L-amino acid-producing bacterium and a method for producing an L-amino acid

• US 9,458,206 B2
• Filed: 02/16/2010
• Issued: 10/04/2016
• Est. Priority Date: 02/16/2009
• Status: Active Grant

• Current Assignee: Ajinomoto Co., Inc.
• Original Assignee: Ajinomoto Co., Inc.
• Inventors: Takumi, Kazuhiro, Nonaka, Gen
• Primary Examiner(s): Saidha, Tekchand
• Application Number:
  US12/706,032
• Publication Number:
  US 20100209977A1
• Time in Patent Office: 2,422 Days
• Field of Search: 435/107
• US Class Current: 1/1
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C12P 13/12 Methionine; Cysteine; Cystine

Abstract

A bacterium belonging to the family Enterobacteriaceae, which has an ability to produce an amino acid such as L-cysteine and has been modified to have specific mutation in the yeas gene, is cultured in a medium, and the L-amino acid is collected from the medium.

Claims

1. A method for producing an L-amino acid comprising:

a) culturing in a medium a bacterium belonging to the family Enterobacteriaceae, which is able to produce an L-amino acid, and has been modified to have a mutation in a yeaS gene selected from the group consisting of;

(I) replacing the amino acid residue at position 28 of SEQ ID NO;

2 with an amino acid residue other than threonine,

(II) replacing the amino acid residue at position 137 of SEQ ID NO;

2 with an amino acid residue other than phenylalanine residue,

(III) replacing the amino acid residue at position 188 of SEQ ID NO;

2 with an amino acid residue other than leucine, and,

(IV) combinations thereof; and

b) collecting the L-amino acid from the medium;

wherein the yeaS gene without said mutation encodes a protein selected from the group consisting of;

(A) a protein comprising the amino acid sequence of SEQ ID NO;

2, and

(B) a protein comprising the amino acid sequence of SEQ ID NO;

2, but wherein 1 to 10 amino acid residues are substituted, and the protein has an activity of improving an L-cysteine-producing ability in the bacterium belonging to the family Enterobacteriaceae as compared to a non-modified bacterium when expression of the protein is increased in the bacterium.

2. The method according to claim 1, wherein the yeaS gene without said mutation is selected from the group consisting of:

(a) a DNA comprising the nucleotide sequence of SEQ ID NO;

1, and

(b) a DNA which is able to hybridize with a sequence complementary to the nucleotide sequence of SEQ ID NO;

1, or a probe which can be prepared from the nucleotide sequence, under stringent conditions, and wherein said DNA encodes a protein having an activity of improving an L-cysteine-producing ability in the bacterium belonging to the family Enterobacteriaceae as compared to a non-modified strain when expression the protein is increased in the bacterium.

3. The method according to claim 1, wherein

(i) said amino acid residue other than threonine is asparagine,

(ii) said amino acid residue other than phenylalanine is selected from the group consisting of serine, glutamine, alanine, histidine, cysteine, and glycine, and

(iii) said amino acid residue other than leucine is glutamine.

4. The method according to claim 3, wherein said amino acid other than phenylalanine is serine or glutamine.

5. The method according to claim 1, wherein the L-amino acid is selected from the group consisting of L-cysteine, L-leucine, L-threonine, L-serine, L-methionine, L-histidine, L-valine, L-glutamic acid, L-arginine, L-isoleucine, L-phenylalanine, L-tyrosine, L-tryptophan, and L-proline.

6. The method according to claim 5, wherein the L-amino acid is L-cysteine.

7. The method according to claim 6, wherein the bacterium has been further modified to increase activity of an L-cysteine biosynthesis system enzyme.

8. The method according to claim 7, wherein the L-cysteine biosynthesis system enzyme is serine acetyltransferase.

9. The method according to claim 8, wherein feedback inhibition of serine acetyltransferase by L-cysteine is reduced.

10. The method according to claim 1, wherein the bacterium is a Pantoea bacterium.

11. The method according to claim 10, wherein the bacterium is Pantoea ananatis.

12. The method according to claim 1, wherein the bacterium is Escherichia coli.

13. A bacterium belonging to the family Enterobacteriaceae, which is able to produce an L-amino acid, and has been modified to have a mutation in a yeaS gene selected from the group consisting of:

(I) replacing the threonine residue at position 28 of SEQ ID NO;

2 with an amino acid residue other than threonine,

(II) replacing the phenylalanine residue at position 137 of SEQ ID NO;

2 with an amino acid residue other than phenylalanine, and

(III) replacing the leucine residue at position 188 of SEQ ID NO;

2 with an amino acid residue other than leucine, and,

(IV) combinations thereof; and

wherein the yeaS gene without said mutation encodes a protein selected from the group consisting of;

(A) a protein comprising the amino acid sequence of SEQ ID NO;

2, and

(B) a protein comprising the amino acid sequence of SEQ ID NO;

2, but wherein 1 to 10 amino acid residues are substituted, and the protein has an activity of improving the ability to produce L-cysteine when overexpressed in a bacterium belonging to the family Enterobacteriaceae as compared to a bacterium in which the protein is not overexpressed.

14. The bacterium according to claim 13, wherein the L-amino acid is L-cysteine.

15. A DNA which encodes a protein selected from the group consisting of:

(A) a protein comprising the amino acid sequence of SEQ ID NO;

2, and

(B) a protein comprising the amino acid sequence of SEQ ID NO;

2, but wherein 1 to 10 amino acid residues are substituted, and the protein has an activity of improving the ability to produce L-cysteine when overexpressed in a bacterium belonging to the family Enterobacteriaceae as compared to a bacterium in which the protein is not overexpressed,

wherein the protein has a mutation selected from the group consisting of;

(I) replacing the threonine residue at position 28 of SEQ ID NO;

2 with an amino acid residue other than threonine,

(II) replacing the phenylalanine residue at position 137 of SEQ ID NO;

2 with an amino acid residue other than phenylalanine,

(III) replacing the leucine residue at position 188 of SEQ ID NO;

2 with an amino acid residue other than leucine.