Engineering and enhancement of Bst DNA polymerase variants toward improved diagnostic applications: A review

Abstract

Bst DNA polymerase, the key enzyme in loop-mediated isothermal amplification (LAMP), is pivotal for point-of-care diagnostics due to its strand displacement activity. Aiming to optimize Bst DNA polymerase for faster, more accurate, and more robust nucleic acid detection in clinical diagnostics and various point-of-care testing scenarios, this review summarizes current research focused on engineering Bst DNA polymerase to enhance its functional properties. We systematically analyze key mutation sites within its structural domains, detailing how specific amino acid substitutions influence thermostability, strand displacement efficiency, fidelity, inhibitor tolerance, and nucleotide selectivity. Additionally, we explore advancements on fusion protein strategies aimed at enhancing various properties of Bst DNA polymerase, including DNA affinity, processivity, salt tolerance, thermostability, and resistance to inhibitors. We also discuss the challenges and future directions of domain-specific engineering and emphasize the potential of integrating artificial intelligence and high-throughput screening for rapid mutant development.

Keywords: Bst DNA polymerase; Domain-specific engineering; Enzyme engineering; Loop-mediated isothermal amplification; Point-of-care testing.