Bt Research 2025, Vol.16, No.2, 55-62 http://microbescipublisher.com/index.php/bt 60 8 Future Research Directions 8.1 Integration of computational design and structural biology in toxin engineering The structure and function of Bt toxins are closely related. Protein engineering combined with structural biology provides a theoretical basis and technical methods for toxin modification. By using three-dimensional structure modeling, molecular docking and rational design, optimization can be carried out for specific receptor sites, thereby improving the specificity and toxicity of toxins. For instance, the three-domain structure model of Cry toxin has been widely applied to guide directed mutation and structural optimization. These modifications can enhance insecticidal activity while delaying the evolution of pest resistance (Kashyap et al., 2012). In addition, a deeper analysis of the binding interface between toxins and receptors is conducive to the development of novel toxins or the acquisition of functional diversification through domain exchange (Li et al., 2024). In the future, if high-throughput computational screening can be combined with experimental verification, it will greatly accelerate the development of highly efficient Bt toxins (Flórez et al., 2012). 8.2 Expanding Bt applications beyond agriculture The application scope of Bt toxin is expanding from traditional agricultural pest control to more fields. Bt and its toxins have been used to control some sanitary pests, such as mosquitoes and black flies, showing great potential in vector control (Wang et al., 2024). Furthermore, the high specificity and low environmental risk of Bt toxin make it have application prospects in animal parasite prevention and control, food safety and environmental protection, etc. (Bangaru, 2025). In the future, Bt toxins are also expected to be optimized through protein engineering for use in medicine, such as the development of new antibacterial or anti-tumor agents (Aswathi et al., 2024). 8.3 Development of next-generation biopesticides with multifunctional traits The research direction of the next generation of Bt biopesticides is multi-functionalization. Through protein engineering and genetic recombination, complex toxins with multiple insecticidal mechanisms can be designed. This type of toxin can not only deal with more pests, but also delay the development of resistance (Aswathi et al., 2024). For example, the delivery of Bt toxins and RNA interference molecules together using nano-carriers can significantly improve the insecticidal effect and reduce the use of chemical pesticides at the same time (Wang et al., 2024). Furthermore, the synergistic effect of Bt toxins and other bioactive molecules also provides new ideas for the development of environmentally friendly and highly targeted multifunctional biopesticides (Flórez et al., 2012; Aswathi et al., 2024; Wang et al., 2024). 9 Conclusion Bt toxin engineering has adopted a variety of new methods, including toxin truncation, domain exchange, site-directed mutagenesis and fusion protein design, etc. These methods have significantly enhanced the insecticidal effect on the target pests and also expanded the range of action. The control of Bt crops against resistant pests has also been significantly enhanced through multi-toxin gene pyramiding and fusion proteins (such as BtRB). These technologies have enabled Bt crops to be widely applied globally, reducing the use of chemical pesticides and bringing environmental and economic benefits. However, issues such as the evolution of pest resistance, the influence of non-target organisms, and gene flow still require continuous attention. Bt crops have become an important part of sustainable pest management. In many countries, the cultivation of Bt crops has reduced the use of pesticides and achieved a significant decline in the number of regional pests. Studies have shown that measures such as high-dose/shelter strategies, multi-toxin superposition, resistance monitoring, and integrated management can all delay the development of resistance and help Bt crops maintain long-term benefits. In the future, if the research on resistance mechanisms at the molecular level and new technologies such as RNAi can be combined, it is expected to establish a more scientific resistance management system, further ensuring the sustainable utilization of Bt crops. To promote the innovation of Bt toxins, it is also necessary to combine basic research with industrial application. Universities, research institutions and enterprises can accelerate the molecular design, field validation and commercialization process of new Bt toxins through cooperation. At the same time, the development of
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