(288c) Production of Psilocybin and Norbaeocystin in E. coli Enable the Discovery of an Entourage Effect in Psilocybe, “Magic”, Mushrooms

Since the beginning of the COVID-19 pandemic, the prevalence and demand for treatment of depression and anxiety have skyrocketed. To meet this demand, scientists must work to find new ways to tackle these complex disease states. Psilocybin, the main psychoactive molecule in “magic” mushrooms, is being studied for its ability to serve as a fast-acting treatment for a range of neurological diseases. Encouraged by recent successes in producing psilocybin from a recombinant E. coli host, we developed a novel production method for another phosphorylated tryptamine mushroom metabolite, norbaeocystin.

Norbaeocystin’s structural similarities to psilocybin and the scarcity of information about this molecule in the peer reviewed literature motivated our development of an E. coli production platform. Due to many pathway, substrate, and cofactor similarities to psilocybin, we utilized similar strain construction and optimization techniques to those explored for psilocybin production. Surprisingly, the results of our transcriptional balancing study revealed a different optimal solution, suggesting there is still much to be learned before a priori pathway optimization studies become a reality. Furthermore, to enhance production of norbaeocystin to industrially viable levels, we performed a range of benchtop bioreactor studies varying key media components and control setpoints. Due to high metabolic demand for serine in the pathway, we hypothesized that the exogenous pathway would deplete the natural supply. However, we found the addition of serine to the culture media had no significant impact on titer upon scale-up; therefore, resulting in 1.58 ± 0.08 g/L of norbaeocystin, the highest production levels reported to date for this molecule.

The high production of our strain allowed us to test the pharmacological response to both psilocybin and norbaeocystin in Long-Evans rats using a head twitch response (HTR) model. HTRs are high-frequency paroxysmal head and body rotations that occur in rodents after 5-HT2A receptor activation. These behaviors are widely used as an indicator of hallucinogenic effects, and are one of the only behaviors that can reliably distinguish hallucinogenic from non-hallucinogenic 5-HT2A agonists. Results from HTR studies showed that psilocybin alone elicits a HTR while norbaeocystin does not. More interestingly, we found that when dosed in combination, psilocybin + norbaeocystin causes an increased HTR when compared to psilocybin alone. This discovery suggesting a natural synergy, or entourage effect, between naturally occurring mushroom metabolites. These results could have implications in the ongoing development of novel tryptamine therapeutics for mental health treatment.