(469d) Characterization of the Biosynthetic Pathway of Physostigmine

Physostigmine is a tryptophan-derived natural product with a rare pyrroloindole skeleton. Because of its ability to reversibly inhibit acetylcholinesterase, physostigmine has been used to treat a variety of neurological disorders, including glaucoma and Alzheimer’s disease. However, despite its medicinal uses, little is known about the biosynthesis of physostigmine and pyrroloindole alkaloids in general. In this work, we identified the biosynthetic gene cluster for physostigmine in the producer, Streptomyces griseofuscus, and confirmed its function by heterologous expression.  Gene deletion mutants and in vitro assays with purified enzymes further enabled us to identify the set of enzymes essential for physostigmine biosynthesis and propose an enzymatic pathway for the transformation of tryptophan into physostigmine. We find that the formation of the pyrroloindole skeleton proceeds by an unexpected N-acetylated intermediate that is necessary for methylation of C-3 of the indole ring although the subsequent ring closure occurs with the hydrolysis of the acetyl group. This characterization of the physostigmine biosynthetic pathway provides the basis for future work in engineering the biosynthesis of physostigmine intermediates and analogues.