Identification of a Cyclosporine-Specific P450 Hydroxylase Gene through Targeted Cytochrome P450 Complement (CYPome) Disruption in Sebekia benihana

Author:

Lee Mi-Jin1,Kim Hyun-Bum1,Yoon Yeo Joon2,Han Kyuboem3,Kim Eung-Soo1

Affiliation:

1. Department of Biological Engineering, Inha University, Incheon, South Korea

2. Department of Chemistry and Nano Science, Ewha Woman's University, Seoul, South Korea

3. Cha Bio & Diostech Bio Pharmaceutical Research Institute, Gangnam-gu, Seoul, South Korea

Abstract

ABSTRACT 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.

Publisher

American Society for Microbiology

Subject

Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology

Reference55 articles.

1. Enhanced Heterologous Expression of Two Streptomyces griseolus Cytochrome P450s and Streptomyces coelicolor Ferredoxin Reductase as Potentially Efficient Hydroxylation Catalysts

2. Bioconversion of monensin by a soil bacterium, Sebekia benihana.;Vaufrey F;J. Antibiot.,1990

3. The cytochrome P450 complement (CYPome) of Streptomyces coelicolor A3(2);Lamb DC;J. Biol. Chem.,2002

4. HopwoodDABibbMJChaterKFKieserTBrutonCJKieserHMLydiateDJSmithCPWardJMSchrempfH (ed). 1985. Genetic manipulation of Streptomyces: a laboratory manual. John Innes Foundation Norwich United Kingdom.

5. Microbial conversion of nigericin in three successive steps, by Sebekia benihana.;Delort AM;J. Antibiot.,1988

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