Phylogenomics and metabolic engineering reveal a conserved gene cluster in Solanaceae plants for withanolide biosynthesis

Withanolides are steroidal lactones from nightshade (Solanaceae) plants. Of the over 1,200 known representatives, many possess potent biological activities, but their drug potential has not been fully realised up until now. A central obstacle is the limited availability of minor withanolides, caused by a lack of knowledge about the underlying biosynthetic pathways. Here, we combine phylogenomics with metabolic engineering to overcome this limitation. By sequencing the genome of the medicinal plant and archetypical withanolide producer ashwagandha ( Withania somnifera ) and comparing the genome sequences of nine Solanaceae species, we discovered a conserved gene cluster for withanolide biosynthesis, consisting of two sub-gene clusters which differ in their expression patterns. To investigate the functions of the encoded enzymes, we established metabolic engineering platforms in yeast ( Saccharomyces cerevisiae ) and the model plant Nicotiana benthamiana . This allowed us to reconstitute the first three oxidative steps of withanolide biosynthesis, catalysed by the cytochrome P450 monooxygenases CYP87G1, CYP88C7, and CYP749B2, leading to the aglycone of the known compound withanoside V. Our work sets the basis for the biotechnological production of withanolides in heterologous hosts and will therefore help to fully harness the drug potential of these plant steroids in the future.