Pipecolinic acid 4-hydroxylase and method for producing 4-hydroxy amino acid using same
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.
1. A method for producing a 4-hydroxy amino acid, said method comprising reacting:
a pipecolic acid 4-hydroxylase protein, 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 in the presence of 2-oxoglutaric acid and iron(II) ions to produce the 4-hydroxy amino acid;
wherein the pipecolic acid 4-hydroxylase protein is selected from the group consisting of:
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, or 18; and
(B) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, or 18 except that 1 to 20 amino acids are deleted, substituted, and/or added, and having pipecolic acid 4-hydroxylase activity.
2. The method for producing a 4-hydroxy amino acid according to claim 1 , wherein said α-amino acid has 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 has 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.
3. The method for producing a 4-hydroxy amino acid according to claim 1 , wherein said α-amino acid is pipecolic acid, and said 4-hydroxy amino acid is 4-hydroxy-L-pipecolic acid.
4. The method for producing a 4-hydroxy amino acid according to claim 1 , wherein said cell comprising pipecolic acid 4-hydroxylase is a cell transformed with a DNA encoding pipecolic acid 4-hydroxylase protein.
5. The method for producing a 4-hydroxy amino acid according to claim 4 , wherein said DNA encoding pipecolic acid 4-hydroxylase protein is selected from the group consisting of:
(D) a DNA comprising the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17; and
(E) a DNA comprising the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17 except that 1 to 60 nucleotides are substituted, deleted, and/or added, and encoding a polypeptide having pipecolic acid 4-hydroxylase activity.