Psilocybin, the prodrug to the psychoactive compound in 'magic' mushrooms, is currently being studied in clinical trials as a treatment for severe mental health conditions, such as depression and anxiety. Previous reports of psilocybin biosynthesis as reconstituted in E. coli reported maximum titers of 1.16 g/L, exclusively using genes from the most common recreationally used mushroom, Psilocybe cubensis. This study explores the effect of gene species variation on psilocybin and baeocystin production using various exogenous genes sourced from psilocybin-producing mushrooms Psilocybe cubensis, Psilocybe cyanescens, Panaeolus cyanescens, and Gymnopilus dilepis. The psiD and psiK genes sourced from P. cubensis demonstrated unequivocally superior performance, while psiM showed varied production levels of psilocybin and the pathway intermediate baeocystin with changes in gene source. Strains containing a psiM gene sourced from Psilocybe cyanescens demonstrated a higher degree of baeocystin selectivity as compared to other psiM genes, demonstrating a key difference between species. Most notably, the strain Gymdi30, containing psiM sourced from G. dilepis, achieved a psilocybin titer of 1.46 ± 0.13 g/L, the highest reported to date. Comparative proteomic analysis of Gymdi30 during periods of high and low productivity was also performed to investigate bottlenecks in cellular metabolism, which could be limiting strain performance. This work represents a significant improvement in psilocybin biosynthesis, a key step towards the development of a biosynthetic manufacturing route for psilocybin.
