Production method for l-cyclic amino acids

Patent metadata

Field	Value
Publication number	US-2022275410-A1
Application number	US-202017606123-A
Country	US
Kind code	A1
Filing date	May 15, 2020
Priority date	Apr 25, 2019
Publication date	Sep 1, 2022
Grant date	—

Abstract

An object of the present invention is to provide a method of industrially producing a high-purity L-cyclic amino acid more inexpensively and with a high efficiency, from a cyclic amino acid having a double bond at the 1-position. The present invention provides a method in which an L-cyclic amino acid is produced by allowing a cyclic amino acid having a double bond at the 1-position to react with a specific enzyme having a catalytic ability to reduce a cyclic amino acid having a double bond at the 1-position to produce an L-cyclic amino acid.

First claim

1 . A method of producing an L-cyclic amino acid, the method comprising bringing a cyclic amino acid having a double bond at the 1-position and represented by the following general formula (I):

wherein A represents an alkylene chain which has a chain length of from 1 to 4 atoms, which optionally contains at least one hetero atom selected from the group consisting of a sulfur atom, an oxygen atom and a nitrogen atom, in the chain or at the end of the chain, and which optionally has a substituent,
into contact with a polypeptide shown in (A), (B) or (C) below, a microorganism or cell having the ability to produce said polypeptide or comprising said polypeptide, a processed product of the microorganism or the cell, and/or a culture liquid obtained by culturing the microorganism or the cell and comprising said polypeptide, to produce an L-cyclic amino acid represented by the following general formula (II)

wherein A is the same as defined above:
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12;
(B) a polypeptide which comprises the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12 except that one or several amino acids are deleted, substituted and/or added, and which has the ability to catalyze the reaction represented by the following formula (1) to produce the L-cyclic amino acid:

wherein A is the same as defined above;
or
(C) a polypeptide which comprises an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12, and which has the ability to catalyze the reaction represented by the formula (1) to produce the L-cyclic amino acid.

2 . The method of producing an L-cyclic amino acid according to claim 1 , wherein the polypeptide is encoded by a nucleic acid shown in (D), (E) or (F) below:
(D) a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11; (E) a nucleic acid which comprises the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11 except that one or several nucleotides are substituted, deleted and/or added, and which encodes a polypeptide having the ability to catalyze the reaction represented by the formula (1) to produce the L-cyclic amino acid; or (F) a nucleic acid which comprises a nucleotide sequence that hybridizes with a complementary strand of the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11 under stringent conditions, and which encodes a polypeptide having the ability to catalyze the reaction represented by the formula (1) to produce the L-cyclic amino acid.

3 . A method of producing an L-cyclic amino acid, the method comprising:
allowing an acyclic α, ω-diamino acid represented by the following general formula (III):

wherein A represents an alkylene chain which has a chain length of from 1 to 4 atoms, which optionally contains at least one hetero atom selected from the group consisting of a sulfur atom, an oxygen atom and a nitrogen atom, in the chain or at the end of the chain, and which optionally has a substituent,
to react with an enzyme capable of converting the amino group at the α-position of the diamino acid to a keto group and producing an α-keto acid, to produce a cyclic amino acid having a double bond at the 1-position and represented by the following general formula (I):

wherein A is the same as defined above;

and
then producing an L-cyclic amino acid represented by the following general formula (II):

wherein A is the same as defined above,
from the resulting cyclic amino acid having a double bond at the 1-position by the method according to claim 1 .

4 . The method of producing an L-cyclic amino acid according to claim 3 , wherein the enzyme capable of converting the amino group at the α-position of the diamino acid to a keto group and producing an α-keto acid is one or more enzymes selected from the group consisting of a D-amino acid oxidase, an L-amino acid oxidase, a D-amino acid dehydrogenase, an L-amino acid dehydrogenase, a D-amino acid aminotransferase and an L-amino acid aminotransferase.

5 . The method of producing an L-cyclic amino acid according to claim 1 , wherein the cyclic amino acid having a double bond at the 1-position and represented by the general formula (I) is Δ 1 -piperidine-2-carboxylic acid, and the L-cyclic amino acid represented by the general formula (II) is L-pipecolic acid.

6 . A polypeptide shown in (a), (b) or (c) below:
(a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10 or 12; (b) a polypeptide which comprises the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12 except that one or several amino acids are deleted, substituted and/or added, and which has the ability to catalyze the reaction represented by the following formula (1) to produce the L-cyclic amino acid:

wherein A represents an alkylene chain which has a chain length of from 1 to 4 atoms, which optionally contains at least one hetero atom selected from the group consisting of a sulfur atom, an oxygen atom and a nitrogen atom, in the chain or at the end of the chain, and which optionally has a substituent;

or
(c) a polypeptide which comprises an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12, and which has the ability to catalyze the reaction represented by the formula (1) to produce the L-cyclic amino acid.

7 . A nucleic acid encoding the polypeptide according to claim 6 .

8 . The nucleic acid according to claim 7 , wherein the nucleic acid is derived from a plant.

9 . The nucleic acid according to claim 8 , wherein the plant is a plant belonging to the genus Morus or Lathyrus japonicus.

10 . The nucleic acid according to claim 7 ,
wherein the nucleic acid is a nucleic acid shown in (d), (e) or (f) below: (d) a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 3, 5, 7, 9 or 11; (e) a nucleic acid which comprises the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11 except that one or several nucleotides are substituted, deleted and/or added, and which encodes a polypeptide having the ability to catalyze the reaction represented by the formula (1) to produce the L-cyclic amino acid; or (f) a nucleic acid which comprises a nucleotide sequence that hybridizes with a complementary strand of the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11 under stringent conditions, and which encodes a polypeptide having the ability to catalyze the reaction represented by the formula (1) to produce the L-cyclic amino acid.

11 . A recombinant vector comprising the nucleic acid according to claim 7 .

12 . A transformant comprising the recombinant vector according to claim 11 .

Assignees

• Api Corp

Inventors

• Mizutani Masaharu
• Miyake Ryoma
• Kawabata Hiroshi

Classifications

• C12N9/1096

  transferring nitrogenous groups (2.6) · CPC title

• C12Y206/01

  Transaminases (2.6.1) · CPC title

• C40B40/08

  Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries · CPC title

• C12Y105/01021

  DELTA1-piperideine-2-carboxylate reductase (1.5.1.21) · CPC title

• C12P13/04 Primary

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