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

Patent metadata

Field	Value
Publication number	US-10273508-B2
Application number	US-201615148759-A
Country	US
Kind code	B2
Filing date	May 6, 2016
Priority date	Jun 4, 2009
Publication date	Apr 30, 2019
Grant date	Apr 30, 2019

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 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:succinat

Assignees

• 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

• C07D207/267

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

• C07D307/08

  Preparation of tetrahydrofuran · CPC title

• C12P17/10

  Nitrogen as only ring hetero atom · CPC title

• C07D307/33

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

• C12P17/04

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