Enhanced toxicity of Bacillus thuringiensis Cry3A δ-endotoxin in coleopterans by mutagenesis in a receptor binding loop

We used site-directed mutagenesis to modify the Bacillus thuringiensis cry3A gene in amino acid residues 350-354. Two mutant toxins, Al (R(345)A,Y350F,Y351F) and A2 (R(345)A,Delta Y-350,Delta Y-351), showed significantly improved toxicity against Tenebrio molitor (yellow mealworm). The mutant toxin Al was also more potent against both Leptinotarsa decemlineata (Colorado potato beetle) and Chrysomela scripta (cottonwood leaf beetle), while A2 displayed enhanced toxicity only in L, decemlineata, Competitive binding assays of L, decemlineata brush border membrane vesicles (BBMV) revealed that binding affinities for the Al and A2 mutant toxins were ca, 2.5-fold higher than for the wild-type Cry3 toxin, Similar binding assays with C. scripta BBMV revealed a ca, 5-fold lower dissociation rate for the Al mutant as compared to that of Cry3A, (C) 2000 Federation of European Biochemical Societies.