Rational Engineering of Carboxylic Acid Reductase for Improved Catalytic Efficiency and Substrate Tolerance Enabling Green Vanillin Biosynthesis

Abstract Vanillin is a high-value natural aromatic compound widely used in food, flavor, and pharmaceutical fields. Carboxylic acid reductase (CAR)-catalyzed vanillic acid bioreduction offers a green vanillin biosynthesis route, yet low catalytic activity and severe substrate inhibition limit its industrial application. Herein, we screened 10 bacterial CAR homologues and constructed superior chimeric CAR templates via domain recombination. Combined with structure-guided directed evolution and substrate tunnel remodeling, the optimized quadruple mutant M4 was obtained, exhibiting 4-fold higher catalytic efficiency compared with the wild-type MavCAR (and 1.6-fold higher than the parental chimera M0) and partially relieved activity decline at elevated substrate concentrations. Structural simulations clarified that tunnel expansion and hydrogen bond remodeling synergistically stabilized enzyme–substrate binding. The M4-based whole-cell system achieved 95% vanillin conversion (8.7 g·L–1) from 10 g·L–1 vanillic acid, and a two-step cascade system realized efficient de novo vanillin synthesis from glucose. This work provides a robust engineered CAR biocatalyst for green vanillin preparative-scale biosynthesis.