Elucidation of gene clusters underlying withanolide biosynthesis in ashwagandha

Abstract

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. In this thesis, we report a chromosome-scale assembly of the W. somnifera genome, which we use for biosynthetic gene cluster mining. We identify two biosynthetic gene clusters likely involved in withanolide biosynthesis and explore some aspects of their evolution. The identified clusters are among the largest identified in plants to date and they exhibit an unusual tissue-specific subcluster structure. Next, we characterize the genes in the identified biosynthetic gene clusters using heterologous expression in yeast and tobacco, in conjunction with in vitro enzyme assays. We discover two cytochromes P450 (CYP87G1 and CYP749B2) and a short-chain dehydrogenase (SDH2) responsible for formation of the lactone ring on the sterol side chain, a key chemical feature of withanolides. Two additional P450s (CYP88C7 and CYP88C10) and a sulfotransferase (SULF1) generate the characteristic A-ring structure of withanolides, featuring a C₁ ketone and C₂-C₃ unsaturation. The discovery of SULF1 as a core withanolide pathway enzyme challenges the conventional view of sulfotransferases as tailoring enzymes and suggests a wider role for this enzyme family in plant secondary metabolism. This work opens new avenues for the sustainable production of withanolides through biomanufacturing and for drug development leveraging the withanolide scaffold.