Growth-coupled continuous directed evolution by MutaT7 enables efficient and automated enzyme engineering

Enzyme engineering aims to develop enzymes with improved or novel traits, but traditional methods are slow and require repetitive manual steps. This study presents a faster, automated protein engineering approach. We utilized an <i>in vivo</i> mutagenesis technique, MutaT7 tools, to induce mutations in living bacteria and established a direct link between enzyme activity and bacterial growth. A continuous culture setup enables automated mutagenesis and growth-coupled selection of better-performing variants in real time. Bacteria with improved enzymes grew faster, selecting superior variants without manual intervention. Using this method, we engineered CelB with better performance at lower temperatures while maintaining thermal stability. By combining high-throughput mutagenesis and selection in a single process, this system bypasses iterative cycles of error-prone PCR, transformation, and screening. Our approach is adaptable to various enzymes, providing a faster and more efficient solution for enzyme engineering.