Gram-scale microbial synthesis of hypericin, a low-abundance complex phytochemical drug candidate

Hypericin is a promising Phase II/III drug candidate for oncology and psoriasis, yet its sustainable supply is problematic: multi-step synthesis is resource-intensive, and heterologous expression is blocked by the unknown plant pathway. Here, we re-engineer the polyketide biosynthetic pathways in Cladosporium fulvum and overcome substrate inhibition and metabolic inefficiency, achieving scalable and plant-pathway-independent biomanufacturing of hypericin. In particular, we redesigned the catalytic cavity of the critical P450 enzyme RugG to yield a variant (RugGT452P-R160D) with enhanced activity and reduced substrate inhibition. This achievement was combined with the suppression of shunt product formation to redirect metabolic flux toward emodin bianthrone—an indispensable hypericin precursor. These efforts enabled us to construct an engineered strain, ΔclaC-ΔclaH2-rugGT452P-R160D-claA-claE, which produced 1.87 g/L hypericin in a 5-L bioreactor after photocatalytic treatment. Collectively, this work provides an effective strategy for alleviating substrate inhibition and redirecting metabolic flux in the biomanufacturing of complex, high-value natural products.