Engineering a Substrate-Affinitive and Thermostable YqhD Variant for Efficient Bioconversion of Perillyl Aldehyde to Perillyl Alcohol

Perillyl alcohol is a valuable monoterpenoid, but its production is limited by low extraction yields and inefficient chemical synthesis. Here, we have a novel biocatalytic strategy for converting perillyl aldehyde to perillyl alcohol using the endogenous NADPH-dependent oxidoreductase YqhD from <i>Escherichia coli</i>. Structure-guided rational and semirational design yielded the double mutant M2 (Q262G/M268F) with 2.61-fold higher activity and 2.11-fold improved <i>k</i>_cat/<i>K</i>_m. Introduction of A166P further increased the optimal reaction temperature by 10 °C, and triple mutant M3 (A166P/Q262G/M268F) exhibited a 9.45-fold longer half-life at 40 °C. Molecular dynamics simulations indicate that a more hydrophobic and rigid active-site microenvironment underlies these improvements. In an engineered mevalonate pathway strain, M3 raised perillyl alcohol titers from 291.19 to 388.97 mg/L at 30 °C and enabled 107.48 mg/L production at 40 °C while reducing perillyl aldehyde accumulation, providing a framework for engineering YqhD toward hydrophobic monoterpenoids.