The research in this manuscript focuses on the rapid boosting of the activity of enzymes lacking crystal structure by a combinatorial computer approach Our results are validated by a model enzyme D-pa

This manuscript presents research that focuses on rapidly enhancing the activity of enzymes lacking crystal structure using a combinatorial computer approach. Our findings are validated by a model enzyme, D-pantothenate lactone hydrolase, and demonstrate that the Csfp strategy accelerates the improvement of d-lactate hydrolase activity while minimizing the need for activity screening of mutant proteins. This provides a universal pipeline for the directed evolution of enzymes without known structures and clear catalytic mechanisms, facilitating their application in synthetic biology. We believe that our work will be of interest to a wide audience and will make a significant contribution to the scientific community