COMPOSITIONS AND METHODS FOR THE BIOSYNTHESIS OF 1,4-BUTANEDIOL AND ITS PRECURSORS

• US 20090075351A1
• Filed: 03/14/2008
• Published: 03/19/2009
• Est. Priority Date: 03/16/2007
• Status: Active Grant

• Current Assignee: Genomatica Incorporated
• Original Assignee: Genomatica Incorporated
• Inventors: Burgard, Anthony P., Burk, Mark J., Niu, Wei, Van Dien, Stephen J.
• Granted Patent: US 8,067,214 B2
• Time in Patent Office: Days
• US Class Current: 435/141
• CPC Class Codes: B01D 3/002 by continuous methods
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12P 17/04 containing a five-membered hetero ring, e.g. griseofulvin , vitamin C
• CPC Class Codes: C12P 7/18 polyhydric
• CPC Class Codes: C12P 7/42 Hydroxy-carboxylic acids
• CPC Class Codes: C12P 7/52 Propionic acid; Butyric acids
• CPC Class Codes: C12Y 101/01061 4-Hydroxybutyrate dehydrogenase (1.1.1.61)
• CPC Class Codes: C12Y 102/01076 Succinate-semialdehyde dehydrogenase (acetylating) (1.2.1.76)
• CPC Class Codes: C12Y 401/01071 2-Oxoglutarate decarboxylase (4.1.1.71)
• CPC Class Codes: C12Y 602/01004 Succinate-CoA ligase (GDP-forming) (6.2.1.4)
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

The invention provides a non-naturally occurring microbial biocatalyst including a microbial organism having a 4-hydroxybutanoic acid (4-HB) biosynthetic pathway having at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, or α-ketoglutarate decarboxylase, wherein the exogenous nucleic acid is expressed in sufficient amounts to produce monomeric 4-hydroxybutanoic acid (4-HB). Also provided is a non-naturally occurring microbial biocatalyst including a microbial organism having 4-hydroxybutanoic acid (4-HB) and 1,4-butanediol (BDO) biosynthetic pathways, the pathways include at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:CoA transferase, 4-butyrate kinase, phosphotransbutyrylase, α-ketoglutarate decarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase or an aldehyde/alcohol dehydrogenase, wherein the exogenous nucleic acid is expressed in sufficient amounts to produce 1,4-butanediol (BDO). Additionally provided is a method for the production of 4-HB. The method includes culturing a non-naturally occurring microbial organism having a 4-hydroxybutanoic acid (4-HB) biosynthetic pathway including at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase or α-ketoglutarate decarboxylase under substantially anaerobic conditions for a sufficient period of time to produce monomeric 4-hydroxybutanoic acid (4-HB). Further provided is a method for the production of BDO. The method includes culturing a non-naturally occurring microbial biocatalyst, comprising a microbial organism having 4-hydroxybutanoic acid (4-HB) and 1,4-butanediol (BDO) biosynthetic pathways, the pathways including at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:CoA transferase, 4-hydroxybutyrate kinase, phosphotranshydroxybutyrylase, α-ketoglutarate decarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase or an aldehyde/alcohol dehydrogenase for a sufficient period of time to produce 1,4-butanediol (BDO). The 4-HB and/or BDO products can be secreted into the culture medium.

Claims

1. A non-naturally occurring microbial biocatalyst, comprising a microbial organism having a 4-hydroxybutanoic acid (4-HB) biosynthetic pathway comprising at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, or α

-ketoglutarate decarboxylase, wherein said exogenous nucleic acid is expressed in sufficient amounts to secrete monomeric 4-hydroxybutanoic acid (4-HB).

2. The non-naturally occurring microbial biocatalyst of claim 1, wherein said 4-HB biosynthetic pathway comprises 4-hydroxybutanoate dehydrogenase and succinyl-CoA synthetase and CoA-dependent succinic semialdehyde dehydrogenase, or α

-ketoglutarate decarboxylase.

3. The non-naturally occurring microbial biocatalyst of claim 1, wherein said exogenous nucleic acid encodes 4-hydroxybutanoate dehydrogenase.

4. The non-naturally occurring microbial biocatalyst of claim 3, further comprising a nucleic acid encoding an exogenous α

-ketoglutarate decarboxylase.

5. The non-naturally occurring microbial biocatalyst of claim 1, wherein said at least one exogenous nucleic acid further comprises two or more exogenous nucleic acids.

6. The non-naturally occurring microbial biocatalyst of claim 3, further comprising a nucleic acid encoding an exogenous succinyl-CoA synthetase, exogenous CoA-dependent succinic semialdehyde dehydrogenase or exogenous succinyl-CoA synthetase and exogenous CoA-dependent succinic semialdehyde dehydrogenase.

7. The non-naturally occurring microbial biocatalyst of claim 1, wherein said microbial organism lacks an endogenous 4-HB biosynthetic activity selected from 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, and α

-ketoglutarate decarboxylase.

8. The non-naturally occurring microbial biocatalyst of claim 1, wherein said at least one exogenous nucleic acid further comprises a heterologous encoding nucleic acid.

9. The non-naturally occurring microbial biocatalyst of claim 1, wherein said monomeric 4-HB is expressed at an intracellular concentration of at least about 5 mM.

10. The non-naturally occurring microbial biocatalyst of claim 9, further comprising an intracellular concentration of said monomeric 4-HB of about 10 mM or more.

11. The non-naturally occurring microbial biocatalyst of claim 1, further comprising a substantially anaerobic culture medium.

12. A non-naturally occurring microbial biocatalyst, comprising a microbial organism having 4-hydroxybutanoic acid (4-HB) and 1,4-butanediol (BDO) biosynthetic pathways, said pathways comprise at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:

CoA transferase, 4-butyrate kinase, phosphotransbutyrylase, α

-ketoglutarate decarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase or an aldehyde/alcohol dehydrogenase, wherein said exogenous nucleic acid is expressed in sufficient amounts to produce 1,4-butanediol (BDO).

13. The non-naturally occurring microbial biocatalyst of claim 12, wherein said 4-HB biosynthetic pathway comprises 4-hydroxybutanoate dehydrogenase and succinyl-CoA synthetase and CoA-dependent succinic semialdehyde dehydrogenase or α

-ketoglutarate decarboxylase.

14. The non-naturally occurring microbial biocatalyst of claim 12, wherein said exogenous nucleic acid encodes 4-hydroxybutanoate dehydrogenase.

15. The non-naturally occurring microbial biocatalyst of claim 14, further comprising a nucleic acid encoding an exogenous α

-ketoglutarate decarboxylase.

16. The non-naturally occurring microbial biocatalyst of claim 12, wherein said at least one exogenous nucleic acid further comprises two or more exogenous nucleic acids.

17. The non-naturally occurring microbial biocatalyst of claim 14, further comprising a nucleic acid encoding an exogenous succinyl-CoA synthetase, exogenous CoA-dependent succinic semialdehyde dehydrogenase or exogenous succinyl-CoA synthetase and exogenous CoA-dependent succinic semialdehyde dehydrogenase.

18. The non-naturally occurring microbial biocatalyst of claim 12, wherein said microbial organism lacks an endogenous 4-HB biosynthetic activity selected from 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase CoA-dependent succinic semialdehyde dehydrogenase and α

-ketoglutarate decarboxylase.

19. The non-naturally occurring microbial biocatalyst of claim 12, wherein said at least one exogenous nucleic acid further comprises a heterologous encoding nucleic acid.

20. The non-naturally occurring microbial biocatalyst of claims 14, 15 or 17, wherein said exogenous nucleic acid is expressed in sufficient amounts to produce monomeric 4-hydroxybutanoic acid.

21. The non-naturally occurring microbial biocatalyst of claim 12, wherein said BDO biosynthetic pathway comprises aldehyde dehydrogenase, and alcohol dehydrogenase or aldehyde/alcohol dehydrogenase.

22. The non-naturally occurring microbial biocatalyst of claim 21, wherein said BDO biosynthetic pathway further comprises 4-hydroxybutyrate:

CoA transferase or 4-butyrate kinase and phosphotransbutyrylase.

23. The non-naturally occurring microbial biocatalyst of claim 21, wherein said exogenous nucleic acid encodes an aldehyde/alcohol dehydrogenase.

24. The non-naturally occurring microbial biocatalyst of claim 21, wherein said at least one exogenous nucleic acid further comprises two or more exogenous nucleic acids.

25. The non-naturally occurring microbial biocatalyst of claim 21 or 22, wherein said microbial organism lacks an endogenous BDO biosynthetic activity selected from 4-hydroxybutyrate:

CoA transferase, 4-butyrate kinase, phosphotransbutyrylase, aldehyde dehydrogenase, alcohol dehydrogenase and aldehyde/alcohol dehydrogenase.

26. The non-naturally occurring microbial biocatalyst of claim 21, wherein said at least one exogenous nucleic acid further comprises a heterologous encoding nucleic acid.

27. The non-naturally occurring microbial biocatalyst of claim 12, further comprising a substantially anaerobic culture medium.

28. The non-naturally occurring microbial biocatalyst of claim 25, wherein said monomeric BDO is expressed at an intracellular concentration of at least about 5 mM.

29. The non-naturally occurring microbial biocatalyst of claim 28, further comprising an intracellular concentration of said monomeric BDO of about 10 mM or more.

30. A method for the production of 4-HB, comprising culturing a non-naturally occurring microbial organism having a 4-hydroxybutanoic acid (4-HB) biosynthetic pathway comprising at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, or α

-ketoglutarate decarboxylase under substantially anaerobic conditions for a sufficient period of time to produce monomeric 4-hydroxybutanoic acid (4-HB).

31. The method of claim 30, wherein said 4-HB biosynthetic pathway comprises 4-hydroxybutanoate dehydrogenase and succinyl-CoA synthetase and CoA-dependent succinic semialdehyde dehydrogenase or α

-ketoglutarate decarboxylase.

32. The method of claim 30, wherein said exogenous nucleic acid encodes 4-hydroxybutanoate dehydrogenase.

33. The method of claim 32, wherein said non-naturally occurring microbial organism further comprises a nucleic acid encoding an exogenous α

-ketoglutarate decarboxylase.

34. The method of claim 30, wherein said at least one exogenous nucleic acid further comprises two or more exogenous nucleic acids.

35. The method of claim 32, further comprising a nucleic acid encoding an exogenous succinyl-CoA synthetase, exogenous CoA-dependent succinic semialdehyde dehydrogenase or exogenous succinyl-CoA synthetase and exogenous CoA-dependent succinic semialdehyde dehydrogenase.

36. The method of claim 30, wherein said non-naturally occurring microbial organism lacks an endogenous 4-HB biosynthetic activity selected from 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase and α

-ketoglutarate decarboxylase.

37. The method of claim 30, wherein said at least one exogenous nucleic acid further comprises a heterologous encoding nucleic acid.

38. The method of claim 30, wherein said monomeric 4-HB is expressed at an intracellular concentration of at least about 5 mM.

39. The method of claim 38, further comprising an intracellular concentration of said monomeric 4-HB of about 10 mM or more.

40. The method of claim 30, further comprising isolating 4-HB.

41. The method of claim 30, further comprising a culture medium having a pH of about 7.5 or less.

42. The method of claim 41, further comprising isolating γ

-butyrolactone (GBL).

43. The method of claim 42, wherein said isolation of GBL comprises separation of GBL.

44. The method of claim 43, further comprising distillation to produce substantially pure GBL.

45. The method of claim 30 or 44, further comprising chemical hydrogenation of 4-HB, GBL or a mixture thereof to produce 1-4 butanediol (BDO).

46. The method of claim 30 or 44, further comprising chemical hydrogenation of 4-HB, GBL or a mixture thereof to produce tetrahydrofuran (THF).

47. A method for the production of BDO, comprising culturing a non-naturally occurring microbial biocatalyst, comprising a microbial organism having 4-hydroxybutanoic acid (4-HB) and 1,4-butanediol (BDO) biosynthetic pathways, said pathways comprising at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:

CoA transferase, 4-butyrate kinase, phosphotransbutyrylase, α

-ketoglutarate decarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase or an aldehyde/alcohol dehydrogenase for a sufficient period of time to produce 1,4-butanediol (BDO).

48. The method of claim 47, wherein said 4-HB biosynthetic pathway comprises 4-hydroxybutanoate dehydrogenase and succinyl-CoA synthetase and CoA-dependent succinic semialdehyde dehydrogenase or α

-ketoglutarate decarboxylase.

49. The method of claim 47, wherein said exogenous nucleic acid encodes 4-hydroxybutanoate dehydrogenase.

50. The method of claim 49, further comprising a nucleic acid encoding an exogenous α

-ketoglutarate decarboxylase.

51. The method of claim 47, wherein said at least one exogenous nucleic acid further comprises two or more exogenous nucleic acids.

52. The method of claim 49, further comprising a nucleic acid encoding an exogenous succinyl-CoA synthetase, exogenous CoA-dependent succinic semialdehyde dehydrogenase or exogenous succinyl-CoA synthetase and exogenous CoA-dependent succinic semialdehyde dehydrogenase.

53. The method of claim 47, wherein said microbial organism lacks an endogenous 4-HB biosynthetic activity selected from 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase and α

-ketoglutarate decarboxylase.

54. The method of claim 47, wherein said at least one exogenous nucleic acid further comprises a heterologous encoding nucleic acid.

55. The method of claims 49, 50, or 52, wherein said exogenous nucleic acid is expressed in sufficient amounts to produce monomeric 4-hydroxybutanoic acid.

56. The method of claim 47, wherein said BDO biosynthetic pathway comprises aldehyde dehydrogenase and alcohol dehydrogenase or an aldehyde/alcohol dehydrogenase.

57. The method of claim 56, wherein said BDO biosynthetic pathway further comprises 4-hydroxybutyrate:

CoA transferase or 4-butyrate kinase and phosphotransbutyrylase.

58. The method of claim 56, wherein said at least one exogenous nucleic acid further comprises two or more exogenous nucleic acids.

59. The method of claim 56 or 57, wherein said microbial organism lacks an endogenous BDO biosynthetic activity selected from 4-hydroxybutyrate:

CoA transferase, 4-butyrate kinase, phosphotransbutyrylase, aldehyde dehydrogenase, alcohol dehydrogenase and aldehyde/alcohol dehydrogenase.

60. The method of claim 56, wherein said at least one exogenous nucleic acid further comprises a heterologous encoding nucleic acid.

61. The method of claim 47, further comprising a substantially anaerobic culture medium.

62. The method of claim 59, wherein said monomeric BDO is expressed at an intracellular concentration of at least about 5 mM.

63. The method of claim 62, further comprising an intracellular concentration of said monomeric BDO of about 10 mM or more.