Abstract
3D-Cry toxins, produced by the entomopathogenic bacterium Bacillus thuringiensis, have been extensively mutated in order to elucidate their elegant and complex mechanism of action necessary to kill susceptible insects. Together with the study of the resistant insects, 3D-Cry toxin mutants represent one of the pillars to understanding how these toxins exert their activity on their host. The principle is simple, if an amino acid is involved and essential in the mechanism of action, when substituted, the activity of the toxin will be diminished. However, some of the constructed 3D-Cry toxin mutants have shown an enhanced activity against their target insects compared to the parental toxins, suggesting that it is possible to produce novel versions of the natural toxins with an improved performance in the laboratory. In this report, all mutants with an enhanced activity obtained by accident in mutagenesis studies, together with all the variants obtained by rational design or by directed mutagenesis, were compiled. A description of the improved mutants was made considering their historical context and the parallel development of the protein engineering techniques that have been used to obtain them. This report demonstrates that artificial 3D-Cry toxins made in laboratories are a real alternative to natural toxins.
Highlights
Sporulating Bacillus thurigiensis produces four non-phylogenetically related insecticidal protein families, the three domain Cry toxins or 3D-Cry toxins, the mosquitocidal Mtx, the binary-like (Bin), and Cyt toxins
These mutants were not as useful as the impaired mutant to elucidate the mechanism of action of 3D-Cry toxins, but served to settle the concept that activity improvement was possible with only the change of a single amino acid
The use of several molecular techniques has allowed researchers to obtain 3D-Cry toxin mutants with improved activities compared to natural toxins
Summary
Sporulating Bacillus thurigiensis produces four non-phylogenetically related insecticidal protein families, the three domain Cry toxins or 3D-Cry toxins, the mosquitocidal Mtx, the binary-like (Bin), and Cyt toxins. They determined that when a 241 nt segment from cryIIA was inserted on the cryIIB gene, the lepidopteran toxin showed a broader insect spectrum, becoming active against Diptera They even narrowed down the region responsible for the specificity of CryIIA protein toward mosquitoes to 76 amino acids. This represented the first proof that by changing specific areas in the sequence, was it a means of modifying the specificity of the 3D-Cry toxins, and a way of increasing their activity At this point in 3D-Cry toxin history, researchers started to have the overall view that 3D-Cry toxins were modular structures and that their function could be manipulated by exchanging parts of the molecules
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