Improvement of cyclosporin a hydroxylation in Sebekia benihana by conjugational transfer of streptomyces coelicolor SC04967, a secondary metabolite regulatory gene

Actinomycetes are Gram-positive soil bacteria and one of the most important industrial microorganisms due to superior biosynthetic capabilities of many valuable secondary metabolites as well as production of various valuable bioconversion enzymes. Among them are cytochrome P450 hydroxylase (CYP), which are hemoproteins encoded by a super family of genes, are universally distributed in most of the organisms from all biological kingdoms. Actinomycetes are a rich source of soluble CYP enzymes, which play critical roles in the bioactivation and detoxification of a wide variety of metabolite biosynthesis and xenobiotic transformation. Cyclosporin A (CyA), one of the most commonly-prescribed immunosuppressive drugs, was previously reported to be hydroxylated at the position of 4th N-methyl leucine by a rare actinomycetes called Sebekia benihana, leading to display different biological activity spectrum such as loss of immunosuppressive activities yet retaining hair growth-stimulating side effect. In order to improve this regio-selective CyA hydroxylation in S. benihana, previously-identified several secondary metabolite up-regulatory genes from Streptomyces coelicolor and S. avermitilis were heterologously overexpressed in 5. benihana using an ermE* promoter-containing Streptomyces integrative expression vector. Among tested, SCO4967 encoding a conserved hypothetical protein significantly stimulated region-specific CyA hydroxylation in S. benihana, implying that some common regulatory systems functioning in both biosynthesis and bioconversion of secondary metabolite might be present in different actinomycetes species.