By deleting genes involved in L-tryptophan catabolism, psilocybin production was increased fivefold in baker's yeast and tenfold in the filamentous fungus Aspergillus nidulans. Process optimization in A. nidulans batch cultures yielded a final psilocybin titre of 267 mg/L with a space-time-yield of 3.7 mg/L/h. The engineered strain demonstrates suitability as a production chassis for psilocybin and other tryptamine-derived pharmaceuticals.
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.