Car enzymes and improved production of fatty alcohols

Patent metadata

Field	Value
Publication number	US-9340801-B2
Application number	US-201314390350-A
Country	US
Kind code	B2
Filing date	Apr 2, 2013
Priority date	Apr 2, 2012
Publication date	May 17, 2016
Grant date	May 17, 2016

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 90% sequence identity to 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 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 said variant CAR polypeptide in a recombinant host cell results in a higher titer of fatty alcohol compositions compared to a host cell expressing a corresponding wild type polypeptide.

3. The variant CAR polypeptide of claim 1 , wherein said 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, I870L, R873S, V926A, V926E, S927K, S927G, M930K, M930R and L1128W.

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

6. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations E20R, F288G, Q473I and A535S.

7. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations E20R, F288G, Q473H, A535S, R827A and S927G.

8. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations E20R, S22R, F288G, Q473H, A535S, R827A and S927G.

9. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations S3R, E20R, S22R, F288G, Q473H, A535S, R873S, S927G, M930R and L1128W.

10. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations E20R, S22R, F288G, Q473H, A535S, R873S, S927G, M930R and L1128W.

11. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations D18R, E20R, S22R, F288G, Q473I, A535S, S927G, M930K and L1128W.

12. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations E20R, S22R, F288G, Q473I, A535S, R827C, V926E, S927K and M930R.

13. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations D18R, E20R, 288G, Q473I, A535S, R827C, V926E, M930K and L1128W.

14. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations E20R, S22R, F288G, Q473H, A535S, R827C, V926A, S927K and M930R.

15. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations E20R, S22R, F288G, Q473H, A535S and R827C.

16. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations E20R, S22R, F288G, Q473I, A535S, R827C and M930R.

17. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations E20R, S22R, F288G, Q473I, A535S, I870L, S927G and M930R.

18. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations E20R, S22R, F288G, Q473I, A535S, I870L and S927G.

19. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations D18R, E20R, S22R, F288G, Q473I, A535S, R827C, I870L, V926A and S927G.

20. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations E20R, S22R, F288G, Q473H, A535S, R827C, I870L and L1128W.

21. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations D18R, E20R, S22R, F288G, Q473H, A535S, R827C, I870L, S927G and L1128W.

22. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations E20R, S22R, F288G, Q473I, A535S, R827C, I870L, S927G and L1128W.

23. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations E20R, S22R, F288G, Q473I, A535S, R827C, I870L, S927G, M930K and L1128W.

24. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations E20R, S22R, F288G, Q473H, A535S, I870L, S927G and M930K.

25. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations E20R, F288G, Q473I, A535S, I870L, M930K.

26. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations E20R, S22R, F288G, Q473H, A535S, S927G, M930K and L1128W.

27. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations D18R, E20R, S22R, F288G, Q473I, A535S, S927G and L1128W.

28. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations E20R, S22R, F288G, Q473I, A535S, R827C, I870L and S927G.

29. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations D18R, E20R, S22R, F288G, Q473I, A535S, R827C, I870L, S927G and L1128W.

30. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations D18R, E20R, S22R, F288G, Q473I, A535S, S927G, M930R and L1128W.

31. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations E20R, S22R, F288G, Q473H, A535S, V926E, S927G and M930R.

32. The variant CAR polypeptide of claim 4 , wherein said variant polypeptide comprises mutations E20R, S22R, F288G, Q473H, A535S, R827C, I870L, V926A and L1128W.

33. A recombinant host cell comprising a polynucleotide sequence encoding a variant carboxylic acid reductase (CAR) polypeptide having at least 90% 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, 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 said variant CAR polypeptide.

34. The recombinant host cell of claim 33 , wherein said SEQ ID NO: 7 is the corresponding wild type CAR polypeptide.

35. The recombinant host cell of claim 33 , further comprising a polynucleotide encoding a thioesterase polypeptide.

36. The recombinant host cell of claim 35 , further comprising a polynucleotide encoding a FabB polypeptide and a FadR polypeptide.

37. The recombinant host cell of claim 33 , wherein said recombinant host cell has a titer that is at least 3 times greater than the titer of a host cell expressing the corresponding wild type CAR polypeptide when cultured under the same conditions as the recombinant host cell.

38. The recombinant host cell of claim 37 , wherein said recombinant host cell has a titer of from about 30 g/L to about 250 g/L.

39. The recombinant host cell of claim 38 , wherein said recombinant host cell has a titer of from about 90 g/L to about 120 g/L.

40. The recombinant host cell of claim 33 , wherein said recombinant host cell has a yield f

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

• C12Y102/99006

  Carboxylate reductase (1.2.99.6) · CPC title

• C12N9/0008 Primary

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

• C12P7/04 Primary

  acyclic · CPC title

• Y02P20/52

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

• C12N9/16

  acting on ester bonds (3.1) · CPC title