Focused rational iterative site-specific mutagenesis (FRISM): A powerful method for enzyme engineering

The FRISM strategy has emerged as one of the most efficient methods for protein engineering. It involves the identification of hotspots, which is usually rationalized through computer-aided technologies, followed by the generation of a focused mutant library. By reducing the size of the mutation library and the screening efforts, the FRISM strategy has been successfully employed in engineering a wide range of enzymes with enhanced catalytic performance, improved enantio‑, regio-, and chemoselectivities. This review discusses the underlying principle, historical development and diverse applications of the FRISM strategy. We hope that this review will inspire more research groups to embrace and apply FRISM in their protein engineering endeavors.