Incorporation of nitrogen in antinutritional Solanum alkaloid biosynthesis.

Steroidal glycoalkaloids (SGAs) are specialized metabolites produced by hundreds of Solanum species including food crops, such as tomato, potato and eggplant. Unlike true alkaloids, nitrogen is introduced at a late stage of SGA biosynthesis through an unknown transamination reaction. Here, we reveal the mechanism by which GLYCOALKALOID METABOLISM12 (GAME12) directs the biosynthesis of nitrogen-containing steroidal alkaloid aglycone in Solanum. We report that GAME12, a neofunctionalized γ-aminobutyric acid (GABA) transaminase, undergoes changes in both active site specificity and subcellular localization to switch from its renown and generic activity in core metabolism to function in a specialized metabolic pathway. Moreover, overexpression of GAME12 alone in engineered S. nigrum leaves is sufficient for de novo production of nitrogen-containing SGAs. Our results highlight how hijacking a core metabolism GABA shunt enzyme is crucial in numerous Solanum species for incorporating a nitrogen to a steroidal-specialized metabolite backbone and form defensive alkaloids.

A scaffold protein manages the biosynthesis of steroidal defense metabolites in plants.

Solanaceae plants produce two major classes of valuable sterol derived natural products-steroidal glycoalkaloids and steroidal saponins-from a common cholesterol precursor. Attempts to heterologously produce these molecules have consistently failed, although the genes responsible for each biosynthetic step have been identified. Here we identify a cellulose synthase like protein, an unexpected biosynthetic component that interacts with the early pathway enzymes, enabling steroidal scaffolds production in plants. Moreover, knockout of this gene in black nightshade, Solanum nigrum resulted in plants lacking both steroidal alkaloids and saponins. Unexpectedly, these knockout plants also revealed that steroidal saponins deter serious agricultural insect pests. This discovery provides the missing link to engineer these high value steroidal molecules, and also pinpoints the ecological role for the steroidal saponins.

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

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.