Reprogramming a Baeyer-Villiger monooxygenase for improved synthesis of 3-hydroxypropionic acid precursor by structure-guided mutagenesis

Baeyer-Villiger monooxygenases (BVMOs) are attractive oxidative biocatalysts for the synthesis of 3-hydroxypropionic acid (3-HP) precursors from alkyl levulinates, but their broader application is often limited by insufficient operational robustness, uncoupling side reactions, and incomplete control over product distribution. Here, the Baeyer-Villiger monooxygenase from Acinetobacter radioresistens (Ar-BVMO) was engineered for improved oxidation of butyl levulinate through a structure-guided hotspot transfer strategy inspired by cyclopentanone monooxygenase (CPMO) from Comamonas sp. Structural alignment identified the Y141/Y142 region of Ar-BVMO as a catalytically sensitive site, and two single mutants, Y141L and Y142L, were generated. Their structural and functional effects were investigated by molecular dynamics simulations, stopped-flow measurements, steady-state kinetic analysis, coupling efficiency evaluation, and whole-cell biotransformations. Both substitutions increased the conformational mobility of the Ar-BVMO scaffold and altered NADPH-dependent catalytic behaviour. Among the two mutants, Y142L showed the best overall performance toward butyl levulinate, with 2.5-fold increase in catalytic efficiency (from 4.0 to 10.2 min−1 mM−1), ≃ 6% increase in coupling efficiency, and 54% increase in total turnover number. In contrast, Y141L displayed lower productivity and coupling efficiency, but complete regioselectivity toward the desired 3-acetoxypropionate product. Whole-cell biotransformations with Y142L confirmed the improved phenotype, achieving >95% substrate consumption within 24 h. Overall, this targeted enzyme-engineering study shows that hotspot transfer to the Ar-BVMO Y141/Y142 region can separate productivity from regioselectivity during butyl levulinate oxidation, providing a focused framework for further optimization of BVMOs for sustainable 3-HP precursor synthesis.