Method for Manufacturing Useful Substance

Patent metadata

Field	Value
Publication number	US-2016130618-A1
Application number	US-201614990047-A
Country	US
Kind code	A1
Filing date	Jan 7, 2016
Priority date	Jul 9, 2013
Publication date	May 12, 2016
Grant date	—

Abstract

A method for producing an objective substance is provided. An objective substance is produced by culturing a microorganism which has been modified so that the activity of a dicarboxylic acid exporter protein is reduced in a medium, and collecting the objective substance from the medium.

First claim

1 . A method for producing an objective substance, the method comprising:
culturing a microorganism having an objective substance-producing ability in a medium to produce and accumulate the objective substance in the medium or in cells of the microorganism; and collecting the objective substance from the medium or the cells, wherein the microorganism has been modified so that the activity of a dicarboxylic acid exporter protein is reduced.

2 . The method according to claim 1 , wherein the activity of the dicarboxylic acid exporter protein is reduced by attenuating the expression of a gene encoding the dicarboxylic acid exporter protein or by deleting the gene.

3 . The method according to claim 2 , wherein the gene encoding the dicarboxylic acid exporter protein is selected from the group consisting of yjjP gene, yjjB gene, yeeA gene, ynfM gene, sucE1 gene, and combinations thereof.

4 . The method according to claim 3 , wherein the yjjP gene is a DNA selected from the group consisting of:
(A) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 158 or 160; (B) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 158 or 160, but including substitution, deletion, insertion, or addition of one or several amino acid residues, the protein having an activity to export a dicarboxylic acid; (C) a DNA comprising the nucleotide sequence of SEQ ID NO: 157 or 159; and (D) a DNA able to hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 157 or 159, or with a probe that can be prepared from the complementary nucleotide sequence, and encoding a protein having an activity to export a dicarboxylic acid.

5 . The method according to claim 3 , wherein the yjjB gene is a DNA selected from the group consisting of:
(A) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 162 or 164; (B) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 162 or 164, but including substitution, deletion, insertion, or addition of one or several amino acid residues, the protein having an activity to export a dicarboxylic acid; (C) DNA comprising the nucleotide sequence of SEQ ID NO: 161 or 163; and (D) DNA able to hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 161 or 163, or with a probe that can be prepared from the complementary nucleotide sequence, and encoding a protein having an activity to export a dicarboxylic acid.

6 . The method according to claim 3 , wherein the yeeA gene is a DNA selected from the group consisting of:
(A) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 166, 168, or 170; (B) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 166, 168, or 170, but including substitution, deletion, insertion, or addition of one or several amino acid residues, the protein having an activity to export a dicarboxylic acid; (C) DNA comprising the nucleotide sequence of SEQ ID NO: 165, 167, or 169; and (D) DNA able to hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 165, 167, or 169, or with a probe that can be prepared from the complementary nucleotide sequence, and encoding a protein having an activity to export a dicarboxylic acid.

7 . The method according to claim 3 , wherein the ynfM gene is a DNA selected from the group consisting of:
(A) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 172, 174, 176, 178, or 180; (B) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 172, 174, 176, 178, or 180, but including substitution, deletion, insertion, or addition of one or several amino acid residues, the protein having an activity to export a dicarboxylic acid; (C) DNA comprising the nucleotide sequence of SEQ ID NO: 171, 173, 175, 177, or 179; and (D) DNA able to hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 171, 173, 175, 177, or 179, or with a probe that can be prepared from the complementary nucleotide sequence, and encoding a protein having an activity to export a dicarboxylic acid.

8 . The method according to claim 3 , wherein the sucE1 gene is a DNA selected from the group consisting of:
(A) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 278 or 280; (B) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 278 or 280, but including substitution, deletion, insertion, or addition of one or several amino acid residues, the protein having an activity to export a dicarboxylic acid; (C) DNA comprising the nucleotide sequence of SEQ ID NO: 277 or 279; and (D) DNA able to hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 277 or 279, or with a probe that can be prepared from the complementary nucleotide sequence, and encoding a protein having an activity to export a dicarboxylic acid.

9 . The method according to claim 1 , wherein the objective substance is a metabolite derived from acetyl-CoA and/or an L-amino acid.

10 . The method according to claim 9 , wherein the metabolite derived from acetyl-CoA and/or the L-amino acid is selected from the group consisting of isopropyl alcohol, ethanol, acetone, propylene, isoprene, 1,3-butanediol, 1,4-butanediol, 1-propanol, 1,3-propanediol, 1,2-propanediol, ethylene glycol, isobutanol, and combinations thereof.

11 . The method according to claim 9 , wherein the metabolite derived from acetyl-CoA and/or the L-amino acid is selected from the group consisting of citric acid, itaconic acid, acetic acid, butyric acid, 3-hydroxybutyric acid, polyhydroxybutyric acid, 3-hydroxyisobutyric acid, 3-aminoisobutyric acid, 2-hydroxyisobutyric acid, methacrylic acid, 6-aminocaproic acid, and combinations thereof.

12 . The method according to claim 9 , wherein the metabolite derived from acetyl-CoA and/or the L-amino acid is selected from the group consisting of polyglutamic acid, L-glutamic acid, L-glutamine, L-arginine, L-ornithine, L-citrulline, L-leucine, L-isoleucine, L-valine, L-cysteine, L-serine, L-proline, and combinations thereof.

13 . The method according to claim 12 , wherein the L-glutamic acid is monoammonium L-glutamate or monosodium L-glutamate.

14 . The method according to claim 1 , wherein the microorganism is a coryneform bacterium or a bacterium belonging to the family Enterobacteriaceae.

15 . The method according to claim 14 , wherein the coryneform bacterium is Corynebacterium glutamicum.

16 . The method according to claim 14 , wherein the bacterium belonging to the family Enterobacteriaceae is Escherichia coli, Pantoea ananatis , or Enterobacter aerogenes.

17 . The method according to claim 1 , wherein the dicarboxylic acid is selected from the group consisting of malic acid, succinic acid, fumaric acid, 2-hydroxyglutaric acid, and α-ketoglutaric acid.

18 . The method according to claim 1 , wherein the microorganism has been further modified so that malyl-CoA-producing ability is increased.

19 . The method according to claim 1 , wherein the microorganism has been further modified so that α-ketoglutarate synthase activity is increased.

Assignees

• Ajinomoto Kk

Inventors

• Hara Yoshihiko
• Fukui Keita
• Chinen Akito
• Yamakami Suguru

Classifications

• C12P13/04

  Alpha- or beta- amino acids {(other amino acids C12P13/005)} · CPC title

• C12P13/14 Primary

  Glutamic acid; Glutamine · CPC title

• C12P13/001

  Amines; Imines · CPC title

• C12P7/00

  Preparation of oxygen-containing organic compounds · CPC title