A strategy to enhance the insecticidal potency of Vip3Aa by introducing additional cleavage sites to increase its proteolytic activation efficiency

•Vip3Aa has multiple cleavage activation sites in the loop region between domain I and domain II.•Introducing additional cleavage sites between domain I and domain II of vip3Aa can significantly increase its proteolytic efficiency by midgut juice.•Increasing the proteolytic efficiency of vip3Aa can promote its insecticidal potency. Microbially derived, protein-based biopesticides have become a vital element in pest management strategies. Vip3 family proteins from Bacillus thuringiensis have distinct characteristics from known insecticidal Cry toxins and show efficient insecticidal activity against several detrimental lepidopteran pests. They are considered to be a promising toxic candidate for the management of various detrimental pests. In this study, we found that in addition to the preliminary digestion sites lysine, there are multiple cleavage activation sites in the linker region between domain I (DI) and DII of Vip3Aa. We further demonstrated that by adding more cleavage sites between DI and DII of Vip3Aa, its proteolysis efficiency by midgut proteases can be significantly increased, and correspondingly enhance its insecticidal activity against Spodoptera frugiperda and Helicoverpa armigera larvae. Our study promotes the understanding of the insecticidal mechanism of Vip3 proteins and illustrates an easily implementable strategy to increase the insecticidal potency of Vip3Aa. This facilitates their potential future development and efficient application for sustainable agriculture. [Display omitted]