#200-102 Elucidation and engineering of withanolide biosynthesis in the medicinal plant ashwagandha

Withanolides are a class of steroidal lactone plant natural products that show promise for the treatment of neurodegenerative disease and cancer. However, only one enzyme in the biosynthetic pathway has been characterized: a sterol isomerase converting 24-methylene cholesterol to 24-methyldesmosterol, representing the first committed step in withanolide biosynthesis. Elucidation of the remaining steps would enable withanolide production in a heterologous host such as Saccharomyces cerevisiae and open the door for the development of withanolide-based drugs. We used a combination of tissue-specific transcriptomics and biosynthetic gene cluster analysis on the de novo assembled genome of ashwagandha (Withania somnifera) to identify candidate enzymes. We characterized the candidate enzymes using transient expression in Nicotiana benthamiana and pathway reconstruction in S. cerevisiae. In addition, we used viral induced gene silencing in Withania somnifera to investigate the in vivo activity of candidate enzymes. We identified five cytochrome P450s that are involved in the withanolide biosynthetic pathway. We purified the products of the first two enzymes from large scale yeast culture and structurally elucidated them using NMR. The insights into withanolide biosynthesis gained from our study pave the way for an efficient biomanufacturing process for withanolides and will eventually enable the development and application of withanolide-derived drugs.