Bt Research 2025, Vol.16 http://microbescipublisher.com/index.php.bt © 2025 MicroSci Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher.
Bt Research 2025, Vol.16 http://microbescipublisher.com/index.php.bt © 2025 MicroSci Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher. MicroSci Publisher is an international Open Access publisher specializing in microbiology, bacteriology, mycology, molecular and cellular biology and virology registered at the publishing platform that is operated by Sophia Publishing Group (SPG), founded in British Columbia of Canada. Publisher MicroSci Publisher Editedby Editorial Team of Bt Research Email: edit@bt.microbescipublisher.com Website: http://microbescipublisher.com/index.php/bt Address: 11388 Stevenston Hwy, PO Box 96016, Richmond, V7A 5J5, British Columbia Canada Bt Research (ISSN 1925-1939) is an open access, peer reviewed journal published online by MicroSciPublisher. The journal is publishing high quality original research on all aspects of Bacillus thuringiensis and their toxins affecting the living organisms, as well as environmental risk and public policy relevant to Bt modified organisms. Topics include (but are not limited to) Bt strain identification, novel Bt toxin discovery and bioassay, transgenic Bt plants, insecticidal mechanism of Bt toxin as well as resistant mechanisms of target-insect to Bt toxin. All the articles published in Bt Research are Open Access, and are distributed 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. MicroSciPublisher uses CrossCheck service to identify academic plagiarism through the world’s leading plagiarism prevention tool, iParadigms, and to protect the original authors’ copyrights.
Bt Research (online), 2025, Vol. 16, No. 4 ISSN 1925-1939 http://microbescipublisher.com/index.php/bt © 2025 MicroSci Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher. Latest Content Engineering of Bt Plasmids to Enhance Their Insecticidal Activity Delong Wang, Jiong Fu Bt Research, 2025, Vol. 16, No. 4, 125-135 Bioinformatics Tools for Bt Genome Data Analysis Shusheng Liu, Chunyang Zhan Bt Research, 2025, Vol. 16, No. 4, 136-146 Research on Public Awareness and Acceptance of Bt-Related Public Health Measures Hongwei Liu, Qikun Huang Bt Research, 2025, Vol. 16, No. 4, 147-156 Engineering Bt Strains Using Synthetic Biology Approaches to Enhance Efficacy Xiazhen Huang, Kaiwen Liang Bt Research, 2025, Vol. 16, No. 4, 157-167 Development and Field Performance of Bt Transgenic Crops Zhonggang Li, Xing Zhao Bt Research, 2025, Vol. 16, No. 4, 168-181
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.
Bt Research 2025, Vol.16, No.4, 125-135 http://microbescipublisher.com/index.php/bt 126 2 The Structure and Function of the Bt Plasmid 2.1 Bt plasmid types, replication mechanisms and their stability in host strains Bt strains usually contain multiple plasmids, including large plasmids, which vary in size and carry different genetic information. People can classify plasmids based on their size, the genes they contain, and whether they can coexist in the same bacterium. For instance, through genome-wide analysis of Bt strains, researchers discovered several large plasmids (also known as giant plasmids), each carrying different insecticidal genes and genes that make bacteria toxic (Li, 2024). The rich plasmid types enable Bt strains to adapt to different living environments and parasitic insects by obtaining or losing specific plasmids (Pacheco et al., 2021). The replication mode of Bt plasmids is crucial for their retention and stability in bacteria. The presence of Rep protein and the biased analysis of GC content indicated that many Bt giant plasmids were replicated through the bidirectional theta mode. The stability of these plasmids in the host strain is also assisted by some genes-these genes are involved in the binding and division processes of plasmids, ensuring that when bacteria divide, the plasmids can be accurately passed on to the next generation of bacteria. In addition, some plasmids have the CRISPR-Cas system, which may further stabilize the plasmids by resisting foreign DNA (Navas et al., 2017). 2.2 Distribution and regulation of insecticidal genes (Cry, Cyt, Vip, etc.) on plasmids Insecticidal genes such as cry, cyt and vip are mainly located on Bt plasmids and are usually concentrated in large pathogenic gene regions. These concentrated gene groups may contain multiple toxin genes. For instance, in some Bt strains, as many as 13 genes capable of producing insecticidal proteins are aggregated on a single plasmid, forming "toxin islands", which are similar to the pathogenic gene regions in other bacteria (Zhou et al., 2024). These genes are arranged in such a way that it is conducive to the mutual coordination of the expressions of different toxins and may also produce a synergistic effect among them (Wang et al., 2020). The control process of these insecticidal genes is rather complex and involves the promoter and regulatory components of the plasmid itself. The function of genes is also influenced by environmental conditions and the physiological state of bacteria. For instance, researchers discovered through proteomic analysis that multiple toxin genes in the plasmid gene population can undergo transcription and translation, indicating that these genes are being actively controlled at specific stages of bacterial growth (Pacheco et al., 2021; Zhou et al., 2024). In addition, genetic recombination can also place new toxin genes at the existing positions of plasmids, thus enabling Bt to have the ability to kill more types of pests (Wang et al., 2020). 2.3 The contribution of plasmids to Bt toxicity and the co-expression of multiple toxin genes Plasmids play a core role in the toxicity of Bt, as most of the insecticidal proteins that make Bt effective against pests are synthesized under the guidance of genes on plasmids. If there are multiple toxin genes on a plasmid, it can produce various toxins, enabling Bt to target more types of insects and enhancing its overall toxicity (Navas et al., 2017). For instance, studies have found that when the cry gene and the vip gene are located on the same plasmid, they can produce a synergistic insecticidal effect, making the killing power of Bt strains against resistant pests stronger (Pacheco et al., 2021). Simultaneous expression of multiple toxin genes is a key method to delay the development of resistance in target insects. Proteomic analysis and functional studies have shown that Bt strains that can express multiple Cry proteins, or simultaneously express Cry proteins and Vip proteins, have stronger toxicity and a wider range of parasitic insects (Wang et al., 2020; Pacheco et al., 2021). This mode of simultaneous expression of multiple genes not only enables better pest control but also reduces the probability of insects developing resistance, thus allowing BT-based biopesticides to exert their effects for a long time and continuously. 3 The Key Strategies for Bt Plasmid Engineering 3.1 Design of recombinant toxin genes and co-expression of multiple toxins Simultaneously activating multiple Bt toxin genes such as Cry1Ac and Cry3A in the same host bacteria or the same plasmid is an effective approach to expand the insecticidal range and better address pest resistance. Research
Bt Research 2025, Vol.16, No.4, 125-135 http://microbescipublisher.com/index.php/bt 127 on genetically modified plants has shown that allowing plants to simultaneously express two Bt toxin genes can effectively resist more types of pests, including Lepidoptera and Coleoptera insects. For example, there is a poplar variety called Populus x euramericana, which can express both Cry1Ac and Cry3Agenes simultaneously and has strong resistance to various insect larvae. Both of these two genes can undergo normal transcription and translation in plants (Ren et al., 2021). This method not only enhances the effectiveness of pest control but also slows down the rate at which the target pests develop resistance. However, due to the differences in structure and regulatory elements among different toxin genes, even when expressed together, their yields will be significantly different. In the above-mentioned study, the content of Cry3A protein was significantly higher than that of Cry1Ac, which might affect the overall insecticidal effect of transgenic organisms (Ren et al., 2021). Therefore, it is necessary to rationally design the structure of the recombinant gene, such as optimizing the codon and adjusting the regulatory elements, to make the expression more balanced. This is very important for achieving the ideal expression ratio and maximizing the synergy. 3.2 Optimize regulatory elements to enhance the expression of toxin genes To increase the expression level of Bt toxin genes, it largely depends on optimizing regulatory elements, such as promoters, ribosome binding sites (RBS), and transcriptional regulatory factors. The selection of different promoters and RBS will have a significant impact on the transcriptional and translation efficiency of each toxin gene. Just as observed in transgenic lines, different promoter types can lead to differences in the contents of Cry1Ac and Cry3A proteins (Ren et al., 2021). If the promoter used is suitable for the host's transcriptional mechanism, has high intensity, can continuously function or can be induced to activate, it can significantly promote the production of toxins. In addition, regulatory elements must be carefully matched with the host strain and specific toxin genes to avoid gene silencing or imposing additional metabolic burdens on the host. The stability and high-level expression of exogenous genes may be affected by environmental factors or changes in host growth and development, resulting in a decrease in insect resistance over time (Ren et al., 2021). Future genetic engineering work should focus on adjusting the details of regulatory sequences and utilize synthetic biology tools to ensure that multiple toxin genes can be stably, efficiently and continuously expressed. 3.3 Enhance plasmid stability and host adaptability The stability of the plasmid is the key for the continuous functioning of the Bt toxin gene, especially when there is no pressure from antibiotic screening. Research has found that adding a toxin-antitoxin (TA) system to the plasmid structure can effectively ensure the long-term existence of plasmids in the host microbiota. At present, different types of TA systems have been developed, such as those based on ccd modules or prcA/prcT modules. They can kill the progeny bacteria that did not obtain the plasmid, thus ensuring that the modified plasmid is stably passed on to the next generation and retained for a long time (Wright et al., 2015; Fraikin and Van Melderen, 2023; Liu, 2024). The stabilizing effects of these systems can be compared with the effect of integrating genes into chromosomes, and at the same time, they can maintain a high expression level of exogenous genes (Takashima et al., 2021; Ren et al., 2023). In addition, optimizing the replication origin (ori) of plasmids and adopting host-dependent replication systems can further enhance the stability and adaptability of plasmids. By using conditional replication starting points and modular integration systems, plasmids can also be restricted to transfer only within the specified host bacteria, thereby reducing the risk of gene-level transfer to other bacteria and diffusion to the environment. By combining these methods with the TA system, stable and safe Bt plasmid vectors suitable for agricultural and environmental applications can be designed. 4 Evaluation of the Efficacy after Enhanced Insecticidal Activity 4.1 Laboratory bioassay of target insects and expansion of the insecticidal range Conducting biological experiments in the laboratory is an important step in determining the insecticidal efficacy
Bt Research 2025, Vol.16, No.4, 125-135 http://microbescipublisher.com/index.php/bt 128 of Bt enhanced preparations and Bt modified strains. For instance, when Bt is used in combination with other microbial preparations or with modified Bt toxins (such as Vip3Aa mutants), the toxicity to major pests (such as armyworms and cotton bollworms) can be significantly enhanced - in laboratory tests, Its virulence is 8 times higher than that of natural Bt protein (Yang et al., 2022; Chang et al., 2024). These experiments also show that mixing different components together or using modified toxins can reduce the amount of Bt required to kill pests. This indicates that transgenic Bt has improved in both the types and effects of pest control (Figure 1). Figure 1 Histopathological effects on the midgut tissue of third-instar S. frugiperda larvae (Adopted from Chang et al., 2024) Image caption: (a) non-treatment, (b) Bacillus thuringiensis subsp. kurstaki (Bt), (c) detail of the section from the single treatment with Bt, (d) Photorhabdus luminescens (Pl) ATCC 29 999 in whole broth (after 120 h of incubation), (e) detail of the section from the single treatment with Pl ATCC 29 999, (f) Pl 2103-UV in whole broth (after 120 h incubation), (g) detail of the section from the single treatment with Pl 2103-UV, (h) combined treatment of Pl ATCC 29 999 and Bt (mixed at a ratio of 1:5), and (i) combined treatment of Pl 2103-UV and Bt (mixed at a ratio of 1:5). Scale bar = 90 µm. Am, apical membrane; Bm, basal membrane; Lu, lumen; N, nucleus; V, vesicle formation (Adopted from Chang et al., 2024) In addition, laboratory studies have found that treating Bt toxins with encapsulation technology or adding substances that can promote biofilm formation can help Bt toxins resist environmental damage, thereby enhancing the control ability for more pests (Jalali et al., 2023; Zhao et al., 2025). These methods can not only directly increase the toxicity of Bt, but also enable Bt products to maintain their activity for a longer time under experimental conditions simulating harsh environments. 4.2 Field trials, long-term effect evaluation and drug resistance management Field trials are crucial for verifying laboratory results and confirming the practical application effects of improved Bt products. Studies have shown that mixing Bt with bacterial metabolites that can inhibit insect immunity, or using Bt products with optimized formulas, can achieve good control effects in both greenhouse and field environments, especially against lepidoptera pests such as noctuids and armyworms (Hrithik et al., 2022; Chang et al., 2024). These results prove that under different environmental conditions, the improved Bt formula can control pests more stably and effectively. Long-term field monitoring can also assess whether strategies for managing pest resistance can be used in the long term. Research has found that growing Bt crops, using Bt products that can express multiple toxins, or employing crops with novel modes of action can delay the development of pest resistance in actual environments - especially when these methods are used in combination with "refuge strategies" and integrated pest management measures, the effect is more obvious. These methods can help Bt technology remain effective throughout multiple growing seasons.
Bt Research 2025, Vol.16, No.4, 125-135 http://microbescipublisher.com/index.php/bt 129 4.3 The role of multiple toxin expressions in restoring insect sensitivity and delaying resistance Making Bt express multiple toxins with different modes of action simultaneously is a proven effective strategy - it can not only make insects that have already developed resistance sensitive to Bt again, but also delay the development of insect resistance. Both laboratory and field data show that Bt crops or Bt products that can produce multiple toxins can kill pests multiple times. Even if insects develop resistance to one toxin, they are still sensitive to the other, which reduces the probability of insect resistance being fixed (Tabashnik et al., 2013; Tabashnik et al., 2015). For instance, studies have found that the proprotein form of Cry protein is more effective against resistant insect strains than its activated form, which also demonstrates the value of allowing Bt to express multiple toxins. In addition, by using the modified toxins or the combined application of multiple toxins, not only can the control range of Bt over pests be expanded, but also the difficulty for pests to develop resistance can be increased. This comprehensive strategy is of great significance for ensuring the long-term effectiveness of BT-based agricultural pest control techniques (Deist et al., 2014). 5 Application Cases and Commercial Development 5.1 Characteristics of successfully engineered Bt strains and plasmid design The improvement of Bt strains is aimed at making up for the shortcomings of natural Bt strains, such as the limited variety of pests they can kill and the fact that pests often develop resistance very quickly. With the help of recombinant DNA technology, researchers can replicate genes that produce insecticidal proteins (such as the cry gene), and then put these genes into Bt strains or other hosts. In this way, strains with stronger insecticidal effects and better pest control capabilities can be obtained. For instance, there was a study that used electroporation technology to introduce the Cry3Aa7 gene into wild Bt strains, resulting in the recombinant strain G033A. This strain is highly toxic to lepidoptera pests and is also the first transgenic Bt strain registered as a pesticide in China (Li et al., 2022). These modified strains, usually with specially designed plasmids, can effectively express the target genes, remain stable, and transfer between different hosts, thus expanding their application possibilities in commercial biopesticide formulations (Figure 2) (Nakamura, 2020; Li et al., 2022). Figure 2 Environmental behaviors of Bt protein (A) and its three-dimensional structures (B). I, II, and III: domains I, II, and III (Adopted from Li et al., 2022) In the plasmid design of these strains, selectable markers, optimized promoters and regulatory sequences are usually included to ensure the stable production of large amounts of toxins. There are also some technological advancements, such as the construction of temperature-sensitive recombinant plasmids or the replacement of antibiotic resistance markers (for instance, using tetracycline resistance instead of erythromycin resistance), which have enriched the tools of genetic engineering and enabled the development of safer and more widely applicable strains for commercial use (Yanhua et al., 2008).
Bt Research 2025, Vol.16, No.4, 125-135 http://microbescipublisher.com/index.php/bt 130 5.2 Integration with genetically modified crops Integrating the Bt gene into the genome of crops has completely transformed the way pests are controlled in agriculture. Bt genetically modified crops, such as cotton and corn, are all designed to express insecticidal proteins that protect the crops from major pests throughout the growing season. These crops are widely cultivated all over the world. Since 1996, the planting area of Bt crops has increased by more than 100 times and exceeded 190 million hectares by 2019 (Li et al., 2022). The Bt gene in these crops can be stably passed on to the offspring and continuously expressed. This not only improves the agronomic traits of the crops, but also reduces the use of pesticides and increases the economic value. When using Bt genes to modify plants, the codons of the genes are usually optimized, and plant-specific promoters are also employed to enable the Bt genes to be better expressed and exert better effects. The Bt protein produced by genetically modified crops is different in structure and function from that produced by natural Bt bacteria. Therefore, a comprehensive biosafety assessment and environmental impact assessment must be conducted before commercial promotion. 5.3 Industrial practice of plasmid engineering and market performance of commercial strains The large-scale industrial production of Bt biopesticides cannot do without reliable plasmid engineering technology, which can ensure high output, stable and safe products. Innovations in strain and vector engineering - such as optimizing the plasmid's skeleton structure to make it more stable, have higher yields, and facilitate subsequent purification - have all enhanced the cost-effectiveness of Bt biopesticide production and made it easier to scale up production (Bower and Prather, 2009). In addition, the use of efficient transformation systems (such as electroporation technology) and the development of high-yield production strains have further enhanced the commercial feasibility of the modified Bt products (Nakamura, 2020). In the commercial market, BT-based biopesticides and genetically modified crops occupy a major position in global microbial pest control, accounting for approximately 90% of global biopesticide usage (Li et al., 2022). These products have been successfully promoted because they are only effective against specific pests, have high safety and a wide range of applications. The current focus of research is to continue improving plasmid design, enhancing environmental safety, and optimizing resistance management methods. This can not only keep the market growing continuously, but also ensure the long-term effectiveness of the product. 6 Current Challenges and Limitations 6.1 Biosafety issues of engineering plasmids and potential risks of gene transfer The application of engineered Bt plasmids in agriculture has raised significant biosafety issues, particularly regarding how long insecticidal genes can remain in the environment and whether gene transfer is possible. Bt proteins produced by modified strains and transgenic plants are regarded as foreign substances in the environment, and their structures and functions are different from those of natural Bt toxins. These proteins can remain in the soil for a period of time, combine with organic matter, and maintain insecticidal activity for a relatively long time. This may affect the variety and quantity of microorganisms in the soil as well as the normal operation of the entire ecosystem. Therefore, before releasing BT-modified organisms into the wild or conducting commercial production, a comprehensive biosafety assessment must be carried out to determine their changes in the environment and their ecological impact (Li et al., 2022). The main risk associated with engineered plasmids is the possibility that their genes may be transferred to non-target microorganisms. Plasmids (especially those with binding elements) may allow insecticidal genes or resistance genes to spread into host organisms they were not originally intended to act on, which has raised concerns - fears of unexpected ecological consequences and even the emergence of new, potentially harmful microbial strains. Current regulatory rules are becoming increasingly strict, requiring the removal of antibiotic resistance markers and binding systems from large engineering plasmids to reduce these risks. Meanwhile, researchers are also developing new plasmid designs centered on biosafety to limit gene transfer and ensure that genes do not spread at will.
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
Bt Research 2025, Vol.16, No.4, 125-135 http://microbescipublisher.com/index.php/bt 132 copy number of each bacterial cell can be flexibly adjusted between 1 and 800, which provides an effective means to balance bacterial metabolic stress and maximize toxin production (Rouches et al., 2021). This fine regulatory ability is of great significance for improving the performance and stability of engineered Bt strains in different environments. 7.2 Develop an environment-responsive expression system to control toxin release Another promising direction is to create a "system that can adjust its expression in response to the environment", allowing Bt toxins to be regulated and activated for release only when needed. Researchers can add some regulatory components that are sensitive to environmental signals to the plasmid, such as those sensitive to light, temperature or specific chemicals. This way, "temporal and spatial control" of the expression of toxin genes can be achieved. For instance, light-controlled plasmids have been developed: they feature a modified promoter that can be cut off by light, enabling remote activation of gene expression without direct manipulation of bacteria (Chung and Booth, 2023). This method not only reduces the impact on non-target organisms but also only produces toxins when necessary, thereby enhancing overall biological safety. This response system can also be designed to "sense the presence of pests" or "sense the signals of plant stress" - only activating the expression of insecticidal genes when necessary. This strategy can reduce the unnecessary metabolic burden on host bacteria, lower the exposure to Bt toxins in the environment, address key biosafety and ecological issues, and at the same time maintain efficient pest control effects. 7.3 Integrate multi-omics and genome editing to construct broad-spectrum and highly efficient Bt strains Combining "multi-omics methods" (such as genomics, transcriptomics, and proteomics) with advanced genome editing technologies offers new opportunities for constructing Bt strains with "stronger insecticidal activity and the ability to target more pests". Multi-omics analysis can comprehensively reveal gene functions, regulatory networks and metabolic pathways, providing guidance for the rational design of plasmids and the selection of the optimal combination of toxin genes. This system-level understanding can help researchers identify new renovation targets and predict possible unexpected impacts. Genome editing tools (including CRISPR/Cas systems) can precisely modify plasmid and chromosomal DNA to achieve the insertion, deletion or replacement of toxin genes and regulatory elements. These techniques have been successfully used to design "plasmids that can be used in multiple hosts" and to knock out or inhibit specific genes-fully demonstrating their role in fine-tuning the performance of Bt strains (Leonard et al., 2018). Combining multi-omics data with genome editing will accelerate the development of "the next generation of efficient and environmentally friendly" Bt strains. 8 Concluding Remarks Engineering Bt plasmids have become an important foundation for promoting biological pest control. They can help cultivate strains and crops with stronger insecticidal effects, control more types of pests, and better deal with new problems constantly emerging in agriculture. With the help of DNA recombination technology, people can efficiently introduce and express new insecticidal protein genes, thereby developing Bt pesticides and genetically modified crops with more comprehensive functions and better effects. This not only reduces the reliance on chemical pesticides but also provides support for the sustainable development of agriculture. In order to smoothly design and promote the use of enhanced Bt plasmids, close cooperation among fields such as molecular biology, ecology, agronomy and regulatory science is required. Innovative approaches such as protein engineering, advanced transformation methods, and systematic biosafety assessment have all played a significant role in addressing technical challenges, reducing ecological risks, and controlling pest resistance. Multidisciplinary collaboration will promote the development of the new generation of Bt technology, ensuring that it functions efficiently while also protecting the environment. With the continuous advancement of genetic engineering, protein modification and integrated pest management technologies, transgenic Bt strains and crops will play a greater role in sustainable pest control. They only act on
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Bt Research 2025, Vol.16, No.4, 136-146 http://microbescipublisher.com/index.php/bt 136 Feature Review Open Access Bioinformatics Tools for Bt Genome Data Analysis Shusheng Liu 1, Chunyang Zhan2 1 Tropical Microbial 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.4 doi: 10.5376/bt.2025.16.0017 Received: 15 May, 2025 Accepted: 20 Jun., 2025 Published: 08 Jul., 2025 Copyright © 2025 Liu 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: Liu S.S., and Zhan C.Y., 2025, Engineering of Bt plasmids to enhance their insecticidal activity, Bt Research, 16(4): 136-146 (doi: 10.5376/bt.2025.16.0017) Abstract As an important microbial insecticide, Bacillus thuringiensis is of great significance to understanding the Bt insecticidal mechanism and improving the performance of strains. This study reviews the commonly used bioinformatics tools and methods in Bt genome data analysis in recent years, including genome assembly and annotation, transcriptome expression analysis, mining of virulence genes and their regulatory elements, comparative genome and evolutionary analysis, protein structure function prediction, and multiomics data integration and visualization platform. Through the application of these tools, researchers can more comprehensively analyze the genomic characteristics of Bt, reveal the expression and regulation mechanism of functional genes such as Cry toxin, compare the differences between Bt and other Bacillus, and explore its evolutionary laws, predict the structure and receptor interaction of toxin proteins, and then guide the design and application of new insecticidal strains. This study looks forward to the trend of bioinformatics integrating multiomics data and artificial intelligence analysis in Bt research, aiming to provide systematic tool guides and references for researchers engaged in Bt genome research and application. Keywords Bacillus thuringiensis; Genome; Bioinformatics tools; Toxin genes; Comparative genomics 1 Introduction Bacillus thuringiensis is a Gram-positive Bacillus widely used in agricultural and sanitary pest control, and is also a recognized safe and efficient microbial insecticide. During the spore formation process, Bt can produce consorubic crystal toxins with specific insecticidal activity, which has an efficient lethal effect on hundreds of insect larvae such as Lepidoptera, Diptera, Coleoptera, and at the same time is safe for humans and animals (Reyaz et al., 2019; Peralta et al., 2021). Genetically modified insect-resistant crops developed based on the Bt insecticidal gene have been widely planted and have played an important role in increasing crop yields and reducing the use of chemical pesticides. Bioinformatics plays an indispensable role in Bt genome research. Modern high-throughput sequencing technology allows us to obtain a large number of Bt strains' whole genome sequences and transcriptome, proteome and other omic data, but these data are huge and complex, and require processing and analysis with the help of bioinformatics tools. Through genome assembly and annotation, functional genes in the Bt genome, including insecticidal crystal protein genes and other secondary metabolite synthesis gene clusters (Yılmaz et al., 2024), can be comprehensively identified, thereby providing a basis for screening and modifying highly efficient strains. This study systematically sorts out the commonly used bioinformatics tools and their application progress in Bt genomic data analysis in the past five years, from the acquisition of original genomic data, efficient assembly and annotation, to the comparative assembly and difference analysis of transcriptomic data, to the mining of toxin gene families and regulatory elements, comparative genomics and evolutionary analysis methods, and toxin protein structure and function prediction, and finally discusses platform tools for integrating multiomics data and visualization of results. Through these contents, we strive to reflect the latest application progress and future trends of bioinformatics in Bt genome analysis, and provide a reference basis for further Bt functional gene discovery and strain molecular improvement.
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