Cloning and expression of a cytochrome P450 hydroxylase gene from Amycolatopsis orientalis: hydroxylation of epothilone B for the production of epothilone F.
Degenerate PCR primers were used to amplify cytochrome P450 gene fragments from the high-GC gram-negative bacteria Amycolatopsis orientalis, which catalyzes the hydroxylation of epothilone B to produce epothilone F. The amplified fragments were used as hybridization probes to identify and clone two intact cytochrome P450 genes. The expression of one of the cloned genes in a Streptomyces lividans transformant resulted in the biotransformation of epothilone B to epothilone F. The conversion of epothilone B to epothilone F by the S. lividans transformant was confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy.
Basch
Jonathan
J
Technical Operations, Bristol-Myers Squibb Company, P.O. Box 4755, Syracuse, NY 13221-4755, USA. Jonathan.basch@bms.com
Chiang
Shu-Jen
SJ
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001709:Biotechnology / Q000379:methods; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D017931:DNA Primers; D034261:Epothilones / Q000096:biosynthesis* / Q000378:metabolism*; D006900:Hydroxylation; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D048170:Streptomyces lividans / Q000201:enzymology / Q000235:genetics / Q000254:growth & development
D017931:DNA Primers; D034261:Epothilones; C426625:epothilone F; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C093788:epothilone B