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

Withanolides are medicinally relevant steroidal lactones produced by Withania somnifera (ashwagandha) amongst other Solanaceae family plants. However, the biosynthetic pathway to withanolides is largely unknown, preventing scale-up and hindering pharmaceutical applications. We sequenced the genome of W. somnifera and identified two biosynthetic gene clusters exhibiting a segmented tissue-specific expression pattern. We characterized the cluster enzymes through stepwise pathway reconstitution in yeast and transient expression in Nicotiana benthamiana , leading to the identification of three cytochrome P450s (CYP87G1, CYP88C7, and CYP749B2) and a short-chain dehydrogenase that produce a lactone ring- containing intermediate when co-expressed. A fourth cytochrome P450 (CYP88C10) and a sulfotransferase convert this into an intermediate with the characteristic withanolide A-ring structure, featuring a C ₁ ketone and C ₂ -C ₃ unsaturation. The discovery of the sulfotransferase as a core pathway enzyme challenges the conventional paradigm of sulfotransferases as tailoring enzymes. These insights pave the way for an efficient biomanufacturing process for withanolides and future development of withanolide-derived drugs.