The Cry11 family belongs to a large group of δ-endotoxins that share three distinct structural domains. Among the dipteran-active toxins referred to as three-domain Cry11 toxins, the Cry11Aa protein from Bacillus thuringiensis subsp. israelensis (Bti) has been the most extensively studied. Despite the potential of Bti as an effective biological control agent, the understanding of Cry11 toxins remains incomplete. In this study, five Cry11 variants obtained via DNA shuffling displayed toxic activity against Aedes aegypti and Culex quinquefasciatus. Three of these Cry11 variants (8, 23, and 79) were characterized via 3D modeling and analysis of docking with ALP1. The relevant mutations in these variants, such as deletions, insertions and point mutations, are discussed in relation to their structural domains, toxic activities and toxin-receptor interactions. Importantly, deletion of the N-terminal segment in domain I was not associated with any change in toxic activity, and domain III exhibited higher sequence variability than domains I and II. Variant 8 exhibited up to 3.78- and 6.09-fold higher toxicity to A. aegypti than Cry11Bb and Cry11Aa, respectively. Importantly, variant 79 showed an α-helix conformation at the C-terminus and formed crystals retaining toxic activity. These findings indicate that five Cry11 variants were preferentially reassembled from the cry11Aa gene during DNA shuffling. The mutations described in loop 2 and loop 3 of domain II provide valuable information regarding the activity of Cry11 toxins against A. aegypti and C. quinquefasciatus larvae and reveal new insights into the application of directed evolution strategies to study the genetic variability of specific domains in cry11 family genes.

The Cry1Ab δ-endotoxin V171C mutant protein exhibits a 25-fold increase in toxicity against Lymantria dispar , which correlates with a faster rate of partitioning into the midgut membrane and slightly decreased protein stability. This is an insect-specific mechanism; similar results were not observed in Manduca sexta , another Cry1Ab δ-endotoxin-susceptible insect.

The Cry toxins, or δ-endotoxins, are a diverse group of proteins produced by Bacillus thuringiensis. While DNA secondary structures are biologically relevant, it is unknown if such structures are formed in regions encoding conserved domains of Cry toxins under shuffling conditions. We analyzed 5 holotypes that encode Cry toxins and that grouped into 4 clusters according to their phylogenetic closeness. The mean number of DNA secondary structures that formed and the mean Gibbs free energy $$ \left(\overline{\varDelta G}\right) $$ were determined by an in silico analysis using different experimental DNA shuffling scenarios. In terms of spontaneity, shuffling efficiency was directly proportional to the formation of secondary structures but inversely proportional to ∆G. The results showed a shared thermodynamic pattern for each cluster and relationships among sequences that are phylogenetically close at the protein level. The regions of the cry11Aa, Ba and Bb genes that encode domain I showed more spontaneity and thus a greater tendency to form secondary structures (<∆G). In the region of domain III; this tendency was lower (>∆G) in the cry11Ba and Bb genes. Proteins that are phylogenetically closer to Cry11Ba and Cry11Bb, such as Cry2Aa and Cry18Aa, maintained the same thermodynamic pattern. More distant proteins, such as Cry1Aa, Cry1Ab, Cry30Aa and Cry30Ca, featured different thermodynamic patterns in their DNA. These results suggest the presence of thermodynamic variations associated to the formation of secondary structures and an evolutionary relationship with regions that encode highly conserved domains in Cry proteins. The findings of this study may have a role in the in silico design of cry gene assembly by DNA shuffling techniques.