Bt Research 2025, Vol.16, No.2, 55-62 http://microbescipublisher.com/index.php/bt 55 Research Perspective Open Access Future Directions in Bt Toxin Engineering for Enhanced Efficacy Xueyan Chen1, Chunyang Zhan2 1 Tropical Animal Resources Research Center, Hainan Institute of Tropical Agricultural Resources, Sanya, 572025, Hainan, China 2 Hainan Institute of Biotechnology, Haikou, 570206, Hainan, China Corresponding author: chunyang.zhan@hitar.org Bt Research, 2025, Vol.16, No.2 doi: 10.5376/bt.2025.16.0007 Received: 20 Jan., 2025 Accepted: 28 Feb., 2025 Published: 10 Mar., 2025 Copyright © 2025 Chen and Zhan, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Chen X.Y., and Zhan C.Y., 2025, Future directions in Bt toxin engineering for enhanced efficacy, Bt Research, 16(2): 55-62 (doi: 10.5376/bt.2025.16.0007) Abstract Bt (Bacillus thuringiensis) toxin is currently one of the most widely used biological pesticides worldwide and has played a significant role in agricultural pest control. However, some problems have also been encountered in the application process. For instance, pests may develop resistance, the toxin has a limited range of action, and the effect in the field does not last long. This study summarizes the progress made in the engineering of Bt toxins in recent years. The main methods include domain recombination, directed evolution, and chimeric toxin design. These methods can enhance the insecticidal effect, expand the applicable objects, and delay the development of resistance in pests. It also explores the combined application of Bt toxins with RNA interference, beneficial microorganisms, and multi-gene superposition technologies. And the ecological risks and regulatory issues they may bring. This study aims to provide some ideas and references for the modification and sustainable utilization of Bt toxins. Keywords Bt toxins; Resistance management; Host range expansion; Sustainable pest control; Biopesticides 1 Introduction Bt (Bacillus thuringiensis) toxins are a type of protein derived from soil bacteria and are widely used in the biological control of agricultural pests. The most important Bt toxins are the Cry and Cyt families. These toxins have strong specificity and insecticidal effects on specific insects, so they are often used in sprays and genetically modified crops (Zhang, 2024). They can effectively control the number of many major agricultural pests, reduce the use of chemical pesticides, and bring obvious benefits to agricultural production and the ecological environment (Soberón et al., 2007; Deist et al., 2014; Badran et al., 2016). However, Bt toxins have also encountered many problems in application. An important issue is that the target pests are gradually developing resistance, which has become a major threat to the long-term effectiveness of Bt crops worldwide (Coates, 2016; Zhao et al., 2020). In addition, the host range of Bt toxin is limited, and its natural toxicity to some important pests (such as hemipteran pests) is relatively weak, which limits its application (Chougule et al., 2013). Meanwhile, the stability of Bt toxin in the environment is insufficient, and its expression level in crops fluctuates. Coupled with its interaction with external factors, all these will affect field persistence and selectivity (Olsen et al., 2005; Then, 2009). This study summarizes the major progress in Bt toxin engineering in recent years, introduces strategies for enhancing the insecticidal effect of Bt toxins, expanding the host range, delaying pest resistance and improving environmental stability through methods such as protein engineering and domain exchange, and also discusses the application prospects of new engineered Bt toxins in resistance management as well as the possible risks. This study aims to provide theoretical support and technical references for the rational modification and sustainable utilization of Bt toxins. 2 Molecular Basis of Bt Toxin Structure and Function 2.1 Structural domains of Cry and Cyt toxins Cry toxin (crystalline protein) is the most common and important insecticidal protein in Bt. A typical Cry toxin has three domains (3D structure). The first region is at the N-terminal and is an α -helical structure, mainly responsible for inserting into the cell membrane and forming pores. The second region is a β -folded structure that
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