Reshaping Interface Interactions of <i>P. litoralis</i> Acyltransferase for Efficient Chemoenzymatic Epoxidation in Aqueous Phase

Epoxides, a class of ethers with a three-membered ring structure, are widely used in the textile, pharmaceutical, and packaging industries. Chemoenzymatic epoxidation presents a promising method for synthesizing epoxides. However, its epoxidation efficiency is hindered by low chemoselective perhydrolysis, which is caused by the hydrolysis side reaction in the aqueous phase. In this study, a chemoenzymatic epoxidation process in the aqueous phase was developed by utilizing an acyltransferase from <i>P. litoralis</i> (PlAcT) for its chemoselective perhydrolysis. Crystal structure analysis, molecular dynamics simulations, and quantum mechanics calculations, along with site-specific mutagenesis, revealed that the selectivity of perhydrolysis is due to a lower energy barrier in the acyl transfer step compared to that in hydrolysis. Furthermore, the mutant PlAcT^M3-2 exhibited a 7.6-fold improvement in solvent stability and a 1.3-fold increase in perhydrolysis activity compared to the wild type, achieved by reshaping interface interactions. As a result, the engineered strain Y07, harboring PlAcT^M3-2, successfully synthesized compounds <b>3a</b>-<b>3n</b> with conversions ranging from 11-99%, and the titers of compounds α-pinene oxide<b>(3i),</b> β-pinene oxide<b>(3j),</b> 3-carene oxide<b>(3k)</b>, and limonene dioxide<b>(3l-3)</b> reached 55.8, 16.7, 75.2, and 21.4 g/L, respectively. These results demonstrate a sustainable method for chemoenzymatic epoxidation in the aqueous phase.