Biosynthesis of medicinal tropane alkaloids in yeast

Tropane alkaloids (TAs) from nightshade plants are neurotransmitter inhibitors used for treating neuromuscular disorders and are classified as essential medicines by the World Health Organization(1,2). Global supply challenges have resulted in frequent drug shortages(3,4). Further supply-chain vulnerabilities are revealed by events like the Australian wildfires(5) and the COVID-19 pandemic(6). Rapidly deployable production strategies that are robust to environmental and socioeconomic upheaval(7,8) are needed. Here, we engineered baker’s yeast to produce the medicinal TAs hyoscyamine and scopolamine starting from simple sugars and amino acids. We combined functional genomics to identify a missing pathway enzyme, protein engineering to enable functional acyltransferase expression via trafficking to the vacuole, heterologous transporters to facilitate intracellular routing, and strain optimization to improve titers. Our integrated system positions >20 proteins adapted from yeast, bacteria, plants, and animals across six sub-cellular locations to recapitulate the spatial organization of TA biosynthesis in plants. Microbial biosynthesis platforms can facilitate discovery of TA derivatives as novel therapeutics for neurological disease and, once scaled, enable robust and agile supply of these essential medicines.