Enhanced Psilocybin Biosynthesis through Genomic Pathway Integration

Psychedelic compounds, including psilocybin and DMT, have demonstrated success in clinical trials treating individuals with neuropsychiatric conditions. The pharmaceutical potential of these compounds have motivated the development of more economic production processes. Metabolic engineering—a discipline that uses genetic engineering to modify an organism’s metabolism to enhance chemical production—has enabled high psilocybin production in Escherichia coli using plasmid-based pathway expression systems. Advancement in genome editing tools has opened new doors to potentially improve heterologous pathway function and expression in E. coli. 

Initial efforts to integrate psilocybin pathway genes at four locations in the E. coli genome proved successful, however, psilocybin titers were greatly reduced. Further characterization of the promoters used in the integrated system revealed significant expression deviations from the characterization of these promoters in the plasmid-based system. Here, we describe the development and characterization of new promoter mutants that, when expressed on the genome, result in a more complete range of expression levels. These new promoters were then cloned into transcriptional libraries controlling expression of psilocybin and DMT production pathway genes. The resulting E. coli genome integrated strains were assessed for psilocybin and DMT production in a 48-well plate fermentation study.