De novo biosynthesis of antiarrhythmic alkaloid ajmaline

Abstract The antiarrhythmic drug ajmaline is a monoterpenoid indole alkaloid (MIA) isolated from the Ayurvedic plant Rauvolfia serpentina (Indian Snakeroot). Research into the biosynthesis of ajmaline and another renowned MIA chemotherapeutic drug vinblastine has yielded pivotal advancements in the fields of plant specialized metabolism and engineering over recent decades. While the majority of vinblastine biosynthesis has been recently elucidated, the quest for comprehending ajmaline biosynthesis remains incomplete, marked by the absence of two critical enzymes. Here, we show the discovery and characterization of these two elusive reductases, alongside the identification of two physiologically relevant esterases that complete the biosynthesis of ajmaline. We show that ajmaline biosynthesis proceeds with vomilenine 1,2( R )-reduction followed by its 19,20( S )-reduction. This process is further modulated by two root-expressing esterases that deacetylate 17- O -acetylnorajmaline. Expanding upon the successful completion of the ajmaline biosynthetic pathway, we engineer the de novo biosynthesis of ajmaline in Baker’s yeast.