Target Discovery of\nDhilirane-Type Meroterpenoids\nby Biosynthesis Guidance and Tailoring Enzyme Catalysis

Dhilirane-type meroterpenoids (DMs) featuring a 6/6/6/5/5\nring\nsystem represent a rare group of fungal meroterpenoids. To date, merely\n11 DMs have been isolated or derived, leaving their chemical diversity\npredominantly unexplored. Herein, we leverage an understanding of\nbiosynthesis to develop a workflow for discovery of DMs by genome\nmining, metabolite analysis, and tailoring enzyme catalysis. Twenty-three\nnew DMs, including seven unprecedented scaffolds, were consequently\nidentified. An α-ketoglutarate (α-KG)-dependent oxygenase\nDhiD was found to catalyze the stereodivergent ring contraction of\ndhilirolide D to form the dhilirane skeleton; while the cytochrome\nP450 DhiH reshaped the structural diversity by establishing diverse\nC–C bonds and oxidation. Crystallographic and mutagenesis experiments\nprovide a molecular basis for the DhiD reaction and its stereodivergent\nproducts. Notably, DhiD exhibits substrate-controlled catalytic versatility\nin the chemical expansion of DMs through ring contraction, hydroxylation,\ndehydrogenation, epoxidation, isomerization, epimerization, and α-ketol\ncleavage. Bioassay results demonstrated that the obtained meroterpenoids\nexhibited anti-inflammatory and insecticidal activities. Our work\nprovides insight into nature’s arsenal for DM biosynthesis\nand the functional versatility of α-KG-dependent oxygenase and\nP450, which can be applied for target discovery and diversification\nof DM-type natural products.