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

), ≃ 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.