises branched chain fatty alcohols, the fatty aldehyde composition comprises branched chain fatty aldehydes and the composition that is a mixture of fatty aldehydes and fatty alcohols compr

Assignees

• Reg Life Sciences Llc

Inventors

• Greenfield Derek L
• Clarke Elizabeth J
• Groban Eli S
• Arlagadda Vikranth
• Lee Sungwon
• Li Xuezhi
• Hu Zhihao
• Zhu Baolong

Classifications

• C12P7/6409

  Fatty acids · CPC title

• C12P7/04

  acyclic · CPC title

• C12Y102/99006

  Carboxylate reductase (1.2.99.6) · CPC title

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12N9/0008 Primary

  acting on the aldehyde or oxo group of donors (1.2) · CPC title