Here, we present our progress towards the development of a modular biosynthetic route for the production of the hallucinogenic natural product, psilocybin, in the model microbe, <i>Escherichia coli</i>. The development of a psilocybin production platform in a highly engineerable microbe could lead to rapid advances towards the bioproduction of psilocybin for use in ongoing clinical trials for treatment resistant depression, post-traumatic stress disorder (PTSD), and addiction. Efforts to improve pathway performance through genetic and fermentation conditions optimization will be presented. These efforts include the construction, evaluation, and comparison of a combinatorically defined copy number library and a series of random ePathOptimize promoter libraries. Through these genetic optimization approaches, we are able to show a 55-fold improvement in psilocybin titer. Further improvements in this genetically-optimized base strain were achieved through optimization of fermentation conditions, including fermentation temperature, media composition, induction timing, as well as inducer, supplement and precursor concentrations. The combination of genetic and fermentation optimization resulted in an over 200-fold improvement in psilocybin titer. Ongoing and future efforts to deregulate and enhance intracellular precursor and co-factor flux in the psilocybin production host will also be presented. The evaluation of strains and conditions was accomplished through the use of medium-throughput screening in 48-well plates coupled with HPLC analysis of metabolite concentrations. We discuss our work with this unique approach to develop and characterize the production landscape of our novel production host.

Biosynthesis of the hallucinogenic natural product psilocybin in <i>E. coli</i>

2019 Spring National Meeting

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Biosynthesis of the hallucinogenic natural product psilocybin in E. coli