Hydroxylase gene and use thereof
A vitamin D 3 hydroxylase is purified from Pseudonocardia autotrophica cell, and a primer is designed based on amino acid sequence obtained from hydroxylase. Subsequently, PCR is conducted using genomic DNA of Pseudonocardia autotrophica as a template to clone a gene for the vitamin D 3 hydroxylase. By conducting a conversion reaction using a microorganism in which the vitamin D 3 hydroxylase gene is expressed using a proper expression system, a hydroxide of vitamin D or the like (e.g., hydroxy vitamin D 3 ) can be produced with high efficiency.
1. An isolated polynucleotide encoding a hydroxylase selected from (a)-(c):
(a) an isolated polynucleotide comprising nucleotides 320-1531 of SEQ ID NO: 1; and
(b) an isolated polynucleotide that hybridizes under stringent conditions to a polynucleotide entirely complementary to nucleotides 320-1531 of SEQ ID NO: 1, wherein said hybridization under stringent conditions is hybridization at 65° C. in the presence of 0.7-1.0M sodium chloride; and
(c) an isolated polynucleotide comprising a polynucleotide sequence having at least 90% nucleic acid sequence identity to nucleotides 320-1531 of SEQ ID NO: 1.
2. An isolated host cell transformed with the polynucleotide of claim 1 .
3. The host cell of claim 2 , wherein said host cell is Escherichia coli or an actinomycete.
4. A method for producing a hydroxide of a vitamin D compound or 7-dehydrocholesterol, comprising:
(a) culturing the cell of claim 2 in a culture medium under conditions in which the polypeptide encoded by said polynucleotide is expressed; and
(b) contacting a vitamin D compound or 7-dehydrocholesterol with said culture medium, or with a purified extract of said culture medium containing said polypeptide, whereby a hydroxide of said vitamin D compound or 7-dehydrocholesterol is produced.
5. The method of claim 4 , wherein said cell further expresses redox partner proteins during said culturing.
6. The method of claim 4 , wherein the hydroxide of the vitamin D compound is 1α, 25-dihydroxy vitamin D 3 .
7. The method of claim 4 , wherein the hydroxide of the vitamin D compound is 25-dihydroxy vitamin D 3 .
8. The method of claim 4 , wherein the hydroxide of the vitamin D compound is 25-dihydroxy vitamin D 2 .
9. An isolated polynucleotide selected from (a) and (b):
(a) an isolated polynucleotide comprising a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2; and
(b) an isolated polynucleotide that hybridizes under stringent conditions to a polynucleotide entirely complementary to a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2, wherein said hybridization under stringent conditions is hybridization at 65° C. in the presence of 0.7-1.0M sodium chloride, wherein the polynucleotide encodes a hydroxylase.