Accelerated Enzyme Engineering: Rapid Activity Boosting without Crystal Structure using a Combinatorial Computer Approach

This manuscript details research focused on rapidly enhancing the activity of enzymes lacking known crystal structures through a novel combinatorial computer approach. We validate our findings using a model enzyme, D-pantothenate lactone hydrolase, demonstrating that the Csfp strategy effectively accelerates the enhancement of d-lactate hydrolase activity. Importantly, this method minimizes the need for extensive activity screening of mutant proteins. Our work presents a universal pipeline for the directed evolution of enzymes without known structures and clear catalytic mechanisms, thus facilitating their application in synthetic biology. We believe that this research will be of significant interest to a wide scientific audience and contribute valuable insights to the field of enzyme engineering.