Vanillin is a valuable natural product that can be used as a fragrance and additive. Recent research in the biosynthesis of vanillin has brought attention to a key enzyme, carboxylic acid reductase (CAR), which catalyzes the reduction of vanillic acid to vanillin. Nevertheless, the biosynthesis of vanillin is hampered by the low activity and stability of CAR. As such, a rational design campaign was conducted on a well-documented carboxylic acid reductase from Segniliparus rugosus (SrCAR), using vanillic acid as the model substrate. After combined active site saturation and iterative site-specific mutagenesis, the best quadruple mutant N292H/K524S/A627L/E1121W (M3) was successfully obtained. In comparison to the wildtype SrCAR, M3 demonstrated a 4.2-fold increase in catalytic efficiency (kcat/Km), and its half-life (t1/2) was enhanced by 3.8 times up to 385.08 minutes at 40 °C. In silico docking and molecular dynamics simulation provided insights into the improved activity and stability. In the subsequent preparative-scale reaction with 100 mM (16.8 g L-1) vanillic acid, the whole cell catalysis utilizing M3 produced 10.15 g·L-1 of vanillin and 1.11 g·L-1 of vanillyl alcohol, respectively. This work demonstrates a dual improvement in the activity and thermal stability of SrCAR, thereby potentially facilitating the application of carboxylic acid reductase in the biosynthesis of vanillin.
