Li Chao, Gao Xin, Qi Hongbin, Zhang Wei, Li Lei, Wei Cancan, Wei Meijing, Sun Xiaoxuan, Wang Shusen, Wang Liyan, Ji Yingbin, Mao Shuhong, Zhu Zhangliang, Tanokura Masaru, Lu Fuping, Qin Hui-Min
Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, National Engineering Laboratory for Industrial Enzymes, Tianjin, 300457, China.
Luoyang BIO-Industry Technology Innovation Center, Luoyang, 471000, Henan, China.
Angew Chem Int Ed Engl. 2023 Mar 1;62(10):e202216721. doi: 10.1002/anie.202216721. Epub 2023 Feb 1.
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.
利用酮糖3-表异构酶（KEases）已实现D-阿洛酮糖的生物合成，但其应用受到催化性能不佳的限制。在本研究中，我们基于D-阿洛酮糖响应转录调节因子重新设计了一种基因编码生物传感器，用于实时监测D-阿洛酮糖。通过与这种检测D-阿洛酮糖的生物传感器耦合，进一步构建了一个基于超高通量液滴的微流控筛选平台，用于KEases的定向进化。费氏中华根瘤菌D-阿洛酮糖3-表异构酶（SfDAE）的结构分析表明，一个高度灵活的螺旋/环区域作为调节底物识别的关键盖子构象，暴露或封闭催化中心。我们利用结构导向的理性设计和定向进化对SfDAE进行了重新编程，其中鉴定出一个催化效率提高17倍的突变体M3-2。我们的研究为基于生物传感器的微滴筛选平台的设计和优化提供了一个范例。
