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.

Growth light substantially affects both primary and secondary metabolic processes in Catharanthus roseus plants

“…It catalyses the formation of strictosidine from tryptamine and secologanin (Stöckigt et al 2008). Strictosidine serves as the central intermediate in indole alkaloid biosynthesis, including, among others, quinine, vinblastine, and vincamine (Loris et al 2007, Boccia et al 2022. Overexpression of these genes may lead to elevated level of alkaloid production, including vindoline, vinblastine, and catharanthine (Sharma et al 2018).…”

Chemistry, bioactivity, biosynthesis, and total synthesis of stemmadenine alkaloids

“…The mechanism of the conversion was hypothesised as follows: the strictosidine aglycone, such as Recently, O'Connor and Caputi reported directed biosynthesis of analogues of alstonine, a Corynanthe alkaloid, and stemmadenine acetate (84) in a heterologous host Nicotiana benthamiana (Scheme 3). 80 They cloned six genes CrSGD, CrGS, CrGo, CrReDOX1, CrReDOX2, and CrSAT, each of which was transformed into an individual A. tumefaciens GV3101 strain. Then the mixture of strains was inltrated in Nicotiana benthamiana for the transient expression.…”

Advances in Metabolic Engineering of Plant Monoterpene Indole Alkaloids

“…Other examples of genetic manipulation include expressing constitutive biosynthetic enzymes, transcription factors, or transporters known to regulate MIA accumulation [ 43 , 45 , 46 ]. While most engineering efforts of the MIA pathways into microbial and plant systems are intended to increase the yield of the desired MIAs, a notable advantage of reconstitution of natural product pathways includes the expansion of chemical diversity, potentially enabling the production of other bioactive MIAs that are not readily available in nature [ 47 , 48 , 49 ].…”

“…Interestingly, besides improving the yield of the desired compound in heterologous plant hosts, transient expression in Nicotiana plants facilitates efficient generation of molecular diversity. For instance, Boccia and colleagues utilized unnatural starting substrates to generate MIAs that do not commonly occur in nature [ 49 ]. The derivatization of natural products in heterologous plant systems lays a substantial foundation for further expansion of metabolic engineering in the development of novel compounds with improved pharmacological properties, or for enhancing the biological activity of the parent compound.…”