Improved insecticidal activity of Vip3Ca protein through enhanced processing and receptor affinity

The Vip3 protein family from Bacillus thuringiensis shows great potential for controlling lepidopteran pests. However, the natural insecticidal activity of the Vip3C subfamily, such as Vip3Ca, is often limited, restricting its application. This study aims to enhance the insecticidal activity of Vip3Ca through modifications targeting processing and receptor binding.

Five Vip3Ca protease cleavage site mutants were constructed, using the protease cleavage sites between domains I and II of Vip3Aa as a reference. Four of these mutants exhibited significantly increased processing in Spodoptera frugiperda midgut protease extracts and enhanced insecticidal activity against S. frugiperda larvae. Three selected cleavage site mutants also showed increased insecticidal activity against the larvae of Helicoverpa armigera, Spodoptera litura, and Spodoptera exigua. Furthermore, substitution of residues G302, N390, or Q468 with lysine improved the binding affinity of Vip3Ca to S. frugiperda brush border membrane vesicles by 1.52- to 1.96-fold, and increased its toxicity against multiple lepidopteran pests. The combined mutants, Vip3Ca^{Q198VKK/G302K} and Vip3Ca^{Q198VKK/N390K}, also showed significantly higher insecticidal activity than Vip3Ca. The insecticidal activity of the combined mutants against larvae of S. frugiperda, H. armigera, S. litura, S. exigua, and Ostrinia furnacalis increased by up to 5.23-, 4.06-, 3.65-, 6.00-, and 12.30-fold, respectively.

This study demonstrates that improving both processing and receptor binding affinity is an effective strategy for enhancing the insecticidal activity of Vip3Ca. The successful engineering of Vip3Ca mutants with significantly improved potency provides valuable insights for the functional optimization of other members within the Vip3 protein family. © 2026 Society of Chemical Industry.