P133: Identification and characterization of Cyclosporine-specific Cytochrome P450 hydroxylase in Pseudonocardia autotrophica

Cyclosporine A(CsA) is one of most commonly-prescribed immunosuppressive compound, and generated non-ribosomally from a multi-functional cyclosporine synthetase enzyme complex by filamentous fungus, Tolypocladium niveum. It was previously proposed that regio-specific hydroxylation of an immunosuppressive CsA at the 4^th N-methyl leucine is mediated by cytochrome P450 hydroxylase (CYP) in the rare actinomycetes named Sebekia benihana, leading to maintaining hair growth promoting side effect without the immunosuppressive activity of CsA. In this study, we confirmed that another rare actinomycetes named Pseudoncardia autotrophica is also able to perform region-specific hydroxylations of CsA. Interestingly, P. autotrophica was revealed to hydroxylate CsA at 4^th N-methyl leucine (mono-hydroxylation) as well as 4^th and 9^th N-methyl leucines (di-hydroxylation). Through P. autotrophica whole genome sequencing and in silico analysis, we classified the entire CYPome including their electron transfer partners such as ferredoxins and ferredoxin reductases. Three putative CYP genes showing high sequence homologies with the CsA-specific CYP in Sebekia benihana were successfully knocked out using Escherichia coli conjugation-based PCR targeted gene disruption. The more detailed results on P. autotrophica CYP disruption and complementation will be further discussed.