Nicotiana benthamiana as a competitive host for large-scale oxidative biotransformations and an efficient chassis for metabolic engineering of CYP-mediated biosynthetic pathways

Because of their remarkable catalytic diversity, human cytochrome P450 enzymes (CYPs) are valuable tools for biotransformation. They enable regio- and stereospecific oxidation of non-activated hydrocarbon bonds, a process often difficult to achieve by conventional chemical methods. Plants serve as versatile host platforms for expressing foreign CYPs, providing access to cofactor flux and a variety of reducing partners and membrane systems suitable for enzyme integration. However, it is essential to adapt heterologous CYPs to the plant metabolic environment to establish this system as a viable option for large-scale biotechnological applications. To achieve this, we tested various approaches aimed at enhancing the functional activity of heterologous CYPs, including co-expression of heterologous redox partners and adapting CYPs for transfer to various subcellular compartments. While compartmentalization did not enhance catalytic function, we found that the transient co-expression of redox partners significantly influenced the performance of human CYPs in a plant production system. While human cytochrome P450 reductase (CPR) typically enhances or maintains enzyme activity, plant-derived CPRs can either enhance or significantly reduce the metabolism of specific drugs. For most CYP/CPR/substrate combinations, we increased product accumulation by 1.8- to 4.3-fold. By swapping the structural domains of human and plant CPR, we created a hybrid CPR variant that demonstrates improved productivity across all tested CYPs. This variant may serve as a potential universal redox partner for human CYPs expressed in plant hosts. Using scaled transient expression of CYPs and the hybrid CPR gene, we performed preparative biotransformation of the pharmaceutically valuable prodrug yohimbine, yielding around 50 mg of 11-hydroxyyohimbine. This represents the highest ever reported CYP-mediated biotransformation of exogenous substrates in a plant system, comparable to that achieved in yeast. To diversify the spectrum of natural products, we integrated human CYP and CPR enzymes into the engineered irregular monoterpene pathways. These secondary metabolites function as pheromones and insecticides, and demonstrate cytotoxic and antibiotic activities. We established a sustainable platform for irregular monoterpene accumulation in Nicotiana benthamiana, achieving the highest level in plant systems. Two CYPs converted branched and cyclopropane monoterpenes into novel hydroxylated derivatives, with human CPR co-expression aiding in product accumulation. Thus, by integrating human CYPs into a network of heterologous genes from different organisms, we initiated new biosynthetic pathways in N. benthamiana and expanded the range of irregular monoterpene structures. Acknowledgements: YVS acknowledges the financial support from the German Research Foundation (DFG), grant number 516587177.