DNA encoding dipeptide-synthesizing enzyme (variants), bacterium belonging to the genus Escherichia, and methods for producing dipeptides using thereof

Patent metadata

Field	Value
Publication number	US-9428783-B2
Application number	US-201514591387-A
Country	US
Kind code	B2
Filing date	Jan 7, 2015
Priority date	Jul 11, 2012
Publication date	Aug 30, 2016
Grant date	Aug 30, 2016

Abstract

The present invention describes novel bacterial L-amino acids α-ligases, which catalyzing reaction of dipeptide formation having an acidic L-amino acid such as L-Asp or L-Glu at the N-terminus. The method for producing dipeptides using said L-amino acids α-ligases and a bacterium of the family Enterobacteriaceae, particularly a bacterium belonging to the genus Escherichia , which has been modified to contain the DNA encoding said L-amino acids α-ligases, is described.

First claim

The invention claimed is:

1. A method for producing a dipeptide or a salt thereof, comprising:
(a) reacting L-amino acids, L-amino acid derivatives, or salts thereof under appropriate conditions in the presence of a protein having dipeptide-synthesizing activity such that a dipeptide or a salt thereof is produced;
(b) accumulating the dipeptide or a salt thereof in an appropriate solvent; and
(c) collecting the dipeptide or a salt thereof from the appropriate solvent,
wherein the protein is selected from the group consisting of:
a protein having the amino acid sequence of SEQ ID NO: 2, 4, or 6;
a variant of a protein having the amino acid sequence of SEQ ID NO: 2, 4, or 6 in which no more than fifteen amino acid residues of the amino acid sequence of SEQ ID NO: 2, 4, or 6 are changed by substitution, deletion, insertion, or addition, and wherein the variant protein has the dipeptide-synthesizing activity; and
a protein having an amino acid sequence having not less than 95% sequence identity to the amino acid sequence of SEQ ID NO: 2, 4, or 6 and wherein the protein has the dipeptide-synthesizing activity,
wherein the dipeptide comprises two L-amino acids, two L-amino acid derivatives, or an L-amino acid and an L-amino acid derivative, and
wherein the L-amino acids or the L-amino acid derivatives are selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and a lower alkyl ester of L-phenylalanine, wherein the lower alkyl ester of L-phenylalanine is methyl, ethyl or propyl ester of the L-phenylalanine.

2. A method for producing a dipeptide or a salt thereof, comprising:
(a) cultivating a bacterium in a culture medium comprising an L-amino acid or an L-amino acid derivative;
(b) accumulating the dipeptide in the bacterium, the culture medium, or both; and
(c) collecting the dipeptide from the bacterium, the culture medium, or both,
wherein the bacterium is a dipeptide-producing bacterium belonging to the genus Escherichia transformed with a recombinant DNA comprising a DNA encoding a protein having dipeptide-synthesizing activity,
wherein the DNA encoding the protein having a dipeptide-synthesizing activity is selected from the group consisting of:
a DNA having the nucleotide sequence of SEQ ID NO: 1, 3, or 5;
a DNA encoding a protein having the amino acid sequence of SEQ ID NO: 2, 4, or 6;
a DNA encoding a variant of a protein having the amino acid sequence of SEQ ID NO: 2, 4, or 6 in which no more than fifteen amino acid residues of the amino acid sequence of SEQ ID NO: 2, 4, or 6 are changed by substitution, deletion, insertion, or addition, and wherein the variant protein has the dipeptide-synthesizing activity; and
a DNA encoding a protein having an amino acid sequence having not less than 95% sequence identity to the amino acid sequence of SEQ ID NO: 2, 4, or 6 and wherein the protein has the dipeptide-synthesizing activity,
wherein the dipeptide comprises two L-amino acids, two L-amino acid derivatives, or an L-amino acid and an L-amino acid derivative, and
wherein the L-amino acids or the L-amino acid derivatives are selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and a lower alkyl ester of L-phenylalanine, wherein the lower alkyl ester of L-phenylalanine is methyl, ethyl or propyl ester of the L-phenylalanine.

3. The method according to claim 1 ,
wherein the dipeptide is represented by the formula:
R1-R2

wherein R1 is an acidic L-amino acid residue,
wherein R2 is an L-amino acid or a derivative of the L-amino acid, and
wherein the L-amino acid or the derivative of the L-amino acid is selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and a lower alkyl ester of L-phenylalanine, wherein the lower alkyl ester of L-phenylalanine is methyl, ethyl or propyl ester of the L-phenylalanine.

4. The method according to claim 3 ,
wherein R1 is L-aspartic acid or L-glutamic acid, and
wherein R2 is L-glutamic acid, L-isoleucine, L-phenylalanine, L-tryptophan, L-valine or a lower alkyl ester of L-phenylalanine, and wherein the lower alkyl ester of L-phenylalanine is methyl, ethyl or propyl ester of the L-phenylalanine.

5. The method according to claim 3 ,
wherein R1 is L-aspartic acid, and
wherein R2 is L-phenylalanine or a lower alkyl ester of L-phenylalanine, and wherein the lower alkyl ester of L-phenylalanine is methyl, ethyl or propyl ester of the L-phenylalanine.

6. The method according to claim 2 , wherein the bacterium is the species Escherichia coli .

7. The method according to claim 2 ,
wherein a chromosomal gene encoding a protein having peptidase activity is attenuated or inactivated in the bacterium.

8. The method according to claim 7 ,
wherein the gene encoding a protein having peptidase activity is selected from the group consisting of pepA, pepB, pepD, pepE, pepP, pepQ, pepN, pepT, iadA, iaaA (ybiK), and dapE.

9. The method according to claim 1 ,
wherein the protein having dipeptide-synthesizing activity is at least one of:
a protein having the amino acid sequence of SEQ ID NO: 2, 4, or 6, and
a protein having an amino acid sequence having not less than 95% sequence identity to the amino acid sequence of SEQ ID NO: 2, 4, or 6 and wherein the protein has the dipeptide-synthesizing activity.

10. The method according to claim 9 ,
wherein the dipeptide is represented by the formula:
R1-R2

wherein R1 is L-aspartic acid or L-glutamic acid, and
wherein R2 is L-glutamic acid, L-isoleucine, L-phenylalanine, L-tryptophan, L-valine or a lower alkyl ester of L-phenylalanine, and wherein the lower alkyl ester of L-phenylalanine is methyl, ethyl or propyl ester of the L-phenylalanine.

11. The method according to claim 9 ,
wherein the dipeptide is represented by the formula:
R1-R2

wherein R1 is L-aspartic acid, and
wherein R2 is L-phenylalanine or a lower alkyl ester of L-phenylalanine, and wherein the lower alkyl ester of L-phenylalanine is methyl, ethyl or propyl ester of the L-phenylalanine.

12. The method according to claim 2 ,
wherein the DNA encoding the protein having dipeptide-synthesizing activity is at least one of:
a DNA having the nucleotide sequence of SEQ ID NO: 1, 3, or 5;
a DNA encoding a protein having the amino acid sequence of SEQ ID NO: 2, 4, or 6; and
a DNA encoding a protein having an amino acid sequence having not less than 95% sequence identity to the amino acid sequence of SEQ ID NO: 2, 4, or 6 and wherein the protein has the dipeptide-synthesizing activity.

13. The method according to claim 12 ,
wherein the dipeptide is represented by the formula:
R1-R2

wherein R1 is L-aspartic acid or L-glutamic acid, and
wherein R2 is L-glutamic acid, L-isoleucine, L- phenylalanine, L-tryptophan, L-valine or a lower alkyl ester of L-phenylalanine, and wherein the lower alkyl ester of L-phenylalanine is methyl, ethyl or propyl ester of the L-phenylalanine.

14. The method according to claim 12 ,
wherein the dipeptide is represented by

Assignees

• Ajinomoto Kk

Inventors

• Smirnov Sergey Vasilievich
• Sokolov Pavel Mikhailovich
• Ito Takayuki

Classifications

• C12N9/93 Primary

  Ligases (6) · CPC title

• C07K5/06104

  with the first amino acid being acidic · CPC title

• C07K5/06113

  Asp- or Asn-amino acid · CPC title

• C12Y603/02028

  L-Amino-acid alpha-ligase (6.3.2.28) · CPC title

• C12P21/00 Primary

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title