Method for producing hydroxy-L-pipecolic acid

Patent metadata

Field	Value
Publication number	US-12173330-B2
Application number	US-202318157226-A
Country	US
Kind code	B2
Filing date	Jan 20, 2023
Priority date	Oct 2, 2015
Publication date	Dec 24, 2024
Grant date	Dec 24, 2024

Abstract

A method of producing high-purity hydroxy-L-pipecolic acids that includes allowing an L-pipecolic acid hydroxylase, a microorganism or cell having the ability to produce the enzyme, a processed product of the microorganism or cell, and/or a culture liquid comprising the enzyme and obtained by culturing the microorganism or cell, to act on L-pipecolic acid as a substrate in the presence of 2-oxoglutaric acid and ferrous ion, wherein the L-pipecolic acid hydroxylase (1) acts on L-pipecolic acid in the presence of 2-oxoglutaric acid and ferrous ion to add a hydroxy group to the carbon atom at positions 3, 4, and/or 5 of L-pipecolic acid; and (2) has a catalytic efficiency (kcat/Km) with L-proline that is equal to or less than 7 times the catalytic efficiency (kcat/Km) with L-pipecolic acid.

First claim

The invention claimed is:

1. A method of producing a (5S)-hydroxy-L-pipecolic acid without producing a hydroxy-L-proline, the method comprising:
allowing an L-pipecolic acid hydroxylase, a microorganism or cell having the ability to produce the enzyme, a processed product of the microorganism or cell, and/or a culture liquid comprising the enzyme and obtained by culturing the microorganism or cell, to act on L-pipecolic acid as a substrate in the presence of 2-oxoglutaric acid and ferrous ion,
wherein the L-pipecolic acid hydroxylase has properties (1) and (2) below:
(1) the enzyme can act on L-pipecolic acid in the presence of 2-oxoglutaric acid and ferrous ion to add a hydroxy group to the carbon atom at positions 3, 4, and/or 5 of L-pipecolic acid; and
(2) the enzyme has a catalytic efficiency (kcat/Km) with L-proline that is equal to or less than 7 times the catalytic efficiency (kcat/Km) with L-pipecolic acid,

wherein the content of L-proline in the L-pipecolic acid as the substrate is not more than 10% (w/w), and
wherein the L-pipecolic acid hydroxylase comprises one or more proteins selected from (A), (B), and (C) below:
(A) a protein consisting of the amino acid sequence of SEQ ID NO: 2;
(B) a protein having the same amino acid sequence as the amino acid sequence of SEQ ID NO: 2, except that 1 to 10 amino acids are deleted, substituted, inserted, and/or added, which protein has L-pipecolic acid hydroxylase activity; and
(C) a protein having an amino acid sequence with an identity of not less than 90% to the amino acid sequence of SEQ ID NO: 2, which protein has the L-pipecolic acid hydroxylase activity.

2. The method of producing a hydroxy-L-pipecolic acid according to claim 1 , wherein the L-pipecolic acid as the substrate is produced by:
allowing L-lysine and/or DL-lysine to react with:
(i-1) one or more enzymes selected from the group consisting of an L-amino acid oxidase, an L-amino acid dehydrogenase and an L-amino acid aminotransferase, or
(i-2) an amino acid racemase and one or more enzymes selected from the group consisting of a D-amino acid oxidase, a D-amino acid dehydrogenase and a D-amino acid aminotransferase,

for the production of 3,4,5,6-tetrahydropyridine-2-carboxylic acid; and subsequently
allowing an N-methyl-L-amino acid dehydrogenase to act on the 3,4,5,6-tetrahydropyridine-2-carboxylic acid for the production of the L-pipecolic acid.

3. The method of producing a hydroxy-L-pipecolic acid according to claim 1 , wherein the L-pipecolic acid as the substrate is produced by:
allowing L-lysine to react with one or more enzymes selected from the group consisting of an L-lysine 6-oxidase, an L-lysine 6-dehydrogenase and an L-lysine 6-aminotransferase, for the production of 2,3,4,5-tetrahydropyridine-2-carboxylic acid; and subsequently
allowing a pyrroline-5-carboxylate reductase to act on the 2,3,4,5-tetrahydropyridine-2-carboxylic acid for the production of the L-pipecolic acid.

4. The method of producing a hydroxy-L-pipecolic acid according to claim 1 , wherein the L-pipecolic acid as the substrate is produced by allowing a lysine cyclodeaminase to act on L-lysine for the production of the L-pipecolic acid.

5. The method of producing a (5S)-hydroxy-L-pipecolic acid according to claim 1 , wherein the microorganism or cell having the ability to produce the enzyme is a microorganism or cell transformed with DNA encoding the L-pipecolic acid hydroxylase comprises one or more DNAs selected from (D), (E), and (F) below:
(D) DNA having a nucleotide sequence of SEQ ID NO: 1;
(E) DNA comprising the same nucleotide sequence as the nucleotide sequence of SEQ ID NO: 1, except that 1 to 30 bases are deleted, substituted, inserted, and/or added, which DNA encodes a protein having the L-pipecolic acid hydroxylase activity; and
(F) DNA comprising a nucleotide sequence which hybridizes with a complementary strand of the nucleotide sequence of SEQ ID NO: 1 under stringent conditions, wherein the conditions comprise a hybridization at 65° C. in the presence of 0.7 mol/L to 1.0 mol/L sodium chloride aqueous solution and a washing under a temperature condition of 65° C. by using one solution with an SSC concentration of 0.1×SSC (the composition of 1×SSC: 150 mmol/L sodium chloride aqueous solution, 15 mmol/L sodium citrate aqueous solution), which DNA encodes a protein having the L-pipecolic acid hydroxylase activity.

6. The method of producing a hydroxy-L-pipecolic acid according to claim 1 , wherein the protein (C) is a protein having an amino acid sequence with an identity of not less than 95% to the amino acid sequence of SEQ ID NO: 2, which protein has the L-pipecolic acid hydroxylase activity.

Assignees

• Api Corp

Inventors

• Miyake Ryoma
• Kawabata Hiroshi

Classifications

• C12Y114/11028

  Proline 3-hydroxylase (1.14.11.28) · CPC title

• C12P17/12 Primary

  containing a six-membered hetero ring · CPC title

• C12N9/0022

  with oxygen as acceptor (1.4.3) · CPC title

• C12N9/0016

  with NAD or NADP as acceptor (1.4.1) · CPC title

• C12N9/1096

  transferring nitrogenous groups (2.6) · CPC title