Engineering and Analysis of a Self-Sufficient Biosynthetic Cytochrome P450 PikC Fused to the RhFRED Reductase Domain

Cytochrome P450 enzymes mediate important oxidative processes in biological systems including regio- and stereospecific hydroxylation and epoxidation reactions. The inherent requirement of these biomolecules for separate redox partner(s) significantly limits their application in biotechnology. To address this challenge, naturally occurring and/or bioengineered self-sufficient P450 systems with covalently fused redox partners have been utilized to harness their catalytic power. In this study, we describe the first in vitro characterization of a bacterial biosynthetic cytochrome P450 PikC fused to a heterologous reductase domain RhFRED that demonstrates single-component self-sufficiency. This novel fusion system not only produces a more active and effective biocatalyst but also suggests a general design for a universal reductase to generate diverse self-sufficient fusions for functional identification or industrial applications of biosynthetic P450s.