Transient expression of human cytochrome P450s 2D6 and 3A4 in Nicotiana benthamiana provides a possibility for rapid substrate testing and production of novel compounds.

Employment of transient expression of foreign genes for bioconversion of pharmaceutically valuable low-molecular-weight compounds, including plant secondary metabolites, is an enticing trend still scantily explored in plant biotechnology. In the present work, we put forth an efficient protocol for rapid assessment of synthetic and plant-derived metabolites as potential substrates for human P450s (CYP2D6 and CYP3A4) via Agrobacterium-mediated transient expression in Nicotiana benthamiana. Animal P450s with broad substrate specificity are promising candidates for transformation of diverse metabolites. The efficiency of P450s in heterologous surroundings is not always satisfactory and depends on the availability of an associated electron-transfer enzyme. Plants represent an attractive assortment of prospective hosts for foreign P450s expression. We selected the optimal composition of genetic blocks providing the highest transient expression efficiency, designed and validated an effective substrate administration scheme and tested biological activity of the investigated P450s against loratadine and several indole alkaloids with different molecular scaffold structures. A novel indole alkaloid, 11-hydroxycorynanthine, was isolated from N. benthamiana plants transiently expressing CYP2D6 and supplemented with corynanthine, and its structure was elucidated. The proposed technique might be of value in realization of combinatorial biosynthesis concept comprising the junction of heterologous enzymes and substrates in different metabolic surroundings