Methods and organisms for the growth-coupled production of 1,4-butanediol

• US 20090047719A1
• Filed: 08/10/2007
• Published: 02/19/2009
• Est. Priority Date: 08/10/2007
• Status: Active Grant

• Current Assignee: Genomatica Incorporated
• Original Assignee: Genomatica Incorporated
• Inventors: Burk, Mark, Burgard, Anthony P., Van Dien, Stephen J.
• Granted Patent: US 7,947,483 B2
• Time in Patent Office: Days
• US Class Current: 435/158
• CPC Class Codes: B01D 3/002 by continuous methods
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12P 7/18 polyhydric
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The invention provides a non-naturally occurring microorganism comprising one or more gene disruptions, the one or more gene disruptions occurring in genes encoding an enzyme obligatory to coupling 1,4-butanediol production to growth of the microorganism when the gene disruption reduces an activity of the enzyme, whereby theone or more gene disruptions confers stable growth-coupled production of 1,4-butanediol onto the non-naturally occurring microorganism. The microorganism can further comprise a gene encoding an enzyme in a 1,4-butanediol (BDO) biosynthetic pathway. The invention additionally relates to methods of using microorganisms to produce BDO.

Claims

1. A non-naturally occurring microorganism comprising one or more gene disruptions, said one or more gene disruptions occurring in genes encoding an enzyme obligatory to coupling 1,4-butanediol production to growth of said microorganism when said gene disruption reduces an activity of said enzyme, whereby said one or more gene disruptions confers stable growth-coupled production of 1,4-butanediol onto said non-naturally occurring microorganism.

2. The non-naturally occurring microorganism of claim 1, wherein said one or more gene disruptions comprise a metabolic modification listed in Table 6 or 7.

3. The non-naturally occurring microorganism of claim 1, wherein said one or more gene disruptions comprise a deletion of said one or more genes.

4. The non-naturally occurring microorganism of claim 1, wherein said non-naturally occurring microorganism is selected from the group of microorganisms having a metabolic modification listed in Table 6 or 7.

5. The non-naturally occurring microorganism of claim 1, wherein said microorganism comprises a bacterium, yeast or fungus.

6. The non-naturally occurring microorganism of claim 5, wherein said bacteria comprises a species selected from Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.

7. The non-naturally occurring microorganism of claim 5, wherein said yeast comprises a species selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, and Pichia pastoris.

8. A non-naturally occurring microorganism comprising a set of metabolic modifications obligatory to coupling 1,4-butanediol production to growth of said microorganism, said set of metabolic modifications comprising disruption of one or more genes, or an ortholog thereof, wherein said set of metabolic modifications comprises disruption of adhE and ldhA, wherein said microorganism exhibits stable growth-coupled production of 1,4-butanediol.

9. The non-naturally occurring microorganism of claim 8, wherein said set of metabolic modifications further comprise disruption of mdh.

10. The non-naturally occurring microorganism of claim 9, wherein said set of metabolic modifications further comprise disruption of one or more genes selected from the set of genes comprising mqo, aspA, sfcA, maeB, pntAB, and gdhA.

11. The non-naturally occurring microorganism of claim 10, wherein said set of metabolic modifications comprise disruption of sfcA and maeB.

12. The non-naturally occurring microorganism of claim 9, wherein said set of metabolic modifications further comprise disruption of one or more genes selected from the set of genes comprising pykA, pykF, dhaKLM, deoC, edd, yiaE, ycdW, prpC, and gsk.

13. The non-naturally occurring microorganism of claim 12, wherein said set of metabolic modifications comprise disruption of pykA, pykF, dhaKLM, deoC, edd, yiaE and ycdW.

14. The non-naturally occurring microorganism of claim 13, wherein said set of metabolic modifications further comprise disruption of prpC and gsk.

15. The non-naturally occurring microorganism of any of claims 9-14, wherein said set of metabolic modifications further comprise disruption of pflAB.

16. The non-naturally occurring microorganism of claim 8, wherein said set of metabolic modifications comprise disruption of one or more genes selected from the set of genes comprising adhE, ldhA, pflAB, mdh, and aspA.

17. The method of claim 8, wherein said set of metabolic modifications comprise disruption of the genes adhE, ldhA, pflAB, mdh, and aspA.

18. The non-naturally occurring microorganism of claim 8, wherein said microorganism further comprises a 1,4-butanediol (BDO) biosynthetic pathway, said pathway comprising at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, CoA-independent succinic semialdehyde dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:

CoA transferase, glutamate;

succinic semialdehyde transaminase, glutamate decarboxylase, CoA-independent aldehyde dehydrogenase, CoA-dependent aldehyde dehydrogenase or alcohol dehydrogenase, wherein said exogenous nucleic acid is expressed in sufficient amounts to produce 1,4-butanediol (BDO).

19. The non-naturally occurring microorganism of claim 8, wherein said disruption of one or more genes comprises a deletion.

20. The non-naturally occurring microorganism of claim 8, wherein said microorganism comprises a bacterium, yeast or fungus.

21. The non-naturally occurring microorganism of claim 20, wherein said bacteria comprises a species selected from E. coli, K. oxytoca, A. succiniciproducens, A. succinogenes, M. succiniciproducens, R. etli, B. subtilis, C. glutamicum, G. oxydans, Z. mobilis, L. lactis, L. plantarum, S. coelicolor, C. acetobutylicum, P. fluorescens, and P. putida.

22. The non-naturally occurring microorganism of claim 20, wherein said yeast comprises a species selected from S. cerevisiae, S. pombe, K. lactis, K. marxianus, A. terreus, A. niger, and P. pastoris.

23. A method of producing a non-naturally occurring microorganism having stable growth-coupled production of 1,4-butanediol, comprising:

(a) identifying in silico a set of metabolic modifications requiring 1,4-butanediol production during exponential growth, and

(b) genetically modifying a microorganism to contain said set of metabolic modifications requiring 1,4-butanediol production.

24. The method of claim 23, wherein said set of metabolic modifications are identified by an in silico method selected from OptKnock or SimPheny.

25. The method of claim 23, wherein said set of metabolic modifications comprise functional disruption of one or more metabolic reactions.

26. The method of claim 25, wherein said metabolic modifications are disrupted by gene deletion.

27. The method of claim 25, wherein said non-naturally occurring microorganism comprises a microorganism having a metabolic modification selected from the set of metabolic modifications listed in Table 6 or 7.

28. The method of claim 23, further comprising culturing said genetically modified microorganism.

29. The method of claim 28, further comprising adaptively evolving said genetically modified microorganism under conditions requiring 1,4-butanediol production.

30. The method of claim 23, wherein said non-naturally occurring microorganism comprises a bacterium, yeast or fungus.

31. The method of claim 30, wherein said bacteria comprises a species selected from E. coli, K. oxytoca, A. succiniciproducens, A. succinogenes, M. succiniciproducens, R. etli, B. subtilis, C. glutamicum, G. oxydans, Z. mobilis, L. lactis, L. plantarum, S. coelicolor, C. acetobutylicum, P. fluorescens, and P. putida.

32. The non-naturally occurring microorganism of claim 30, wherein said yeast comprises a species selected from S. cerevisiae, S. pombe, K. lactis, K. marxianus, A. terreus, A. niger, and P. pastoris.

33. A microorganism produced by the method of claim 23 or 29.

34. A method of producing 1,4-butanediol coupled to the growth of a microorganism, comprising:

(a) culturing under exponential growth phase in a sufficient amount of nutrients and media a non-naturally occurring microorganism comprising a set of metabolic modifications obligatorily coupling 1,4-butanediol production to growth of said microorganism, said set of metabolic modifications comprising disruption of one or more genes, or an ortholog thereof, wherein said set of metabolic modifications comprises disruption of adhE and ldhA, wherein said microorganism exhibits stable growth-coupled production of 1,4-butanediol; and

(b) isolating 1,4-butanediol produced from said non-naturally occurring microorganism.

35. The method of claim 34, wherein said set of metabolic modifications further comprise disruption of mdh.

36. The method of claim 35, wherein said set of metabolic modifications further comprise disruption of one or more genes selected from the set of genes comprising mqo, aspA, sfcA, maeB, pntAB, and gdhA.

37. The method of claim 36, wherein said set of metabolic modifications comprise disruption of sfcA and maeB.

38. The method of claim 35, wherein said set of metabolic modifications further comprise disruption of one or more genes selected from the set of genes comprising pyk, pykF, dhaKLM, deoC, edd, yiaE, ycdW, prpC, and gsk.

39. The method of claim 38, wherein said set of metabolic modifications comprise disruption of pykA, pykF, dhaKLM, deoC, edd, yiaE and ycdW.

40. The method of claim 39, wherein said set of metabolic modifications further comprise disruption of prpC and gsk.

41. The method of any of claims 34-40, wherein said set of metabolic modifications further comprise disruption of pflAB.

42. The method of claim 34, wherein said set of metabolic modifications comprise disruption of one or more genes selected from the set of genes comprising adhE, ldhA, pflAB, mdh, and aspA.

43. The method of claim 34, wherein said set of metabolic modifications comprise disruption of the genes adhE, ldhA, pflAB, mdh, and aspA.

44. The method of claim 34, wherein said non-naturally occurring microorganism further comprises a 1,4-butanediol (BDO) biosynthetic pathway, said pathway comprising at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, CoA-independent succinic semialdehyde dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:

CoA transferase, glutamate;

succinic semialdehyde transaminase, glutamate decarboxylase, CoA-independent aldehyde dehydrogenase, CoA-dependent aldehyde dehydrogenase or alcohol dehydrogenase, wherein said exogenous nucleic acid is expressed in sufficient amounts to produce 1,4-butanediol (BDO).