MICROORGANISMS AND METHODS FOR THE COPRODUCTION 1,4-BUTANEDIOL AND GAMMA-BUTYROLACTONE

• US 20110217742A1
• Filed: 11/04/2010
• Published: 09/08/2011
• Est. Priority Date: 11/25/2009
• Status: Active Grant

• Current Assignee: Genomatica Incorporated
• Original Assignee: Genomatica Incorporated
• Inventors: Osterhout, Robin E., Sun, Jun, Burgard, Anthony P., Burk, Mark J., Trawick, John D., Niu, Wei, Haselbeck, Robert
• Granted Patent: US 8,530,210 B2
• Time in Patent Office: Days
• US Class Current: 435/126
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12P 17/04 containing a five-membered hetero ring, e.g. griseofulvin , vitamin C
• CPC Class Codes: C12P 7/18 polyhydric

Abstract

The invention provides non-naturally occurring microbial organisms comprising 1,4-butanediol (14-BDO) and gamma-butyrolactone (GBL) pathways comprising at least one exogenous nucleic acid encoding a 14-BDO and GBL pathway enzyme expressed in a sufficient amount to produce 14-BDO and GBL. The invention additionally provides methods of using such microbial organisms to produce 14-BDO and GBL.

Claims

1. A method for producing 1,4-butanediol (14-BDO) and gamma-butyrolactone (GBL), comprising culturing a non-naturally occurring microbial organism under conditions and for a sufficient period of time to produce 14-BDO and 4-hydroxybutyryl-CoA followed by spontaneous lactonization of 4-hydroxybutyryl-CoA to form GBL, wherein said non-naturally occurring microbial organism comprise a microbial organism having a 14-BDO pathway and a 4-hydroxybutyryl-CoA pathway, said 14-BDO pathway comprising at least one exogenous nucleic acid encoding a 14-BDO pathway enzyme expressed in a sufficient amount to produce 14-BDO, said 14-BDO pathway comprising:

a 4-hydroxybutyryl-CoA reductase (alcohol forming), a succinyl-CoA reductase (aldehyde forming), an alpha-ketoglutarate decarboxylase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate reductase, a 1,4-butanediol dehydrogenase, a succinate reductase, a succinyl-CoA transferase, a succinyl-CoA hydrolase, a succinyl-CoA synthetase, a glutamate dehydrogenase, a glutamate transaminase, a glutamate decarboxylase, an 4-aminobutyrate dehydrogenase, an 4-aminobutyrate transaminase, a 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA reductase (aldehyde forming), a 4-hydroxybutyryl-phosphate reductase, a succinyl-CoA reductase (alcohol forming), a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase, an alpha-ketoglutarate reductase, a 5-hydroxy-2-oxopentanoate dehydrogenase, a 5-hydroxy-2-oxopentanoate decarboxylase or a 5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation),

said 4-hydroxybutyryl-CoA pathway comprising at least one exogenous nucleic acid encoding an 4-hydroxybutyryl-CoA pathway enzyme expressed in a sufficient amount to produce 4-hydroxybutyryl-CoA, said 4-hydroxybutyryl-CoA pathway comprising;

a 4-hydroxybutyryl-CoA transferase, a succinyl-CoA reductase (aldehyde forming), an alpha-ketoglutarate decarboxylase, a 4-hydroxybutyrate dehydrogenase, a succinate reductase, a succinyl-CoA transferase, a succinyl-CoA hydrolase, a succinyl-CoA synthetase, a glutamate dehydrogenase, a glutamate transaminase, a glutamate decarboxylase, a 4-aminobutyrate dehydrogenase, a 4-aminobutyrate transaminase, a 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a succinyl-CoA reductase (alcohol forming), a 4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase, an alpha-ketoglutarate reductase, a 5-hydroxy-2-oxopentanoate dehydrogenase, or a 5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation).

2. The method of claim 1, wherein the 14-BDO pathway comprises a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate reductase; and

a 1,4-butanediol dehydrogenase.

3. The method of claim 1, wherein the 14-BDO pathway comprises a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase.

4. The method of claim 1, wherein the 14-BDO pathway comprises a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a 4-hydroxybutyryl-phosphate reductase; and

a 1,4-butanediol dehydrogenase.

5. The method of claim 1, wherein the 14-BDO pathway comprises a succinyl-CoA transferase, a succinyl-CoA hydrolase, or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase.

6. The method of claim 1, wherein the 14-BDO pathway comprises a succinyl-CoA transferase, a succinyl-CoA hydrolase or succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and

a 4-hydroxybutyryl-CoA reductase (alcohol forming).

7. The method of claim 1, wherein the 14-BDO pathway comprises a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase; and

a 4-hydroxybutyryl-CoA reductase (alcohol forming).

20. The method of claim 1, wherein the 14-BDO pathway comprises an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate reductase; and

1,4-butanediol dehydrogenase.

21. The method of claim 1, wherein the 14-BDO pathway comprises an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase.

22. The method of claim 1, wherein the 14-BDO pathway comprises an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase and 4-hydroxybutyryl-CoA reductase (alcohol forming).

23. The method of claim 1, wherein the 14-BDO pathway comprises an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a 4-hydroxybutyryl-phosphate reductase; and

a 1,4-butanediol dehydrogenase.

24. The method of claim 1, wherein the 14-BDO pathway comprises an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or 4-hydroxybutyryl-CoA synthetase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase.

25. The method of claim 1, wherein the 14-BDO pathway comprises an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and

4-hydroxybutyryl-CoA reductase (alcohol forming).

35. The method of claim 1, wherein the 4-hydroxybutyryl-CoA pathway comprises a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase; and

a phosphotrans-4-hydroxybutyrylase.

36. The method of claim 1, wherein the 4-hydroxybutyryl-CoA pathway comprises a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase; and

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase.

41. The method of claim 1, wherein the 4-hydroxybutyryl-CoA pathway comprises an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase; and

a phosphotrans-4-hydroxybutyrylase.

42. The method of claim 1, wherein the 4-hydroxybutyryl-CoA pathway comprises an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase; and

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase.

47. The method of claim 1, wherein said conditions comprise substantially anaerobic culture conditions.

48. The method of claim 1, wherein said exogenous nucleic acid is a heterologous nucleic acid.

49. The method of claim 1, wherein said 14-BDO and GBL are produced at a predetermined ratio.

50. The method of claim 49, wherein said predetermined ratio of 14-BDO to GBL is selected from the group consisting of 1:

1, 6;

1 and 7;

1.

8-19. -19. (canceled)

26-34. -34. (canceled)

37-40. -40. (canceled)

43-45. -45. (canceled)

46. A method for producing 1,4-butanediol (14-BDO) and gamma-butyrolactone (GBL), comprising culturing a non-naturally occurring microbial organism under conditions and for a sufficient period of time to produce 14-BDO and 4-hydroxybutyryl-CoA followed by spontaneous lactonization of 4-hydroxybutyryl-CoA to form GBL, wherein said non-naturally occurring microbial organism comprise a microbial organism having a 14-BDO pathway and a 4-hydroxybutyryl-CoA pathway, said 14-BDO pathway comprising a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, said first set is selected from the group consisting of:

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate reductase; and

a 1,4-butanediol dehydrogenase)];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a 4-hydroxybutyryl-phosphate reductase; and

a 1,4-butanediol dehydrogenase];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase, or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and

a 4-hydroxybutyryl-CoA reductase (alcohol forming)];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase; and

a 4-hydroxybutyryl-CoA reductase (alcohol forming)];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (alcohol forming);

a 4-hydroxybutyrate reductase; and

1,4-butanediol dehydrogenase];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (alcohol forming);

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or succinyl-CoA synthetase;

a succinyl-CoA reductase (alcohol forming), a 4-hydroxybutyrate kinase, a 4-hydroxybutyryl-phosphate reductase; and

a 1,4-butanediol dehydrogenase];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (alcohol forming);

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (alcohol forming);

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and

a 4-hydroxybutyryl-CoA reductase (alcohol forming)];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (alcohol forming);

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase; and

a 4-hydroxybutyryl-CoA reductase (alcohol forming)];

[a succinate reductase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate reductase; and

1,4-butanediol dehydrogenase];

[a succinate reductase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase];

[a succinate reductase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase; and

a 4-hydroxybutyryl-CoA reductase (alcohol forming)];

[a succinate reductase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a 4-hydroxybutyryl-phosphate reductase; and

1,4-butanediol dehydrogenase];

[a succinate reductase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

1,4-butanediol dehydrogenase];

[a succinate reductase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and

a 4-hydroxybutyryl-CoA reductase (alcohol forming)];

[an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate reductase; and

1,4-butanediol dehydrogenase];

[an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase];

[an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase and 4-hydroxybutyryl-CoA reductase (alcohol forming)];

[an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a 4-hydroxybutyryl-phosphate reductase; and

a 1,4-butanediol dehydrogenase];

[an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or 4-hydroxybutyryl-CoA synthetase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase];

[an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and

4-hydroxybutyryl-CoA reductase (alcohol forming)];

[a glutamate dehydrogenase or a glutamate transaminase;

a glutamate decarboxylase;

a 4-aminobutyrate dehydrogenase or 4-aminobutyrate transaminase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate reductase; and

a 1,4-butanediol dehydrogenase];

[a glutamate dehydrogenase or a glutamate transaminase;

a glutamate decarboxylase;

a 4-aminobutyrate dehydrogenase or a 4-aminobutyrate transaminase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase];

[a glutamate dehydrogenase or glutamate transaminase;

a glutamate decarboxylase;

a 4-aminobutyrate dehydrogenase or a 4-aminobutyrate transaminase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase; and

a 4-hydroxybutyryl-CoA reductase (alcohol forming)];

[a glutamate dehydrogenase or a glutamate transaminase;

a glutamate decarboxylase;

a 4-aminobutyrate dehydrogenase or a 4-aminobutyrate transaminase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a 4-hydroxybutyryl-phosphate reductase; and

a 1,4-butanediol dehydrogenase];

[a glutamate dehydrogenase or a glutamate transaminase;

a glutamate decarboxylase;

a 4-aminobutyrate dehydrogenase or a 4-aminobutyrate transaminase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase];

[a glutamate dehydrogenase or a glutamate transaminase;

a glutamate decarboxylase;

a 4-aminobutyrate dehydrogenase or a 4-aminobutyrate transaminase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and

a 4-hydroxybutyryl-CoA reductase (alcohol forming)];

[an alpha-ketoglutarate reductase;

a 5-hydroxy-2-oxopentanoate dehydrogenase;

a 5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation);

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase];

[an alpha-ketoglutarate reductase;

a 5-hydroxy-2-oxopentanoate dehydrogenase;

a 5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation); and

a 4-hydroxybutyryl-CoA reductase (alcohol forming)]; and

[an alpha-ketoglutarate reductase;

a 5-hydroxy-2-oxopentanoate dehydrogenase;

a 5-hydroxy-2-oxopentanoate decarboxylase; and

a 1,4-butanediol dehydrogenase],

said 4-hydroxybutyryl-CoA pathway comprising a second set of exogenous nucleic acids encoding 4-hydroxybutyryl-CoA pathway enzymes expressed in a sufficient amount to produce 4-hydroxybutyryl-CoA, said second set selected from the group consisting of;

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase; and

a phosphotrans-4-hydroxybutyrylase];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase; and

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (alcohol forming);

a 4-hydroxybutyrate kinase; and

a phosphotrans-4-hydroxybutyrylase];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (alcohol forming); and

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase];

[a succinate reductase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase and a phosphotrans-4-hydroxybutyrylase];

[a succinate reductase;

a 4-hydroxybutyrate dehydrogenase; and

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase];

[an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase; and

a phosphotrans-4-hydroxybutyrylase];

[an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase; and

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase];

[a glutamate dehydrogenase or a glutamate transaminase;

a glutamate decarboxylase;

an 4-aminobutyrate dehydrogenase or an 4-aminobutyrate transaminase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase; and

a phosphotrans-4-hydroxybutyrylase];

[a glutamate dehydrogenase or a glutamate transaminase;

a glutamate decarboxylase;

an 4-aminobutyrate dehydrogenase or an 4-aminobutyrate transaminase;

a 4-hydroxybutyrate dehydrogenase; and

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase]; and

an alpha-ketoglutarate reductase;

a 5-hydroxy-2-oxopentanoate dehydrogenase and a 5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation)].

51-58. -58. (canceled)

59. A method for producing 1,4-butanediol (14-BDO) and gamma-butyrolactone (GBL), comprising culturing a non-naturally occurring microbial organism under conditions and for a sufficient period of time to produce 14-BDO and 4-hydroxybutyryl-phosphate followed by spontaneous lactonization of 4-hydroxybutyryl-phosphate to form GBL, wherein said non-naturally occurring microbial organism comprise a microbial organism having a 14-BDO pathway and a 4-hydroxybutyryl-phosphate pathway, said 14-BDO pathway comprising at least one exogenous nucleic acid encoding a 14-BDO pathway enzyme expressed in a sufficient amount to produce 14-BDO, said 14-BDO pathway comprising:

a 4-hydroxybutyryl-CoA reductase (alcohol forming), a succinyl-CoA reductase (aldehyde forming), an alpha-ketoglutarate decarboxylase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate reductase, a 1,4-butanediol dehydrogenase, a succinate reductase, a succinyl-CoA transferase, a succinyl-CoA hydrolase, a succinyl-CoA synthetase, a glutamate dehydrogenase, a glutamate transaminase, a glutamate decarboxylase, an 4-aminobutyrate dehydrogenase, an 4-aminobutyrate transaminase, a 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA reductase (aldehyde forming), a 4-hydroxybutyryl-phosphate reductase, a succinyl-CoA reductase (alcohol forming), a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase, an alpha-ketoglutarate reductase, a 5-hydroxy-2-oxopentanoate dehydrogenase, a 5-hydroxy-2-oxopentanoate decarboxylase or a 5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation),

said 4-hydroxybutyryl-phosphate pathway comprising at least one exogenous nucleic acid encoding an 4-hydroxybutyryl-phosphate pathway enzyme expressed in a sufficient amount to produce 4-hydroxybutyryl-phosphate, said 4-hydroxybutyryl-phosphate pathway comprising;

a 4-hydroxybutyrate kinase, a succinyl-CoA reductase (aldehyde forming), an alpha-ketoglutarate decarboxylase, a 4-hydroxybutyrate dehydrogenase, a succinate reductase, a succinyl-CoA transferase, a succinyl-CoA hydrolase, a succinyl-CoA synthetase, a glutamate dehydrogenase, a glutamate transaminase, a glutamate decarboxylase, a 4-aminobutyrate dehydrogenase, a 4-aminobutyrate transaminase or a succinyl-CoA reductase (alcohol forming).

60. The method of claim 59, wherein the 14-BDO pathway comprises a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate reductase; and

a 1,4-butanediol dehydrogenase.

61. The method of claim 59, wherein the 14-BDO pathway comprises a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase.

62. The method of claim 59, wherein the 14-BDO pathway comprises a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a 4-hydroxybutyryl-phosphate reductase; and

a 1,4-butanediol dehydrogenase.

63. The method of claim 59, wherein the 14-BDO pathway comprises a succinyl-CoA transferase, a succinyl-CoA hydrolase, or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase.

64. The method of claim 59, wherein the 14-BDO pathway comprises a succinyl-CoA transferase, a succinyl-CoA hydrolase or succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and

a 4-hydroxybutyryl-CoA reductase (alcohol forming).

65. The method of claim 59, wherein the 14-BDO pathway comprises a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase; and

a 4-hydroxybutyryl-CoA reductase (alcohol forming).

78. The method of claim 59, wherein the 14-BDO pathway comprises an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate reductase; and

1,4-butanediol dehydrogenase.

79. The method of claim 59, wherein the 14-BDO pathway comprises an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase.

80. The method of claim 59, wherein the 14-BDO pathway comprises an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase and 4-hydroxybutyryl-CoA reductase (alcohol forming).

81. The method of claim 59, wherein the 14-BDO pathway comprises an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a 4-hydroxybutyryl-phosphate reductase; and

a 1,4-butanediol dehydrogenase.

82. The method of claim 59, wherein the 14-BDO pathway comprises an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or 4-hydroxybutyryl-CoA synthetase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase.

83. The method of claim 59, wherein the 14-BDO pathway comprises an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and

4-hydroxybutyryl-CoA reductase (alcohol forming).

93. The method of claim 59, wherein the 4-hydroxybutyryl-phosphate pathway comprises a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase; and

4-hydroxybutyrate kinase.

96. The method of claim 59, wherein the 4-hydroxybutyryl-phosphate pathway comprises an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase; and

a 4-hydroxybutyrate kinase.

99. The method of claim 59, wherein said conditions comprise substantially anaerobic culture conditions.

100. The method of claim 59, wherein said exogenous nucleic acid is a heterologous nucleic acid.

101. The method of claim 59, wherein said 14-BDO and GBL are produced at a predetermined ratio.

102. The method of claim 101, wherein said predetermined ratio of 14-BDO to GBL is selected from the group consisting of 1:

1, 6;

1 and 7;

1.

66-77. -77. (canceled)

84-92. -92. (canceled)

94-95. -95. (canceled)

97. (canceled)

98. A method for producing 1,4-butanediol (14-BDO) and gamma-butyrolactone (GBL), comprising culturing a non-naturally occurring microbial organism under conditions and for a sufficient period of time to produce 14-BDO and 4-hydroxybutyryl-phosphate followed by spontaneous lactonization of 4-hydroxybutyryl-phosphate to form GBL, wherein said non-naturally occurring microbial organism comprise a microbial organism having a 14-BDO pathway and a 4-hydroxybutyryl-phosphate pathway, said 14-BDO pathway comprising a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, said first set is selected from the group consisting of:

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate reductase; and

a 1,4-butanediol dehydrogenase)];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a 4-hydroxybutyryl-phosphate reductase; and

a 1,4-butanediol dehydrogenase];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase, or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and

a 4-hydroxybutyryl-CoA reductase (alcohol forming)];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase; and

a 4-hydroxybutyryl-CoA reductase (alcohol forming)];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (alcohol forming);

a 4-hydroxybutyrate reductase; and

1,4-butanediol dehydrogenase];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (alcohol forming);

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or succinyl-CoA synthetase;

a succinyl-CoA reductase (alcohol forming), a 4-hydroxybutyrate kinase, a 4-hydroxybutyryl-phosphate reductase; and

a 1,4-butanediol dehydrogenase];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (alcohol forming);

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (alcohol forming);

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and

a 4-hydroxybutyryl-CoA reductase (alcohol forming)];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (alcohol forming);

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase; and

a 4-hydroxybutyryl-CoA reductase (alcohol forming)];

[a succinate reductase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate reductase; and

1,4-butanediol dehydrogenase];

[a succinate reductase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase];

[a succinate reductase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase; and

a 4-hydroxybutyryl-CoA reductase (alcohol forming)];

[a succinate reductase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a 4-hydroxybutyryl-phosphate reductase; and

1,4-butanediol dehydrogenase];

[a succinate reductase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

1,4-butanediol dehydrogenase];

[a succinate reductase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and

a 4-hydroxybutyryl-CoA reductase (alcohol forming)];

[an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate reductase; and

1,4-butanediol dehydrogenase];

[an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase];

[an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase and 4-hydroxybutyryl-CoA reductase (alcohol forming)];

[an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a 4-hydroxybutyryl-phosphate reductase; and

a 1,4-butanediol dehydrogenase];

[an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or 4-hydroxybutyryl-CoA synthetase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase];

[an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and

4-hydroxybutyryl-CoA reductase (alcohol forming)];

[a glutamate dehydrogenase or a glutamate transaminase;

a glutamate decarboxylase;

a 4-aminobutyrate dehydrogenase or 4-aminobutyrate transaminase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate reductase; and

a 1,4-butanediol dehydrogenase];

[a glutamate dehydrogenase or a glutamate transaminase;

a glutamate decarboxylase;

a 4-aminobutyrate dehydrogenase or a 4-aminobutyrate transaminase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase];

[a glutamate dehydrogenase or glutamate transaminase;

a glutamate decarboxylase;

a 4-aminobutyrate dehydrogenase or a 4-aminobutyrate transaminase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a phosphotrans-4-hydroxybutyrylase; and

a 4-hydroxybutyryl-CoA reductase (alcohol forming)];

[a glutamate dehydrogenase or a glutamate transaminase;

a glutamate decarboxylase;

a 4-aminobutyrate dehydrogenase or a 4-aminobutyrate transaminase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyrate kinase;

a 4-hydroxybutyryl-phosphate reductase; and

a 1,4-butanediol dehydrogenase];

[a glutamate dehydrogenase or a glutamate transaminase;

a glutamate decarboxylase;

a 4-aminobutyrate dehydrogenase or a 4-aminobutyrate transaminase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase;

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase];

[a glutamate dehydrogenase or a glutamate transaminase;

a glutamate decarboxylase;

a 4-aminobutyrate dehydrogenase or a 4-aminobutyrate transaminase;

a 4-hydroxybutyrate dehydrogenase;

a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and

a 4-hydroxybutyryl-CoA reductase (alcohol forming)];

[an alpha-ketoglutarate reductase;

a 5-hydroxy-2-oxopentanoate dehydrogenase;

a 5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation);

a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and

a 1,4-butanediol dehydrogenase];

[an alpha-ketoglutarate reductase;

a 5-hydroxy-2-oxopentanoate dehydrogenase;

a 5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation); and

a 4-hydroxybutyryl-CoA reductase (alcohol forming)]; and

[an alpha-ketoglutarate reductase;

a 5-hydroxy-2-oxopentanoate dehydrogenase;

a 5-hydroxy-2-oxopentanoate decarboxylase; and

a 1,4-butanediol dehydrogenase],

said 4-hydroxybutyryl-phosphate pathway comprising a second set of exogenous nucleic acids encoding 4-hydroxybutyryl-phosphate pathway enzymes expressed in a sufficient amount to produce 4-hydroxybutyryl-phosphate, said second set selected from the group consisting of;

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (aldehyde forming);

a 4-hydroxybutyrate dehydrogenase; and

4-hydroxybutyrate kinase];

[a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;

a succinyl-CoA reductase (alcohol forming); and

a 4-hydroxybutyrate kinase];

[a succinate reductase;

a 4-hydroxybutyrate dehydrogenase; and

a 4-hydroxybutyrate kinase];

[an alpha-ketoglutarate decarboxylase;

a 4-hydroxybutyrate dehydrogenase; and

a 4-hydroxybutyrate kinase]; and

[a glutamate dehydrogenase or a glutamate transaminase;

a glutamate decarboxylase;

an 4-aminobutyrate dehydrogenase or an 4-aminobutyrate transaminase;

a 4-hydroxybutyrate dehydrogenase; and

a 4-hydroxybutyrate kinase].

103-157. -157. (canceled)