Evaluation of Biosynthetic Approaches Toward the Production of Non-Natural Psilocybin Derivatives

Psilocybin is the psychoactive prodrug of psilocin that has been shown to lessen the negative effects of PTSD, depression, and addiction. While there are multiple established process for the synthesis of psilocybin including extraction methods and traditional chemical synthesis, they have proven to be tedious, time-consuming, and expensive. However, my lab has championed the production of psilocybin with a fermentation-based approached using genetically engineered E. coli. Psilocybin treatment is, however, not perfect. It has been shown to be safe and nonaddictive but there is one significant side effect that cannot be ignored...a roughly 6-hour hallucinogenic experience immediately after consuming the drug. Neuroscientists are beginning to unravel the mechanism of mental health treatment by psychedelics and have suggested that the presence of a hallucinogenic experience is not necessarily connected to the positive mental health impacts. Here, we evaluate four different approaches towards the production of non-natural psilocybin derivatives to create a larger drug candidate pool with diverse efficacies and psychedelic properties. The first approach expands upon our previously reported psilocybin production pathway to develop a route for the de novo co-culture biosynthesis of this compound. Incorporating a P450 monooxygenase, PsiH, to catalyze the hydroxylation of tryptamine intermediate compounds enables both a route to de novo psilocybin biosynthesis as well as a study of the substrate promiscuity for the full psilocybin biosynthesis pathway through supplementation of a library of single substituted indoles. A second approach will explore the production of psilocybin derivatives through supplementation of disubstituted indoles, bypassing the low-yield PsiH catalyzed step. These starting substrates will be supplied to a previously optimized psilocybin production strain in monoculture. A third approach will expand the current psilocybin biosynthesis pathway through use of a new type class of enzymes to the Jones Lab, halogenases. Specifically, we will focus on site-specific chlorination enzymes which have previously been shown to have activity towards tryptophan substrates in prokaryotic hosts. Finally, the last approach will combine the first and third approaches into a single study, enabling de novo psilocybin derivative production by expansion of the psilocybin biosynthesis pathway to include both P450 monooxygenase and halogenase modules.