In Vitro Psilocybin Synthesis by Co‐Immobilized Enzymes

Abstract Advanced clinical trials investigate the Psilocybe magic mushroom natural product psilocybin as a treatment against major depressive disorder. Currently, synthetic material is used to meet the demand for legitimate pharmaceutical purposes. Here, we report an in vitro approach to biocatalytically produce psilocybin on a solid‐phase matrix charged with five covalently bound biosynthetic enzymes. These enzymes include three Psilocybe enzymes: IasA*, an engineered l ‐tryptophan decarboxylase/aromatic aldehyde synthase, the 4‐hydroxytryptamine kinase PsiK and the norbaeocystin methyltransferase PsiM, along with Escherichia coli nucleosidase MtnN and adenine deaminase Ade. In a proof‐of‐principle experiment, this enzyme‐charged resin allowed for quantitative turnover of 4‐hydroxy‐ l ‐tryptophan into psilocybin. This facile process i) represents a sustainable approach with reusable enzymes, ii) circumvents the drawbacks of in vivo processes while harnessing the selectivity of enzymatic catalysis and iii) helps access an urgently needed drug candidate.