De Novo Biosynthesis of Antidepressant Psilocybin in Escherichia coli

Psilocybin, a tryptamine-derived alkaloid with Breakthrough Therapy designation for treatment-resistant depression, was synthesized de novo in Escherichia coli by engineering the fungal cytochrome P450 enzyme PsiH. Modifications to PsiH's N-terminal domain and expression of the entire biosynthetic pathway at low temperature improved enzyme activity. Enhancing precursor supply and the P450 electron transfer chain increased norbaeocystin titers 33-fold (to 105.3 mg/L) and psilocybin titers 17-fold (to 14 mg/L). Overexpressing an extra copy of the methyltransferase gene psiM further boosted psilocybin production. Optimizing flask fermentation conditions yielded 79.4 mg/L of psilocybin, a 100-fold improvement over the starting strain, advancing sustainable microbial production of this antidepressant.

Study at a glance

Abstract

ABSTRACT Psilocybin, a tryptamine‐derived alkaloid, has been granted Breakthrough Therapy designation by the U.S. FDA for treatment‐resistant depression, underscoring its clinical importance. Therefore, sustainable and economic production is urgently needed. Manufacturing of psilocybin in Escherichia coli has drawn great attention. However, due to the low expression and activity of the eukaryotic cytochrome P450 enzyme PsiH in the psilocybin biosynthetic pathway, de novo synthesis of psilocybin in prokaryotic cells has been hampered. To overcome this dilemma, we herein demonstrated de novo synthesis of psilocybin in E. coli by constructing PsiH variants with N‐terminal domain modifications and expressing the entire biosynthetic pathway at a concordantly low temperature. Improving the supply of precursor and engineering the P450 electron transfer chain resulted in a 33‐fold increase in the titre of norbaeocystin (105.3 mg/L), a key intermediate of psilocybin biosynthesis, and a 17‐fold increase in the titre of psilocybin (14 mg/L). Further enhancement of psilocybin production was achieved by converting norbaeocystin to psilocybin by overexpressing an extra copy of the methyltransferase gene psiM . Finally, 79.4 mg/L of psilocybin was produced by optimising flask fermentation conditions, a 100‐fold improvement over the starting strain. Our work demonstrates the successful fungal P450 engineering to improve the catalytic activity in E. coli and will advance the sustainable production of the important antidepressant psilocybin in prokaryotic microbial cells.