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

Patent metadata

Field	Value
Publication number	US-11401534-B2
Application number	US-201916294753-A
Country	US
Kind code	B2
Filing date	Mar 6, 2019
Priority date	Jun 4, 2009
Publication date	Aug 2, 2022
Grant date	Aug 2, 2022

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 bacterial organism, comprising a bacterial 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 bacterial organism is genetically modified to express an exogenous succinyl-CoA synthetase; to express an exogenous alpha-ketoglutarate decarboxylase; to express an exogenous succinate semialdehyde dehydrogenase and a 4-hydroxybutyrate dehydrogenase and optionally a 4-hydroxybutyryl-CoA/acetyl-CoA transferase; to express an exogenous butyrate kinase and a phosphotransbutyrylase; to express an exogenous 4-hydroxybutyryl-CoA reductase; to express an exogenous 4-hydroxybutanal reductase; to express an exogenous pyruvate dehydrogenase; to disrupt a gene encoding an aerobic respiratory control regulatory system; to express an exogenous NADH insensitive citrate synthase; and to express an exogenous phosphoenolpyruvate carboxykinase.

2. The non-naturally occurring bacterial 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.

3. The non-naturally occurring bacterial organism of claim 1 , wherein said BDO pathway comprises 4-aminobutyrate CoA transferase, 4-aminobutyryl-CoA hydrolase, 4-aminobutyrate-CoA ligase, 4-aminobutyryl-CoA oxidoreductase (deaminating), 4-aminobutyryl-CoA transaminase, or 4-hydroxybutyryl-CoA dehydrogenase.

4. The non-naturally occurring bacterial organism of claim 3 , wherein said BDO pathway further comprises 4-hydroxybutyryl-CoA reductase (alcohol forming), 4-hydroxybutyryl-CoA reductase, or 1,4-butanediol dehydrogenase.

5. The non-naturally occurring bacterial organism of claim 1 , wherein said BDO pathway comprises 4-aminobutyrate CoA transferase, 4-aminobutyryl-CoA hydrolase, 4-aminobutyrate-CoA ligase, 4-aminobutyryl-CoA reductase (alcohol forming), 4-aminobutyryl-CoA reductase, 4-aminobutan-1-ol dehydrogenase, 4-aminobutan-1-ol oxidoreductase (deaminating) or 4-aminobutan-1-ol transaminase.

6. The non-naturally occurring bacterial organism of claim 1 , wherein said BDO pathway comprises 4-aminobutyrate kinase, 4-aminobutyraldehyde dehydrogenase (phosphorylating), 4-aminobutan-1-ol dehydrogenase, 4-aminobutan-1-ol oxidoreductase (deaminating), 4-aminobutan-1-ol transaminase, [(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase (deaminating), [(4-aminobutanolyl)oxy]phosphonic acid transaminase, 4-hydroxybutyryl-phosphate dehydrogenase, or 4-hydroxybutyraldehyde dehydrogenase (phosphorylating).

7. The non-naturally occurring bacterial organism of claim 6 , wherein said BDO pathway further comprises 1,4-butanediol dehydrogenase.

8. The non-naturally occurring bacterial organism of claim 1 , wherein said BDO pathway comprises alpha-ketoglutarate 5-kinase, 2,5-dioxopentanoic semialdehyde dehydrogenase (phosphorylating), 2,5-dioxopentanoic acid reductase, alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase, alpha-ketoglutaryl-CoA ligase, alpha-ketoglutaryl-CoA reductase, 5-hydroxy-2-oxopentanoic acid dehydrogenase, alpha-ketoglutaryl-CoA reductase (alcohol forming), 5-hydroxy-2-oxopentanoic acid decarboxylase, or 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation).

9. The non-naturally occurring bacterial organism of claim 8 , wherein said BDO pathway further comprises 4-hydroxybutyryl-CoA reductase (alcohol forming), 4-hydroxybutyryl-CoA reductase, or 1,4-butanediol dehydrogenase.

10. The non-naturally occurring bacterial 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).

11. The non-naturally occurring bacterial organism of claim 10 , wherein said BDO pathway further comprises 4-hydroxybutyryl-CoA reductase (alcohol forming), 4-hydroxybutyryl-CoA reductase, or 1,4-butanediol dehydrogenase.

12. The non-naturally occurring bacterial 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.

13. The non-naturally occurring bacterial organism of claim 1 , wherein said BDO pathway comprises homoserine deaminase, homoserine CoA transferase, homoserine-CoA hydrolase, homoserine-CoA ligase, homoserine-CoA deaminase, 4-hydroxybut-2-enoyl-CoA transferase, 4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA ligase, 4-hydroxybut-2-enoate reductase, 4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or 4-hydroxybut-2-enoyl-CoA reductase.

14. The non-naturally occurring bacterial organism of claim 13 , wherein said BDO pathway further comprises 4-hydroxybutyryl-CoA reductase (alcohol forming), 4-hydroxybutyryl-CoA reductase, or 1,4-butanediol dehydrogenase.

15. The non-naturally occurring bacterial 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).

16. The non-naturally occurring bacterial organism of claim 15 , 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.

17. The non-naturally occurring bacterial 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).

18. The non-naturally occurring bacterial organism of claim 17 , 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.

19. A non-naturally occurring bacterial organism, comprising a bacterial 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 bacterial organism is genetically modified and selected from the group consisting of:
a) a non-naturally occurring bacterial organism comprising deletion of endogenous adhE, IdhA, pfIB, and IpdA genes; chromosomal insertion of a Klebsiella pneumoniae IpdA gene encoding a Glu354Lys mutation; and a vector comprising E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd, P. gingivalis Cat2, and C. acetobutylicum adhE2 genes;
b) a non-naturally occurring bacter

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

• C12N15/52 Primary

  Genes encoding for enzymes or proenzymes · CPC title

• C12N9/93

  Ligases (6) · CPC title

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• C12N9/0006

  acting on CH-OH groups as donors (1.1) · CPC title

• C07D307/08

  Preparation of tetrahydrofuran · CPC title