Microorganism producing 4-hydroxybutyrate and a method for producing 4-hydroxybutyrate in anaerobic condition using the same

Patent metadata

Field	Value
Publication number	US-9447439-B2
Application number	US-201414468067-A
Country	US
Kind code	B2
Filing date	Aug 25, 2014
Priority date	Aug 23, 2013
Publication date	Sep 20, 2016
Grant date	Sep 20, 2016

Abstract

A genetically modified microorganism comprising a polynucleotide encoding α-ketoglutarate synthase or a mutant thereof, and a polynucleotide encoding pyruvate carboxylase or a mutant thereof; wherein the genetically modified microorganism has decreased malate quinone oxidoreductase activity and/or decreased phosphoenolpyruvate carboxykinase activity compared to an unmodified microorganism of the same type, and wherein the genetically modified microorganism produces 4-hydroxybutyrate.

First claim

What is claimed is:

1. A genetically modified microorganism comprising
a polynucleotide encoding exogenous α-ketoglutarate synthase, and
a polynucleotide encoding endogenous pyruvate carboxylase or a mutant thereof;
wherein the genetically modified microorganism has decreased malate quinone oxidoreductase activity, decreased phosphoenolpyruvate carboxykinase activity, or a combination thereof, compared to an unmodified microorganism of the same type,
wherein the genetically modified microorganism produces 4-hydroxybutyrate, and
wherein the genetically modified microorganism is a strain selected from the group consisting of lumen bacteria, Corynebacterium genus, Brevibacterium genus, and Escherichia coli.

2. The genetically modified microorganism of claim 1 , wherein the microorganism comprises a polynucleotide encoding succinyl-CoA:coenzyme A transferase or a mutant thereof, a polynucleotide encoding coenzyme A-dependent succinate semialdehyde dehydrogenase or a mutant thereof, and a polynucleotide encoding 4-hydroxybutyrate dehydrogenase or a mutant thereof.

3. The genetically modified microorganism of claim 1 , wherein the microorganism has decreased succinate semialdehyde dehydrogenase activity compared to an unmodified microorganism of the same type.

4. The genetically modified microorganism of claim 1 , wherein one or more of NCg10049, NCg10463, and NCg12619 genes in the microorganism has an addition, substitution, or deletion mutation that eliminates succinate semialdehyde dehydrogenase activity, wherein the NCg10049 gene comprises the nucleic acid sequence of SEQ ID NO: 22, the NCg10463 gene comprises the nucleic acid sequence of SEQ ID NO: 23, and the NCg12619 gene comprises the nucleic acid sequence of SEQ ID NO: 24.

5. The genetically modified microorganism of claim 1 , wherein the microorganism additionally comprises a polynucleotide encoding pyruvate dehydrogenase or a mutant thereof.

6. The genetically modified microorganism of claim 1 , wherein the microorganism additionally comprises a gene encoding dihydrolipoyl dehydrogenase (E3), a gene encoding pyruvate dehydrogenase (E1), and a gene encoding dihydrolipoyl transacetylase (E2).

7. The genetically modified microorganism of claim 6 , wherein the gene encoding dihydrolipoyl dehydrogenase (E3) comprises the nucleic acid sequence of SEQ ID NO: 12, the gene encoding pyruvate dehydrogenase (E1) comprises the nucleic acid sequence of SEQ ID NO: 13, and the gene dihydrolipoyl transacetylase (E2) comprises the nucleic acid sequence of SEQ ID NO: 14.

8. The genetically modified microorganism of claim 1 , wherein the microorganism additionally comprises a polynucleotide encoding formate dehydrogenase or a mutant thereof.

9. The genetically modified microorganism of claim 1 , wherein the microorganism is a strain of the Corynebacterium genus.

10. The genetically modified microorganism of claim 9 , wherein the strain of Corynebacterium genus is Corynebacterium glutamicum.

11. The genetically modified microorganism of claim 1 , wherein the pyruvate carboxylase comprises the amino acid sequence of SEQ ID NO: 9.

12. The genetically modified microorganism of claim 1 , wherein the mutant pyruvate carboxylase comprises the amino acid sequence of SEQ ID NO: 10.

13. The genetically modified microorganism of claim 1 , wherein the α-ketoglutarate synthase comprises the amino acid sequence of SEQ ID NO: 7.

14. The genetically modified microorganism of claim 2 , wherein the succinyl-CoA:coenzyme A transferase comprises the amino acid sequence of SEQ ID NO: 1, the CoA-dependent succinate semialdehyde dehydrogenase comprises the amino acid sequence of SEQ ID NO: 3, and the 4-hydroxybutyrate dehydrogenase comprises the amino acid sequence of SEQ ID NO: 5.

15. The genetically modified microorganism of claim 8 , wherein the formate dehydrogenase comprises the amino acid sequence of SEQ ID NO: 15.

16. A method of producing a C4-chemical comprising:
culturing the genetically modified microorganism of claim 1 in a cell culture medium, whereby the microorganism produces a C4-chemical; and
recovering the C4-chemical from the cell culture medium.

17. The method of claim 16 , wherein the C4-chemical is 4-hydroxybutyrate.

18. The method of claim 16 , wherein the genetically modified microorganism additionally comprises a polynucleotide encoding pyruvate dehydrogenase or a mutant thereof and a polynucleotide encoding formate dehydrogenase or a mutant thereof.

19. The method of claim 18 , wherein the C4-chemical is selected from the group consisting of succinic acid, 4-hydroxybutyrate, and gamma butyrolactone.

Assignees

• Samsung Electronics Co Ltd

Inventors

• Lee Youngmin
• Lee Wooyong
• Kim Jinwoo
• Park Jaechan
• Park Jinhwan
• Cho Hwayoung

Classifications

• C12Y401/01

  Carboxy-lyases (4.1.1) · CPC title

• C12Y103/05

  with a quinone or related compound as acceptor (1.3.5) · CPC title

• C12Y102/07003

  2-Oxoglutarate synthase (1.2.7.3) · CPC title

• C12Y102/01024

  Succinate-semialdehyde dehydrogenase (NAD+) (1.2.1.24) · CPC title

• C12Y101/01027

  L-Lactate dehydrogenase (1.1.1.27) · CPC title