Substantial Improvement of an Epimerase for the Synthesis of D‐Allulose by Biosensor‐Based High‐Throughput Microdroplet Screening

Biosynthesis of D‐allulose has been achieved using ketose 3‐epimerases (KEases), but its application is limited by poor catalytic performance. In this study, we redesigned a genetically encoded biosensor based on a D‐allulose‐responsive transcriptional regulator for real‐time monitoring of D‐allulose. An ultrahigh‐throughput droplet‐based microfluidic screening platform was further constructed by coupling with this D‐allulose‐detecting biosensor for the directed evolution of the KEases. Structural analysis of Sinorhizobium fredii D‐allulose 3‐epimerase (SfDAE) revealed that a highly flexible helix/loop region exposes or occludes the catalytic center as an essential lid conformation regulating substrate recognition. We reprogrammed SfDAE using structure‐guided rational design and directed evolution, in which a mutant M3‐2 was identified with 17‐fold enhanced catalytic efficiency. Our research offers a paradigm for the design and optimization of a biosensor‐based microdroplet screening platform. A modified D‐allulose biosensor was combined with a microdroplet screening system to establish an ultrahigh‐throughput screening strategy. Structure‐guided rational design and directed evolution were conducted to remodel the SfDAE active pocket and its lid region for enhanced catalytic performance for the synthesis of D‐allulose. This approach shows great potential for the design and optimization of biosensor‐based microdroplet screening platforms.