Nicotiana benthamiana is a suitable host for the generation of cannabinoid producing enzymes. To attain whole pathway integration, careful analysis of subcellular localization is necessary.
Based on findings described herein, we contend that the reduction of vomilenine en route to antiarrhythmic ajmaline in planta might proceed via an alternative, novel sequence of biosynthetic steps. In the genus Rauvolfia, monoterpenoid indole alkaloids (MIAs) are formed via complex biosynthetic sequences. Despite the wealth of information about the biochemistry and molecular genetics underlying these processes, many reaction steps involving oxygenases and oxidoreductases are still elusive. Here, we describe molecular cloning and characterization of three cinnamyl alcohol dehydrogenase (CAD)-like reductases from Rauvolfia serpentina cell culture and R. tetraphylla roots. Functional analysis of the recombinant proteins, with a set of MIAs as potential substrates, led to identification of one of the enzymes as a CAD, putatively involved in lignin formation. The two remaining reductases comprise isoenzymes derived from orthologous genes of the investigated alternative Rauvolfia species. Their catalytic activity consists of specific conversion of vomilenine to 19,20-dihydrovomilenine, thus proving their exclusive involvement in MIA biosynthesis. The obtained data suggest the existence of a previously unknown bypass in the biosynthetic route to ajmaline further expanding structural diversity within the MIA family of specialized plant metabolites.
Human drug‐metabolizing cytochrome P450 monooxygenases (CYPs) have enormous substrate promiscuity; this makes them promising tools for the expansion of natural product diversity. Here, we used CYP3A4 for the targeted diversification of a plant biosynthetic route leading to monoterpenoid indole alkaloids. In silico, in vitro and in planta studies proved that CYP3A4 was able to convert the indole alkaloid vinorine into vomilenine, the former being one of the central intermediates in the ajmaline pathway in the medicinal plant
Rauvolfia serpentina
(L.) Benth. ex Kurz. However, to a much larger extent, the investigated conversion yielded vinorine (19
R
,20R)‐epoxide, a new metabolite with an epoxide functional group that is rare for indole alkaloids. The described work represents a successful example of combinatorial biosynthesis towards an increase in biodiversity of natural metabolites. Moreover, characterisation of the products of the in vitro and in planta transformation of potential pharmaceuticals with human CYPs might be indicative of the route of their conversion in the human organism.
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