Method for producing L-amino acid

Patent metadata

Field	Value
Publication number	US-10787691-B2
Application number	US-202016774075-A
Country	US
Kind code	B2
Filing date	Jan 28, 2020
Priority date	Jan 28, 2019
Publication date	Sep 29, 2020
Grant date	Sep 29, 2020

Abstract

A method for producing an L-amino acid such as L-glutamic acid is provided. An L-amino acid is produced by culturing in a medium a bacterium having an L-amino acid-producing ability, which has been modified so that the activity of a c1795 protein is reduced or the activity of a protein of which the expression is repressed by a c1795 protein is increased, and collecting the L-amino acid from the medium or cells of the bacterium.

First claim

The invention claimed is:

1. A bacterium belonging to the family Enterobacteriaceae and having an L-amino acid-producing ability,
wherein the bacterium has a feature selected from the group consisting of:
(A) the bacterium has been modified so that the activity of a c1795 protein is reduced; and
(B) the bacterium has been modified so that the activity of a protein P is increased, wherein the expression of protein P is repressed by a c1795 protein;
wherein the c1795 protein and the protein P are native to a bacterium belonging to the genus Pantoea.

2. The bacterium according to claim 1 , wherein the protein P is a protein selected from the group consisting of a PAJ_1175 protein, a PAJ_1174 protein, a PAJ_1173 protein, and combinations thereof.

3. The bacterium according to claim 2 , wherein at least the activity of the PAJ_1175 protein is increased, or at least the activities of the PAJ_1174 protein and PAJ_1173 protein are increased.

4. The bacterium according to claim 1 , wherein the activity of the protein P is increased by increasing the expression of a gene encoding the protein P.

5. The bacterium according to claim 4 , wherein the expression of the gene encoding the protein P is increased by a method selected from the group consisting of:
(1) increasing the copy number of the gene encoding the protein P;
(2) modifying an expression control sequence of the gene encoding the protein P;
(3) reducing the activity of the c1795 protein; and
(4) combinations thereof.

6. The bacterium according to claim 1 , wherein the activity of the c1795 protein is reduced by reducing the expression of a c1795 gene and/or disrupting a c1795 gene.

7. The bacterium according to claim 6 , wherein the expression of the c1795 gene is reduced by modifying an expression control sequence of the c1795 gene.

8. The bacterium according to claim 1 , wherein the activity of the c1795 protein is reduced by deleting a part of or the entirety of the amino acid sequence of the c1795 protein.

9. The bacterium according to claim 8 , wherein the activity of the c1795 protein is reduced by a method selected from the group consisting of:
A) deletion of a partial or the entire region of the coding region of the c1795 gene,
B) introduction of a stop codon into the coding region of the c1795 gene,
C) introduction of a frame shift into the coding region of the c1795 gene, and
D) combinations thereof.

10. The bacterium according to claim 1 , wherein the c1795 protein is selected from the group consisting of:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 2;
(b) a protein comprising the amino acid sequence of SEQ ID NO: 2, but which includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and having a function of a c1795 protein; and
(c) a protein comprising an amino acid sequence having an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 2, and having a function of a c1795 protein;
wherein the function of a c1795 protein is as a transcriptional regulator of the Rrf2 family.

11. The bacterium according to claim 2 , wherein the PAJ_1175 protein is selected from the group consisting of:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 4;
(b) a protein comprising the amino acid sequence of SEQ ID NO: 4, but which includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and having a function of a PAJ_1175 protein; and
(c) a protein comprising an amino acid sequence having an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 4, and having a function of a PAJ_1175 protein;
wherein the function of a PAJ_1175 protein is as a transcriptional regulatory of the AraC family.

12. The bacterium according to claim 2 , wherein the PAJ_1174 protein is selected from the group consisting of:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 6;
(b) a protein comprising the amino acid sequence of SEQ ID NO: 6, but which includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and having a function of a PAJ_1174 protein; and
(c) a protein comprising an amino acid sequence having an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 6, and having a function of a PAJ_1174 protein;
wherein the function of a PAJ_1174 protein is as a periplasm adapter subunit of a multi-drug efflux transporter belonging to the RND (resistance-nodulation-cell division) superfamily.

13. The bacterium according to claim 2 , wherein the PAJ_1173 protein is selected from the group consisting of:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 8;
(b) a protein comprising the amino acid sequence of SEQ ID NO: 8, but which includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and having a function of a PAJ_1173 protein; and
(c) a protein comprising an amino acid sequence having an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 8, and having a function of a PAJ_1173 protein;
wherein the function of a PAJ_1173 protein is as a permease subunit of a multi-drug efflux transporter belonging to the RND (resistance-nodulation-cell division) superfamily.

14. The bacterium according to claim 1 , wherein the bacterium is a Pantoea bacterium or an Escherichia bacterium.

15. The bacterium according to claim 14 , wherein the bacterium is Pantoea ananatis or Escherichia coli.

16. A method for producing an L-amino acid, the method comprising:
culturing the bacterium according to claim 1 in a medium to accumulate the L-amino acid in the medium and/or cells of the bacterium; and
collecting the L-amino acid from the medium and/or the cells.

17. The method according to claim 16 , wherein the L-amino acid is selected from the group consisting of L-glutamic acid, L-lysine, L-threonine, L-arginine, L-histidine, L-isoleucine, L-valine, L-leucine, L-phenylalanine, L-tyrosine, L-tryptophan, L-cysteine, and combinations thereof.

18. The method according to claim 16 , wherein the L-amino acid is selected from the group consisting of L-glutamic acid, L-lysine, L-threonine, L-tryptophan, and combinations thereof.

19. The method according to claim 16 , wherein the L-amino acid is L-glutamic acid.

20. The method according to claim 17 , wherein the L-glutamic acid is monoammonium L-glutamate or monosodium L-glutamate.

21. A bacterium belonging to the family Enterobacteriaceae and having an L-amino acid-producing ability,
wherein the bacterium has a feature selected from the group consisting of:
(A) the bacterium has been modified so that the activity of a c1795 protein is reduced; and
(B) the bacterium has been modified so that the activity of a protein P is increased, wherein the expression of protein P is repressed by a c1795 protein,
wherein the protein P is a protein selected from the group consisting of a PAJ_1175 protein, a PAJ_1174 protein, a PAJ_1173 protein, and combinations thereof,
wherein the c1795 protein is selected from the group consisting of:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 2;
(b) a protein comprising the amino acid sequence of SEQ ID NO: 2, but which includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and having a function of a c1795 protein; and
(c) a protein comprising an amino acid sequence having an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 2, and having a function of a c1

Assignees

• Ajinomoto Kk

Inventors

• Noguchi Hiromi
• Nishiyama Yo
• Ito Yoshihiro

Classifications

• C12P13/04

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

• C07K14/245

  Escherichia (G) · CPC title

• C07K14/24 Primary

  from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia · CPC title

• C07K14/195

  from bacteria · CPC title

• C12P13/14 Primary

  Glutamic acid; Glutamine · CPC title