Reconstitution of the in vitro activity of the cyclosporine-specific P450 hydroxylase from Sebekia benihana and development of a heterologous whole-cell biotransformation system

Li Ma, Lei Du, Hui Chen, Yue Sun, Shan Huang, Xianliang Zheng, Eung Soo Kim*, Shengying Li

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

The cytochrome P450 enzyme CYP-sb21 from Sebekia benihana is capable of catalyzing the site-specific hydroxylation of the immunosuppressant cyclosporine (CsA), leading to the single product γ-hydroxy-N-methyl-L-Leu -CsA (CsA-4-OH). Unlike authentic CsA, this hydroxylated CsA shows significantly reduced immunosuppressive activity while it retains a side effect of CsA, the hair growth stimulation effect. Although CYP-sb21 was previously identified to be responsible for CsA-specific hydroxylation in vivo, the in vitro activity of CYP-sb21 has yet to be established for a deeper understanding of this P450 enzyme and further reaction optimization. In this study, we reconstituted the in vitro activity of CYP-sb21 by using surrogate redox partner proteins of bacterial and cyanobacterial origins. The highest CsA site-specific hydroxylation activity by CYP-sb21 was observed when it was partnered with the cyanobacterial redox system composed of seFdx and seFdR from Synechococcus elongatus PCC 7942. The best bioconversion yields were obtained in the presence of 10% methanol as a cosolvent and an NADPH regeneration system. A heterologous whole-cell biocatalyst using Escherichia coli was also constructed, and the permeability problem was solved by using N-cetyl-N,N,N-trimethylammonium bromide (CTAB). This work provides a useful example for reconstituting a hybrid P450 system and developing it into a promising biocatalyst for industrial application.

Original languageEnglish
JournalApplied and Environmental Microbiology
Volume81
Issue number18
Pages (from-to)6268-6275
Number of pages8
ISSN0099-2240
DOIs
Publication statusPublished - 2015
Externally publishedYes

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