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.