Reshaping Interface Interactions of P. litoralis Acyltransferase for Efficient Chemoenzymatic Epoxidation in Aqueous Phase

Abstract

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 P. litoralis (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 3a–3n with conversions ranging from 11–99%, and the titers of compounds α-pinene oxide(3i), β-pinene oxide(3j), 3-carene oxide(3k), and limonene dioxide(3l–3) 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.