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

Patent metadata

Field	Value
Publication number	US-9434964-B2
Application number	US-201213361799-A
Country	US
Kind code	B2
Filing date	Jan 30, 2012
Priority date	Jun 4, 2009
Publication date	Sep 6, 2016
Grant date	Sep 6, 2016

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.

First claim

What is claimed is:

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 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; 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 said E. coli comprises disruption of a gene encoding a protein in an aerobic respiratory control regulatory system.

3. The non-naturally occurring E. coli of claim 2 , wherein said gene encoding the protein in the aerobic respiratory control regulatory system is an arcA gene.

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

5. The non-naturally occurring E. coli of claim 4 , 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.

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

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

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

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

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

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

12. The non-naturally occurring E. coli of claim 11 , wherein one or more genes encoding pyruvate dehydrogenase subunits is under the control of a pyruvate formate lyase promoter.

13. The non-naturally occurring E. coli of claim 11 , wherein said exogenous pyruvate dehydrogenase is NADH insensitive.

14. The non-naturally occurring E. coli of claim 11 , wherein said exogenous pyruvate dehydrogenase is encoded by the Klebsiella pneumonia lpdA gene.

15. 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.

16. The non-naturally occurring E. coli of claim 1 , wherein said 4-hydroxybutyryl-CoA reductase is encoded by the Clostridium beijerinckii ald gene.

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

18. The non-naturally occurring E. coli of claim 17 , wherein said succinyl-CoA synthetase is encoded by the Escherichia coli sucCD genes.

19. The non-naturally occurring E. coli of claim 1 , wherein said alpha-ketoglutarate decarboxylase is encoded by the Mycobacterium bovis sucA gene.

20. The non-naturally occurring E. coli of claim 1 , wherein said 4-hydroxybutyrate kinase and said phosphotrans-4-hydroxybutyrylase are encoded by the Clostridium acetobutylicum buk1 and ptb genes.

21. The non-naturally occurring E. coli of claim 1 , wherein said 4-hydroxybutyryl-CoA reductase is encoded by the Clostridium beijerinckii ald gene.

22. The non-naturally occurring E. coli of claim 1 , wherein said 4-hydroxybutyraldehyde reductase is encoded by the Geobacillus thermoglucosidasius adh1 gene.

23. The non-naturally occurring E. coli of claim 6 , wherein said phosphoenolpyruvate carboxykinase is encoded by the Haemophilus influenza phosphoenolpyruvate carboxykinase gene.

Assignees

• Van Dien Stephen J
• Burgard Anthony P
• Haselbeck Robert
• Pujol-Baxley Catherine J
• Niu Wei
• Trawick John D
• Yim Harry
• Burk Mark J
• Osterhout Robin E
• Sun Jun
• Genomatica Inc

Inventors

• Van Dien Stephen J
• Burgard Anthony P
• Haselbeck Robert
• Pujol-Baxley Catherine J
• Niu Wei
• Trawick John D
• Yim Harry
• Burk Mark J
• Osterhout Robin E
• Sun Jun

Classifications

• C12P17/04

  containing a five-membered hetero ring, e.g. griseofulvin {, vitamin C} · CPC title

• C07D307/08

  Preparation of tetrahydrofuran · CPC title

• C07D307/33

  in position 2, the oxygen atom being in its keto or unsubstituted enol form · CPC title

• C12P17/10

  Nitrogen as only ring hetero atom · CPC title

• C07D207/267

  with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to the ring nitrogen atom · CPC title