"Biosynthesis of psilocybin and its nonnatural derivatives by a promiscuous psilocybin synthesis pathway in Escherichia coli".

Traditional psychedelics are undergoing a transformation from recreational drugs, to promising pharmaceutical drug candidates with the potential to provide an alternative treatment option for individuals struggling with mental illness. Sustainable and economic production methods are thus needed to facilitate enhanced study of these drug candidates to support future clinical efforts. Here, we expand upon current bacterial psilocybin biosynthesis by incorporating the cytochrome P450 monooxygenase, PsiH, to enable the de novo production of psilocybin as well as the biosynthesis of 13 psilocybin derivatives. The substrate promiscuity of the psilocybin biosynthesis pathway was comprehensively probed by using a library of 49 single-substituted indole derivatives, providing biophysical insights to this understudied metabolic pathway and opening the door to the in vivo biological synthesis of a library of previously unstudied pharmaceutical drug candidates.

Evaluation of TrpM and PsiD substrate promiscuity reveals new biocatalytic capabilities.

N-methylated tryptamines, such as the hallucinogenic natural products, psilocybin and N,N-dimethyltryptamine (DMT), are gaining interest from the medical community due to their potential as next generation treatments for mental health disorders. The clinical relevance of these compounds has driven scientists to develop biosynthetic production routes to a number of tryptamine drug candidates, and efforts are ongoing to expand and further develop these biosynthetic capabilities. To that end, we have further characterized the substrate preferences of two enzymes involved in tryptamine biosynthesis: TrpM, a tryptophan N-methyltransferase from Psilocybe serbica, and PsiD, the gateway decarboxylase of the psilocybin biosynthesis pathway. Here, we show that TrpM can N-methylate the non-native amino acid substrate, 4-hydroxytryptophan, a key intermediate in the Escherichia coli-based recombinant psilocybin biosynthesis pathway. However, the ability to incorporate TrpM into a functional psilocybin biosynthesis pathway was thwarted by PsiD's inability to use N,N-dimethyl-4-hydroxytryptophan as substrate, under the culturing conditions tested, despite demonstrating activity on N-methylated and 4-hydroxylated tryptophan derivatives individually. Taken together, this work expands upon the known substrates for TrpM and PsiD, further increasing the diversity of tryptamine biosynthetic products.