PMID 42716166
TITLE Balanced regulation of UvsX activity and thermostability based on active learning.
JOURNAL International journal of biological macromolecules 2026
DOI 10.1016/j.ijbiomac.2026.154387
PMCID 

ABSTRACT
Recombinase polymerase amplification (RPA) enables isothermal nucleic acid detection. However, the key enzyme UvsX of RPA exhibits suboptimal efficiency at low-temperature conditions and low template concentration. Here, we employed a directed engineering strategy combining energy-based screening and protein language models (PLMs). Through semi rational design and iterative optimization with EVOLVEpro, we obtained a triple mutant, TIG (S233T/A311I/A341G), which achieved 1.89-fold RPA relative activity of the wild-type (WT) at a template concentration of 1 × 105 copies reaction-1, and showed higher amplification activity to WT across 37.5-44.3 °C at a template concentration of 1 × 104 copies reaction-1. Molecular dynamics simulations revealed that TIG enhances residue flexibility and remodels the ATP-binding pocket, thereby improving DNA-binding pathway connectivity and strengthening the coupling between ATP hydrolysis and DNA strand exchange. Per-residue energy decomposition and protein structure network analyses further elucidate how key mutations modulate DNA-binding pathways. Finally, under optimized expression conditions (20 °C, 12 h, 0.1 mM IPTG), the yield reached 206.87 ± 3.56 mg L-1 of TIG. Collectively, this study provides a strategy for UvsX engineering and establishes a foundation for the development of more robust RPA assays.
