文献类型：期刊文献

英文题名：FADS-Based Directed Evolution of a Robust Bst DNA Polymerase Adapting High-Temperature Loop-Mediated Isothermal Amplification (HT-LAMP)

作者：Li, Xiao[1];Tang, Qiongwei[2];Jiang, Jingjie[1,6];Shang, Yuepeng[2];Lu, Zelin[1];Chen, Mingli[1];He, Jiajia[1];Liu, Feng[3];Zhu, Sisi[2];Zhang, Zengping[2];Han, Hui[3];Yu, Xixi[2];Li, Qiuxian[3];Xiu, Yuansong[2];Yang, Yuhong[3];Gui, Ping[1];Wang, Xuefeng[1];Lu, Feng[1];Jing, Wei[1];Xu, Langping[1,4];Lin, Yanna[4];Wang, Xinglong[1];Quan, Shu[5];Liu, Xiang[2,3];Yin, Huancai[1];Ma, Fuqiang[1,4]

机构：[1]Chinese Acad Sci, Suzhou Inst Biomed Engn & Technol, Med Enzyme Engn Ctr, CAS Key Lab Biomed Diagnost, Suzhou 215613, Peoples R China;[2]Yeasen Biotechnol Shanghai Co Ltd, Shanghai 200032, Peoples R China;[3]Molefuture Biotechnol Shanghai Co Ltd, Shanghai 200032, Peoples R China;[4]Shandong Lab Adv Biomat & Med Devices Weihai, Weihai 264210, Peoples R China;[5]East China Univ Sci & Technol, Sch Biotechnol, Shanghai 200237, Peoples R China;[6]Univ Sci & Technol China, Suzhou Inst Adv Res, Suzhou 215123, Peoples R China

年份：2025

卷号：15

期号：7

起止页码：5391

外文期刊名：ACS CATALYSIS

收录：;EI(收录号：20251218079243);WOS:【SCI-EXPANDED(收录号:WOS:001448291000001)】；

基金：This work is supported by the Projects of the National Key R&D Program of China [2022YFF0710101]; National Natural Science Foundation of China [32471321]; Shandong Provincial Laboratory Project [SYS202209]; Chinese Academy of Sciences [YJKYYQ20210032]; Youth Innovation Promotion Association Fellowship Program, CAS [2022327]; Natural Science Foundation of Shandong Province, China, [ZR2023QB240, ZR2023QC257]; China Postdoctoral Science Foundation [2022M722337]; Excellent Postdoctoral Program in Jiangsu Province [2022ZB874, 2023ZB046]; Jiangsu Province Innovation and Entrepreneurship Leading Talent Project [JSJCRC2021548]; Youth Leading Talent Program of Gusu [ZXL2022477]; Guangdong Provincial Key R&D Programme [2022B1111050001]; and the Innovation and Entrepreneurship Team of Jiangsu Province [JSSCTD202145].

语种：英文

外文关键词：FADS; Bst DNA polymerase; HT-LAMP; strand displacement index; hydrophobic blade

摘要：Bst DNA polymerase is a key enzyme used in both molecular diagnosis and scientific research. Employing the fluorescence-activated droplet sorting (FADS) technique, we successfully evolved a suboptimal wild-type Bst DNA polymerase into practically valuable mutants through directed evolution. The mutants exhibited significantly improved thermostability and strand displacement capability, enabling much better loop-mediated isothermal amplification (LAMP) performance, with a faster reaction speed (reduced from 40 to 10 min) and a highly stable solid reagent that remained stable for 2 months at 50 degrees C. Moreover, these robust mutants facilitated high-temperature LAMP assays at 70 degrees C, thereby eliminating the common issue of false positives in LAMP assays. To better understand the molecular mechanism behind the strand displacement capability, we proposed the strand displacement index (SDI) as a parameter to quantify this property. We also proposed the "hydrophobic blade" hypothesis, providing insights into the mechanism underlying enhanced strand displacement capability. This work serves as a successful example of molecular engineering and LAMP applications of the Bst DNA polymerase.