Engineering artificial biosynthetic pathways for efficient microbial production of psilocybin and psilocin

Psychedelic-assisted therapy is emerging as a highly promising approach for treating depression, with psilocybin, a psychoactive compound in magic mushrooms, gaining the most recognition for its efficacy in treating post-traumatic stress disorder and treatment-resistant depression. However, its low natural abundance makes extraction costly, necessitating alternative production methods. While engineered microbial production has been explored, dependence on the CYP450 hydroxylase (PsiH) in the natural biosynthetic pathway remains a major bottleneck, limiting production efficiency. Here, we report the design, validation, and optimization of artificial biosynthetic pathways in Escherichia coli that bypass PsiH, enabling efficient psilocybin and psilocin production. De novo biosynthesis of psilocybin achieved record titers of 557.91 mg/L in shake flasks and 2.00 g/L in a bioreactor, outperforming previous microbial engineering efforts. This work demonstrates the great commercial potential of microbial psilocybin production via combinatorial metabolic engineering and synthetic biology approaches.