Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine¹. As MIAs are difficult to chemically synthesize, the world’s supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale^2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.

单萜吲哚生物碱 (MIA) 是复杂植物次级代谢产物的一个多样化家族，具有许多药用特性，包括必需的抗癌疗法长春花碱和长春新碱¹。由于 MIA 难以化学合成，世界上的长春碱供应链依赖于从长春花植物中低产地提取和纯化前体文多灵和长春质，然后通过简单的体外化学偶联和还原形成长春碱工业规模^2,3. 在这里，我们展示了使用高度工程化的酵母从头微生物生物合成文多灵和长春花碱，以及与长春花碱的体外化学偶联。该研究展示了一个很长的生物合成途径，该途径被重构为微生物细胞工厂，包括酵母天然代谢物香叶基焦磷酸和色氨酸到长春质碱和文多灵之外的 30 个酶促步骤。总共进行了 56 次基因编辑，包括来自植物的 34 个异源基因的表达，以及 10 个酵母基因的缺失、敲低和过表达，以改善前体供应，以从头生产长春花碱和文多灵，从中半合成长春碱发生。由于长春碱途径是最长的 MIA 生物合成途径之一，本研究将酵母定位为可扩展平台，以生产超过 3，