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 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,
wherein the pipecolic acid 4-hydroxylase protein is selected from the group consisting of the following (A), (B-1), (B-2), (B-3), (B-4), (C-1), and (C-2), and the pipecolic acid 4-hydroxylase protein is a recombinant non-glycosylated protein:
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or 18;
(B-1) a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, 6, 12, or 18 except that one to ten amino acids are deleted, substituted, and/or added, and having pipecolic acid 4-hydroxylase activity;
(B-2) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 except that one to five amino acids are deleted, substituted, and/or added, and having pipecolic acid 4-hydroxylase activity;
(B-3) a polypeptide comprising the amino acid sequence of SEQ ID NO: 8 or 10 except that one amino acid is deleted, substituted, and/or added, and having pipecolic acid 4-hydroxylase activity;
(B-4) a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 except that one to five amino acids are deleted, substituted, and/or added, and having pipecolic acid 4-hydroxylase activity;
(C-1) a polypeptide comprising an amino acid sequence that is not less than 80% but not 100% identical to the amino acid sequence of SEQ ID NO: 4, 6, 12, or 18, and having pipecolic acid 4-hydroxylase activity; and
(C-2) a polypeptide comprising an amino acid sequence that is not less than 95% but not 100% identical to the amino acid sequence of SEQ ID NO: 2, and having pipecolic acid 4-hydroxylase activity.
2. A microorganism having no glycosylation ability transformed with a DNA encoding a pipecolic acid 4-hydroxylase protein selected from the group consisting of the following (D), (E-1), (E-2), (E-3), and (E-4):
(D) a DNA comprising the nucleotide sequence of SEQ ID NO: 15 or 17;
(E-1) a DNA comprising the nucleotide sequence of SEQ ID NO: 3, 5, 11, or 17 except that one to three nucleotides are substituted, deleted, and/or added, and encoding a polypeptide having pipecolic acid 4-hydroxylase activity;
(E-2) a DNA comprising the nucleotide sequence of SEQ ID NO: 1 except that one to three nucleotides are substituted, deleted, and/or added, and encoding a polypeptide having pipecolic acid 4-hydroxylase activity;
(E-3) a DNA comprising the nucleotide sequence of SEQ ID NO: 7 or 9 except that one nucleotide is substituted, deleted, and/or added, and encoding a polypeptide having pipecolic acid 4-hydroxylase activity; and
(E-4) a DNA comprising the nucleotide sequence of SEQ ID NO: 15 except that one to three nucleotides are substituted, deleted, and/or added, and encoding a polypeptide having pipecolic acid 4-hydroxylase activity.
3. The microorganism according to claim 2 , which is selected from the group consisting of the genera Escherichia, Bacillus, Pseudomonas , and Corynebacterium.