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.睡茄内酯（Withanolides）是一类具有重要药用价值的甾体内酯类化合物，由南非醉茄（Withania somnifera，又称ashwagandha）及其他茄科（Solanaceae）植物合成，这类化合物以抗炎、抗癌及适应原调节活性而闻名。然而，睡茄内酯的生物合成途径尚未完全阐明，这阻碍了其规模化生产与医药应用。本研究完成了南非醉茄的染色体级基因组组装，并鉴定出两个具备分段组织特异性表达模式的睡茄内酯生物合成基因簇。本研究通过酵母代谢工程技术，辅以本氏烟草（Nicotiana benthamiana）异源表达以及南非醉茄中的病毒诱导基因沉默实验，阐明了睡茄内酯的生物合成途径，获得了具备睡茄内酯全部典型化学特征的中间产物。本研究鉴定出两种细胞色素P450（CYP87G1与CYP749B2）及一种短链脱氢酶（SDH2），它们负责内酯环的形成；同时还鉴定出另外两种细胞色素P450（CYP88C7与CYP88C10）及一种磺基转移酶（SULF1），可构建睡茄内酯标志性的A环结构，该结构带有C1位酮基与C2-C3位不饱和键。将SULF1鉴定为该生物合成途径的核心酶，这一发现挑战了"磺基转移酶仅作为修饰酶"的传统认知，并暗示该酶家族在植物特化代谢中具有更广泛的作用。本研究为通过生物制造技术可持续生产睡茄内酯，以及利用睡茄内酯骨架开展药物开发开辟了全新路径。