Compositions and methods for the biosynthesis of 1,4-butanediol and its precursors

Patent metadata

Field	Value
Publication number	US-8969054-B2
Application number	US-201213717350-A
Country	US
Kind code	B2
Filing date	Dec 17, 2012
Priority date	Mar 16, 2007
Publication date	Mar 3, 2015
Grant date	Mar 3, 2015

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 are methods for the production of 4-HB and BDO.

First claim

What is claimed is:

1. A non-naturally occurring microbial organism having 4-hydroxybutanoic acid (4-HB) and 1,4-butanediol (1,4-BDO) biosynthetic pathways, said pathways comprising exogenous nucleic acids encoding a) an α-ketoglutarate decarboxylase; b) a 4-hydroxybutanoate dehydrogenase; c) a 4-hydroxybutyryl-CoA:acetyl-CoA transferase, or a butyrate kinase and a phosphotransbutyrylase; d) an aldehyde dehydrogenase; and e) an alcohol dehydrogenase, wherein said exogenous nucleic acids are expressed in sufficient amounts to produce 1,4-butanediol (1,4-BDO).

2. The non-naturally occurring microbial organism of claim 1 , wherein said pathways comprise a) an α-ketoglutarate decarboxylase; b) a 4-hydroxybutanoate dehydrogenase; c) a 4-hydroxybutyryl-CoA:acetyl-CoA transferase; d) an aldehyde dehydrogenase; and e) an alcohol dehydrogenase.

3. The non-naturally occurring microbial organism of claim 2 , further comprising a CoA-dependent succinic semialdehyde dehydrogenase.

4. The non-naturally occurring microbial organism of claim 2 , wherein said exogenous nucleic acids comprise at least one heterologous nucleic acid.

5. The non-naturally occurring microbial organism of claim 2 , wherein the microbial organism is in a substantially anaerobic culture medium.

6. The non-naturally occurring microbial organism of claim 1 , wherein said pathways comprise a) an α-ketoglutarate decarboxylase; b) a 4-hydroxybutanoate dehydrogenase; c) a butyrate kinase and a phosphotransbutyrylase; d) an aldehyde dehydrogenase; and e) an alcohol dehydrogenase.

7. The non-naturally occurring microbial organism of claim 6 , further comprising a CoA-dependent succinic semialdehyde dehydrogenase.

8. The non-naturally occurring microbial organism of claim 6 , wherein said exogenous nucleic acids comprise at least one heterologous nucleic acid.

9. The non-naturally occurring microbial organism of claim 6 , wherein the microbial organism is in a substantially anaerobic culture medium.

10. A non-naturally occurring microbial organism having 4-hydroxybutanoic acid (4-HB) and 1,4-butanediol (1,4-BDO) biosynthetic pathways, said pathways comprising exogenous nucleic acids encoding a) an α-ketoglutarate dehydrogenase and a CoA-dependent succinic semialdehyde dehydrogenase; b) a 4-hydroxybutanoate dehydrogenase; c) a 4-hydroxybutyryl-CoA:acetyl-CoA transferase, or a butyrate kinase and a phosphotransbutyrylase; d) an aldehyde dehydrogenase; and e) an alcohol dehydrogenase, wherein said exogenous nucleic acids are expressed in sufficient amounts to produce 1,4-butanediol (1,4-BDO).

11. The non-naturally occurring microbial organism of claim 10 , wherein said pathways comprise a) an α-ketoglutarate dehydrogenase and a CoA-dependent succinic semialdehyde dehydrogenase; b) a 4-hydroxybutanoate dehydrogenase; c) a 4-hydroxybutyryl-CoA:acetyl-CoA transferase; d) an aldehyde dehydrogenase; and e) an alcohol dehydrogenase.

12. The non-naturally occurring microbial organism of claim 11 , wherein said exogenous nucleic acids comprise at least one heterologous nucleic acid.

13. The non-naturally occurring microbial organism of claim 11 , wherein the microbial organism is in a substantially anaerobic culture medium.

14. The non-naturally occurring microbial organism of claim 10 , wherein said pathways comprise a) an α-ketoglutarate dehydrogenase and a CoA-dependent succinic semialdehyde dehydrogenase; b) a 4-hydroxybutanoate dehydrogenase; c) a butyrate kinase and a phosphotransbutyrylase; d) an aldehyde dehydrogenase; and e) an alcohol dehydrogenase.

15. The non-naturally occurring microbial organism of claim 14 , wherein said exogenous nucleic acids comprise at least one heterologous nucleic acid.

16. The non-naturally occurring microbial organism of claim 14 , wherein the microbial organism is in a substantially anaerobic culture medium.

17. A non-naturally occurring microbial organism having 4-hydroxybutanoic acid (4-HB) and 1,4-butanediol (1,4-BDO) biosynthetic pathways, said pathways comprising exogenous nucleic acids encoding a) a glutamate:succinate semialdehyde transaminase and a glutamate decarboxylase; b) a 4-hydroxybutanoate dehydrogenase; c) a 4-hydroxybutyryl-CoA:acetyl-CoA transferase, or a butyrate kinase and a phosphotransbutyrylase; d) an aldehyde dehydrogenase; and e) an alcohol dehydrogenase, wherein said exogenous nucleic acids are expressed in sufficient amounts to produce 1,4-butanediol (1,4-BDO).

18. The microbial organism of claim 17 , wherein said pathways comprise a) a glutamate: succinate semialdehyde transaminase and a glutamate decarboxylase; b) a 4-hydroxybutanoate dehydrogenase; c) a 4-hydroxybutyryl-CoA:acetyl-CoA transferase; d) an aldehyde dehydrogenase; and e) an alcohol dehydrogenase.

19. The non-naturally occurring microbial organism of claim 18 , further comprising a CoA-dependent succinic semialdehyde dehydrogenase.

20. The non-naturally occurring microbial organism of claim 18 , wherein said exogenous nucleic acids comprise at least one heterologous nucleic acid.

21. The non-naturally occurring microbial organism of claim 18 , wherein the microbial organism is in a substantially anaerobic culture medium.

22. The non-naturally occurring microbial organism of claim 17 , wherein said pathways comprise a) a glutamate:succinate semialdehyde transaminase and a glutamate decarboxylase; b) a 4-hydroxybutanoate dehydrogenase; c) a butyrate kinase and a phosphotransbutyrylase; d) an aldehyde dehydrogenase; and e) an alcohol dehydrogenase.

23. The non-naturally occurring microbial organism of claim 22 , further comprising a CoA-dependent succinic semialdehyde dehydrogenase.

24. The non-naturally occurring microbial organism of claim 22 , wherein said exogenous nucleic acids comprise at least one heterologous nucleic acid.

25. The non-naturally occurring microbial organism of claim 22 , wherein the microbial organism is in a substantially anaerobic culture medium.

26. A method for the production of 1,4-BDO comprising, culturing the non-naturally occurring microbial organism of claim 1 under conditions and for a sufficient period of time to produce 1,4-BDO.

27. The method of claim 26 , wherein said pathways comprise a) an α-ketoglutarate decarboxylase; b) a 4-hydroxybutanoate dehydrogenase; c) a 4-hydroxybutyryl-CoA:acetyl-CoA transferase; d) an aldehyde dehydrogenase; and e) an alcohol dehydrogenase.

28. The method of claim 27 , wherein the microbial organism further comprises a CoA-dependent succinic semialdehyde dehydrogenase.

29. The method of claim 27 , wherein said exogenous nucleic acids comprise at least one heterologous nucleic acid.

30. The method of claim 27 , wherein the microbial organism is in a substantially anaerobic culture medium.

31. The method of claim 26 , wherein said pathways comprise a) an α-ketoglutarate decarboxylase; b) a 4-hydroxybutanoate dehydrogenase; c) a butyrate kinase and a phosphotransbutyrylase; d) an aldehyde dehydrogenase; and e) an alcohol dehydrogenase.

32. The method of claim 31 , wherein the microbial organism further comprises a CoA-dependent succinic semialdehyde dehydrogenase.

33. The method of claim 31 , wherein said exogenous nucleic acids comprise at least one heterologous nucleic acid.

34. The method of claim 31 , wherein the microb

Assignees

• Genomatica Inc

Inventors

• Burk Mark J
• Van Dien Stephen J
• Burgard Anthony P
• Niu Wei

Classifications

• C12Y602/01004

  Succinate-CoA ligase (GDP-forming) (6.2.1.4) · CPC title

• C12P7/42 Primary

  Hydroxy-carboxylic acids · CPC title

• C12N15/52 Primary

  Genes encoding for enzymes or proenzymes · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N9/88

  Lyases (4.) · CPC title