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Transient Expression of Human Cytochrome P450s 2D6 and 3A4 in Nicotiana benthamiana Provides a Possibility for Rapid Substrate Testing and Production of Novel Compounds
Author(s) -
Sheludko Yuriy V.,
Gerasymenko Iryna M.,
Warzecha Heribert
Publication year - 2018
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201700696
Subject(s) - nicotiana benthamiana , heterologous expression , biochemistry , heterologous , biology , agrobacterium , indole alkaloid , substrate (aquarium) , transformation (genetics) , cytochrome p450 , computational biology , indole test , enzyme , gene , recombinant dna , ecology
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, 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 is put forth. 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. The optimal composition of genetic blocks providing the highest transient expression efficiency is designed, an effective substrate administration scheme is validated, and biological activity of the investigated P450s against loratadine and several indole alkaloids with different molecular scaffold structures is tested. A novel indole alkaloid, 11‐hydroxycorynanthine, is 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.