AbstractPharmaceutical industry is progressively replacing the chemical synthesis of cholesterol‐lowering agents by enzymatic processes. The directed evolution of acyltransferase LovD was a breakthrough in the synthesis of simvastatin, although little is known about how the in vitro evolution path raised up an engineered variant (LovD9) with excellent biocatalytic properties (high catalytic efficiency and stability under reaction conditions). In this study, we unveil how different mutation clusters scattered across LovD9 primary sequence specifically contribute to enhance both enzyme kinetics and stability. To this aim, simvastatin synthetic and hydrolytic activities, kinetic parameters and thermostability of several engineered variants were assessed. Through a rational combination of those clusters of mutations, we generated the variant LovD−BuCh2 whose catalytic efficiency is around 90 % of that obtained with LovD9 but with 15 less mutations. Supported by molecular dynamics simulations, this work demonstrates the cumulative effect of mutations at both the active site and the substrate entrance channel to enhance binding of the acyl donor and speed up the acyl transfer step from the acyl‐enzyme complex to monacolin J acid, while simultaneously minimizing detrimental side‐reaction pathways, substrate inhibition and increasing thermostability.