Optimized psilocybin production in tryptophan catabolism‐repressed fungi

The high therapeutic potential of psilocybin, a prodrug of the psychotropic psilocin, holds great promise for the treatment of mental disorders such as therapy‐refractory depression, alcohol use disorder and anorexia nervosa. Psilocybin has been designated a ‘Breakthrough Therapy’ by the US Food and Drug Administration, and therefore a sustainable production process must be established to meet future market demands. Here, we present the development of an in vivo psilocybin production chassis based on repression of l‐tryptophan catabolism. We demonstrate the proof of principle in Saccharomyces cerevisiae expressing the psilocybin biosynthetic genes. Deletion of the two aminotransferase genes ARO8/9 and the indoleamine 2,3‐dioxygenase gene BNA2 yielded a fivefold increase of psilocybin titre. We transferred this knowledge to the filamentous fungus Aspergillus nidulans and identified functional ARO8/9 orthologs involved in fungal l‐tryptophan catabolism by genome mining and cross‐complementation. The double deletion mutant of A. nidulans resulted in a 10‐fold increased psilocybin production. Process optimization based on respiratory activity measurements led to a final psilocybin titre of 267 mg/L in batch cultures with a space–time‐yield of 3.7 mg/L/h. These results demonstrate the suitability of our engineered A. nidulans to serve as a production strain for psilocybin and other tryptamine‐derived pharmaceuticals. Here, we developed an in vivo psilocybin production chassis, based on repression of l‐tryptophan catabolism. By genome mining, we identified two aminotransferase ARO8/9 orthologs in Aspergillus nidulans involved in l‐tryptophan catabolism. Double deletion of the aminotransferase genes aroH1 and aroH2 resulted in a 10‐fold increased psilocybin production.