Self-sufficient P450-reductase chimeras for biocatalysis

In recent years, cytochromes P450 have emerged as powerful, versatile biocatalysts for the site-selective functionalization of small molecules. Catalyzing an impressive range of chemical transformations, these enzymes have been widely used to effect C-H oxidation, biaryl coupling, and carbon-heteroatom bond formation, among many other reactions. However, the majority of P450s are multi-protein systems that employ secondary redox partners in key steps of the catalytic cycle, which limits their broader applicability. In response, the discovery of self-sufficient P450s, such as P450_BM3 and P450_RhF, has provided a template for the construction of artificial, self-sufficient P450-reductase fusions. In this chapter, we describe a procedure for the design, assembly, and application of two engineered, self-sufficient P450s of Streptomyces origin via fusion with an exogenous reductase domain. In particular, we generated artificial chimeras of P450s PtmO5 and TleB by linking them covalently with the reductase domain of P450_RhF. Upon verification of their activities, both enzymes were employed in preparative-scale biocatalytic reactions. This approach can feasibly be applied to any P450 of interest, thereby laying the groundwork for the production of self-sufficient P450s for diverse chemical applications.