苦参酮类 (Cardenolides) 是一类特殊的类固醇代谢产物，在各类植物科中广泛产生[1,2]。苦参酮类在植物中具有保护作用，但这些分子，包括指南针（Digitalis spp.）中的洋地黄毒甙（Digoxin），更为人所熟知的是用于治疗先天性心力衰竭、心房心律失常、各种癌症和其他慢性疾病[3-9]。然而，植物是如何从甾醇前体合成“高价值”的复杂苦参酮结构，目前尚不清楚。本研究在两个系统发育上远的植物紫地胆（Digitalis purpurea）和甘树胶（Calotropis procera）中鉴定出两种作用于胆固醇和植物甾醇（甘油甾醇和β-谷甾醇）的细胞色素P450酶87家族A亚科（CYP87A）酶，形成孕烯醇，这是苦参酮生物合成中的第一个确定步骤。过表达这些CYP87A酶的拟南芥植株在异位上积累了孕烯醇，而通过RNA干扰沉默D. purpurea叶中的CYP87A，则大幅减少了孕烯醇和苦参酮。我们的研究揭示了进入苦参酮途径的关键入口点，扩展了通过合成生物学可持续生产高价值植物类固醇的工具箱©2023. 作者。

Cardenolides are specialized, steroidal metabolites produced in a wide array of plant families1,2. Cardenolides play protective roles in plants, but these molecules, including digoxin from foxglove (Digitalis spp.), are better known for treatment of congenital heart failure, atrial arrhythmia, various cancers and other chronic diseases3-9. However, it is still unknown how plants synthesize 'high-value', complex cardenolide structures from, presumably, a sterol precursor. Here we identify two cytochrome P450, family 87, subfamily A (CYP87A) enzymes that act on both cholesterol and phytosterols (campesterol and β-sitosterol) to form pregnenolone, the first committed step in cardenolide biosynthesis in the two phylogenetically distant plants Digitalis purpurea and Calotropis procera. Arabidopsis plants overexpressing these CYP87A enzymes ectopically accumulated pregnenolone, whereas silencing of CYP87A in D. purpurea leaves by RNA interference resulted in substantial reduction of pregnenolone and cardenolides. Our work uncovers the key entry point to the cardenolide pathway, and expands the toolbox for sustainable production of high-value plant steroids via synthetic biology.© 2023. The Author(s).