Microorganisms for the production of 1,4-butanediol and related methods

1. A non-naturally occurring Escherichia coli , comprising a 1,4-butanediol (BDO) pathway comprising one or more heterologous polynucleotides encoding BDO pathway enzymes expressed in a sufficient amount to produce BDO, wherein said E. coli:

(A) comprises a BDO pathway selected from the group consisting of:

(i) a BDO pathway comprising:

(a) alpha-ketoglutarate decarboxylase; or

alpha-ketoglutarate dehydrogenase and CoA-dependent succinate semialdehyde dehydrogenase; or

glutamate: succinate semialdehyde transaminase and glutamate decarboxylase;

(b) 4-hydroxybutyrate dehydrogenase;

(c) 4-hydroxybutyryl-CoA transferase; or

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

(d) 4-hydroxybutyryl-CoA reductase and 4-hydroxybutyraldehyde reductase; or

aldehyde/alcohol dehydrogenase, said aldehyde/alcohol dehydrogenase converting 4-hydroxybutyryl-CoA to 1,4-butanediol;

(ii) a BDO pathway comprising:

(a) alpha-ketoglutarate decarboxylase; or

succinyl-CoA synthetase and CoA-dependent succinate semialdehyde dehydrogenase;

(b) 4-hydroxybutyrate dehydrogenase;

(c) 4-hydroxybutyryl-CoA transferase; or

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

(d) aldehyde dehydrogenase, said aldehyde dehydrogenase converting 4-hydroxybutyryl-CoA to 4-hydroxybutyraldehyde; and alcohol dehydrogenase, said alcohol dehydrogenase converting 4-hydroxybutyraldehyde to 1,4-butanediol; or

aldehyde/alcohol dehydrogenase, said aldehyde/alcohol dehydrogenase converting 4-hydroxybutyryl-CoA to 1,4-butanediol;

(iii) a BDO pathway comprising:

(a) alpha-ketoglutarate decarboxylase; or

glutamate dehydrogenase, glutamate decarboxylase, and deaminating 4-aminobutyrate oxidoreductase or 4-aminobutyrate transaminase; or

alpha-ketoglutarate dehydrogenase and CoA-dependent succinate semialdehyde dehydrogenase;

(b) 4-hydroxybutyrate dehydrogenase; and

(c) 4-hydroxybutyrate kinase, phosphotrans-4-hydroxybutyrylase, 4-hydroxybutyryl-CoA reductase, and 4-hydroxybutyraldehyde reductase; or

4-hydroxybutyrate kinase, phosphorylating 4-hydroxybutanal dehydrogenase, and 4-hydroxybutyraldehyde reductase; or

4-hydroxybutyrate kinase, phosphotrans-4-hydroxybutyrylase, and alcohol forming 4-hydroxybutyryl-CoA reductase; or

4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA hydrolase or 4-hydroxybutyryl-CoA ligase, 4-hydroxybutyryl-CoA reductase, and 4-hydroxybutyraldehyde reductase; or

4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA hydrolase or 4-hydroxybutyryl-CoA ligase, and alcohol forming 4-hydroxybutyryl-CoA reductase; and

(iv) a BDO pathway comprising:

(a) glutamate CoA transferase or glutamyl-CoA hydrolase or glutamyl-CoA ligase; glutamyl-CoA reductase, and glutamate-5-semialdehyde reductase; or

glutamate CoA transferase or glutamyl-CoA hydrolase or glutamyl-CoA ligase, and alcohol forming glutamyl-CoA reductase; or

glutamate 5-kinase, phosphorylating glutamate-5-semialdehyde dehydrogenase, and glutamate-5-semialdehyde reductase;

(b) deaminating 2-amino-5-hydroxypentanoic acid oxidoreductase or 2-amino-5-hydroxypentanoic acid transaminase; and

(c) 5-hydroxy-2-oxopentanoic acid decarboxylase, and 4-hydroxybutyraldehyde reductase; or

decarboxylating 5-hydroxy-2-oxopentanoic acid dehydrogenase, 4-hydroxybutyryl-CoA reductase, and 4-hydroxybutyraldehyde reductase; or

decarboxylating 5-hydroxy-2-oxopentanoic acid dehydrogenase and alcohol forming 4-hydroxybutyryl-CoA reductase; and

(B) comprises disruption of a gene encoding a protein in an aerobic respiratory control regulatory system; or expresses an exogenous NADH insensitive citrate synthase.

2. The non-naturally occurring E. coli of claim 1 , wherein the BDO pathway comprises:

(a) alpha-ketoglutarate decarboxylase;

(b) 4-hydroxybutyrate dehydrogenase;

(c) 4-hydroxybutyryl-CoA transferase; and

(d) 4-hydroxybutyryl-CoA reductase and 4-hydroxybutyraldehyde reductase.

3. The non-naturally occurring E. coli of claim 1 , wherein the BDO pathway comprises:

(a) alpha-ketoglutarate dehydrogenase and CoA-dependent succinate semialdehyde dehydrogenase;

(b) 4-hydroxybutyrate dehydrogenase;

(c) 4-hydroxybutyryl-CoA transferase; and

(d) 4-hydroxybutyryl-CoA reductase and 4-hydroxybutyraldehyde reductase.

4. The non-naturally occurring E. coli of claim 1 , wherein the BDO pathway comprises:

(a) glutamate:succinate semialdehyde transaminase and glutamate decarboxylase;

(b) 4-hydroxybutyrate dehydrogenase;

(c) 4-hydroxybutyryl-CoA transferase; and

(d) 4-hydroxybutyryl-CoA reductase and 4-hydroxybutyraldehyde reductase.

5. The non-naturally occurring E. coli of claim 1 , wherein the BDO pathway comprises:

(a) alpha-ketoglutarate decarboxylase;

(b) 4-hydroxybutyrate dehydrogenase;

(c) 4-hydroxybutyrate kinase and phosphotrans-4-hydroxybutyrylase;

(d) 4-hydroxybutyryl-CoA reductase and 4-hydroxybutyraldehyde reductase.

6. The non-naturally occurring E. coli of claim 1 , wherein the BDO pathway comprises:

(a) alpha-ketoglutarate dehydrogenase and CoA-dependent succinate semialdehyde dehydrogenase;

(b) 4-hydroxybutyrate dehydrogenase;

(c) 4-hydroxybutyrate kinase and phosphotrans-4-hydroxybutyrylase; and

(d) 4-hydroxybutyryl-CoA reductase and 4-hydroxybutyraldehyde reductase.

7. The non-naturally occurring E. coli of claim 1 , wherein the BDO pathway comprises:

(a) glutamate:succinate semialdehyde transaminase and glutamate decarboxylase;

(b) 4-hydroxybutyrate dehydrogenase;

(c) 4-hydroxybutyrate kinase and phosphotrans-4-hydroxybutyrylase; and

(d) 4-hydroxybutyryl-CoA reductase and 4-hydroxybutyraldehyde reductase.

8. The non-naturally occurring E. coli of claim 1 , wherein the BDO pathway comprises:

(a) alpha-ketoglutarate decarboxylase;

(b) 4-hydroxybutyrate dehydrogenase;

(c) 4-hydroxybutyryl-CoA transferase; and

(d) aldehyde/alcohol dehydrogenase, said aldehyde/alcohol dehydrogenase converting 4-hydroxybutyryl-CoA to 1,4-butanediol.

9. The non-naturally occurring E. coli of claim 1 , wherein the BDO pathway comprises:

(a) alpha-ketoglutarate dehydrogenase and CoA-dependent succinate semialdehyde dehydrogenase;

(b) 4-hydroxybutyrate dehydrogenase;

(c) 4-hydroxybutyryl-CoA transferase; and

(d) aldehyde/alcohol dehydrogenase, said aldehyde/alcohol dehydrogenase converting 4-hydroxybutyryl-CoA to 1,4-butanediol.

10. The non-naturally occurring E. coli of claim 1 , wherein the BDO pathway comprises:

(a) glutamate:succinate semialdehyde transaminase and glutamate decarboxylase;

(b) 4-hydroxybutyrate dehydrogenase;

(c) 4-hydroxybutyryl-CoA transferase; and

(d) aldehyde/alcohol dehydrogenase, said aldehyde/alcohol dehydrogenase converting 4-hydroxybutyryl-CoA to 1,4-butanediol.

11. The non-naturally occurring E. coli of claim 1 , wherein the BDO pathway comprises:

(a) alpha-ketoglutarate decarboxylase;

(b) 4-hydroxybutyrate dehydrogenase;

(c) 4-hydroxybutyrate kinase and phosphotrans-4-hydroxybutyrylase; and

(d) aldehyde/alcohol dehydrogenase, said aldehyde/alcohol dehydrogenase converting 4-hydroxybutyryl-CoA to 1,4-butanediol.

12. The non-naturally occurring E. coli of claim 1 , wherein the BDO pathway comprises:

(a) alpha-ketoglutarate dehydrogenase and CoA-dependent succinate semialdehyde dehydrogenase;

(b) 4-hydroxybutyrate dehydrogenase;

(c) 4-hydroxybutyrate kinase and phosphotrans-4-hydroxybutyrylase; and

(d) aldehyde/alcohol dehydrogenase, said aldehyde/alcohol dehydrogenase converting 4-hydroxybutyryl-CoA to 1,4-butanediol.

13. The non-naturally occurring E. coli of claim 1 , wherein the BDO pathway comprises:

(a) glutamate:succinate semialdehyde transaminase and glutamate decarboxylase;

(b) 4-hydroxybutyrate dehydrogenase;

(c) 4-hydroxybutyrate kinase and phosphotrans-4-hydroxybutyrylase; and

(d) aldehyde/alcohol dehydrogenase, said aldehyde/alcohol dehydrogenase converting 4-hydroxybutyryl-CoA to 1,4-butanediol.

14. The non-naturally occurring E. coli of claim 1 , wherein the BDO pathway comprises:

(a) alpha-ketoglutarate decarboxylase; or

succinyl-CoA synthetase and CoA-dependent succinate semialdehyde dehydrogenase;

(b) 4-hydroxybutyrate dehydrogenase;

(c) 4-hydroxybutyryl-CoA transferase; or

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

(d) aldehyde dehydrogenase, said aldehyde dehydrogenase converting 4-hydroxybutyryl-CoA to 4-hydroxybutyraldehyde; and alcohol dehydrogenase, said alcohol dehydrogenase converting 4-hydroxybutyraldehyde to 1,4-butanediol; or

aldehyde/alcohol dehydrogenase, said aldehyde/alcohol dehydrogenase converting 4-hydroxybutyryl-CoA to 1,4-butanediol.

15. The non-naturally occurring E. coli of claim 1 , wherein the BDO pathway comprises:

(a) alpha-ketoglutarate decarboxylase; or

glutamate dehydrogenase, glutamate decarboxylase, and deaminating 4-aminobutyrate oxidoreductase or 4-aminobutyrate transaminase; or

alpha-ketoglutarate dehydrogenase and CoA-dependent succinate semialdehyde dehydrogenase;

(b) 4-hydroxybutyrate dehydrogenase; and

(c) 4-hydroxybutyrate kinase, phosphotrans-4-hydroxybutyrylase, 4-hydroxybutyryl-CoA reductase, and 4-hydroxybutyraldehyde reductase; or

4-hydroxybutyrate kinase, phosphorylating 4-hydroxybutanal dehydrogenase, and 4-hydroxybutyraldehyde reductase; or

4-hydroxybutyrate kinase, phosphotrans-4-hydroxybutyrylase, and alcohol forming 4-hydroxybutyryl-CoA reductase; or

4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA hydrolase or 4-hydroxybutyryl-CoA ligase, 4-hydroxybutyryl-CoA reductase, and 4-hydroxybutyraldehyde reductase; or

4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA hydrolase or 4-hydroxybutyryl-CoA ligase, and alcohol forming 4-hydroxybutyryl-CoA reductase.

16. The non-naturally occurring E. coli of claim 1 , wherein the BDO pathway comprises:

(a) glutamate CoA transferase or glutamyl-CoA hydrolase or glutamyl-CoA ligase, glutamyl-CoA reductase, and glutamate-5-semialdehyde reductase; or

glutamate CoA transferase or glutamyl-CoA hydrolase or glutamyl-CoA ligase, and alcohol forming glutamyl-CoA reductase; or

glutamate 5-kinase, phosphorylating glutamate-5-semialdehyde dehydrogenase, and glutamate-5-semialdehyde reductase;

(b) deaminating 2-amino-5-hydroxypentanoic acid oxidoreductase or 2-amino-5-hydroxypentanoic acid transaminase; and

(c) 5-hydroxy-2-oxopentanoic acid decarboxylase, and 4-hydroxybutyraldehyde reductase; or

decarboxylating 5-hydroxy-2-oxopentanoic acid dehydrogenase, 4-hydroxybutyryl-CoA reductase, and 4-hydroxybutyraldehyde reductase; or

decarboxylating 5-hydroxy-2-oxopentanoic acid dehydrogenase and alcohol forming 4-hydroxybutyryl-CoA reductase.

17. The non-naturally occurring E. coli of claim 1 , wherein said E. coli comprises disruption of a gene encoding a protein in an aerobic respiratory control regulatory system.

18. The non-naturally occurring E. coli of claim 17 , wherein said gene encoding the protein in the aerobic respiratory control regulatory system is an arcA gene.

19. The non-naturally occurring E. coli of claim 1 , wherein said E. coli expresses an exogenous NADH insensitive citrate synthase.

20. The non-naturally occurring E. coli of claim 19 , wherein said NADH insensitive citrate synthase is encoded by a gltA gene or a mutant gltA gene encoding an R163L mutant NADH insensitive citrate synthase.

21. The non-naturally occurring E. coli of claim 20 , wherein said NADH insensitive citrate synthase is encoded by a gltA gene.

22. The non-naturally occurring E. coli of claim 20 , wherein said NADH insensitive citrate synthase is encoded by a mutant gltA gene encoding an R163L mutant NADH insensitive citrate synthase.

23. The non-naturally occurring E. coli of claim 1 , wherein said E. coli comprises disruption of a gene encoding a protein in an aerobic respiratory control regulatory system and expresses an exogenous NADH insensitive citrate synthase.

24. The non-naturally occurring E. coli of claim 3 , wherein said E. coli comprises disruption of a gene encoding a protein in an aerobic respiratory control regulatory system and expresses an exogenous NADH insensitive citrate synthase.

25. The non-naturally occurring E. coli of claim 1 , wherein said E. coli further expresses an exogenous phosphoenolpyruvate carboxykinase.

26. The non-naturally occurring E. coli of claim 1 , further comprising disruption of a gene encoding malate dehydrogenase.

27. The non-naturally occurring E. coli of claim 1 , wherein one or more of said one or more heterologous polynucleotides encoding BDO pathway enzymes is integrated into the fimD locus of the E. coli.

28. The non-naturally occurring E. coli of claim 1 , wherein said E. coli further expresses an exogenous non-phosphotransferase sucrose uptake system.

29. The non-naturally occurring E. coli of claim 1 , wherein said E. coli further comprises disruption of an endogenous lactate dehydrogenase, endogenous alcohol dehydrogenase, and endogenous pyruvate formate lyase.

30. The non-naturally E. coli of claim 1 , wherein said E. coli further expresses an exogenous pyruvate dehydrogenase.

31. The non-naturally occurring E. coli of claim 30 , wherein one or more genes encoding pyruvate dehydrogenase subunits is under the control of a pyruvate formate lyase promoter.

32. The non-naturally occurring E. coli of claim 30 , wherein said exogenous pyruvate dehydrogenase is NADH insensitive.

33. The non-naturally occurring E. coli of claim 30 , wherein said exogenous pyruvate dehydrogenase is encoded by the Klebsiella pneumonia lpdA gene.

34. The non-naturally occurring E. coli of claim 1 , wherein said succinate semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase and 4-hydroxybutyryl-CoA/acetyl-CoA transferase are encoded by Porphyromonas gingivalis W83 genes.

35. The non-naturally occurring E. coli of claim 1 , wherein said E. coli further expresses an exogenous succinyl-CoA synthetase.

36. The non-naturally occurring E. coli of claim 35 , wherein said succinyl-CoA synthetase is encoded by the Escherichia coli sucCD genes.

37. The non-naturally occurring E. coli of claim 1 , wherein said 4-hydroxybutyryl-CoA reductase is encoded by Clostridium beijerinckii ald gene; said alpha-ketoglutarate decarboxylase is encoded by the Mycobacterium bovis sucA gene; said 4-hydroxybutyrate kinase and said phosphotrans-4-hydroxybutyrylase are encoded by the Clostridium acetobutylicum buk1 and ptb genes; said 4-hydroxybutyraldehyde reductase is encoded by the Geobacillus thermoglucosidasius adh1 gene; or said phosphoenolpyruvate carboxykinase is encoded by the Haemophilus influenza phosphoenolpyruvate carboxykinase gene.

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

39. The method of claim 38 , further comprising the step of isolating the BDO.

40. The method of claim 39 , wherein the BDO is isolated by distillation.

41. A method for producing 1,4-butanediol (BDO), comprising culturing the non-naturally occurring microbial organism of claim 3 under conditions and for a sufficient period of time to produce BDO.

42. The method of claim 41 , further comprising the step of isolating the BDO.

43. The method of claim 42 , wherein the BDO is isolated by distillation.