Multi-Modal Engineering of Bst DNA Polymerase for Thermostability in Ultra-Fast Lamp Reactions

Despite the fact that strand-displacing activity is of great utility for a variety of applications, including isothermal amplification assays, there are relatively few strand-displacing DNA polymerases. In particular, the thermotolerant DNA polymerase from Geobacillus stearothermophilus (previously Bacillus stearothermophilus), Bst DNA polymerase (Bst DNAP), is used in a variety of assays, including loop-mediated isothermal amplification. However, despite its wide use, its properties remain open to improvement, as has been demonstrated by a variety of engineering efforts, including the identification of point mutations that impact its robustness, strand-displacement capabilities, and nascent reverse transcriptase activity.

Engineering enzymes is typically undertaken either via high-throughput screening or directed evolution. Instead, we have brought to bear several rational design tools (domain addition, machine learning, and supercharging) to quickly and efficiently generate a vastly improved version of Bst DNAP, termed Br512g3, that is extremely robust and that in consequence can perform ultrafast (6’) isothermal amplification. It far exceeds the capabilities of its parental enzyme, Bst-LF, and a widely (almost exclusively) used engineered commercial variant, Bst 2.0, from New England Biolabs