Rapid Enzyme Engineering: Boosting Activity Without Crystal Structure Using a Combinatorial Computer Approach

This manuscript introduces a novel combinatorial computer approach for rapidly enhancing the activity of enzymes, particularly those lacking a resolved crystal structure. Our research focuses on a strategy called 'Csfp' and validates its effectiveness using the model enzyme D-pantothenate lactone hydrolase. Results demonstrate that the Csfp strategy significantly accelerates the improvement of d-lactate hydrolase activity while simultaneously minimizing the need for extensive activity screening of mutant proteins. This efficient approach offers a universal pipeline for the directed evolution of enzymes, even in cases where structural information and catalytic mechanisms remain unclear. By facilitating the engineering of enzymes without known structures, our method paves the way for expanded applications in synthetic biology and beyond. We believe this work holds significant interest for a broad scientific audience and contributes a valuable tool for enzyme engineering and biotechnology.