Method for producing an L-amino acid using a bacterium of the family Enterobacteriaceae having enhanced expression of the flagella formation and motility cascade genes

Patent metadata

Field	Value
Publication number	US-9284584-B2
Application number	US-201414177538-A
Country	US
Kind code	B2
Filing date	Feb 11, 2014
Priority date	Aug 18, 2011
Publication date	Mar 15, 2016
Grant date	Mar 15, 2016

Abstract

The present invention provides a method for producing L-amino acid using a bacterium belonging to the family Enterobacteriaceae, particularly a motile bacterium belonging to the genus Escherichia, Enterobacter or Pantoea , wherein the bacterium has been modified so that expression of at least one gene of the flagella formation and motility cascade is enhanced.

First claim

The invention claimed is:

1. A method for producing an L-amino acid comprising culturing an Escherichia coli bacterium in a medium, and collecting the L-amino acid from the medium,
wherein the bacterium is able to produce an L-amino acid and has been modified so that expression of either the flhD gene or flhC gene, or both, is/are enhanced by a method selected from the group consisting of: a) increasing the copy number of the gene(s), b) modifying an expression control region of the gene(s), and c) combinations thereof;
wherein the flhD gene encodes a protein selected from the group consisting of:
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 2;
(B) a protein comprising the amino acid sequence shown in SEQ ID NO: 2, but wherein one to five amino acid residues are substituted, deleted, inserted, added or inverted, and said protein has DNA-binding transcriptional dual regulator activity according to the amino acid sequence of SEQ ID NO: 2, 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 said protein has DNA-binding transcriptional dual regulator activity according to the amino acid sequence of SEQ ID NO: 2, and
(D) combinations thereof;
wherein the flhC gene encodes a protein selected from the group consisting of:
(i) a protein comprising the amino acid sequence shown in SEQ ID NO: 4,
(ii) a protein comprising the amino acid sequence shown in SEQ ID NO: 4, but wherein one to five amino acid residues are substituted, deleted, inserted, added or inverted, and said protein has DNA-binding transcriptional dual regulator activity according to the amino acid sequence of SEQ ID NO: 4,
(iii) 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 said protein has DNA-binding transcriptional dual regulator activity according to the amino acid sequence of SEQ ID NO: 4, and
(iv) combinations thereof.

2. The method according to claim 1 , wherein the bacterium has been further modified to enhance expression of at least one gene selected from the group consisting of a) the yhjH gene, b)the fliZ gene, and c) combinations thereof.

3. The method according to claim 2 , wherein the yhjH gene encodes a protein selected from the group consisting of:
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 6,
(B) a protein comprising the amino acid sequence shown in SEQ ID NO: 6, but wherein one to five amino acid residues are substituted, deleted, inserted, added or inverted, and said protein has cyclic-di-GMP phosphodiesterase activity according to the amino acid sequence of SEQ ID NO: 6,
(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 said protein has cyclic-di-GMP phosphodiesterase activity according to the amino acid sequence of SEQ ID NO: 6, and
(D) combinations thereof.

4. The method according to claim 2 , wherein the fliZ gene encodes a protein selected from the group consisting of:
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 8,
(B) a protein comprising the amino acid sequence shown in SEQ ID NO: 8, but wherein one to five amino acid residues are substituted, deleted, inserted, added or inverted, and said protein has regulator activity according to the amino acid sequence of SEQ ID NO: 8,
(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 said protein has regulator activity according to the amino acid sequence of SEQ ID NO: 8, and
(C) combinations thereof.

5. The method according to claim 1 , wherein said L-amino acid is an aromatic L-amino acid.

6. The method according to claim 5 , wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine, L-tryptophan, and combinations thereof.

7. The method according to claim 1 , wherein said L-amino acid is a non-aromatic L-amino acid.

8. The method according to claim 7 , wherein said non-aromatic L-amino acid is selected from the group consisting of L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, L-arginine, glycine, and combinations thereof.

9. The method according to claim 1 , wherein said L-amino acid is L-phenylalanine.

10. The method according to claim 1 , wherein said L-amino acid is L-threonine.

Assignees

• Ajinomoto Kk

Inventors

• Altman Irina Borisovna
• Yampolskaya Tatyana Abramovna
• Ptitsyn Leonid Romanovich

Classifications

• C12P13/08 Primary

  Lysine; Diaminopimelic acid; Threonine; Valine · CPC title

• C12P13/222

  Phenylalanine · CPC title

• C07K14/245

  Escherichia (G) · CPC title

• C12P13/04

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

• C07K14/24

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