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
Cited by
14 articles.
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