Expansion of Non-Natural Psilocybin Derivative Synthesis in E. coli

Psychedelic drugs have garnered much clinical attention for their promising therapeutic potential in the treatment of mental health conditions. However, the presence of hallucinogenic side effects remains an issue that greatly limits their clinical use and legalization. It is suspected that derivatives of psychedelics may elicit the observed positive mental health effects while minimizing the negative hallucinogenic side effects. Production of a strong drug candidate pool is fundamental to the testing and discovery of such drugs. Recent derivatization of psilocybin already expanded this candidate pool to non-natural psychedelics; however, derivative synthesis was limited by the promiscuity of the traditional enzymatic pathway. Synthetic bottlenecks have been observed at tryptophan synthase (TrpB) and L-tryptophan decarboxylase (PsiD), thus alternative enzymes were explored. Incorporating TrpB variants Q90, Tri, and Azul and PsiD variant RgnTDC has enabled the expansion of derivative production from monosubstituted indoles beyond what was previously observed. Both tri-culture and co-culture designs were employed using optimized psilocybin production strains in conjunction with the new alternate enzymes in either wild-type E. coli or E. coli containing genomic integration of the traditional pathway enzymes. These systems were found to be successful in expanding non-natural derivative synthesis. They enabled production of seven new tryptophans and four new tryptamines and greatly enhanced the production of 18 more. The tri-culture setup successfully produced three new psilocybin derivatives, 7-aldehyde-psilocybin, 7-cyano-psilocybin, and 7-isopropyl-psilocybin, and the genome-integrated co-culture has produced 5-chloro-psilocybin. This testing primarily focuses on the use of Q90 and RgnTDC and suggests that use of these alternative enzymes allows for synthesis of bulkier indole derivatives than observed with traditional pathway enzymes. Further testing of monosubstituted indoles is required to realize the complete potential of the integrated co-culture setup.