Domain-guided engineering of a thermoresistant Vip3A toxin for enhanced functional robustness.

Vip3A toxins from Bacillus thuringiensis are effective insecticidal agents for lepidopteran pest control, but their application is limited by poor thermal stability and loss of activity during storage. In this study, we engineered a thermoresistant Vip3Aa64 variant using a combined rational design and directed-evolution approach. Structural analysis and thermal profiling identified domains IV and V as the primary determinants of instability. Rational design targeting domain V yielded stabilizing substitutions that enhanced interdomain interactions, while error-prone mutagenesis of domain IV coupled with high-throughput nanoscale differential scanning fluorimetry screening identified additional mutations conferring increased thermal resistance. Combining the most effective substitutions and removing a mutation that reduced toxicity produced the variant Vip3A-TR6 (I408E/M755K/N633V/G580E), which exhibited a 5.1 °C increase in melting temperature without compromising insecticidal activity against Spodoptera exigua. Vip3A-TR6 displayed enhanced resistance to heat-induced aggregation and retained bioactivity after prolonged storage at 25 °C and 37 °C. Importantly, the variant was efficiently produced and secreted by B. thuringiensis. These results demonstrate a robust strategy for improving the robustness of Vip3A toxins and support the development of more durable Vip3A-based biopesticides.