PRODUCTION OF SATURATED FATTY ALCOHOLS FROM ENGINEERED MICROORGANISMS

• US 20140336423A1
• Filed: 12/13/2012
• Published: 11/13/2014
• Est. Priority Date: 12/20/2011
• Status: Active Grant

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, Inc.
• Inventors: Valle, Fernando, Balatskaya, Svetlana, Clark, Louis, Vroom, Jonathan, Barak, Yoram, Karlshoej, Kristian, Choudhary, Patricia, Cho, Catherine, Chan, Kaman
• Granted Patent: US 9,447,436 B2
• Time in Patent Office: Days
• US Class Current: 568/840
• CPC Class Codes: A61K 2800/10 General cosmetic use
• CPC Class Codes: A61K 8/342 Alcohols having more than seven atoms in an unbroken chain
• CPC Class Codes: A61Q 19/00 Preparations for care of the skin
• CPC Class Codes: A61Q 5/00 Preparations for care of the hair
• CPC Class Codes: C07C 31/125 containing five to twenty-two carbon atoms
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12P 7/04 acyclic
• CPC Class Codes: C12Y 102/01 with NAD+ or NADP+ as acceptor (1.2.1)
• CPC Class Codes: C12Y 102/0105 Long-chain-fatty-acyl-CoA reductase (1.2.1.50)
• CPC Class Codes: C12Y 102/0108 Long-chain acyl-[acyl-carrier-protein] reductase (1.2.1.80)
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

Recombinant bacterial microorganisms are provided which comprise heterologous fatty acyl reductases (“FAR”) polypeptides wherein said microorganisms have been engineered to produce increased amounts of saturated fatty alcohols and methods of making saturated fatty alcohols using the recombinant bacterial microorganisms.

Claims

1. An engineered bacterial microorganism comprising at least one polynucleotide encoding at least one heterologous fatty acyl-ACP reductase enzyme (FAR) and a first recombinant polynucleotide encoding a FabZ enzyme wherein the engineered bacterial microorganism produces a fatty alcohol composition comprising C10 to C18 fatty alcohols.

2. The engineered bacterial microorganism according to claim 1 further comprising a second recombinant polynucleotide encoding a FabI enzyme.

4. The engineered bacterial microorganism of claim 1, wherein the FAR enzyme has an amino acid sequence comprising at least about 80% sequence identity to SEQ ID NOs:

2, 4, 6, 18, and/or 28.

7. The engineered bacterial microorganism of claim 1, wherein the FabZ enzyme comprises an amino acid sequence comprising at least about 80% sequence identity to SEQ ID NO:

8 or SEQ ID NO;

26.

8. The engineered bacterial microorganism of claim 2, wherein the FabI enzyme comprises an amino acid sequence comprising at least about 80% sequence identity to SEQ ID NO:

12.

39. A fatty alcohol composition produced by the engineered microbial cell of claim 1.

40. A method of producing a fatty alcohol composition comprising:

a) providing an engineered microorganism of claim 1;

b) culturing the engineered microorganism in a culture medium under suitable culture conditions for the production of fatty alcohols or derivatives thereof;

c) allowing production of the fatty alcohols or derivatives thereof; and

d) optionally recovering the produced fatty alcohols or derivative thereof from the engineered microorganism.

41. The method according to claim 40, wherein the fatty alcohol composition comprises at least 60% of C12:

0, C14;

0, C16;

0 and C18;

0 fatty alcohols.

43. The method according to claim 40, wherein the % of saturated fatty alcohols in the fatty alcohol composition is at least 60%.

45. The method according to claim 40, wherein the culturing is at a temperature of about 25°

C. to about 45°

C.

46. A detergent, personal care, or surfactant composition comprising a fatty alcohol composition produced by the method of claim 40.

49. The method according to claim 40, wherein at least 2 g/L of fatty alcohols are produced.

53. A method for producing a fatty alcohol composition comprising at least 60% saturated fatty alcohols comprising:

a) providing an engineered bacterial microorganisms of claim 1;

b) culturing the engineered microorganism in a culture medium under suitable culture conditions for the production of fatty alcohols;

c) allowing production of the fatty alcohols; and

d) optionally recovering the produced fatty alcohols.

54. The method according to claim 53, wherein the fatty alcohol composition comprises at least 60% of saturated C12, C14, C16 and/or C18 fatty alcohols.

63. The method according to claim 53, wherein at least 2 g/L of fatty alcohols are produced.

3. (canceled)

5. (canceled)

6. (canceled)

9. An engineered bacterial microorganism comprising a polynucleotide encoding at least one heterologous fatty acyl-ACP reductase enzyme (FAR) and a first recombinant polynucleotide encoding a FabA enzyme wherein the engineered bacterial microorganism produces a fatty alcohol composition comprising C10 to C18 fatty alcohols.

10. The engineered bacterial microorganism according to claim 9 further comprising a second recombinant polynucleotide encoding a FabI enzyme.

12. The engineered bacterial microorganism of claim 9, wherein the FAR enzyme has an amino acid sequence comprising at least about 80% sequence identity to SEQ ID NOs:

2, 4, 6, 18, and/or 28.

13. The engineered bacterial microorganism of claim 9, wherein the FAR enzyme has an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NOs:

2, 4, 6, 18, and/or 28.

15. The engineered bacterial microorganism of claim 9, wherein the FabA enzyme has an amino acid sequence comprising at least about 80% sequence identity to SEQ ID NO:

10.

16. The engineered bacterial microorganism of claim 10, wherein the FabI enzyme has an amino acid sequence comprising at least 80% sequence identity to SEQ ID NO:

12.

11. (canceled)

14. (canceled)

17. An engineered bacterial microorganism comprising a polynucleotide encoding a heterologous fatty acyl-ACP reductase enzyme (FAR) and an inactivated endogenous fabF gene encoding a FabF enzyme, wherein the engineered bacterial microorganism produces a fatty alcohol composition comprising C10 to C18 fatty alcohols.

19. The engineered bacterial microorganism of claim 17, wherein the FAR enzyme has an amino acid sequence comprising at least 80% sequence identity to SEQ ID NOs:

2, 4, 6, 18 or 28.

22. The engineered bacterial microorganism of claim 17, wherein the FabF has an amino acid sequence comprising at least 90% sequence identity to SEQ ID NO:

14.

18. (canceled)

20. (canceled)

21. (canceled)

23. An engineered bacterial microorganism comprising a polynucleotide encoding a heterologous fatty acyl-ACP reductase enzyme (FAR) and an inactivated endogenous fadR gene encoding a FadR enzyme, wherein the engineered bacterial microorganism produces a fatty alcohol composition comprising C10 to C18 fatty alcohols.

25. The engineered bacterial microorganism of claim 23, wherein the FAR enzyme has an amino acid sequence comprising at least 80% sequence identity to SEQ ID NOs:

2, 4, 6, 18 or 28.

28. The engineered bacterial microorganism of claim 23, wherein the FadR has an amino acid sequence comprising at least 90% sequence identity to SEQ ID NO:

16.

24. (canceled)

26. (canceled)

27. (canceled)

29. An engineered bacterial microorganism comprising a polynucleotide encoding at least one heterologous fatty acyl-ACP reductase enzyme (FAR) and a first recombinant polynucleotide encoding a FabI enzyme wherein the engineered bacterial microorganism produces a fatty alcohol composition comprising C10 to C18 fatty alcohols.

31. The engineered bacterial microorganism of claim 29, wherein the FAR enzyme has an amino acid sequence comprising at least about 80% sequence identity to SEQ ID NOs:

2, 4, 6, 18, and/or 28.

34. The engineered bacterial microorganism of claim 29, wherein the FabI enzyme has an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO:

12.

30. (canceled)

32. (canceled)

33. (canceled)

35. An engineered bacterial microorganism comprising a polynucleotide encoding i) a heterologous fatty acyl-ACP reductase enzyme having at least 95% sequence identity to SEQ ID NO:

28, ii) a first recombinant polynucleotide sequence encoding a FabZ enzyme and iii) a second recombinant polynucleotide sequence encoding a FabI enzyme, wherein the engineered bacterial microorganism is capable of producing a fatty alcohol composition.

37. The engineered bacterial microorganism of claim 35, wherein the FabZ enzyme comprises at least 95% sequence identity to SEQ ID NO:

8 or SEQ ID NO;

26.

38. The engineered bacterial microorganism of claim 35, wherein the FabI enzyme comprises at least 95% sequence identity to SEQ ID NO:

12.

36. (canceled)

42. (canceled)

44. (canceled)

47. (canceled)

48. (canceled)

50. (canceled)

51. (canceled)

52. (canceled)

55. (canceled)

56. (canceled)

57. (canceled)

58. (canceled)

59. (canceled)

60. (canceled)

61. (canceled)

62. (canceled)

64. (canceled)