Optimizing the De Novo Production of Psilocybin in E. coli

E. coli-based psilocybin production begins through supplementation of 4-hydroxyindole to the fermentation media, enabling the production of 4-hydroxytryptophan through the promiscuous action of tryptophan synthase, TrpB. The removal of 4-hydroxyindole supplementation could enable efficient de novo biosynthesis of psilocybin, representing a more economical and sustainable pathway for its biosynthesis. This can be accomplished by incorporating a previously identified P450 monooxygenase, PsiH, into the recombinant strain. Initial efforts, although successful at producing psilocybin, have resulted in substantially lower titers than the 4-hydroxyindole supplemented approach.

PsiH selectively hydroxylates the 4-position on tryptamine, forming 4-hydroxytryptamine, which is then phosphorylated by a kinase (PsiK), and then methylated by the SAM-dependent methyltransferase (PsiM) two times to form psilocybin. In the native mushroom, the PsiH is bound to the endoplasmic reticulum membrane. Moving the PsiH to E. coli (a host that lacks membrane-bound organelles) results in decreased function and expression of the protein. To optimize the P450 function in E. coli, codon optimization, truncations to the transmembrane region of the protein, and N-terminal modifications were performed. Using a bioinformatics tool, the PsiH transmembrane region was identified and replaced by four different N-terminal modifications with the goal of increasing soluble expression and activity of the P450 in our E. coli host.

The same four N-terminal changes were performed on five PsiHs from different mushroom species as well as one species of cytochrome P450 reductase (CPR). For the P450 monooxygenase to function properly, it must be coupled with a CPR, which donates electrons to maintain a redox balance. The 80 different combinations of PsiH and CPR were cloned and tested to reveal several key combinations capable of enhanced 4-hydroxytryptamine production. Current work is underway to test these PsiH/CPR modules in conjunction with the current best psilocybin production pathway to enable enhanced de novo psilocybin production. After the top strain has been identified, further optimization will be performed looking at different fermentation conditions.