Abstract
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.
Highlights
Cry toxins or δ-endotoxins secreted by the gram-positive bacterium Bacillus thuringiensis (Bt) are effectively applied to control crop pests and disease vectors due to their specificity and toxicity toward certain insect orders
It has been suggested that following ingestion by insect pests, Cry toxins are activated after solubilization by midgut proteases and interact with different receptors located at the surface of the epithelial cells [3,30,36,37,38,39,40]
Phage display and DNA shuffling techniques have been widely used on Cry toxins to explore specific residues and interaction sites involved in their insecticidal activity
Summary
Cry toxins or δ-endotoxins secreted by the gram-positive bacterium Bacillus thuringiensis (Bt) are effectively applied to control crop pests and disease vectors due to their specificity and toxicity toward certain insect orders. Domain II is organized into three antiparallel β-sheets and two shorter α-helices; this domain is related to receptor binding specificity [12]. In vitro directed evolution to improve the insecticidal activity of Cry toxins has been used with significant success [13,14,15]. We describe the strategies used to obtain Cry variants from two different toxin families: Cry1I and Cry8Ka. we discuss preliminary results from in silico evaluations of specific mutations from variants obtained using phage display and DNA shuffling techniques on three. Cry toxins (Cry1A, Cry1Ia12 and Cry8Ka1) to determine their contribution on improved insecticidal activity and to direct the design of future studies to elucidate the mechanism of Cry toxins
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