Bt Research 2025, Vol.16, No.4, 125-135 http://microbescipublisher.com/index.php/bt 125 Case Study Open Access Engineering of Bt Plasmids to Enhance Their Insecticidal Activity Delong Wang, Jiong Fu Hainan Provincial Key Laboratory of Crop Molecular Breeding, Sanya, 572025, Hainan, China Corresponding author: jiong.fu@hitar.org Bt Research, 2025, Vol.16, No.4 doi: 10.5376/bt.2025.16.0016 Received: 10 May, 2025 Accepted: 15 Jun., 2025 Published: 02 Jul., 2025 Copyright © 2025 Wang and Fu, 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: Wang D.L., and Fu J., 2025, Engineering of Bt plasmids to enhance their insecticidal activity, Bt Research, 16(4): 125-135 (doi: 10.5376/bt.2025.16.0016) Abstract This study explored the key role of Bacillus thuringiensis (Bt) plasmids in the production of insecticidal proteins and the overall insecticidal effect, and summarized the latest progress in modifying Bt plasmids by engineering methods to enhance insecticidal ability. This article also explores the main methods to enhance pest resistance from aspects such as redesigning toxin genes, enabling multiple toxins to work together, optimizing regulatory components, and improving plasmid stability. It also mentioned the application of genetically modified Bt crops and biopesticides, as well as the possible risks of genetically modified plasmids in terms of biosafety. This study aims to integrate synthetic biology, environmental response expression systems, multi-omics analysis and genome editing technologies to promote the efficient, safe and sustainable development of BT-engineered strains. Keywords Bacillus thuringiensis; Plasmid engineering; Insecticidal protein expression; Resistance management; Synthetic biology 1 Introduction Bacillus thuriensis (Bt) is a soil-dwelling bacterium that is best known for its ability to produce proteins that kill pests, especially the toxins Cry and Cyt. During the process of Bt forming spores, these toxins will turn into crystals attached to the spores. These proteins are highly toxic to many agricultural pests and only target specific species of pests, such as Lepidoptera, Coleoptera and diptera insects, all of which fall within their range of action. This characteristic has made Bt the most successful and widely used microbial insecticide in both traditional insecticides and genetically modified crops (Van Frankenhuyzen, 2009; Dominguez-Arrizabalaga et al., 2020). Products with Bt as the main component have been commercially used worldwide for more than 40 years. It is an environmentally friendly alternative to chemical pesticides and has played a significant role in protecting crops and increasing grain production (Jouzani et al., 2017; Pineda and Castellanos-Rozo, 2025). Bt mainly kills pests by its own genes, most of which are located on large plasmids with strong activity. These plasmids can not only control the synthesis of various toxins such as Cry and Cyt, but also generate other biologically active molecules. These products played an important role when Bt adapted to different insect hosts and various environmental conditions (Chelliah et al., 2019; Fayad et al., 2020; Guerrero et al., 2024). The toxin genes on the plasmid can be transferred among different bacteria, which helps Bt acquire the ability to kill more types of pests, enabling the strain to deal with more pests or have a stronger insecticidal effect (Li et al., 2017; Wang et al., 2020). Therefore, the structural characteristics and transfer ability of plasmids lay the foundation for the diversity and effectiveness of Bt as a biological control tool. This study will explore the challenges brought about by the resistance of the target insect population to the long-term insecticidal action of Bacillus thuringiensis (Bt). Although Bt has achieved remarkable results in the prevention and control of agricultural pests and diseases, both field investigations and laboratory studies have shown that insects are gradually developing resistance to Bt preparations and Bt toxins in genetically modified crops. To solve this problem, plasmid engineering technology has been developed. This technology encompasses methods such as the utilization of recombinant DNA, gene modification, and the combined use of multiple toxins. Researchers are maintaining and enhancing the effectiveness and long-term application potential of Bt in integrated pest management by accurately adjusting the plasmid structure and optimizing the expression levels of toxin genes.
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