Co-expression of cytochrome P450 reductases modulates the activity and selectivity of human cytochrome P450 enzymes transiently expressed in Nicotiana benthamiana

The transient co-expression of various redox partners affects the efficiency of human hepatic cytochrome P450 enzymes (CYP) in a plant-based biotransformation system. Using Nicotiana benthamiana as a host, we investigated the interaction of CYP1A2, CYP2D6, and CYP3A4 with different cytochrome P450 reductases (CPRs) derived from animal and plant sources. While human CPR typically enhance or maintain CYP activity, plant-derived CPRs can either enhance or significantly inhibit the metabolism of specific drugs. Additionally, the co-expression of an alternative redox partner, cytochrome b5, can enhance, inhibit, or have no effect on human CYP catalysis, depending on the specific CYP involved. For the majority of CYP/CPR/substrate combinations, we selected variants beneficial for CYP-mediated biotransformation, resulting in a 1.8-4.3-fold increase in product accumulation. We also developed chimeric human-plant CPR variants by swapping structural domains, thereby allowing identification of specific regions responsible for different levels of CYP2D6 productivity. This approach led to the creation of an enhanced CPR variant with a modified transmembrane domain and improved characteristics compared to the native versions.