Elucidation of gene clusters underlying withanolide biosynthesis in ashwagandha through yeast metabolic engineering

Withanolides are medicinally important steroidal lactones produced by Withania somnifera (ashwagandha) amongst other Solanaceae family plants, known for their anti-inflammatory, anti-cancer, and adaptogenic properties. However, the biosynthetic pathway to withanolides is largely unknown, preventing scale-up and hindering pharmaceutical applications. Here, we generate a chromosome-scale assembly of the W. somnifera genome and identify two withanolide biosynthetic gene clusters that exhibit a segmented tissue-specific expression pattern. Using metabolic engineering in yeast, complemented by heterologous expression in Nicotiana benthamiana and virus-induced gene silencing in W. somnifera, we elucidate the withanolide biosynthetic pathway to an intermediate with all characteristic chemical features of withanolides. We report two cytochromes P450 (CYP87G1 and CYP749B2) and a short-chain dehydrogenase (SDH2) responsible for lactone ring formation and two P450s (CYP88C7 and CYP88C10) and a sulfotransferase (SULF1) that generate the characteristic A-ring structure of withanolides, featuring a C1 ketone and C2-C3 unsaturation. Identifying SULF1 as a core pathway enzyme challenges the conventional paradigm of sulfotransferases as tailoring enzymes and suggests a wider role for this enzyme family in plant specialized metabolism. This work opens new avenues for the sustainable production of withanolides through biomanufacturing and for drug development leveraging the withanolide scaffold. Overall design: RNA-seq profiling of leaf, young leaf (<2 cm size), root, and berry tissue of Withania somnifera. Biological replication n=3.