Identification of a cyclosporine-specific P450 hydroxylase gene through targeted cytochrome P450 complement (CYPome) disruption in Sebekia benihana.
It was previously proposed that regio-specific hydroxylation of an immunosuppressive cyclosporine (CsA) at the 4th N-methyl leucine is mediated by cytochrome P450 hydroxylase (CYP) in the rare actinomycete Sebekia benihana. This modification is thought to be the reason for the hair growth-promoting side effect without the immunosuppressive activity of CsA. Through S. benihana genome sequencing and in silico analysis, we identified the complete cytochrome P450 complement (CYPome) of S. benihana, including 21 CYPs and their electron transfer partners, consisting of 7 ferredoxins (FDs) and 4 ferredoxin reductases (FDRs). Using Escherichia coli conjugation-based S. benihana CYPome-targeted disruption, all of the identified CYP, FD, and FDR genes in S. benihana were individually inactivated. Among the 32 S. benihana exconjugant mutants tested, only a single S. benihana CYP mutant, ΔCYP-sb21, failed to exhibit CsA hydroxylation activity. The hydroxylation was restored by CYP-sb21 gene complementation. Since all S. benihana FD and FDR disruption mutants maintained CsA hydroxylation activity, it can be concluded that CYP-sb21, a new member of the bacterial CYP107 family, is the only essential component of the in vivo regio-specific CsA hydroxylation process in S. benihana. Moreover, expression of an extra copy of the CYP-sb21 gene increased CsA hydroxylation in wild-type S. benihana and an NADPH-enriched Streptomyces coelicolor mutant, by 2-fold and 1.5-fold, respectively. These results show for the first time that regio-specific hydroxylation of CsA is carried out by a specific P450 hydroxylase present in S. benihana, and they set the stage for the biotechnological application of regio-specific CsA hydroxylation through heterologous CYP-sb21 expression.
Department of Biological Engineering, Inha University, Incheon, South Korea.
Mi-Jin
MJ
Lee
Hyun-Bum
HB
Kim
Yeo Joon
YJ
Yoon
Kyuboem
K
Han
Eung-Soo
ES
Kim
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D019295:Computational Biology; D016572:Cyclosporine / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D016680:Genome, Bacterial; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D048372:Streptomyces coelicolor / Q000235:genetics
D004269:DNA, Bacterial; D016572:Cyclosporine; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases