Identification and Characterization of Cyclosporin A-specific Hydroxylation System in a Rare Actinomycetes, Sebekia benihana

초록

The cyclic undecapeptide, cyclosporin A (CsA), is one of the most commonly-prescribed immunosuppressive drugs, and generated non-ribosomally from a multi-functional cyclosporin synthetase enzyme complex by the filamentous fungus, Tolypocladium niveum. Previously, a rare actinomycetes named Sebekia benihana was identified to be able to hydroxylate CsA at the position of 4th N-methyl leucine, of which the regiospecically-modified CsA derivative retained hair growth stimulating side effect without immunosuppressive activity. In this work, we identified more than 20 novel Cytochrome P450 hydroxylase (CYP) genes from S. benihana using whole genome scanning, followed by their complete sequencing and in silico characterization. Since a rare actinomycetes species such as S. benihana was not feasible for routine genetic manipulation, we first established some of the most commonly-practiced genome engineering techniques including foreign gene transformation, expression vector system development, and specific targeted-gene disruption. Through individual CYP gene disruption and complementation assays, we finally identified the key CsA-regiospecific CYP gene in S. benihana. The results described here set the stage for maximization of its potential as bioconversion resources of various valuable metabolites including CsA in rare actinomycetes species.