Pipecolinic acid 4-hydroxylase and method for producing 4-hydroxy amino acid using same

Patent metadata

Field	Value
Publication number	US-2016348081-A1
Application number	US-201515115028-A
Country	US
Kind code	A1
Filing date	Jan 27, 2015
Priority date	Jan 31, 2014
Publication date	Dec 1, 2016
Grant date	—

Abstract

The present invention provides a pipecolic acid 4-hydroxylase protein exemplified by the following (A), (B), and (C), having activity to react with L-pipecolic acid in the presence of 2-oxoglutaric acid and iron(II) ions to produce trans-4-hydroxy-L-pipecolic acid, and a method for producing 4-hydroxy amino acid, which method comprises reacting the pipecolic acid 4-hydroxylase protein, cells containing the protein, a treated product of the cells, and/or a culture liquid obtained by culturing the cells, with α-amino acid to produce 4-hydroxy amino acid:
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18; (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18 except that one or several amino acids are deleted, substituted, and/or added, and having pipecolic acid 4-hydroxylase activity; and (C) a polypeptide having an amino acid sequence that is not less than 80% identical to the amino acid sequence represented by SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18, and having pipecolic acid 4-hydroxylase activity.

First claim

1 . A pipecolic acid 4-hydroxylase protein having an activity to react with L-pipecolic acid in the presence of 2-oxoglutaric acid and iron(II) ions, to produce trans-4-hydroxy-L-pipecolic acid.

2 . The pipecolic acid 4-hydroxylase protein according to claim 1 , selected from the group consisting of the following (A), (B), and (C):
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18; (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18 except that one or several amino acids are deleted, substituted, and/or added, and having pipecolic acid 4-hydroxylase activity; and (C) a polypeptide comprising an amino acid sequence that is not less than 80% identical to the amino acid sequence represented by SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18, and having pipecolic acid 4-hydroxylase activity.

3 . The pipecolic acid 4-hydroxylase protein according to claim 2 , which is produced as a recombinant protein by a host having no glycosylation ability.

4 . A method for producing 4-hydroxy amino acid, said method comprising reacting said pipecolic acid 4-hydroxylase protein according to claim 1 , a cell(s) comprising said protein, a treated product of said cell(s), and/or a culture liquid obtained by culturing said cell(s), with α-amino acid to produce 4-hydroxy amino acid.

5 . The method for producing 4-hydroxy amino acid according to claim 4 , wherein said α-amino acid is represented by the following General Formula (I):

(wherein each of R 1 , R 2 , and R 5 represents a hydrogen atom or C 1 -C 3 alkyl; each of R 3 and R 4 represents a hydrogen atom, C 1 -C 3 alkyl, or hydroxyl; and alternatively R 2 may bind to R 5 or the nitrogen atom of the amino group to form a ring structure), and said 4-hydroxy amino acid is represented by the following General Formula (II):

(wherein each of R 1 , R 2 , and R 5 represents a hydrogen atom or C 1 -C 3 alkyl; each of R 3 and R 4 represents a hydrogen atom, C 1 -C 3 alkyl, or hydroxyl; and alternatively R 2 may bind to R 5 or the nitrogen atom of the amino group to form a ring structure).

6 . The method for producing 4-hydroxy amino acid according to claim 4 , wherein said α-amino acid is pipecolic acid, and said 4-hydroxy amino acid is 4-hydroxy-L-pipecolic acid.

7 . The method for producing 4-hydroxy amino acid according to claim 4 , wherein said cell comprising pipecolic acid 4-hydroxylase is a cell transformed with a DNA encoding pipecolic acid 4-hydroxylase protein.

8 . The method for producing 4-hydroxy amino acid according to claim 7 , wherein said DNA encoding pipecolic acid 4-hydroxylase protein is selected from the group consisting of the following (D), (E), and (F):
(D) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17; (E) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17 except that one or several nucleotides are substituted, deleted, and/or added, and encoding a polypeptide having pipecolic acid 4-hydroxylase activity; and (F) a DNA comprising a nucleotide sequence which hybridizes with the complementary strand of the nucleotide sequence represented by SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17 under stringent conditions, and encoding a polypeptide having pipecolic acid 4-hydroxylase activity.

9 . A microorganism transformed with a DNA encoding a pipecolic acid 4-hydroxylase protein selected from the group consisting of the following (D), (E), and (F):
(D) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17; (E) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17 except that one or several nucleotides are substituted, deleted, and/or added, and encoding a polypeptide having pipecolic acid 4-hydroxylase activity; and (F) a DNA comprising a nucleotide sequence which hybridizes with the complementary strand of the nucleotide sequence represented by SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17 under stringent conditions, and encoding a polypeptide having pipecolic acid 4-hydroxylase activity.

10 . The microorganism according to claim 9 , which is selected from the group consisting of the genera Escherichia, Bacillus, Pseudomonas , and Corynebacterium.

Assignees

• Api Corp

Inventors

• Hibi Makoto
• Ogawa Jun
• Miyake Ryoma
• Kawabata Hiroshi

Classifications

• C12P13/04

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

• C12N9/0071 Primary

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

• C12P17/12

  containing a six-membered hetero ring · CPC title

• C12P17/10

  Nitrogen as only ring hetero atom · CPC title

• C12Y114/00

  Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) · CPC title