Characterizing the Biosynthesis of Physostigmine

Physostigmine is a parasympathomimetic drug used to treat a variety of neurological disorders, including Alzheimer's disease and glaucoma. Because of its potent biological activity and unique pyrroloindole skeleton, physostigmine has been the target of many organic syntheses. However, the biosynthesis of physostigmine has been relatively understudied. In this work, we identified a biosynthetic gene cluster for physostigmine by genome mining. The 8.5-kb gene cluster encodes eight proteins (PsmA-H), seven of which are required for the synthesis of physostigmine from 5-hydroxytryptophan as shown by in vitro total reconstitution. Further genetic and enzymatic studies enabled us to delineate the biosynthetic pathway for physostigmine, which features an unusual reaction cascade consisting of highly coordinated methylations and acetylation/deacetylation reactions. Valuable biosynthetic intermediates and analogs were also produced, providing the basis for future work in engineering the biosynthesis of pyrroloindole drugs with improved pharmaceutical properties.