Microbial production of pyruvate and pyruvate derivatives

• US 7,749,740 B2
• Filed: 08/20/2004
• Issued: 07/06/2010
• Est. Priority Date: 02/20/2002
• Status: Expired due to Fees

• Current Assignee: The University of Georgia Research Foundation, Inc.
• Original Assignee: The University of Georgia Research Foundation, Inc.
• Inventors: Altman, Elliot, Eiteman, Mark A.
• Primary Examiner(s): Fronda; Christian L
• Application Number:
  US10/923,458
• Publication Number:
  US 20050255572A1
• Time in Patent Office: 2,146 Days
• Field of Search: 435/116, 435/136, 435/183, 435/190, 435/252.3, 435/320, 435/320.1, 536/23.2
• US Class Current: 435/136
• CPC Class Codes: C12P 13/06 Alanine; Leucine; Isoleucine; Serine; Homoserine
• CPC Class Codes: C12P 7/26 Ketones
• CPC Class Codes: C12P 7/40 containing a carboxyl group including Peroxycarboxylic acids
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Microbial production of pyruvate and metabolites derived from pyruvate in cells exhibiting reduced pyruvate dehydrogenase activity compared to wild-type cells. Acetate and glucose are supplied as a carbon sources.

Claims

1. A method for making a metabolite comprising pyruvate or a pyruvate derivative, the method comprising:

providing a bacterial cell exhibiting reduced activity of at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes, compared to a wild-type bacterial cell; and

culturing the bacterial cell in the presence of glucose and acetate under conditions and for a time effective to accumulate the metabolite to a concentration of at least about 3.3 g/L.

2. The method of claim 1 wherein the metabolite comprises pyruvate or diacetyl.

3. The method of claim 1 wherein the bacterial cell further exhibits added or increased NADH oxidase activity compared to a wild-type bacterial cell.

4. The method of claim 1 wherein the metabolite comprises alanine.

5. The method of claim 1 further comprising culturing the bacterial cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of the bacterial cell.

6. The method of claim 5 wherein the additional carbon source comprises succinate.

7. The method of claim 1 wherein the metabolite comprises pyruvate.

8. The method of claim 7 wherein pyruvate is produced in an amount of at least about 30 g/L.

9. The method of claim 7 wherein the pyruvate yield is at least about 0.70.

10. The method of claim 1 wherein the PDH activity in the bacterial cell is undetectable.

11. A method for making a metabolite comprising pyruvate or a pyruvate derivative, the method comprising:

providing an E. coli cell exhibiting reduced activity of at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes, compared to a wild-type E. coli cell; and

culturing the E. coli cell in the presence of glucose and acetate to yield the metabolite.

12. The method of claim 11 wherein the metabolite comprises pyruvate or diacetyl.

13. The method of claim 11 wherein the E. coli cell further exhibits added NADH oxidase activity.

14. The method of claim 11 wherein the metabolite comprises alanine.

15. The method of claim 11 further comprising culturing the E. coli cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of the E. coli cell.

16. The method of claim 15 wherein the additional carbon source comprises succinate.

17. The method of claim 11 wherein the metabolite comprises pyruvate.

18. A method for making a metabolite comprising pyruvate or a pyruvate derivative, the method comprising:

providing a bacterial cell exhibiting

(a) reduced activity of at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes compared to a wild-type bacterial cell, and

(b) reduced activity of phosphoenolpyruvate carboxylase (PEP carboxylase) compared to a wild-type bacterial cell; and

culturing the bacterial cell in the presence of glucose and acetate to yield the metabolite.

19. The method of claim 18 further comprising culturing the bacterial cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of the bacterial cell.

20. The method of claim 19 wherein the additional carbon source comprises succinate.

21. The method of claim 19 wherein the metabolite comprises pyruvate.

22. A method for making a metabolite comprising pyruvate or a pyruvate derivative, the method comprising:

providing a bacterial cell exhibiting

(a) reduced activity of at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes compared to a wild-type bacterial cell, and

(b) added or increased NADH oxidase activity; and

culturing the bacterial cell in the presence of glucose and acetate to yield the metabolite.

23. The method of claim 22 further comprising culturing the bacterial cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of the bacterial cell.

24. The method of claim 23 wherein the additional carbon source comprises succinate.

25. The method of claim 23 wherein the metabolite comprises pyruvate.

26. A method for making a metabolite comprising pyruvate or a pyruvate derivative, the method comprising:

providing a bacterial cell exhibiting

(a) reduced activity of at least one enzyme in the pyruvate dehydrogenase(PDH) complex of enzymes, compared to a wild-type bacterial cell, and

(b) reduced activity of pyruvate oxidase compared to a wild-type bacterial cell; and

culturing the bacterial cell in the presence of glucose and acetate to yield the metabolite.

27. The method of claim 26 further comprising culturing the bacterial cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of the bacterial cell.

28. The method of claim 27 wherein the additional carbon source comprises succinate.

29. The method of claim 27 wherein the metabolite comprises pyruvate.

30. A method for making a metabolite comprising pyruvate or a pyruvate derivative, the method comprising:

providing a bacterial cell exhibiting

(a) reduced activity of at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes, compared to a wild-type bacterial cell,

(b) reduced activity of phosphoenolpyruvate carboxylase (PEP carboxylase) compared to a wild-type bacterial cell, and

(c) reduced activity of pyruvate oxidase compared to a wild-type bacterial cell; and

culturing the bacterial cell in the presence of glucose and acetate to yield the metabolite.

31. The method of claim 30 further comprising culturing the bacterial cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of the bacterial cell.

32. The method of claim 31 wherein the additional carbon source comprises succinate.

33. The method of claim 30 wherein the metabolite comprises pyruvate.

34. A method for making a metabolite comprising pyruvate or a pyruvate derivative, the method comprising:

providing a bacterial cell exhibiting

(a) reduced activity of at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes, compared to a wild-type bacterial cell,

(b) reduced activity of phosphoenolpyruvate carboxylase (PEP carboxylase) compared to a wild-type bacterial cell,

(c) reduced activity of pyruvate oxidase compared to a wild-type bacterial cell, and

(d) added or increased NADH oxidase activity; and

culturing the bacterial cell in the presence of glucose and acetate to yield the metabolite.

35. The method of claim 34 further comprising culturing the bacterial cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of the bacterial cell.

36. The method of claim 35 wherein the additional carbon source comprises succinate.

37. The method of claim 34 wherein the metabolite comprises pyruvate.

38. A method for making pyruvate comprising:

providing a bacterial cell wherein the gene encoding at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes is knocked out; and

culturing the bacterial cell in the presence of glucose and acetate to yield the metabolite.

39. The method of claim 38 further comprising culturing the bacterial cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of the bacterial cell.

40. The method of claim 39 wherein the additional carbon source comprises succinate.

41. The method of claim 38 wherein the bacterial cell further exhibits reduced activity of pyruvate oxidase.

42. The method of claim 38 wherein the bacterial cell further exhibits reduced activity of phosphoenolpyruvate carboxylase (PEP carboxylase).

43. The method of claim 38 wherein the bacterial cell further exhibits increased or added activity of NADH oxidase.

44. The method of claim 38 wherein the bacterial cell further exhibits reduced activity of pyruvate oxidase and reduced activity of PEP carboxylase.

45. The method of claim 38 further wherein the bacterial cell further exhibits reduced activity of pyruvate oxidase, reduced activity of PEP carboxylase, and increased or added activity of NADH oxidase.

46. The method of claim 38 wherein the metabolite comprises pyruvate.

47. A method for making a metabolite comprising pyruvate or a pyruvate derivative, the method comprising:

providing an E. coli cell wherein the gene encoding at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes is knocked out; and

culturing the E. coli cell in the presence of glucose and acetate to yield the metabolite.

48. The method of claim 47 further comprising culturing the E. coli cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of E. coli.

49. The method of claim 47 wherein the additional carbon source comprises succinate.

50. The method of claim 47 wherein the metabolite comprises pyruvate.

51. A method for making alanine comprising:

providing a bacterial cell exhibiting

(a) reduced activity of at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes, compared to a wild-type bacterial cell, and

(b) added or increased alanine dehydrogenase activity compared to a wild-type bacterial cell; and

culturing the bacterial cell in the presence of glucose and acetate to yield alanine.

52. The method of claim 51 further comprising culturing the bacterial cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of the bacterial cell.

53. The method of claim 52 wherein the additional carbon source comprises succinate.

54. A method for making alanine comprising:

providing an E. coli cell exhibiting

(a) reduced activity of at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes, compared to a wild-type bacterial cell, and

(b) added or increased alanine dehydrogenase activity compared to a wild-type E. coli cell; and

culturing the E. coli cell in the presence of a glucose and acetate to yield alanine.

55. The method of claim 54 further comprising culturing the E. coli cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of E. coli.

56. The method of claim 55 wherein the additional carbon source comprises succinate.

57. A method for making diacetyl comprising:

providing a bacterial cell exhibiting

(a) reduced activity of at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes, compared to a wild-type bacterial cell, and

(b) added or increased acetolactate synthase activity; and

culturing the bacterial cell in the presence of glucose and acetate to yield diacetyl.

58. The method of claim 57 further comprising culturing the bacterial cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of the bacterial cell.

59. The method of claim 58 wherein the additional carbon source comprises succinate.

60. The method of claim 57 wherein the bacterial cell further exhibits added or increased NADH oxidase activity.

61. A method for making diacetyl comprising:

providing an E. coli cell exhibiting

(a) reduced activity of at least one enzyme in the pyruvate dehydrogenase (PDH) complex of enzymes, compared to a wild-type E. coli cell, and

(b) added or increased acetolactate synthase activity; and

culturing the E. coli cell in the presence of glucose and acetate to yield diacetyl.

62. The method of claim 61 further comprising culturing the E. coli cell in the presence of an additional carbon source comprising a compound that is part of the tricarboxylic acid cycle of E. coli.

63. The method of claim 62 wherein the additional carbon source comprises succinate.

64. The method of claim 61 wherein the E. coli cell further exhibits added NADH oxidase activity.