The Bacillus-derived acyltransferase BmmI exhibited broad substrate flexibility, transferring malonyl group not only to the macrolide compounds, but also to the anti-tumor agent glycosylated piericidin A (GPA). In this project, we found that malonylation could improve the cytotoxic activity of GPA, indicating BmmI have a great potential to generate derivatives with higher activities. Therefore, we applied semi-rational directed evolution strategy to engineer BmmI for higher catalytic efficiency toward GPA. Our study provides foundation for further engineering of BmmI to obtain GPA derivatives with better bioactivities.