Prashant D. Sonawane

Summary: The plant family Euphorbiaceae is an abundant source of structurally complex diterpenoids, many of which have reported anticancer, anti‐HIV, and anti‐inflammatory activities. Among these, ingenol‐3‐angelate (1a; tradename: Picato®), isolated from Euphorbia peplus, has potent antitumor activity. We report the discovery and characterization of the first genes linked to committed steps of ingenol‐3‐angelate (1a) biosynthesis in E. peplus. Using pathway reconstitution in Nicotiana benthamiana and in vitro assays with recombinant enzymes, we identified two genes whose products catalyze the addition of angelyl‐CoA (9a) to the ingenol (5) scaffold, producing ingenol‐3‐angelate (1a). We also identified three genes whose products catalyze acetylation of ingenol‐3‐angelate (1a) to ingenol‐3‐angelate‐20‐acetate (2). Virus induced gene silencing (VIGS) suggests considerable functional redundancy in the E. peplus genome for this enzymatic step. We also identified three genes whose products can catalyze acetylation of ingenol‐3‐angelate (1a) to ingenol‐3‐angelate‐20‐acetate (2). In this case, virus‐induced gene silencing (VIGS) indicates considerable functional redundancy in the E. peplus genome of genes encoding this enzymatic step. We demonstrate using VIGS that just one of these genes, EpBAHD‐08, is essential for this angeloylation in E. peplus. VIGS of the second gene, EpBAHD‐06, has a significant effect on jatrophanes rather than ingenanes in E. peplus. This work paves the way for increasing ingenol‐3‐angelate (1a) levels in planta and provides a foundation for the discovery of the remaining genes in the biosynthetic pathway of these important molecules.

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 steroidal saponins.

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.

Cardenolides are specialized, steroidal metabolites produced in a wide array of plant families^1,2. Cardenolides play protective roles in plants, but these molecules, including digoxin from foxglove (Digitalis spp.), are better known for treatment of congenital heart failure, atrial arrhythmia, various cancers and other chronic diseases^3–9. However, it is still unknown how plants synthesize ‘high-value’, complex cardenolide structures from, presumably, a sterol precursor. Here we identify two cytochrome P450, family 87, subfamily A (CYP87A) enzymes that act on both cholesterol and phytosterols (campesterol and β-sitosterol) to form pregnenolone, the first committed step in cardenolide biosynthesis in the two phylogenetically distant plants Digitalis purpurea and Calotropis procera. Arabidopsis plants overexpressing these CYP87A enzymes ectopically accumulated pregnenolone, whereas silencing of CYP87A in D. purpurea leaves by RNA interference resulted in substantial reduction of pregnenolone and cardenolides. Our work uncovers the key entry point to the cardenolide pathway, and expands the toolbox for sustainable production of high-value plant steroids via synthetic biology.

Tomato is the highest value fruit and vegetable crop worldwide, yet produces α-tomatine, a renowned toxic and bitter-tasting anti-nutritional steroidal glycoalkaloid (SGA) involved in plant defense. A suite of modifications during tomato fruit maturation and ripening converts α-tomatine to the non-bitter and less toxic Esculeoside A. This important metabolic shift prevents bitterness and toxicity in ripe tomato fruit. While the enzymes catalyzing glycosylation and hydroxylation reactions in the Esculeoside A pathway have been resolved, the proposed acetylating step remains, to date, elusive. Here, we discovered that GAME36 (GLYCOALKALOID METABOLISM36), a BAHD-type acyltransferase catalyzes SGA-acetylation in cultivated and wild tomatoes. This finding completes the elucidation of the core Esculeoside A biosynthetic pathway in ripe tomato, allowing reconstitution of Esculeoside A production in heterologous microbial and plant hosts. The involvement of GAME36 in bitter SGA detoxification pathway points to a key role in the evolution of sweet-tasting tomato as well as in the domestication and breeding of modern cultivated tomato fruit.