The (seco)iridoids and their derivatives, the monoterpenoid indole alkaloids (MIAs), form two large families of plant-derived bioactive compounds with a wide spectrum of high-value pharmacological and insect-repellent activities. Vinblastine and vincristine, MIAs used as anticancer drugs, are produced by Catharanthus roseus in extremely low levels, leading to high market prices and poor availability. Their biotechnological production is hampered by the fragmentary knowledge of their biosynthesis. Here we report the discovery of the last four missing steps of the (seco)iridoid biosynthesis pathway. Expression of the eight genes encoding this pathway, together with two genes boosting precursor formation and two downstream alkaloid biosynthesis genes, in an alternative plant host, allows the heterologous production of the complex MIA strictosidine. This confirms the functionality of all enzymes of the pathway and highlights their utility for synthetic biology programmes towards a sustainable biotechnological production of valuable (seco)iridoids and alkaloids with pharmaceutical and agricultural applications.

Oleanolic acid (3β-hydroxyolean-12-en-28-oic acid) is a pentacyclic triterpenoid compound with a widespread occurrence throughout the plant kingdom. In nature, the compound exists either as a free acid or as an aglycone precursor for triterpenoid saponins, in which it can be linked to one or more sugar chains. Oleanolic acid and its derivatives possess several promising pharmacological activities, such as hepatoprotective effects, and anti-inflammatory, antioxidant, or anticancer activities. With the recent elucidation of its biosynthesis and the imminent commercialization of the first oleanolic acid-derived drug, the compound promises to remain important for various studies. In this review, the recent progress in understanding the oleanolic acid biosynthesis and its pharmacology are discussed. Furthermore, the importance and potential application of synthetic oleanolic acid derivatives are highlighted, and research perspectives on oleanolic acid are given.

The amount of cholesterol made by many plants is not negligible. Whereas cholesterogenesis in animals was elucidated decades ago, the plant pathway has remained enigmatic. Among other roles, cholesterol is a key precursor for thousands of bioactive plant metabolites, including the well-known Solanum steroidal glycoalkaloids. Integrating tomato transcript and protein co-expression data revealed candidate genes putatively associated with cholesterol biosynthesis. A combination of functional assays including gene silencing, examination of recombinant enzyme activity and yeast mutant complementation suggests the cholesterol pathway comprises 12 enzymes acting in 10 steps. It appears that half of the cholesterogenesis-specific enzymes evolved through gene duplication and divergence from phytosterol biosynthetic enzymes, whereas others act reciprocally in both cholesterol and phytosterol metabolism. Our findings provide a unique example of nature's capacity to exploit existing protein folds and catalytic machineries from primary metabolism to assemble a new, multi-step metabolic pathway. Finally, the engineering of a 'high-cholesterol' model plant underscores the future value of our gene toolbox to produce high-value steroidal compounds via synthetic biology.

Steroidal glycoalkaloids (SGAs) are cholesterol-derived molecules produced by solanaceous species. They contribute to pathogen defence but are toxic to humans and considered as anti-nutritional compounds. Here we show that GLYCOALKALOID METABOLISM 9 (GAME9), an APETALA2/Ethylene Response Factor, related to regulators of alkaloid production in tobacco and Catharanthus roseus, controls SGA biosynthesis. GAME9 knockdown and overexpression in tomato and potato alters expression of SGAs and upstream mevalonate pathway genes including the cholesterol biosynthesis gene STEROL SIDE CHAIN REDUCTASE 2 (SSR2). Levels of SGAs, C24-alkylsterols and the upstream mevalonate and cholesterol pathways intermediates are modified in these plants. Δ(7)-STEROL-C5(6)-DESATURASE (C5-SD) in the hitherto unresolved cholesterol pathway is a direct target of GAME9. Transactivation and promoter-binding assays show that GAME9 exerts its activity either directly or cooperatively with the SlMYC2 transcription factor as in the case of the C5-SD gene promoter. Our findings provide insight into the regulation of SGA biosynthesis and means for manipulating these metabolites in crops.

The saikosaponins comprise oleanane-and ursane-type triterpene saponins that are abundantly present in the roots of the genus Bupleurum widely used in Asian traditional medicine. Here we identified a gene, designated CYP716Y1, encoding a cytochrome P450 monooxygenase from Bupleurum falcatum that catalyzes the C-16α hydroxylation of oleanane-and ursane-type triterpenes. Exploiting this hitherto unavailable enzymatic activity, we launched a combinatorial synthetic biology program in which we combined CYP716Y1 with oxidosqualene cyclase, P450, and glycosyltransferase genes available from other plant species and reconstituted the synthesis of monoglycosylated saponins in yeast. Additionally, we established a culturing strategy in which applying methylated β-cyclodextrin to the culture medium allows the sequestration of heterologous nonvolatile hydrophobic terpenes, such as triterpene sapogenins, from engineered yeast cells into the growth medium, thereby greatly enhancing productivity. Together, our findings provide a sound base for the development of a synthetic biology platform for the production of bioactive triterpene sapo(ge)nins.cyclodextrin | amyrin | metabolic engineering | triterpenoid

Summary Biosynthesis of the sesquiterpene lactone and potent antimalarial drug artemisinin occurs in glandular trichomes of Artemisia annua plants and is subjected to a strict network of developmental and other regulatory cues. The effects of three hormones, jasmonate, gibberellin and cytokinin, were studied at the structural and molecular levels in two different A. annua chemotypes by microscopic analysis of gland development, and by targeted metabolite and transcript profiling. Furthermore, a genome‐wide cDNA‐amplified fragment length polymorphism (AFLP)‐based transcriptome profiling was carried out of jasmonate‐elicited leaves at different developmental stages. Although cytokinin and gibberellin positively affected at least one aspect of gland formation, these two hormones did not stimulate artemisinin biosynthesis. Only jasmonate simultaneously promoted gland formation and coordinated transcriptional activation of biosynthetic gene expression, which ultimately led to increased sesquiterpenoid accumulation with chemotype‐dependent effects on the distinct pathway branches. Transcriptome profiling revealed a trichome‐specific fatty acyl‐ coenzyme A reductase, trichome‐specific fatty acyl‐CoA reductase 1 (TFAR1), the expression of which correlates with trichome development and sesquiterpenoid biosynthesis. TFAR1 is potentially involved in cuticular wax formation during glandular trichome expansion in leaves and flowers of A. annua plants. Analysis of phytohormone‐modulated transcriptional regulons provides clues to dissect the concerted regulation of metabolism and development of plant trichomes.

Plants respond to stresses by producing a broad spectrum of bioactive specialized metabolites. Hormonal elicitors, such as jasmonates, trigger a complex signaling circuit leading to the concerted activation of specific metabolic pathways. However, for many specialized metabolic pathways, the transcription factors involved remain unknown. Here, we report on two homologous jasmonate-inducible transcription factors of the basic helix-loop-helix family, TRITERPENE SAPONIN BIOSYNTHESIS ACTIVATING REGULATOR1 (TSAR1) and TSAR2, which direct triterpene saponin biosynthesis in Medicago truncatula. TSAR1 and TSAR2 are coregulated with and transactivate the genes encoding 3-HYDROXY-3-METHYLGLUTARYL-COENZYME A REDUCTASE1 (HMGR1) and MAKIBISHI1, the rate-limiting enzyme for triterpene biosynthesis and an E3 ubiquitin ligase that controls HMGR1 levels, respectively. Transactivation is mediated by direct binding of TSARs to the N-box in the promoter of HMGR1. In transient expression assays in tobacco (Nicotiana tabacum) protoplasts, TSAR1 and TSAR2 exhibit different patterns of transactivation of downstream triterpene saponin biosynthetic genes, hinting at distinct functionalities within the regulation of the pathway. Correspondingly, overexpression of TSAR1 or TSAR2 in M. truncatula hairy roots resulted in elevated transcript levels of known triterpene saponin biosynthetic genes and strongly increased the accumulation of triterpene saponins. TSAR2 overexpression specifically boosted hemolytic saponin biosynthesis, whereas TSAR1 overexpression primarily stimulated nonhemolytic soyasaponin biosynthesis. Both TSARs also activated all genes of the precursor mevalonate pathway but did not affect sterol biosynthetic genes, pointing to their specific role as regulators of specialized triterpene metabolism in M. truncatula.

Triterpenoids are widespread bioactive plant defence compounds with potential use as pharmaceuticals, pesticides and other high-value products. Enzymes belonging to the cytochrome P450 family have an essential role in creating the immense structural diversity of triterpenoids across the plant kingdom. However, for many triterpenoid oxidation reactions, the corresponding enzyme remains unknown. Here we characterize CYP716 enzymes from different medicinal plant species by heterologous expression in engineered yeasts and report ten hitherto unreported triterpenoid oxidation activities, including a cyclization reaction, leading to a triterpenoid lactone. Kingdom-wide phylogenetic analysis of over 400 CYP716s from over 200 plant species reveals details of their evolution and suggests that in eudicots the CYP716s evolved specifically towards triterpenoid biosynthesis. Our findings underscore the great potential of CYP716s as a source for generating triterpenoid structural diversity and expand the toolbox available for synthetic biology programmes for sustainable production of bioactive plant triterpenoids.