Abstract

The recombination mediator UvsY plays a critical role in recombinase polymerase amplification but is intrinsically limited by poor solubility and aggregation at high protein concentrations. Here, we present a systematic strategy to improve UvsY solubility and functional performance by integrating consensus mutagenesis with virtual saturation mutagenesis. A single substitution, F73P, was identified as a core solubility-enhancing mutation that enables UvsY to remain soluble at concentrations exceeding 137.5 μg/μL and during long-term storage. Molecular dynamics simulations revealed that F73P disrupts a local hydrophobic clustering motif within a surface-exposed loop region, reducing hydrophobic continuity and aggregation propensity without inducing global destabilization. Building on this solubility-enhanced platform, multi-site variants were constructed to further optimize mediator function. The triple mutant F73P/V29P/S119F (FVS) exhibited exceptional solubility and long-term stability, increased ssDNA-binding affinity, and markedly enhanced stimulation of UvsX ATPase activity, resulting in approximately 2-fold improvement in recombination activity relative to wild-type. These results demonstrate that suppressing nonproductive aggregation through targeted local engineering enables subsequent functional enhancement that is otherwise constrained by solubility limitations. This solubility-first, hierarchical design strategy may provide a useful framework for engineering aggregation-prone biomacromolecules with improved stability and activity.