Integrated Engineering of T4 gp32 Achieves Synergistic Improvements in Thermostability, DNA Binding, and Diagnostic Robustness for Enhanced Nucleic Acid Amplification

Single-stranded DNA-binding protein gp32 is essential for DNA replication and widely used in isothermal amplification techniques such as recombinase polymerase amplification (RPA). However, its inherent thermal instability and reaction condition sensitivity restrict its broader applications. Here we develop the Fermentation Integrated Rational Engineering (FIRE-3S) strategy to enhance the functional properties of gp32 through molecular dynamics (MD) simulations, structure-guided fermentation tuning and stability-focused rational design. Starting from a disulfide-bonded mutant (V62C/T80C) that showed enhanced RPA performance but reduced thermostability, MD simulations revealed local structural disruption near the Zn²⁺-binding motif. Then, structure-guided fermentation tuning was applied, which restored structural integrity and enhanced DNA-binding affinity. Building on 2S-step, a computationally guided clique-based design pipeline rapidly identified a multisite mutant, gp32-M1, with elevated thermostability (Δ<i>T</i>_m = +6.03 °C), robust ssDNA-binding, and strong inhibitor tolerance─maintaining >65% activity under 4% isopropanol or ethanol. This work establishes a systematic framework for engineering ssDNA-binding proteins with enhanced biostability and environmental resilience for molecular diagnostics.