Novel hydrolase and method for producing (1s,2s)-1-alkoxycarbonyl-2-vinylcyclopropane carboxylic acid using same

Patent metadata

Field	Value
Publication number	US-2023323317-A1
Application number	US-202218091687-A
Country	US
Kind code	A1
Filing date	Dec 30, 2022
Priority date	Mar 30, 2018
Publication date	Oct 12, 2023
Grant date	—

Abstract

The present invention provides a novel hydrolase that can industrially produce optically highly pure (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropane carboxylic acid with high efficiency at low costs, and a production method using the hydrolase.

First claim

What is claimed is:

1 . A hydrolase comprising a polypeptide of the following (a), (b), or (c):
(a) a polypeptide consisting of an amino acid sequence resulting from the deletion, insertion, substitution and/or addition of one or plural amino acids in the amino acid sequence shown in SEQ ID NO: 4 or 6, and an activity to catalyze the reaction shown in the formula (I)

 wherein R is an alkyl group having 1-6 carbon atoms in the formula (1), and wherein the ratio of Syn isomer to Anti isomer in the formula (II) is equal to or less than 0.2%;
(b) a polypeptide consisting of an amino acid sequence with not less than 95% of sequence identity with the amino acid sequence shown in SEQ ID NO: 4 or 6, and an activity to catalyze the reaction shown in the above-mentioned formula (1);
(c) a polypeptide consisting of an amino acid sequence with not less than 90% of sequence identity with the amino acid sequence shown in SEQ ID NO: 4 or 6, wherein the aforementioned amino acid sequence comprises one or more motif sequences selected from the following motif sequences (i)-(iv):
(i) a sequence consisting of 5 consecutive residues (aspartic acid (D), alanine (A), threonine (T), arginine (R), glycine (G));
(ii) a sequence consisting of 4 consecutive residues (proline (P), tyrosine (Y), glycine (G), phenylalanine (F));
(iii) a sequence consisting of 4 consecutive residues (asparagine (N), tryptophan (W), proline (P), glycine (G));
(iv) a sequence consisting of 5 consecutive residues (proline (P), glycine (G), tryptophan (W), proline (P), glycine (G)).

2 . The hydrolase according to claim 1 , wherein R in the formula (1) is an ethyl group.

3 . A nucleic acid encoding the hydrolase according to claim 1 .

4 . The nucleic acid according to claim 3 , wherein the aforementioned nucleic acid comprises a base sequence of the following (d), (e), (1), or (g):
(d) the base sequence shown in SEQ ID NO: 3 or 5; (e) a nucleic acid consisting of a base sequence resulting from the substitution, deletion, and/or addition of one or plural bases in the base sequence shown in SEQ ID NO: 3 or 5, and encoding a polypeptide having an activity to catalyze the reaction shown in the formula (1)

 wherein R is an alkyl group having 1-6 carbon atoms in the formula (1), and wherein the ratio of Syn isomer to Anti isomer in the formula (II) is equal to or less than 0.2%;
(f) a nucleic acid consisting of a base sequence having not less than 90% of sequence identity with the base sequence shown in SEQ ID NO: 3 or 5, and encoding a polypeptide having an activity to catalyze the reaction shown in the above-mentioned formula (1);
(g) a nucleic acid having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 3 or 5 under stringent conditions, and encoding a polypeptide having an activity to catalyze the reaction shown in the above-mentioned formula (1).

5 . A method for producing (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropane carboxylic acid, comprising bringing the hydrolase according to claim 1 , a microorganism or cell having an ability to produce the aforementioned enzyme, a treated product of the aforementioned microorganism or cell, and/or a culture solution containing the aforementioned enzyme obtained by culturing the aforementioned microorganism or cell into contact with dialkyl 2-vinylcyclopropane-1,1-dicarboxylic acid represented by the formula (1) to produce (1 S,2S)-1-alkoxycarbonyl-2-vinylcyclopropane carboxylic acid represented by the formula (II).

6 . The production method according to claim 5 , wherein R in the formula (1) is an ethyl group.

7 . The production method according to claim 5 , wherein the microorganism or cell is a microorganism or cell transformed with a nucleic acid encoding a hydrolase comprising a polypeptide of the following (a), (b), or (c):
(a) a polypeptide consisting of an amino acid sequence resulting from the deletion, insertion, substitution and/or addition of one or plural amino acids in the amino acid sequence shown in SEQ ID NO: 4 or 6, and an activity to catalyze the reaction shown in the formula (1)

 wherein R is an alkyl group having 1-6 carbon atoms in the formula (1), and wherein the ratio of Syn isomer to Anti isomer in the formula (II) is equal to or less than 0.2%;
(b) a polypeptide consisting of an amino acid sequence with not less than 60% of sequence identity with the amino acid sequence shown in SEQ ID NO: 4 or 6, and an activity to catalyze the reaction shown in the above-mentioned formula (1);
(c) a polypeptide consisting of an amino acid sequence with not less than 60% of sequence identity with the amino acid sequence shown in SEQ ID NO: 4 or 6, wherein the aforementioned amino acid sequence comprises one or more motif sequences selected from the following motif sequences (i)-(iv):
(i) a sequence consisting of 5 consecutive residues (aspartic acid (D)), alanine (A), threonine (T), arginine (R), glycine (G));
(ii) a sequence consisting of 4 consecutive residues (proline (P), tyrosine (Y), glycine (G), phenylalanine (F));
(iii) a sequence consisting of 4 consecutive residues (asparagine (N), tryptophan (W), proline (P), glycine (G));
(iv) a sequence consisting of 5 consecutive residues (proline (P), glycine (G), tryptophan (W), proline (P), glycine (G)).

8 . A recombinant vector comprising the nucleic acid according to claim 3 .

9 . A transformant comprising the recombinant vector according to claim 8 .

10 . The production method according to claim 5 , wherein the microorganism or cell is a microorganism or cell transformed with a nucleic acid comprising a base sequence of the following (d), (e), (f), or (g):
(d) the base sequence shown in SEQ ID NO: 3 or 5; (e) a nucleic acid consisting of a base sequence resulting from the substitution, deletion, and/or addition of one or plural bases in the base sequence shown in SEQ ID NO: 3 or 5, and encoding a polypeptide having an activity to catalyze the reaction shown in the formula (1)

 wherein R is an alkyl group having 1-6 carbon atoms in the formula (1), and wherein the ratio of Syn isomer to Anti isomer in the formula (II) is equal to or less than 0.2%;
(f) a nucleic acid consisting of a base sequence having not less than 60% of sequence identity with the base sequence shown in SEQ ID NO: 3 or 5, and encoding a polypeptide having an activity to catalyze the reaction shown in the above-mentioned formula (1);
(g) a nucleic acid having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 3 or 5 under stringent conditions, and encoding a polypeptide having an activity to catalyze the reaction shown in the above-mentioned formula (1).

11 . A recombinant vector comprising the nucleic acid according to claim 4 .

12 . A nucleic acid encoding the hydrolase according to claim 2 .

13 . The nucleic acid according to claim 12 , wherein the aforementioned nucleic acid comprises a base sequence of the following (d), (e), (f) or (g):
(d) the base sequence shown in SEQ ID NO: 3 or 5; (e) a nucleic acid consisting of

Assignees

• Api Corp

Inventors

• Yoshida Toyokazu
• Ishida Koichi
• Miyake Ryoma
• Iura Takanobu
• Kawabata Hiroshi

Classifications

• C12N9/18 Primary

  Carboxylic ester hydrolases {(3.1.1)} · CPC title

• C12P7/62

  Carboxylic acid esters · CPC title

• C12P41/00

  Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture · CPC title

• C12Y301/01

  Carboxylic ester hydrolases (3.1.1) · CPC title

• C12P41/005 Primary

  by esterification of carboxylic acid groups in the enantiomers or the inverse reaction · CPC title