Engineering a 4-Vinylguaiacol Oxygenase for Cofactor-Free, Cell-Free Vanillin Biosynthesis

Vanillin biosynthesis from ferulic acid offers a sustainable alternative to conventional extraction methods, but the industrial implementation of the coenzyme-independent pathway has been hindered by inefficient oxygenases. This study reports a thermostable 4-vinylguaiacol oxygenase (Vgo), which exhibits soluble expression at 16–30 °C and maintains stable catalytic activity at 50 °C. Through rational design of the substrate-binding pocket, we obtained two optimized variants, VgoM1 (Vgo W335F ) and VgoM10 (Vgo W335F/I404C/V409W ), which demonstrated enhanced thermostability, with T 30,50 increased by 2 °C and catalytic efficiency. Specifically, VgoM1 exhibited a 2-fold lower K m value, while VgoM10 achieved a 4-fold higher k cat and produced 111 mM vanillin in 24 h (89% conversion) in a cell-free system. Molecular simulations revealed (1) strengthened hydrogen-bonding networks, (2) reduced Fe 2+ -substrate distance (12.1 to 8.6 Å), and (3) increased flexibility in gating loops that facilitates substrate access to the active site. Kinetic and thermodynamic analyses confirmed improved substrate-binding stability coupled with optimized conformational dynamics. These findings establish the potential for efficient, sustainable, and scalable cell-free vanillin production.