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

• DOI: 10.1002/anie.202216721
• Publication Date: 2023-01-20T04:03:42Z
• Authors: Chao Li; Xin Gao; Hongbin Qi; Wei Zhang; Lei Li; Cancan Wei; Meijing Wei; Xiaoxuan Sun; Shusen Wang; Liyan Wang; Yingbin Ji; Shuhong Mao; Zhangliang Zhu; Masaru Tanokura; Fuping Lu; Hui‐Min Qin
• Keywords: High-Throughput 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 D-allulose-responsive transcriptional regulator for real-time monitoring D-allulose. An ultrahigh-throughput droplet-based microfluidic screening platform was further constructed coupling with D-allulose-detecting the directed evolution KEases. Structural analysis Sinorhizobium fredii 3-epimerase (SfDAE) revealed that highly flexible helix/loop region exposes or occludes center as an essential lid conformation regulating substrate recognition. We reprogrammed SfDAE structure-guided rational design and evolution, in which mutant M3-2 identified 17-fold enhanced efficiency. Our research offers paradigm optimization biosensor-based microdroplet platform.