Functional Expression and Construction of a Self‐Sufficient Cytochrome P450 Chimera for Efficient Steroidal C14α Hydroxylation in Escherichia coli

ABSTRACT C14‐functionalized steroids enabled diverse biological activities in anti‐gonadotropin and anticancer therapy. However, access to C14‐functionalized steroids was impeded by the deficiency of chemical synthetic methods. Recently, several membrane‐bound fungal cytochrome P450s (CYPs) have been identified with steroid C14α‐hydroxylation activity. However, the lack of efficient heterologous overexpression strategy hampered their further characterization and molecular engineering. In the present study, sequences of fungi‐derived CYP genes encoding putative 14α‐hydroxylase were selected and bioinformatically analyzed. Substitution of the N‐terminal hydrophobic helix by a soluble maltose binding protein tag significantly enhanced the soluble expression level in Escherichia coli . A novel CYP originated from Bipolaris oryzae was discovered with high steroidal C14α‐hydroxylation activity when coupled with the redox partner CPR lun . A catalytically self‐sufficient chimeric CYP‐CPR was built by intramolecular fusion, and the electronic transfer rate was improved. A coenzyme NADPH regeneration system was finally constructed by the co‐expression of glucose dehydrogenase. The developed soluble multi‐enzyme cascade biotransformation system supported the selective C14α‐hydroxylation toward progesterone with a final titer of 34.54 mg/L, the highest level achieved in E. coli ‐based heterologous expression system. This study provides insightful ideas on the functional expression of fungi‐derived CYPs and promises an efficient C14α‐hydroxylation system for steroidal drugs through protein engineering.