Rational Design of UvsX Recombinase Variants for Enhanced Performance in Recombinase Polymerase Amplification Applications

Homologous recombination is a vital biological process for DNA repair, genomic stability, and genetic diversity, driven by the RecA/Rad51 recombinase family. However, as a T4 bacteriophage recombinase homologous to RecA/Rad51, UvsX has limited <i>in vitro</i> performance during recombinase polymerase amplification (RPA) due to ATP utilization and DNA affinity. In this study, UvsX was rationally engineered to enhance these properties through homology modeling, virtual saturation mutations, and consensus mutation strategies. Targeted mutagenesis produced UvsX variants (E198N, E198R, E198K, and K35G) with a 16 ± 4% to 39 ± 6% improvement in RPA activity, while the double mutant K35G/E198R showed an increase of up to 43 ± 4%. Structural analysis revealed that the K35G/E198R mutation enlarged ATP-binding pockets and increased the positive surface potential of DNA-binding sites, resulting in a 12 ± 4% improvement in ATP utilization and more ADP and less AMP generated, a 10 ± 2% enhancement in DNA interaction compared to the wild-type, and better inhibitor tolerance. These findings establish a foundation for the rational optimization of recombinases in nucleic acid amplification and promote their potential for industrial RPA applications.