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

Patent metadata

Field	Value
Publication number	US-9487803-B2
Application number	US-201514603174-A
Country	US
Kind code	B2
Filing date	Jan 22, 2015
Priority date	Mar 16, 2007
Publication date	Nov 8, 2016
Grant date	Nov 8, 2016

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 Escherichia coli 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, or an α-ketoglutarate dehydrogenase and a CoA-dependent succinic semialdehyde dehydrogenase, or 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).

2. The non-naturally occurring microbial organism of claim 1 , wherein said exogenous nucleic acids comprise at least one heterologous nucleic acid.

3. The non-naturally occurring microbial organism of claim 1 , wherein the microbial organism is in a substantially anaerobic culture medium.

4. The non-naturally occurring microbial organism of claim 1 , comprising the α-ketoglutarate decarboxylase.

5. The non-naturally occurring microbial organism of claim 1 , comprising the α-ketoglutarate dehydrogenase and the CoA-dependent succinic semialdehyde dehydrogenase.

6. The non-naturally occurring microbial organism of claim 1 , comprising the glutamate:succinate semialdehyde transaminase and the glutamate decarboxylase.

7. The non-naturally occurring microbial organism of claim 1 , comprising the 4-hydroxybutyryl-CoA:acetyl-CoA transferase.

8. The non-naturally occurring microbial organism of claim 1 , comprising the butyrate kinase and the phosphotransbutyrylase.

9. The non-naturally occurring microbial organism of claim 4 , wherein the microbial organism comprises the α-ketoglutarate decarboxylase and a CoA-dependent succinic semialdehyde dehydrogenase.

10. The non-naturally occurring microbial organism of claim 6 , wherein the microbial organism comprises the glutamate:succinate semialdehyde transaminase, the glutamate decarboxylase and a CoA-dependent succinic semialdehyde dehydrogenase.

11. 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.

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 1 , 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.

14. The non-naturally occurring microbial organism of claim 13 , wherein said exogenous nucleic acids comprise at least one heterologous nucleic acid.

15. The non-naturally occurring microbial organism of claim 1 , 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.

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

17. 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.

18. The non-naturally occurring microbial organism of claim 17 , wherein said exogenous nucleic acids comprise at least one heterologous nucleic acid.

19. The non-naturally occurring microbial organism of claim 1 , 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.

20. The non-naturally occurring microbial organism of claim 19 , wherein said exogenous nucleic acids comprise at least one heterologous nucleic acid.

21. The non-naturally occurring microbial organism of claim 1 , 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.

22. The non-naturally occurring microbial organism of claim 21 , wherein said exogenous nucleic acids comprise at least one heterologous nucleic acid.

23. 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.

24. The method of claim 23 , wherein at least one exogenous nucleic acid comprises a heterologous nucleic acid.

25. The method of claim 23 , wherein the microbial organism is in a substantially anaerobic culture medium.

26. The method of claim 23 , wherein the microbial organism comprises the α-ketoglutarate decarboxylase.

27. The method of claim 23 , wherein the microbial organism comprises the α-ketoglutarate dehydrogenase and the CoA-dependent succinic semialdehyde dehydrogenase.

28. The method of claim 23 , wherein the microbial organism comprises the glutamate:succinate semialdehyde transaminase and the glutamate decarboxylase.

29. The method of claim 23 , wherein the microbial organism comprises the 4-hydroxybutyryl-CoA:acetyl-CoA transferase.

30. The method of claim 23 , wherein the microbial organism comprises the butyrate kinase and the phosphotransbutyrylase.

31. The method of claim 26 , wherein the microbial organism comprises the α-ketoglutarate and a CoA-dependent succinic semialdehyde dehydrogenase.

32. The method of claim 28 , wherein the microbial organism comprises the glutamate:succinate semialdehyde transaminase, the glutamate decarboxylase and a CoA-dependent succinic semialdehyde dehydrogenase.

33. The method of claim 23 , 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.

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

35. The method of claim 23 , 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.

36. The method of claim 35 , wherein said exogenous nucleic acids comprise at least one heterologous nucleic acid.

37. The method of claim 23 , wh

Assignees

• Genomatica Inc

Inventors

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

Classifications

• B01D3/002

  by continuous methods · CPC title

• C12Y401/01071

  2-Oxoglutarate decarboxylase (4.1.1.71) · CPC title

• C12P7/42

  Hydroxy-carboxylic acids · CPC title

• C12N9/0006 Primary

  acting on CH-OH groups as donors (1.1) · CPC title

• C12N9/88

  Lyases (4.) · CPC title