FADS-Based Directed Evolution of a Robust Bst DNA Polymerase Adapting High-Temperature Loop-Mediated Isothermal Amplification (HT-LAMP)
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 °C. Moreover, these robust mutants facilitated high-temperature LAMP assays at 70 °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.
Authors
- Xinglong Wang (ORCID: https://orcid.org/0009-0009-0407-8749)
- Qiongwei Tang (ORCID: https://orcid.org/0000-0003-0359-5655)
- Jing Wei (ORCID: https://orcid.org/0000-0003-1835-2779)
- Yanna Lin
- Qiuxian Li (ORCID: https://orcid.org/0000-0002-7246-5910)
- Long Xu
- Jingjie Jiang
- Fuqiang Ma (ORCID: https://orcid.org/0000-0001-5614-6496)
- Ping Gui (ORCID: https://orcid.org/0000-0003-2049-3001)
- Yuansong Xiu
- Feng Lu (ORCID: https://orcid.org/0000-0001-6799-3252)
- Yue‐Peng Shang (ORCID: https://orcid.org/0000-0002-0923-4124)
- Shu Quan (ORCID: https://orcid.org/0000-0002-6672-4947)
- Sisi Zhu (ORCID: https://orcid.org/0009-0009-7522-159X)
- Zhaohui Lu (ORCID: https://orcid.org/0000-0001-9644-341X)
- Xiang Liu (ORCID: https://orcid.org/0000-0001-8110-7106)
- Xue‐Feng Wang
- Mingli Chen (ORCID: https://orcid.org/0000-0001-5518-8491)
- Feng Liu (ORCID: https://orcid.org/0000-0001-6614-6318)
- J.-J. He (ORCID: https://orcid.org/0000-0002-2021-9929)
- Hui Han (ORCID: https://orcid.org/0000-0002-8890-4295)
- Xiao Li (ORCID: https://orcid.org/0000-0002-4187-1430)
- Yuhong Yang
- Xixi Yu (ORCID: https://orcid.org/0000-0002-9290-5645)
- Zeng-Ping Zhang
- Huancai Yin (ORCID: https://orcid.org/0000-0002-5014-151X)
Institutions
- University of Science and Technology of China (CN)
- East China University of Science and Technology (CN)
- Chinese Academy of Sciences (CN)
- Suzhou Institute of Biomedical Engineering and Technology (CN)
- Shanghai CASB Biotechnology (China) (CN)
- Bio-Medical Science (South Korea) (KR)
Publication Details
- Journal
- ACS Catalysis
- Published
- 2025-03-18
- DOI
- https://doi.org/10.1021/acscatal.4c07614
- Citations
- 3
- Primary Topic
- Biosensors and Analytical Detection
- Type
- article
- Field-Weighted Citation Impact
- 2.02
Funders
- National Natural Science Foundation of China
- Chinese Academy of Sciences
- China Postdoctoral Science Foundation
- Youth Innovation Promotion Association of the Chinese Academy of Sciences
- Natural Science Foundation of Shandong Province
- National Key Research and Development Program of China
- Special Project for Research and Development in Key areas of Guangdong Province