Rewiring Steroidal Metabolic Pathways for Diosgenin Production in <i>Solanum nigrum</i>

Diosgenin is a key starting material for the synthesis of steroidal drugs, such as corticosteroids and sex hormones. While the primary commercial source of diosgenin is the tubers of Dioscorea spp., identifying alternative plant hosts capable of diosgenin biosynthesis could enhance its production. In this study, we present Solanum nigrum, a widely distributed species of the Solanum genus, as a novel platform for diosgenin production. S. nigrum naturally accumulates high concentrations of steroidal glycoalkaloids (SGAs) with a closed F-ring (spirostanol type) in green fruits and steroidal saponins (STSs) with an open F-ring (furostanol type) in leaves. Both classes originate from cholesterol and share the early oxidation steps, followed by specific reactions that redirect distinct metabolic fluxes. In SGAs, the CYP450 enzyme SnGAME4 oxidises C26-OH to an aldehyde, enabling subsequent transamination. In STSs, SnGAME25, a 3β-hydroxysteroid dehydrogenase/isomerase, initiates the reduction reactions at the C5 double bond. Disruption of these two genes shifted the metabolic profiles from the native SGAs and STSs toward furostanol-type proto-diosgenin glycosides. However, these open F-ring structures yield low diosgenin levels during acid hydrolysis. To overcome this limitation, we identified endogenous furostanol glycoside 26-O-β-glucosidases and employed spontaneous fermentation to convert the furostanol structure to the spirostanol structure. Altogether, S. nigrum green fruits yielded diosgenin up to 1% of dry weight. In addition, we engineered S. nigrum to increase fruit number in combination with the SnGAME4 mutation. These results establish S. nigrum as a promising and scalable host for diosgenin production.