Car enzymes and improved production of fatty alcohols

Patent metadata

Field	Value
Publication number	US-9758769-B2
Application number	US-201615154636-A
Country	US
Kind code	B2
Filing date	May 13, 2016
Priority date	Apr 2, 2012
Publication date	Sep 12, 2017
Grant date	Sep 12, 2017

Abstract

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

First claim

What is claimed is:

1. A variant carboxylic acid reductase (CAR) polypeptide comprising an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 7, wherein the variant CAR polypeptide is genetically engineered to have at least one mutation at an amino acid position selected from the group consisting of amino acid positions 3, 18, 20, 22, 80, 87, 191, 288, 473, 535, 750, 827, 870, 873, 926, 927, 930, and 1128.

2. The variant CAR polypeptide of claim 1 , wherein expression of the variant CAR polypeptide in a recombinant host cell results in a higher titer of fatty alcohol compositions compared to a recombinant host cell expressing a corresponding wild type polypeptide.

3. The variant CAR polypeptide of claim 1 , wherein the CAR polypeptide is a CarB polypeptide.

4. The variant CAR polypeptide of claim 1 , wherein the variant CAR polypeptide comprises a mutation 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, 1870L, R873S, V926A, V926E, S927K, S927G, M930K, M930R and L1128W.

5. The variant CAR polypeptide of claim 4 , wherein the variant CAR polypeptide comprises mutation A535S.

6. The variant CAR polypeptide of claim 4 , wherein the variant polypeptide comprises a combination of mutations selected from the group consisting of:
E20R, F288G, Q473I, A535S;
E20R, F288G, Q473H, A535S, R827A, S927G;
E20R, S22R, F288G, Q473H, A535S, R827A, S927G;
S3R, E20R, S22R, F288G, Q473H, A535S, R873S, S927G, M930R, L1128W;
E20R, S22R, F288G, Q473H, A535S, R873S, S927G, M930R, L1128W;
D18R, E20R, S22R, F288G, Q473I, A535S, S927G, M930K, L1128W;
E20R, S22R, F288G, Q473I, A535S, R827C, V926E, S927K, M930R;
D18R, E20R, 288G, Q473I, A535S, R827C, V926E, M930K, L1128W;
E20R, S22R, F288G, Q473H, A535S, R827C, V926A, S927K, M930R;
E20R, S22R, F288G, Q473H, A535S, R827C;
E20R, S22R, F288G, Q473I, A535S, R827C, M930R;
E20R, S22R, F288G, Q473I, A535S, I870L, S927G, M930R;
E20R, S22R, F288G, Q473I, A535S, I870L, S927G;
D18R, E20R, S22R, F288G, Q473I, A535S, R827C, I870L, V926A, S927G;
E20R, S22R, F288G, Q473H, A535S, R827C, I870L, L1128W;
D18R, E20R, S22R, F288G, Q473H, A535S, R827C, I870L, S927G, L1128W;
E20R, S22R, F288G, Q473I, A535S, R827C, I870L, S927G, L1128W;
E20R, S22R, F288G, Q473I, A535S, R827C, I870L, S927G, M930K, L1128W;
E20R, S22R, F288G, Q473H, A535S, I870L, S927G, M930K;
E20R, F288G, Q473I, A535S, I870L, M930K;
E20R, S22R, F288G, Q473H, A535S, S927G, M930K, L1128W;
D18R, E20R, S22R, F288G, Q473I, A535S, S927G, L1128W;
E20R, S22R, F288G, Q473I, A535S, R827C, I870L, S927G;
D18R, E20R, S22R, F288G, Q473I, A535S, R827C, I870L, S927G, L1128W;
D18R, E20R, S22R, F288G, Q473I, A535S, S927G, M930R, L1128W;
E20R, S22R, F288G, Q473H, A535S, V926E, S927G, M930R; and
E20R, S22R, F288G, Q473H, A535S, R827C, I870L, V926A, L1128W.

7. A recombinant host cell comprising an exogenous polynucleotide sequence encoding a variant carboxylic acid reductase (CAR) polypeptide having at least 85% sequence identity to SEQ ID NO: 7 and having at least one mutation at an amino acid position selected from the group consisting of position 3, 18, 20, 22, 80, 87, 191, 288, 473, 535, 750, 827, 870, 873, 926, 927, 930, and 1128, wherein the recombinant host cell produces a fatty alcohol composition at a higher titer or yield than a host cell expressing a corresponding wild type CAR polypeptide when cultured in a medium containing a carbon source under conditions effective to express the variant CAR polypeptide.

8. The recombinant host cell of claim 7 , wherein the SEQ ID NO: 7 is the corresponding wild type CAR polypeptide.

9. The recombinant host cell of claim 7 , further comprising a polynucleotide encoding a) a thioesterase polypeptide; b) a FabB polypeptide and a FadR polypeptide; or c) fatty aldehyde reductase (AlrA) polypeptide.

10. The recombinant host cell of claim 7 , wherein the host cell is selected from the group consisting of a plant cell, an insect cell, a fungal cell, an algal cell, and a bacterial cell.

11. The recombinant host cell of claim 10 , wherein the host cell is a bacterial cell.

12. The recombinant host cell of claim 11 , wherein the bacterial cell is an E. coli cell.

13. A method of making a fatty alcohol composition comprising culturing a recombinant host cell comprising an exogenous polynucleotide sequence encoding a variant carboxylic acid reductase (CAR) polypeptide having at least 85% sequence identity to SEQ ID NO: 7 and having at least one mutation at an amino acid position selected from the group consisting of amino acid positions 3, 18, 20, 22, 80, 87, 191, 288, 473, 535, 750, 827, 870, 873, 926, 927, 930, and 1128, in a culture medium comprising a carbon source under conditions suitable to produce the fatty alcohol composition, wherein the fatty alcohol composition is released from the host cell into the culture medium.

14. The method of claim 13 , wherein the variant CAR polypeptide comprises a mutation 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.

15. The method of claim 14 , wherein the variant CAR polypeptide comprises mutation A535S.

16. The method of claim 14 , wherein the variant CAR polypeptide comprises a combination of mutations selected from the group consisting of:
E20R, F288G, Q473I, A535S;
E20R, F288G, Q473H, A535S, R827A, S927G;
E20R, S22R, F288G, Q473H, A535S, R827A, S927G;
S3R, E20R, S22R, F288G, Q473H, A535S, R873S, S927G, M930R, L1128W;
E20R, S22R, F288G, Q473H, A535S, R873S, S927G, M930R, L1128W;
D18R, E20R, S22R, F288G, Q473I, A535S, S927G, M930K, L1128W;
E20R, S22R, F288G, Q473I, A535S, R827C, V926E, S927K, M930R;
D18R, E20R, 288G, Q473I, A535S, R827C, V926E, M930K, L1128W;
E20R, S22R, F288G, Q473H, A535S, R827C, V926A, S927K, M930R;
E20R, S22R, F288G, Q473H, A535S, R827C;
E20R, S22R, F288G, Q473I, A535S, R827C, M930R;
E20R, S22R, F288G, Q473I, A535S, I870L, S927G, M930R;
E20R, S22R, F288G, Q473I, A535S, I870L, S927G;
D18R, E20R, S22R, F288G, Q473I, A535S, R827C, I870L, V926A, S927G;
E20R, S22R, F288G, Q473H, A535S, R827C, I870L, L1128W;
D18R, E20R, S22R, F288G, Q473H, A535S, R827C, I870L, S927G, L1128W;
E20R, S22R, F288G, Q473I, A535S, R827C, I870L, S927G, L1128W;
E20R, S22R, F288G, Q473I, A535S, R827C, I870L, S927G, M930K, L1128W;
E20R, S22R, F288G, Q473H, A535S, I870L, S927G, M930K;
E20R, F288G, Q473I, A535S, I870L, M930K;
E20R, S22R, F288G, Q473H, A535S, S927G, M930K, L1128W;
D18R, E20R, S22R, F288G, Q473I, A535S, S927G, L1128W;
E20R, S22R, F288G, Q473I, A535S, R827C, I870L, S927G;
D18R, E20R, S22R, F288G, Q473I, A535S, R827C, I870L, S927G, L1128W;
D18R, E20R, S22R, F288G, Q473I, A535S, S927G, M930R, L1128W;
E20R, S22R, F288G, Q473H, A535S, V926E, S927G, M930R; and
E20R, S22R, F288G, Q473H, A535S, R827C, I870L, V926A, L1128W.

17. The method of claim 13 , wherein the host cell further comprises a polynucleotide encoding a) a thioesterase polypeptide; b) a FabB polypeptide and a FadR polypeptide; or c) fatty aldehyde reductase (AlrA) polypeptide.

18. The method of claim 13 , wherein the host cell is selected from the group consisting of a plant cell, an insect cell, a fungal cell, an algal cell, and a bacterial cell.

19. The method of

Assignees

• Reg Life Sciences Llc

Inventors

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

Classifications

• C12P7/04

  acyclic · CPC title

• C12N9/0008 Primary

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

• C12Y102/99006

  Carboxylate reductase (1.2.99.6) · CPC title

• C12P7/6409

  Fatty acids · CPC title

• C12N9/16

  acting on ester bonds (3.1) · CPC title