Regioselective Aromatic <i>O</i> -Demethylation of Lignin-Derived Monomeric Aromatic Ethers with an Engineered P450BM3 Peroxygenase System

Regioselective <i>O</i>-demethylation of lignin-derived aryl ethers is a key yet challenging step in lignin valorization, governing the efficient conversion of lignin into high-value aromatic chemicals. Here, we report an artificial peroxygenase system that integrates engineered P450BM3 with glucose oxidase (GOx) and a dual-functional small molecule (DFSM) to enable efficient <i>O</i>-demethylation driven by in situ H₂O₂ generation. This strategy circumvents the dependence on costly NADPH and improves catalytic sustainability and operational robustness. The engineered F87A/T268D/A328F mutant achieved efficient double <i>O</i>-demethylation of syringol with a turnover number (TON) of 583 ± 7, while the F87A/V78A mutant exhibited a 68.2-fold enhancement in catalytic efficiency toward anisole (TON = 341 ± 8) compared to wild-type P450BM3. Precise regulation of oxidative catalysis and H₂O₂ supply afforded high efficiency, selectivity, and stability. This work establishes a scalable and environmentally benign biocatalytic platform for upgrading lignin-derived aromatic monomers, advancing sustainable biomass utilization.