Computational and Experimental Engineering of trans-Anethole Oxygenase for Enhancing Activity in Industrial Vanillin Biosynthesis

-anethole oxygenase (TAO). However, wild-type TAO shows low activity and stability. This study utilized AlphaFold2/3 modeling, molecular docking, and MD simulations to identify key residues (R86, T90, and H118) involved in substrate binding and catalysis. A multiplatform computational design strategy (PROSS, FireProt, Rosetta, and FoldX) was used to generate a mutation library, from which seven mutants were experimentally tested. Mutants F34Y, G117I, and T138L increased vanillin yield by over 40%, with F34Y reaching 88.7%. Energy decomposition indicated that these mutations enhanced substrate stabilization and heme coordination. F34Y remodeled distal H-bond networks, while G117I and T138L improved HEM stability. In contrast, N144L impaired catalytic efficiency by increasing protein flexibility. This study demonstrates an effective enzyme engineering strategy to improve TAO's catalytic performance and offers insights for optimizing biocatalysts in sustainable vanillin production.