Enabling the De Novo Production of Psilocybin Using an E.coli Co-Culture Approach

Psilocybin is the psychoactive prodrug of psilocin that has been shown to lessen the negative effects of PTSD, depression, and addiction. Biosynthetic production of psilocybin has been previously reported in bacteria, however, this was achieved through the metered supplementation of the starting substrate, 4-hydroxyindole. Here, we demonstrate the first example of de novo biosynthesis of this important molecule in a bacterial host, with all carbon deriving solely from glucose.

De novo synthesis is the process of producing complex biomolecules from simple carbon sources without the addition of supplements or precursors. To enable this process, we used a P450 monooxygenase, psiH, for the selective hydroxylation at the 4-position on tryptamine, resulting in 4-hydroxytryptamine. This intermediate product was then phosphorylated by the kinase, psiK, and dimethylated by a SAM-dependent methyltransferase, psiM, resulting in psilocybin. In order to maintain a redox balance, a cytochrome P450 reductase (CPR) was coupled with psiH; this required screening of multiple CPRs and gene orientations. Once we determined the top producing strain from genetic optimization efforts, we explored a range of fermentation conditions, including induction times, IPTG concentrations, and iron concentrations. Since P450s are heme proteins, they have an iron-porphyrin group. This need for iron acted as motivation to test different iron salts with our media to allow the P450 to fold correctly and carry out its catalytic processes more efficiently. These genetic and fermentation optimizations were performed in a high throughput well-plate format and top strains and conditions were further scaled to shake flask and benchtop bioreactors enabling enhanced pathway performance. To enable rapid evaluation of P450/CPR combinations, a co-culture approach was used in combination with the previously identified psilocybin producing strain, pSilo16, capable of psilocybin production from 4-hydroxyindole.