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 plantCatharanthus 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.

The monoterpene indole alkaloids are a large group of plant-derived specialized metabolites, many of which have valuable pharmaceutical or biological activity. There are ∼3,000 monoterpene indole alkaloids produced by thousands of plant species in numerous families. The diverse chemical structures found in this metabolite class originate from strictosidine, which is the last common biosynthetic intermediate for all monoterpene indole alkaloid enzymatic pathways. Reconstitution of biosynthetic pathways in a heterologous host is a promising strategy for rapid and inexpensive production of complex molecules that are found in plants. Here, we demonstrate how strictosidine can be produced de novo in a Saccharomyces cerevisiae host from 14 known monoterpene indole alkaloid pathway genes, along with an additional seven genes and three gene deletions that enhance secondary metabolism. This system provides an important resource for developing the production of more complex plant-derived alkaloids, engineering of nonnatural derivatives, identification of bottlenecks in monoterpene indole alkaloid biosynthesis, and discovery of new pathway genes in a convenient yeast host. Significance Plants make a wide variety of complex molecules with potent biological activities including several anticancer therapeutics. Unfortunately, plants often produce these molecules in low amounts, making them expensive to obtain. Engineering simpler organisms, such as yeast, to produce these plant-derived compounds provides one solution to production challenges. One group of plant-derived molecules, the monoterpene indole alkaloids, is synthesized from a common intermediate, strictosidine. Here, we report how we developed a yeast strain that produces strictosidine. This required introducing 21 new genes and three gene deletions into the yeast genome. This yeast strain provides an important resource for the production of expensive, complex molecules that plants normally produce in small amounts.

How to make bioactive alkaloids: Vinblastine and vincristine are important, expensive anticancer agents that are produced by dimerization of the plant-derived alkaloids catharanthine and vindoline. The enzymes that transform tabersonine into vindoline are known; however, the mechanism by which the scaffolds of catharanthine and tabersonine are generated has been a mystery. Caputi et al. now describe the biosynthetic genes and corresponding enzymes responsible. This resolves a long-standing question of how plant alkaloid scaffolds are synthesized, which is important not only for vinblastine and vincristine biosynthesis, but also for understanding the many other biologically active alkaloids found throughout nature. Science , this issue p. 1235