Compositions and methods for the biosynthesis of 1,4-butanediol and its precursors
View Patent ↗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.
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 , 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.
38. The method of claim 37 , wherein said exogenous nucleic acids comprise at least one heterologous nucleic acid.
39. The method of claim 23 , 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.
40. The method of claim 39 , wherein said exogenous nucleic acids comprise at least one heterologous nucleic acid.
41. 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 butyrate kinase and a phosphotransbutyrylase; d) an aldehyde dehydrogenase; and e) an alcohol dehydrogenase.
42. The method of claim 41 , wherein said exogenous nucleic acids comprise at least one heterologous nucleic acid.
43. The method of claim 23 , 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.
44. The method of claim 43 , wherein said exogenous nucleic acids comprise at least one heterologous nucleic acid.
45. The method of any one of claims 23 or 33 to 44 , further comprising the step of isolating the 1,4-BDO.
46. The method of claim 45 , wherein the 1,4-BDO is isolated by distillation.