Method for manufacturing cis-5-hydroxy-L-pipecolic acid

Patent metadata

Field	Value
Publication number	US-10087473-B2
Application number	US-201515526031-A
Country	US
Kind code	B2
Filing date	Nov 2, 2015
Priority date	Nov 12, 2014
Publication date	Oct 2, 2018
Grant date	Oct 2, 2018

Abstract

A method for producing cis-5-hydroxy-L-pipecolic acid, the method comprising allowing a 2-oxoglutarate-dependent L-pipecolic acid hydroxylase to act on L-pipecolic acid to generate cis-5-hydroxy-L-pipecolic acid, wherein the 2-oxoglutarate-dependent L-pipecolic acid hydroxylase comprises the polypeptide (A), (B) or (C) below: (A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4 or 11; (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4 or 11 except that one or several amino acids are deleted, substituted, and/or added, which polypeptide has 2-oxoglutarate-dependent L-pipecolic acid hydroxylase activity; or (C) a polypeptide comprising an amino acid sequence with an identity of not less than 60% to the amino acid sequence represented by SEQ ID NO: 4 or 11, which polypeptide has 2-oxoglutarate-dependent L-pipecolic acid hydroxylase activity.

First claim

The invention claimed is:

1. A method for producing cis-5-hydroxy-L-pipecolic acid, the method comprising allowing a 2-oxoglutarate-dependent L-pipecolic acid hydroxylase, a microorganism or cell that produces the enzyme, a processed product of the microorganism or cell having the enzyme, and/or a culture liquid comprising the enzyme and obtained by culturing the microorganism or cell, to act on L-pipecolic acid to generate cis-5-hydroxy-L-pipecolic acid,
wherein the 2-oxoglutarate-dependent L-pipecolic acid hydroxylase comprises:
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or 11;
(B) a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or 11 except that 1 to 10 amino acids are deleted, substituted, and/or added, wherein said polypeptide has 2-oxoglutarate-dependent L-pipecolic acid hydroxylase activity; or
(C) a polypeptide comprising an amino acid sequence with an identity of not less than 90% to the amino acid sequence of SEQ ID NO: 4 or 11, wherein said polypeptide has 2-oxoglutarate-dependent L-pipecolic acid hydroxylase activity.

2. The method for producing cis-5-hydroxy-L-pipecolic acid according to claim 1 , wherein DNA encoding the 2-oxoglutarate-dependent L-pipecolic acid hydroxylase comprises:
(a) DNA comprising the nucleotide sequence of SEQ ID NO: 1 or 10;
(b) DNA comprising the nucleotide sequence of SEQ ID NO: 1 or 10 except that 1 to 30 nucleotides are substituted, deleted, and/or added, wherein said DNA encodes a polypeptide having 2-oxoglutarate-dependent L-pipecolic acid hydroxylase activity; or
(c) DNA which hybridizes with the full-length complementary strand of the polynucleotide of SEQ ID NO: 1 or 10 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 ranging from 0.1 to 2×SSC (the composition of 1×SSC: 150 mmol/L sodium chloride aqueous solution, 15 mmol/L sodium citrate aqueous solution), wherein said DNA encodes a polypeptide having 2-oxoglutarate-dependent L-pipecolic acid hydroxylase activity.

3. The method for producing cis-5-hydroxy-L-pipecolic acid according to claim 1 , wherein the 2-oxoglutarate-dependent L-pipecolic acid hydroxylase, the microorganism or cell that produces the enzyme, the processed product of the microorganism or cell having the enzyme, and/or the culture liquid comprising the enzyme and obtained by culturing the microorganism or cell is/are allowed to act on said L-pipecolic acid in the presence of 2-oxoglutaric acid and a ferrous ion.

4. The method for producing cis-5-hydroxy-L-pipecolic acid according to claim 2 , wherein the 2-oxoglutarate-dependent L-pipecolic acid hydroxylase, the microorganism or cell that produces the enzyme, the processed product of the microorganism or cell having the enzyme, and/or the culture liquid comprising the enzyme and obtained by culturing the microorganism or cell is/are allowed to act on said L-pipecolic acid in the presence of 2-oxoglutaric acid and a ferrous ion.

5. A 2-oxoglutarate-dependent L-pipecolic acid hydroxylase protein having an activity to act on L-pipecolic acid to generate cis-5-hydroxy-L-pipecolic acid, wherein said 2-oxoglutarate-dependent L-pipecolic acid hydroxylase protein comprises:
a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or 11 except that 1-10 amino acids are substituted and/or added, wherein said polypeptide has 2-oxoglutarate-dependent L-pipecolic acid hydroxylase activity.

Assignees

• Api Corp

Inventors

• Miyake Ryoma
• Dekishima Yasumasa

Classifications

• C12N9/0071 Primary

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title

• C12Y114/11

  with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11) · CPC title

• C12P17/12 Primary

  containing a six-membered hetero ring · CPC title

• C12N15/09

  Recombinant DNA-technology · CPC title