Combinatorial Engineering of the Catalytic Pocket and Structural Stability in 4-Vinylguaiacol Oxygenase for Efficient Vanillin Biosynthesis.

The low activity of 4-vinylguaiacol oxygenase severely limits the biocatalytic synthesis of vanillin from ferulic acid. In this study, strategies involving catalytic pocket engineering and machine-learning-assisted structural stability optimization were employed to enhance the activity of oxygenase VgoM1. First, seven rounds of iterative catalytic pocket engineering produced mutant M5, with a kcat/Km value increased by 4.8-fold. Then, fine-tuning the ODM and Trans models on the VgoM1 mutant database enabled the prediction of efficient distal sites of the pocket and the identification of mutant M97021, with a 6.8-fold higher kcat/Km than that of VgoM1. Molecular dynamics simulations demonstrated that distal site regulation enhanced structural stability, characterized by a binding free energy of -22.48 kcal/mol, an enhanced hydrogen-bonding network, RMSD < 0.35 nm, and Rg < 2.24 nm. In vitro catalysis of ferulic acid yielded 19.3 g/L vanillin within 20 h, with a maximum rate of 9.8 mM/h.