Screening of a Psilocybin Pathway Gene Variant Library Leads to Enhanced Strain Performance

Psilocybin is the hallucinogenic compound found in the recreational drug known as magic mushrooms and is currently being studied as a treatment for PTSD, anxiety, and depression. The psilocybin biosynthesis pathway as reconstituted in Escherichia coli contains three exogenous genes: PsiD, a decarboxylase, PsiK, a kinase, and PsiM, a methyltransferase. Previous efforts in the Jones Lab have shown high-titer psilocybin production, up to 1.16 g/L, in E. coli using gene sequences originating from the most common recreationally used psilocybin mushroom, Psilocybe cubensis. Here we attempt to expand our biosynthetic toolbox by testing PsiD, PsiK, and PsiM variants from 3 additional mushroom species: Gymnopilus dilepis, Panaeolus cyanescens, and Psilocybe cyanescens. These variants were identified by sequence homology to the original P. cubensis variant. The gene variants were cloned randomly into a biosynthetic library consisting of 64 (4^3) pathway combinations, four total species variants of three genes. Additionally, a smaller library of only 2 genes, PsiD and PsiK, was assembled and screened for norbaeocystin production. Both libraries yielded top producers that outperformed the production metrics of the original P. cubensis control. The top gene combinations were subjected to promoter optimization to further improve biocatalytic performance. Building upon this work, we look forward to scale up studies in benchtop bioreactors to fully evaluate the potential of the genetically superior strains developed in this work.