Directed Biosynthesis of New to Nature Alkaloids in a Heterologous Nicotiana benthamiana Expression Host

Abstract

Plants produce a wide variety of pharmacologically active molecules classified as natural products. Derivatization of these natural products can modulate or improve the bioactivity of the parent compound. Unfortunately, chemical derivatization of natural products is often difficult or impractical. Here we use the newly discovered biosynthetic genes for two monoterpene indole alkaloids, alstonine and stemmadenine acetate, to generate analogs of these compounds. We reconstitute these biosynthetic genes in the heterologous host Nicotiana benthamiana along with an unnatural starting substrate to produce the corresponding new-to-nature alkaloid product.

Keywords: Nicotiana benthamiana; alstonine; directed biosynthesis; monoterpene indole alkaloid; natural product; new-to-nature products; stemmadenine.

FIGURE 1

Biosynthesis of monoterpene indole alkaloids. (A) Production of strictosidine from tryptamine and secologanin by the action of Catharanthus roseus strictosidine synthase (CrSTR). (B) Biosynthesis of stemmadenine acetate, an intermediate of the pathway for the production of the two anticancer compounds vinblastine and vincristine. The production of stemmadenine acetate involves 6 enzymes from strictosidine: CrSGD, CrGS, CrGO, CrReDOX1, CrReDOX2, and CrSAT. Akuammicine is a spontaneous degradation product of preakuammicine. (C) Biosynthesis of the antipsychotic molecule alstonine in Catharanthus roseus. The production of alstonine involves 3 enzymes from strictosidine: CrSGD, CrTHAS, CrAS.

FIGURE 2

Strictosidine analogs produced in vitro using tryptamine analogs and secologanin. The reaction was catalyzed by CrSTR. Strictosidine analogs shown in red were not turned over by CrSTR or were not produced in high enough levels for isolation.