生物传感器
合理设计
高通量筛选
基质(水族馆)
化学
定向进化
组合化学
调节器
吞吐量
纳米技术
计算生物学
生物化学
突变体
计算机科学
材料科学
生物
无线
基因
电信
生态学
作者
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,Hao Qin
标识
DOI:10.1002/anie.202216721
摘要
Abstract 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.
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