Bt Research 2025, Vol.16, No.4, 125-135 http://microbescipublisher.com/index.php/bt 131 6.2 Metabolic burden and stability issues caused by the expression of multiple toxins Modifying the Bt plasmid to express multiple toxin genes will impose significant metabolic pressure on the host bacteria. Excessive production of several insecticidal proteins may occupy the resources of bacterial cells, causing the growth rate of bacteria to slow down, and also reduce the overall adaptability of modified strains - especially in the absence of screening pressure (such as the absence of antibiotics). This metabolic stress may lead to plasmid instability: either the plasmid is lost over time or it is eliminated in an environment mixed with multiple microorganisms (Rajer and Sandegren, 2022). Moreover, the stability of plasmids may also be affected by gene recombination or fragment deletion, especially for those large plasmids carrying multiple genes, where this problem is more prominent. To address these issues, researchers have adopted various approaches, such as adjusting the expression levels of genes, using regulatory components that can make gene expression more balanced, and removing useless gene fragments to reduce the burden. However, even with these optimizations, bacteria may still lose or destroy toxin genes during their evolution, thereby reducing the cost of adapting to the environment. This leads to the Bt strain, which can produce multiple toxins, being unable to function effectively for a long time. To ensure that these strains maintain good insecticidal effects and plasmid stability in practical use, continuous monitoring is still required, and the design in genetic engineering also needs to be further improved (Porse et al., 2016). 6.3 The continuous development of resistance in the target insect population Although there have been advancements in Bt plasmid engineering, the continuous enhancement of resistance within the target insect population remains a long-term challenge. Because Bt crops and biopesticides are widely and frequently used, several major pests have developed actual resistance, such as the western corn root worm and the small diamond-shaped moth (the original "small diamond-shaped moth" has been corrected to be commonly known as "small diamond-shaped moth"). The development speed of insect resistance may be particularly fast, especially when the structures of multiple toxins produced by Bt are similar. This may cause insects to develop resistance to different Bt products (that is, cross-resistance), thereby reducing the effect of Bt (Jakka et al., 2016; Guo et al., 2019; Carriires and Tabashnik, 2023). Both laboratory and field studies have shown that the development of insect resistance is usually related to genetic changes in receptors in their midgut or other molecular targets. In addition, resistant insects often do not fully resist Bt, and the cost of their survival and adaptation is relatively low, which accelerates the spread of resistance genes. To slow down the emergence of resistance in pests, researchers have come up with many solutions, such as mixing toxins with substances of different modes of action, introducing RNA interference (RNAi) technology, and taking resistance management measures (like establishing "shelters"). However, insect populations will always adapt to the environment. This requires continuous improvement and innovation in Bt plasmid design and resistance monitoring to ensure that pest control technologies relying on Bt can be effective in the long term (Deng et al., 2024). 7 The Future Research Directions of Engineering Bt Plasmids 7.1 Design modular and programmable Bt plasmids using synthetic biology The focus of future Bt plasmid engineering is to create a "assemblable and adjustable" system - simply put, a system that can quickly combine gene fragments and flexibly modify these fragments to meet different needs. Synthetic biology tools are crucial. For instance, platforms that can be used in multiple hosts can be used to assemble plasmid skeletons. They can integrate different promoters, resistance markers and toxin genes, helping to build plasmids suitable for specific uses. For instance, methods such as Kinmen assembly have been successfully used to produce "component replaceable" broad-spectrum host plasmids, making bacterial modification in agriculture and the environment more convenient (Leonard et al., 2018). This modular design can also support rapid testing and optimization of new genetic circuits, accelerating the development speed of Bt strains. Programmable plasmids can further enhance the control ability of copy number, enabling researchers to more precisely regulate the number and expression levels of genes. The latest research shows that the plasmid
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