Regioselective aromatic O-demethylation with an artificial P450BM3/sugar alcohol oxidase peroxygenase system

The enzymatic demethylation of aromatic compounds presents a major challenge in the valorization of lignin. The main goal was to develop an efficient artificial peroxygenase system combining engineered P450BM3 with AldO (sugar alcohol oxidase) and DFSM (dual function small molecule) for the regioselective O-demethylation of lignin-derived aromatic ethers. P450BM3 serves as a versatile biocatalyst, and its engineered variants demonstrate expanded substrate promiscuity toward non-native substrates. AldO, served as the H₂O₂ in situ generation system. The DFSM, a rationally designed catalytic auxiliary, facilitates precise control of enzymatic reactions and enhances the efficiency of O-demethylation. We hypothesize that by combining P450BM3 with AldO and DFSM, we can better control the generation of H₂O₂ and direct the enzymatic system toward efficient O-demethylation. The engineered P450BM3 F87A/V78A/T268D/A328F mutant achieved a TON of 1895 ± 4 for guaiacol, more than double that of the native P450BM3/H₂O₂ system (TON = 872 ± 7). Moreover, the F87A/T268D mutant efficiently catalyzed double-demethylation of syringol, achieving the highest turnover number (TON) of 483 ± 7. This DFSM-assisted P450BM3/AldO system represents a significant advancement in the biocatalytic degradation of lignin and offers a cost-effective and scalable alternative to traditional NADPH-dependent P450 monooxygenases. Our findings open new pathways for sustainable biotechnological applications in lignin valorization and aromatic compound catabolism.