(160ak) Transposon Mediated and Cas9 Assisted Integration and Promoter Optimization of Psilocybin Biosynthetic Pathway

Psychedelics, such as psilocybin, have shown promise for the treatment of several mental health disorders such as post-traumatic stress disorder, anxiety, treatment resistant depression, and addiction. These small biomolecules are difficult to mass produce through traditional chemical synthesis or through extraction from biomass of the native production organism. Recently, psilocybin has been produced at gram per liter titers in E. coli using the cheap substrate, 4-hydroxyindole. Here, we show the integration and subsequent genetic optimization of the psilocybin biosynthetic pathway in the E. coli chromosome. The psilocybin pathway, consisting of PsiD, PsiK, and PsiM in basic operon form, was integrated into the E. coli genome using transposase, with the goal of increasing the stability of the genetic construct and reducing the costs associated with antibiotic selection in large scale bioproduction. Additionally, once a top mutant was identified a λ-Red recombinase system with Cas9 counterselection was used to fine tune the transcriptional optimization through application of a site-specific promoter replacement strategy. In this work, we directly compare genetic optimization processes and optimized psilocybin production strains based on plasmid and chromosomal-based approaches. The genetic stability of these optimized strains was evaluated in a 5-day fermentation using a 2-L benchtop bioreactor. In summary, we present a system for pathway integration and subsequent in vivo optimization leading to high-titer production of psilocybin in E. coli as well as a direct comparison between plasmid and chromosome-based psilocybin production strains.