Method for producing L-amino acid

Patent metadata

Field	Value
Publication number	US-10428359-B2
Application number	US-201715717042-A
Country	US
Kind code	B2
Filing date	Sep 27, 2017
Priority date	Oct 3, 2016
Publication date	Oct 1, 2019
Grant date	Oct 1, 2019

Abstract

A method for producing an L-amino acid such as L-glutamic acid is provided. An L-amino acid is produced by culturing a bacterium having an L-amino acid-producing ability, which has been modified so that the activity of a C4-dicarboxylic acid-uptake carrier such as DctA, DcuA, and DcuB is increased, in a medium, and collecting the L-amino acid from the medium or cells of the bacterium.

First claim

The invention claimed is:

1. A method for producing an L-amino acid, the method comprising the steps of:
A) culturing a bacterium having an L-amino acid-producing ability in a medium resulting in accumulation of an L-amino acid in the medium; and
B) collecting the L-amino acid from the medium;
wherein the bacterium has been modified so that the activity of a C4-dicarboxylic acid-uptake carrier is increased as compared with a non-modified bacterium,
wherein the C4-dicarboxylic acid-uptake carrier is a protein encoded by a gene selected from the group consisting of a dctA gene, a dcuA gene, a dcuB gene, and combinations thereof, and
wherein the L-amino acid is an L-amino acid other than L-aspartic acid,
provided that the L-amino acid is L-glutamic acid when the C4-dicarboxylic acid-uptake carrier is a protein encoded by a dctA gene, and
provided that the bacterium has further been modified so as to harbor a mutant yggB gene when the bacterium is a coryneform bacterium and the C4-dicarboxylic acid-uptake carrier is a protein encoded by a dctA gene.

2. The method according to claim 1 , wherein the dctA gene encodes a protein selected from the group consisting of:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 16 or 18;
(b) a protein comprising the amino acid sequence of SEQ ID NO: 16 or 18, but which includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and wherein said protein has L-aspartic acid-uptake activity;
(c) a protein comprising an amino acid sequence having an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 16 or 18, and wherein said protein has aspartic acid-uptake activity.

3. The method according to claim 1 , wherein the dcuA gene encodes a protein selected from the group consisting of:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 20;
(b) a protein comprising the amino acid sequence of SEQ ID NO: 20, but which includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and wherein said protein has aspartic acid-uptake activity;
(c) a protein comprising an amino acid sequence having an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 20, and wherein said protein has aspartic acid-uptake activity.

4. The method according to claim 1 , wherein the dcuB gene encodes a protein selected from the group consisting of:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 22;
(b) a protein comprising the amino acid sequence of SEQ ID NO: 22, but which includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and wherein said protein has aspartic acid-uptake activity;
(c) a protein comprising an amino acid sequence having an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 22, and wherein said protein has aspartic acid-uptake activity.

5. The method according to claim 1 , wherein the activity of the C4-dicarboxylic acid-uptake carrier is increased by increasing the expression of a gene encoding the C4-dicarboxylic acid-uptake carrier.

6. The method according to claim 5 , wherein the expression of the gene is increased by increasing the copy number of the gene and/or modifying an expression control sequence of the gene.

7. The method according to claim 1 , wherein the bacterium has further been modified so that the activity of phosphoketolase is increased as compared with a non-modified bacterium.

8. The method according to claim 7 , wherein the phosphoketolase is D-xylulose-5-phosphate phosphoketolase and/or fructose-6-phosphate phosphoketolase.

9. The method according to claim 7 , wherein the activity of the phosphoketolase is increased by increasing the expression of a gene encoding the phosphoketolase.

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

11. The method according to claim 1 , wherein the bacterium is a bacterium belonging to the genus Corynebacterium.

12. The method according to claim 11 , wherein the bacterium is Corynebacterium glutamicum.

13. The method according to claim 1 , wherein the bacterium is a bacterium belonging to the genus Pantoea or Escherichia.

14. The method according to claim 13 , wherein the bacterium is Pantoea ananatis or Escherichia coli.

15. The method according to claim 1 , wherein the L-amino acid is an L-amino acid of the glutamate family.

16. The method according to claim 15 , wherein the L-amino acid of the glutamate family is selected from the group consisting of L-glutamic acid, L-glutamine, L-proline, L-arginine, L-citrulline, L-ornithine, and combinations thereof.

17. The method according to claim 15 , wherein the L-amino acid of the glutamate family is L-glutamic acid.

18. The method according to claim 1 , wherein the L-glutamic acid is monoammonium L-glutamate or monosodium L-glutamate.

19. The method according to claim 1 ,
wherein the L-amino acid is an L-amino acid of glutamate family, and
wherein the bacterium has further been modified so that the activity of α-ketoglutarate dehydrogenase and/or succinate dehydrogenase is reduced as compared with a non-modified bacterium.

20. The method according to claim 1 ,
wherein the bacterium is a coryneform bacterium, and
wherein the bacterium has further been modified so as to harbor a mutant yggB gene.

21. The method according to claim 20 , wherein the mutant yggB gene has a mutation that imparts improved L-glutamic acid-producing ability to the coryneform bacterium.

22. The method according to claim 21 , wherein the mutant yggB gene has a mutation selected from the group consisting of:
(1) a mutation in the region coding for the amino acid residues at positions 419 to 533 of SEQ ID NO: 10,
(2) a mutation in the region coding for a transmembrane region of SEQ ID NO: 10, and
(3) a combination thereof.

23. The method according to claim 22 , wherein the wild-type yggB gene encodes a YggB protein that is selected from the group consisting of:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 10;
(b) a protein comprising the amino acid sequence of SEQ ID NO: 10, but which includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and wherein said protein, when overexpressed in the coryneform bacterium, imparts improved L-glutamic acid-producing ability to the coryneform bacterium;
(c) a protein comprising an amino acid sequence having an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 10, and wherein said protein, when overexpressed in the coryneform bacterium, imparts improved L-glutamic acid-producing ability to the coryneform bacterium.

Assignees

• Ajinomoto Kk

Inventors

• Moriya Mika
• Hayashi Kazuyuki
• Yokokawa Akari

Classifications

• C12P13/08

  Lysine; Diaminopimelic acid; Threonine; Valine · CPC title

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• C12P13/14

  Glutamic acid; Glutamine · CPC title

• C07K14/34

  from Corynebacterium (G) · CPC title

• C12N15/66

  General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease · CPC title