De novo biosynthesis of meroterpenoid grifolic acid in Yarrowia lipolytica

Grifolic acid, a potent bioactive meroterpenoid derived from fungi, shows great antitumor and antimicrobial properties. Nevertheless, the difficulty of large-scale chemical production and its minimal presence in natural environments hinder its path toward clinical translation. We developed a de novo grifolic acid biosynthetic route by leveraging the robust metabolic capabilities of the oleaginous yeast Yarrowia lipolytica . By improving the precursor malonyl-CoA supply through ACC1 overexpression and spatially targeting the fungal polyketide synthase ArmB to the vacuolar compartment, we enhanced orsellinic acid accumulation to 8.50 mg/L. We next leveraged AI-guided molecular docking and deep learning kinetic predictions (CataPro and UniKP) to screen candidate enzymes for coupling orsellinic acid with farnesyl pyrophosphate (FPP), identifying StbC from Stachybotrys bisbyi as the optimal prenyltransferase for coupling the polyketide with the terpenoid moieties. Integrating the heterologous module with efflux pump engineering (overexpressing native Pleiotropic Drug Resistance ABC transporters) and blocking the competing β-oxidation pathway (through HBD1 deletion) relieved intracellular toxicity and minimized carbon loss. Under optimized conditions using a two-stage glucose-growth and glycerol-supplementation fed-batch regime, the engineered strain produced grifolic acid at 30.5 mg/L in a shaker flask. This work represents the first successful de novo production of grifolic acid in Y. lipolytica , validating a holistic metabolic engineering strategy for the sustainable synthesis of complex meroterpenoids.