Making 3D-Cry Toxin Mutants: Much More Than a Tool of Understanding Toxins Mechanism of Action

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.

Which Is Stronger? A Continuing Battle Between Cry Toxins and Insects

“…Second, it is also urgent to thoroughly investigate the insect resistance mechanisms, especially on the applications of various high-throughput sequencing technologies and multiomics techniques (e.g., transcriptomic, proteomics, metabonomics, and epigenomics), to enrich the database of insecticidal proteins or rapidly screen the vital resistance genes ( Dhania et al, 2019 ). By artificially selecting new types of resistant insects in the laboratory, one can foresee farther the possible resistant pathways of insects; in the same token, scientists can also use control strategies for insect resistance issue purposefully, such as the use of genetic engineering, synthetic biology, and other technologies ( Vílchez, 2020 ) to carry out a directed evolution of Cry toxins by constructing various Cry mutants for enhancing its virulence, or expanding its insecticidal spectrum; or, for targeting insects, by using CRISPR/Cas9-based gene manipulation technology to restore resistant mutants back to the wild type ( Esvelt et al, 2014 ), as well as using the mating and reproduction characteristics of insects to reduce the number of resistant populations.…”

“…By artificially selecting new types of resistant insects in the laboratory, one can foresee farther the possible resistant pathways of insects; in the same token, scientists can also use control strategies for insect resistance issue purposefully, such as the use of genetic engineering, synthetic biology, and other technologies (Vílchez, 2020) to carry out a directed evolution of Cry toxins by constructing various Cry mutants for enhancing its virulence, or expanding its insecticidal spectrum; or, for targeting insects, by using CRISPR/Cas9-based gene manipulation technology to restore resistant mutants back to the wild type (Esvelt et al, 2014), as well as using the mating and reproduction characteristics of insects to reduce the number of resistant populations.…”

Can (We Make) Bacillus thuringiensis Crystallize More Than Its Toxins?

“…Hundred- to thousand fold increases in mosquitocidal activity could also be obtained by four point mutations in Cry4Ba [ 84 ] and by four point mutations combined with a five-residue deletion in Cry19Aa [ 85 ]. Structural studies, notably based on X-ray crystallography and modeling, greatly help in identifying key residues to be mutated [ 86 , 87 ]. Moreover, structures solved directly from crystals grown in vivo provide insights into the crystallization pathways to design mutations affecting not only toxin activity and host spectrum but also crystal formation and stability.…”

IDOPS, a Profile HMM-Based Tool to Detect Pesticidal Sequences and Compare Their Genetic Context

“…Among those, Bacillus thuringiensis (Bt) strains and their derived crop protection products are safe for humans, highly specific to the targeted pests, and affordable to manufacture in bulk, making Bt the most successful biopesticide implemented worldwide (George and Crickmore, 2012 ; Jouzani et al, 2017 ). Nevertheless, nature fights back and target insects develop resistance mechanisms against Bt, which creates a constant need for novel and improved pest control agents (Vílchez, 2020 ).…”