RESUMEN
Bacillus thuringiensis (Bt) is a gram-positive spore-forming soil bacterium that is distributed worldwide. Originally recognized as a pathogen of the silkworm, several strains were found on epizootic events in insect pests. In the 1960s, Bt began to be successfully used to control insect pests in agriculture, particularly because of its specificity, which reflects directly on their lack of cytotoxicity to human health, non-target organisms and the environment. Since the introduction of transgenic plants expressing Bt genes in the mid-1980s, numerous methodologies have been used to search for and improve toxins derived from native Bt strains. These improvements directly influence the increase in productivity and the decreased use of chemical insecticides on Bt-crops. Recently, DNA shuffling and in silico evaluations are emerging as promising tools for the development and exploration of mutant Bt toxins with enhanced activity against target insect pests. In this report, we describe natural and in vitro evolution of Cry toxins, as well as their relevance in the mechanism of action for insect control. Moreover, the use of DNA shuffling to improve two Bt toxins will be discussed together with in silico analyses of the generated mutations to evaluate their potential effect on protein structure and cytotoxicity.
Asunto(s)
Proteínas Bacterianas , Endotoxinas , Proteínas Hemolisinas , Insecticidas , Animales , Bacillus thuringiensis , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , Endotoxinas/química , Endotoxinas/genética , Endotoxinas/farmacología , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacología , Insectos/efectos de los fármacos , Insecticidas/química , Insecticidas/farmacologíaRESUMEN
BACKGROUND: The cotton boll weevil (Anthonomus grandis) is a serious insect-pest in the Americas, particularly in Brazil. The use of chemical or biological insect control is not effective against the cotton boll weevil because of its endophytic life style. Therefore, the use of biotechnological tools to produce insect-resistant transgenic plants represents an important strategy to reduce the damage to cotton plants caused by the boll weevil. The present study focuses on the identification of novel molecules that show improved toxicity against the cotton boll weevil. In vitro directed molecular evolution through DNA shuffling and phage display screening was applied to enhance the insecticidal activity of variants of the Cry8Ka1 protein of Bacillus thuringiensis. RESULTS: Bioassays carried out with A. grandis larvae revealed that the LC50 of the screened mutant Cry8Ka5 toxin was 3.15-fold higher than the wild-type Cry8Ka1 toxin. Homology modelling of Cry8Ka1 and the Cry8Ka5 mutant suggested that both proteins retained the typical three-domain Cry family structure. The mutated residues were located mostly in loops and appeared unlikely to interfere with molecular stability. CONCLUSIONS: The improved toxicity of the Cry8Ka5 mutant obtained in this study will allow the generation of a transgenic cotton event with improved potential to control A. grandis.