MICROORGANISMS FOR THE PRODUCTION OF 1,4-BUTANEDIOL AND RELATED METHODS

• US 20110045575A1
• Filed: 06/04/2010
• Published: 02/24/2011
• Est. Priority Date: 06/04/2009
• Status: Active Grant

• Current Assignee: Genomatica Incorporated
• Original Assignee: Genomatica Incorporated
• Inventors: Van Dien, Stephen J., Burgard, Anthony P., Haselbeck, Robert, Niu, Wei, Trawick, John D., Yim, Harry, Burk, Mark J., Osterhout, Robin E., Sun, Jun, PUJOL-BAXLEY, CATHERINE J.
• Granted Patent: US 8,129,169 B2
• Time in Patent Office: Days
• US Class Current: 435/252.33
• CPC Class Codes: C07D 207/267 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to the ring nitrogen atom
• CPC Class Codes: C07D 307/08 Preparation of tetrahydrofuran
• CPC Class Codes: C07D 307/33 in position 2, the oxygen atom being in its keto or unsubstituted enol form
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12N 9/001 acting on the CH-CH group of donors (1.3)
• CPC Class Codes: C12N 9/13 transferring sulfur containing groups (2.8)
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12P 17/04 containing a five-membered hetero ring, e.g. griseofulvin , vitamin C
• CPC Class Codes: C12P 17/10 Nitrogen as only ring hetero atom
• CPC Class Codes: C12P 7/18 polyhydric
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

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

Claims

1. A non-naturally occurring microbial organism, comprising a microbial organism having a 1,4-butanediol (BDO) pathway comprising at least one exogenous nucleic acid encoding a BDO pathway enzyme expressed in a sufficient amount to produce BDO, wherein said microbial organism is genetically modified to express exogenous succinyl-CoA synthetase;

to express exogenous alpha-ketoglutarate decarboxylase;

to express exogenous succinate semialdehyde dehydrogenase and 4-hydroxybutyrate dehydrogenase and optionally 4-hydroxybutyryl-CoA/acetyl-CoA transferase;

to express exogenous butyrate kinase and phosphotransbutyrylase;

to express exogenous 4-hydroxybutyryl-CoA reductase; and

to express exogenous 4-hydroxybutanal reductase;

to express exogenous pyruvate dehydrogenase;

to disrupt a gene encoding an aerobic respiratory control regulatory system;

to express an exogenous NADH insensitive citrate synthase;

to express exogenous phosphoenolpyruvate carboxykinase.

2. The non-naturally occurring microbial organism of claim 1, wherein the succinyl-CoA synthetase is encoded by the Escherichia coli sucCD genes;

said alpha-ketoglutarate decarboxylase is encoded by the Mycobacterium bovis sucA gene;

the succinate semialdehyde dehydrogenase (CoA-dependent), 4-hydroxybutyrate dehydrogenase and 4-hydroxybutyryl-CoA/acetyl-CoA transferase are encoded by Porphyromonas gingivalis W83 genes;

the butyrate kinase and phosphotransbutyrylase are encoded by Clostridium acetobutilicum buk1 and ptb genes;

the 4-hydroxybutyryl-CoA reductase is encoded by Clostridium beijerinckii ald gene;

the 4-hydroxybutanal reductase is encoded by Geobacillus thermoglucosidasius adh1 gene;

said exogenous pyruvate dehydrogenase is NADH insensitive;

said exogenous pyruvate dehydrogenase is encoded by the Klebsiella pneumonia lpdA gene;

or said phosphoenolpyruvate carboxykinase is encoded by an Haemophilus influenza phosphoenolpyruvate carboxykinase gene.

10. The non-naturally occurring microbial organism of claim 1, wherein one or more of the pyruvate dehydrogenase subunit genes of said microbial organism is under the control of a pyruvate formate lyase promoter.

11. The non-naturally occurring microbial organism of claim 1, wherein the disruption of said anaerobic respiratory control regulatory system is disruption of the arcA gene.

12. The non-naturally occurring microbial organism of claim 1, further comprising disruption of a gene encoding malate dehydrogenase.

13. The non-naturally occurring microbial organism of claim 1, wherein NADH insensitive citrate synthase is encoded by gltA or an R163L mutant of gltA.

16. The non-naturally occurring microbial organism of claim 1, wherein one or more genes encoding said exogenously expressed enzymes are integrated into the fimD locus of the host organism.

17. The non-naturally occurring microbial organism of claim 1, wherein said organism expresses a non-phosphotransferase sucrose uptake system.

18. The non-naturally occurring microbial organism of claim 1, wherein said organism further comprises disruption of endogenous lactate dehydrogenase, alcohol dehydrogenase and pyruvate formate lyase.

19. The non-naturally occurring microbial organism of claim 1, wherein said BDO pathway comprises 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:

CoA transferase, 4-butyrate kinase, phosphotransbutyrylase, alpha-ketoglutarate decarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase or an aldehyde/alcohol dehydrogenase.

27. The non-naturally occurring microbial organism of claim 1, wherein said BDO pathway comprises glutamate CoA transferase, glutamyl-CoA hydrolase, glutamyl-CoA ligase, glutamate 5-kinase, glutamate-5-semialdehyde dehydrogenase (phosphorylating), glutamyl-CoA reductase, glutamate-5-semialdehyde reductase, glutamyl-CoA reductase (alcohol forming), 2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating), 2-amino-5-hydroxypentanoic acid transaminase, 5-hydroxy-2-oxopentanoic acid decarboxylase, 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation).

28. The non-naturally occurring microbial organism of claim 27, wherein said BDO pathway further comprises 4-hydroxybutyryl-CoA reductase (alcohol forming), 4-hydroxybutyryl-CoA reductase, or 1,4-butanediol dehydrogenase.

29. The non-naturally occurring microbial organism of claim 1, wherein said BDO pathway comprising 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase, vinylacetyl-CoA Δ

-isomerase, or 4-hydroxybutyryl-CoA dehydratase.

32. The non-naturally occurring microbial organism of claim 1, wherein said BDO pathway comprises succinyl-CoA reductase (alcohol forming), 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or 4-hydroxybutanal dehydrogenase (phosphorylating).

33. The non-naturally occurring microbial organism of claim 32, wherein said BDO pathway further comprises succinyl-CoA reductase, 4-hydroxybutyrate dehydrogenase, 4-hydroxybutyryl-CoA transferase, 4-hydroxybutyrate kinase, phosphotrans-4-hydroxybutyrylase, 4-hydroxybutyryl-CoA reductase, 4-hydroxybutyryl-CoA reductase (alcohol forming), or 1,4-butanediol dehydrogenase.

34. The non-naturally occurring microbial organism of claim 1, wherein said BDO pathway comprises glutamate dehydrogenase, 4-aminobutyrate oxidoreductase (deaminating), 4-aminobutyrate transaminase, glutamate decarboxylase, 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or 4-hydroxybutanal dehydrogenase (phosphorylating).

35. The non-naturally occurring microbial organism of claim 34, wherein said BDO pathway further comprises alpha-ketoglutarate decarboxylase, 4-hydroxybutyrate dehydrogenase, 4-hydroxybutyryl-CoA transferase, 4-hydroxybutyrate kinase, phosphotrans-4-hydroxybutyrylase, 4-hydroxybutyryl-CoA reductase, 4-hydroxybutyryl-CoA reductase (alcohol forming), or 1,4-butanediol dehydrogenase.

37. 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.

3-9. -9. (canceled)

14-15. -15. (canceled)

20-26. -26. (canceled)

30-31. -31. (canceled)

36. A non-naturally occurring microbial organism, comprising a microbial organism having a 1,4-butanediol (BDO) pathway comprising at least one exogenous nucleic acid encoding a BDO pathway enzyme expressed in a sufficient amount to produce BDO, wherein said microbial organism is genetically modified as in any of strains 3-20 of Table 28.