Zhao Sijia, Lin Yingwu, Wang Jinghan, Li Dong, Wang Fang, Shoji Osami, Xu Jiakun
State Key Laboratory of Mariculture Biobreeding and Sustainable Goods (Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences), Key Laboratory of Sustainable Development of Polar Fisheries, Ministry of Agriculture and Rural Affairs, Qingdao 266071, China; Laboratory for Marine Drugs and Bioproducts, Qingdao Marine Science and Technology Center, College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.
School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China.
Int J Biol Macromol. 2025 May;309(Pt 1):142768. doi: 10.1016/j.ijbiomac.2025.142768. Epub 2025 Apr 1.
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 HO 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 HO 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/HO 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.
芳香族化合物的酶促脱甲基化是木质素增值过程中的一项重大挑战。主要目标是开发一种高效的人工过氧合酶系统，该系统将工程化的P450BM3与AldO（糖醇氧化酶）和DFSM（双功能小分子）相结合，用于木质素衍生的芳香醚的区域选择性O-脱甲基化。P450BM3是一种多功能生物催化剂，其工程变体对非天然底物表现出扩大的底物选择性。AldO用作原位产生HO的系统。DFSM是一种经过合理设计的催化助剂，有助于精确控制酶促反应并提高O-脱甲基化的效率。我们假设通过将P450BM3与AldO和DFSM相结合，可以更好地控制HO的产生，并使酶促系统朝着高效的O-脱甲基化方向发展。工程化的P450BM3 F87A/V78A/T268D/A328F突变体对愈创木酚的TON达到1895±4，是天然P450BM3/HO系统（TON = 872±7）的两倍多。此外，F87A/T268D突变体有效地催化了丁香酚的双脱甲基化，实现了最高的周转数（TON）483±7。这种DFSM辅助的P450BM3/AldO系统代表了木质素生物催化降解的重大进展，并为传统的依赖NADPH的P450单加氧酶提供了一种经济高效且可扩展的替代方案。我们的发现为木质素增值和芳香族化合物分解代谢中的可持续生物技术应用开辟了新途径。
