(594d) Screening of Norbaeocystin Methyltransferase Variants Enables Enhanced Psilocybin and Baeocystin Production in E. coli

While psilocybin is currently under clinical evaluation for the treatment of severe depression, post-traumatic stress disorder (PTSD), and addiction, anecdotal evidence from recreational users has led some to postulate that the ratio of naturally occurring psychoactive metabolites in various mushroom species may greatly impact the experience and overall effect on the brain. This “Entourage Effect” may necessitate a specific production strain capable of high baeocystin production to meet necessary pharmaceutical demand. Norbaeocystin methyltransferase (psiM) is responsible for the iterative methylation of norbaeocystin, resulting in the production of psilocybin via a monomethylated intermediate, baeocystin. The genetic variation between mushroom species and resulting differences in their metabolite profile, have motivated this study to evaluate a range of psiM variants for their ability to selectively produce the intermediate product baeocystin. Here, we examine the effect of genetic variability of the psiM gene and attempt to create a biosynthetic production platform for producing high titers of baeocystin and higher titers of psilocybin than previously reported. Screening of genes from Psilocybe cubensis, Psilocybe cyanescens, Panaeolus cyanescens and Gymnopilus dilepis in a high throughput well plate assay yielded a production strain capable of producing more than 480 mg/L of psilocybin prior to fermentation optimization and scale up. This surpasses the current best performing psilocybin production strain by an increase of 245% under identical conditions. Additionally, initial screening produced a range of strains with enhanced baeocystin selectivity, with individual titers as high as 124 mg/L. Ongoing efforts to validate, optimize, and scaleup multiple lead strains is currently underway. This work further enhances the viability of industrial batch production of psilocybin using fermentation and provides a starting point for the development of strains capable of selectively producing its precursors for combined medicinal use.