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. 5 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 Metabolic Pathways in Bt: Unveiling the Biochemical Basis of Insect Pathogenicity Jun Wang, Mengyue Chen Bt Research, 2025, Vol. 16, No. 5, 182-193 Bt Gene Regulatory Network: A Comprehensive Genomic Approach Zhongqi Wu , Hui Xiang Bt Research, 2025, Vol. 16, No. 5, 194-203 Combining Bt with Other Biocontrol Agents for Integrated Pest Management Xiaoqing Tang, Fangya Chen Bt Research, 2025, Vol. 16, No. 5, 204-213 Bt Toxins Degradation in Different Environmental Matrices Jiayi Wu, Guanli Fu Bt Research, 2025, Vol. 16, No. 5, 214-223 Impact of Plasmid Loss on the Virulence of Bacillus thuringiensis MingLi Bt Research, 2025, Vol. 16, No. 5, 224-233
Bt Research 2025, Vol.16, No.5, 182-193 http://microbescipublisher.com/index.php/bt 182 Research Report Open Access Metabolic Pathways in Bt: Unveiling the Biochemical Basis of Insect Pathogenicity Jun Wang, Mengyue Chen Animal Science Research Center, Cuixi Academy of Biotechnology, Zhuji, 311800, Zhejiang, China Corresponding author: mengyue.chen@cuixi.org Bt Research, 2025, Vol.16, No.5 doi: 10.5376/bt.2025.16.0021 Received: 03 Jul., 2025 Accepted: 20 Aug., 2025 Published: 05 Sep., 2025 Copyright © 2025 Wang and Chen, 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 J., and Chen M.Y., 2025, Metabolic pathways in Bt: unveiling the biochemical basis of insect pathogenicity, Bt Research, 16(5): 182-193 (doi: 10.5376/bt.2025.16.0021) Abstract Bacillus thuringiensis, as an important microbial insecticide, has an irreplaceable position in agriculture and public health. Bt can produce spores and sporophor crystal toxins during metabolism, and has a highly specific pathogenic activity against a variety of insects and is safe for humans and animals. This study reviews the main metabolic pathways of Bt and their relationship with pathogenicity, including sugar metabolism energy supply, protein and amino acid metabolism regulation, lipid metabolism and cell membrane function, as well as the synthesis mechanism, energy demand and expression regulation of Bt toxins (Cry, Cyt, etc.). We also discussed the role of secondary metabolites produced by Bt (such as antimicrobial peptides, pigments, etc.) in insect lethality, as well as the mechanism of metabolic regulatory networks and signaling in Bt pathogenic processes. This study takes corn borer control as an example to analyze the survival adaptation mechanism and key metabolic links of Bt in the insect intestine, and explores the metabolic optimization strategy of genetically engineered Bt corn, and looks forward to the future prospects and challenges of Bt research in metabolomics, metabolic pathway transformation and green agriculture applications. Keywords Bacillus thuringiensis; Metabolic pathway; Insecticidal mechanism; Secondary metabolites; Insect intestine 1 Introduction Bacillus thuringiensis (Bt) is a Gram-positive Bacillus known for producing crystalline proteins (δ-endotoxins) with insecticidal activity. Because of its high virulence to target insects and relatively safe against non-target organisms and the environment, Bt preparations are considered one of the important alternatives to chemical pesticides. At the same time, the Bt toxin gene was also transferred into crops to cultivate genetically modified insect-resistant crops, which greatly reduced the amount of chemical pesticides (Rajadurai et al., 2023). With the long-term use of Bt preparations and Bt crops, pest resistance problems gradually emerged, and some field populations developed resistance to Bt toxins. According to statistics, as of 2020, more than 10 species of field pests have reported resistance to Bt. This situation has triggered the need for more in-depth research on the Bt insecticidal mechanism and the exploration of how to improve the virility and overcome resistance of Bt strains through metabolic pathway modification (Peralta et al., 2021). The pathogenic mechanism of Bt on insects has long attracted much attention. The currently widely accepted model is that after insects feed on preparations containing Bt spores and crystals, the alkaline intestinal environment dissolves the crystals and releases protoxins, and insect intestinal proteases activate it as toxins. The toxin binds to the midgut epithelial receptor and forms holes, resulting in intestinal cell rupture and insect death. Studies have suggested that the insecticide effect of Bt may not only be the crystal toxin itself: live bacteria proliferate in insects and their secondary metabolites produced may also be involved in the pathogenic process. For example, there is evidence that the existence of insect intestinal flora is closely related to the results of Bt infection. Some scholars have observed that sterile insects have reduced sensitivity to Bt (Xu et al., 2023), and it is speculated that Bt infection may require synergistic effects of insect intestinal flora to cause sepsis and other effects. These controversies suggest that we need to examine the pathogenic mechanism of Bt from a broader perspective, including the role of metabolic pathways in it. The metabolic pathway runs through the entire process of Bt from vegetative growth to sporogenic toxin formation, and has a fundamental impact on its pathogenic properties. This study will review the main metabolic
Bt Research 2025, Vol.16, No.5, 182-193 http://microbescipublisher.com/index.php/bt 183 pathways of Bt and its relationship with insect pathogenicity. First, introduce the main characteristics of Bt strain metabolism, including sugar metabolism, protein and amino acid metabolism, lipid metabolism, etc.; then explore the coupling relationship between Bt crystal toxin synthesis and metabolic pathways; analyze the role of Bt's secondary metabolites in the pathogenic process and the function of the metabolic regulatory network; take Bt's prevention and control of corn borer as a case, and deeply analyze the metabolic mechanism of Bt under specific insect exemption, including Cry1Ab toxin synthesis support and genetic engineering of Bt corn. Through the above content, we hope to deepen our understanding of the biochemical basis of Bt pathogenicity and provide theoretical support for improving the application effect of Bt biopesticides. 2 Main Metabolic Characteristics of Bt 2.1 Sugar metabolism pathways and their energy supply As a heterotrophic bacteria, Bt uses organic matter such as carbohydrates as the main carbon source to obtain energy and intermediate metabolites through central metabolic pathways such as glycolysis (EMP pathway), tricarboxylic acid cycle (TCA cycle) (Wang et al., 2013). Under vegetative conditions, Bt cells break down glucose through the EMP pathway to power and generate mature metabolites through the TCA cycle to meet growth needs. When entering late growth or environmental stress, Bt significantly adjusts the sugar metabolism pathway to adapt to environmental changes. Studies have shown that under alkaline stress, the glycolysis-related genes of Bt are greatly increased, producing a large number of metabolic acids such as lactic acid and malic acid, neutralizing the alkaline environment to help bacteria survive. The accumulation of these organic acids is not only a means for Bt to cope with external stress, but may also indirectly affect its pathogenic process. Sugar metabolism is also closely related to spore production and toxin synthesis. Adequate sugar supply can improve the growth rate and energy reserves of bacteria in the logarithmic phase, but excessively available carbon sources may delay spore production through carbon repression effects, thereby delaying the formation of crystal toxins. Bt cells often begin spore production and synthesize toxins when they enter a stable phase, which is usually accompanied by depletion of sugar sources and reprogramming of metabolic flows (Xie et al., 2019). Therefore, suitable sugar metabolism levels must not only meet the rapid growth in the early stage, but also smoothly transition to toxin synthesis during the spore production period. 2.2 Regulatory effects of protein and amino acid metabolism The need for nitrogen sources in Bt is equally important during growth and spore formation. Protein and amino acid metabolism not only provides structural and functional components, but also affects the expression of virulence-related genes through global regulatory effects. Adequate nitrogen source (amino acid) is conducive to rapid proliferation and accumulation of reserves of bacteria, but excess nitrogen source may inhibit the opening of spore production and toxin genes. This is because the presence of global regulators of nutritional induction in Gram-positive bacteria, such as CodY protein, remains active when amino acid and GTP levels are high, thereby suppressing the transcription of secondary metabolism and sporogenesis-related genes (Qi et al., 2015). When nutrients are exhausted, CodY is inactivated, relieving the inhibition of spore production and toxin synthesis pathways, allowing the bacteria to enter the stage of spore production toxin synthesis. It is expected that there is an amino acid sensing mechanism similar to B. subtilis in Bt. High concentrations of branched chain amino acids can affect the expression balance of genes related to toxin synthesis through regulatory proteins such as CodY. In addition, some specific amino acids are precursors to toxin synthesis. Bt also shows certain ability in decomposing environmental proteins, secreting proteases to degrade proteins in insect carcasses or culture medium to obtain nitrogen sources. However, excessive efflux protease activity in early stages of infection may be detrimental to the toxin's function. 2.3 Lipid metabolism and cell membrane function Lipids are an important part of the cell membrane and cell wall, and are related to the changes and stability of membrane structure when spores and toxin crystals are formed. Bt provides raw materials for cell membrane phospholipids through the fatty acid synthesis pathway during the vegetative growth stage, and can use part of the carbon flow to synthesize energy storage substances when carbon is too high. When entering the spore production
Bt Research 2025, Vol.16, No.5, 182-193 http://microbescipublisher.com/index.php/bt 184 stage, Bt undergoes changes in cell morphology and membrane composition, involving the remodeling of phospholipids and fatty acid composition to adapt to the formation of spore and spore crystals (Wang et al., 2013). Studies have shown that certain membrane enzymes are upregulated during Bt spore production, which may be used to regulate membrane fluidity and assist in the release of intracellular toxic crystals. These lipopeptides have interface activity and antibacterial activity, which can assist Bt in insect colonization and combat intestinal microbial competition. Although the main insecticidal factor of Bt is proteotoxin, the impact of lipid metabolism on virility is indirect and far-reaching. Membrane lipid composition will affect the configuration and function of toxin receptors on the intestinal cell membrane, and thus affect the efficiency of toxin action (Figure 1) (Šolinc et al., 2023); lipid signaling molecules (such as certain lipopeptides) can act as population sensing signals or regulatory molecules to regulate Bt population behavior and toxin synthesis. In addition, Bt can also synthesize pigment secondary metabolites such as melanin under special environmental conditions. As reported, many Bt strains can produce melanin in the presence of high temperature and L-tyrosine. Figure 1 Visualization of Cyt2Aa oligomers (Adopted from Šolinc et al., 2023) Image caption: (A) 5.6 μM of monomeric Cyt2Aa was subjected to different treatments: Untreated monomer, Cyt2Aa incubated for 1 h with 0.25 mM Brij 35 or with 1% Triton X-100, and incubated for 1 h with 5 and 10 mM DOPC:CHOL (13:1) MLVs (respectively). (B) Cyt2Aa incubated for indicated times with 10 mM DOPC:CHOL (13:1) MLVs. Samples were solubilized with 1% Triton X-100 and incubated at 22 °C. (C) Control: cryo-EM micrograph of DOPC:CHOL (13:1) MLVs in the absence of Cyt2Aa (D) Top: representative micrograph of Cyt2Aa incubated with DOPC:CHOL (13:1) MLVs recorded with cryo-EM (Adopted from Šolinc et al., 2023) 3 The Association Between Bt Toxin Synthesis and Metabolic Pathways 3.1 The synthesis mechanism of toxin protein (Cry, Cyt) The most significant insecticidal factor of Bt is the toxin protein contained in its compost crystals, including Cry toxins represented by the three domain structure and a few Cyt toxins. Cry toxin is a high molecular weight protein specifically expressed during Bt spore production, and the encoding gene is usually located on the plasmid
Bt Research 2025, Vol.16, No.5, 182-193 http://microbescipublisher.com/index.php/bt 185 and regulated by the σ factor at the spore production stage. When Bt enters the quiescent phase and begins to form spores, spore-specific transcription factors such as σ^E and σ^K initiate high-level expression of the cry gene, and the folded Cry protein aggregates to form crystals and is encased in the cyst with the spores. This mechanism of synchronous spore production and toxin production ensures that the toxin can enter dormant along with the spores and spread with the spores. During the vegetative period, Cry toxins are generally not expressed or are expressed at very low levels. However, Bt also has the ability to secrete insecticidal proteins during the vegetative phase, such as Vip and Sip proteins. Vip3 proteins are secreted by bacteria into culture medium during the logarithmic growth period of Bt, and are virulent to insects such as Lepidoptera, which is different from the mechanism of action of Cry toxins (Palma et al., 2014; Chen et al., 2022). Sip protein is a protein secreted during the transitional period and is toxic to Coleopteran larvae. 3.2 Energy demand and metabolic support in toxin synthesis The synthesis of insecticidal toxin proteins by Bt is a highly energy-consuming process. It is estimated that Cry toxins may account for more than 20% of the total soluble protein in the bacterial body during the late sporulation stage, and their synthesis requires huge ATP and reducing power (Gong et al., 2012). Therefore, Bt needs to mobilize its metabolic pathways to provide sufficient energy and precursors for toxin synthesis. First, the vigorous operation of sugar metabolism provides ATP for protein biosynthesis. Research shows that by optimizing the medium carbon source and oxygen supply conditions to improve the sugar metabolism rate during Bt fermentation, the final cospore crystal yield of bacteria can significantly increase (López et al., 2023). Secondly, central metabolism requires a large number of amino acids as components of toxin proteins. When the exogenous nitrogen source is insufficient, Bt will initiate its own nitrogen metabolism pathway, including amino acid degradation and mutation, to ensure the supply of raw materials for toxin synthesis. Again, some special metabolic pathways are also related to the efficient expression of toxins. The phenomenon that copper ions promote the synthesis of polyβ-hydroxybutyrate (PHB) in Bt was once thought to increase carbon flow storage, thereby providing a carbon framework and energy for toxin synthesis in the later stage. However, further research found that whether PHB accumulation or not has no direct correlation with toxins and spore formation, and the main effect of Cu2+ still enhances core metabolism and accumulates more raw materials for toxin synthesis. 3.3 Coupling between toxin expression regulation and environmental signal Bt toxin synthesis is strictly regulated at the transcriptional and translational levels, and its temporal and spatial expression is closely related to the environment in which the bacteria are located. The spore production regulation network directly controls the expression of Cry toxin. During the initial stage of sporulation, Spo0A, a key transcription factor in the parent cell, is activated by phosphorylation, which triggers the cascade activation of a series of stage-specific σ factors. Bt also perceives many environmental signals and regulates toxin expression through global regulators. When external carbon sources are abundant, the carbon metabolism repression effect mediated by the carbon metabolism regulator CcpA inhibits the transcription of some sporogenic enzymes and virulence factors. Only when the carbon source is consumed to a certain extent and CcpA inhibition is relieved, can the bacteria enter spore-producing and expressing toxins more actively (López et al., 2023). Some special signaling molecules in the environment can directly influence the toxin synthesis pathway. The study found that the transitional regulatory protein AbrB is very uniquely regulated the expression of a trophic insecticidal protein Sip1Ab1 in Bt. Generally, AbrB is present in large quantities when the nutrients are sufficient and acts as an inhibitor, but in Bt strains, AbrB binds to the sip gene promoter to promote its transcription. This phenomenon shows the sensitivity of Bt regulation network. 4 Secondary Metabolites and Pathogenicity 4.1 Types and characteristics of secondary metabolites (anti-microbial peptides, pigments, etc.) In addition to crystal toxins, Bt can also synthesize a variety of secondary metabolites, including antimicrobial peptides, enzymes, pigments, etc., and has certain biological activity on microorganisms and insects
Bt Research 2025, Vol.16, No.5, 182-193 http://microbescipublisher.com/index.php/bt 186 (Martínez-Zavala et al., 2020). Genomic studies have shown that Bacillus is generally rich in secondary metabolite synthesis gene clusters, and about 5%~8% of the genes in Bt strains are specifically used for the synthesis of secondary metabolites (Yılmaz et al., 2024). Some Bt strains can also produce secondary metabolites with color, such as melanin, flavin, etc. These pigment molecules are not direct insecticide factors, but can confer special functions to bacteria. For example, melanin can improve the tolerance of spores and toxin crystals to ultraviolet rays, thereby increasing the stability of Bt preparations in the field. In addition, Bt also produces enzymes such as chitinase and protease, which can degrade biological macromolecules around insect epidermis or bacterial bodies, and obtain nutrients in habitat. These enzymes are strictly classified proteins rather than "small molecule" secondary metabolites in the classical sense, but their expression usually occurs in the late sporulation stage or when the host nutrient is exhausted, so they can also be regarded as a type of secondary functional product in the late Bt life history (Figure 2). Figure 2 (A) Microscopic view of Bt SY49.1 after spore-staining. Arrows indicate the endospores. (B) Scanning electron micrograph of the different types of crystal proteins and spores (S) produced by Bt SY49.1. B, bipyramidal; C, cubic; Sp, spherical. (C) Gene synteny of the candidate insecticidal genes identified in the Bt SY49.1 genome (Adopted from Yılmaz et al., 2024) 4.2 The role of secondary metabolites in insect lethality The pathogenic effect of Bt on insects is not entirely completed by Cry toxin alone, and multiple secondary metabolites play an auxiliary role at each stage of the infection process. During the initial infection phase, enzyme products secreted by Bt may help break through the insect surface barrier. The midgut of insect larvae has a perimeter composed of chitin and protein. The chitinase secreted by Bt can locally degrade the perimeter, promoting better access to toxins and acting on intestinal epithelial cells. Insects may experience secondary sepsis after the toxins work and cause cells to rupture intestinal walls. At this time, the overgrowth of Bt itself or symbiotic bacteria is one of the important factors that cause host death (Grizanova et al., 2022). The antibacterial peptide secondary metabolites produced by Bt can inhibit some
Bt Research 2025, Vol.16, No.5, 182-193 http://microbescipublisher.com/index.php/bt 187 competitive bacteria or defensive symbiotic bacteria in the insect intestines, thereby allowing itself or harmful bacteria to reproduce faster and spread to the blood cavity to cause septic infection. Small molecule toxins in Bt metabolites such as sulvionin (β-exotoxin) can be absorbed by insects and poison the tissues throughout the body, accelerating insect death. Finally, Bt secondary metabolites may also interfere with insects’ immune systems. For example, some lipopeptide antibiotics can not only kill bacteria, but also induce or deplete immune responses in insects, making it more difficult to remove invading Bt (Hrithik et al., 2022). 4.3 Control of secondary products by metabolic regulatory network The synthesis of Bt secondary metabolites often occurs during secondary growth stages or under stress conditions, and its regulation involves complex metabolic networks and signaling pathways. Generally speaking, when Bt transitions from the exponential growth phase to the stable phase, global metabolic regulation changes, and only then is the secondary metabolic pathway released and inhibited and expressed (Xu, 2024). Only when the carbon and nitrogen source decrease to a certain extent and these inhibitory effects weaken will Bt begin to synthesize large quantities of secondary products such as antibiotics and enzymes. This mechanism can be regarded as a distribution strategy between survival priority and competitive suboptimal. It does not waste resources when growing vigorously, but only uses "chemical weapons" when nutrition is scarce and needs to compete for ecological niches. There are often some specific regulatory genes in the Bt genome that regulate the synthesis of secondary metabolites, such as encoding LysR family regulators or σ^H, which co-regulate multiple secondary metabolic gene clusters (Yi et al., 2020). In addition, the population sensing system of Bt may also be involved in regulating the production components of secondary metabolites. Although Gram-positive bacteria usually use cyclic oligopeptides for population sensing, the possibility of enzyme catalyzing the synthesis of gamma-butyrolactone signaling molecules has also been found in some of the genomes of Bt. 5 Signal Transduction and Metabolic Regulation 5.1 The role of metabolites as signaling molecules Bt cells not only use metabolites as nutrients and energy, but also treat them as information molecules to perceive environmental changes and adjust physiological behavior. A typical example is the role of small molecule metabolites in population sensing and feedback regulation. For Gram-positive bacteria, cyclic peptides are often used as quorum sensing signals, but recent studies have suggested that some metabolites themselves can act as signal molecules. The lactic acid accumulated by Bt in an alkaline environment is not only used to neutralize pH, but may also enable the expression of the corresponding gene by regulating transcription factor LldR or CRP/FNR family regulatory proteins (Peng et al., 2024). Peng et al. (2024) found that the lactic acid metabolism pathway induced by Bt under alkaline stress is controlled by a regulator called LtmR, which is very sensitive to changes in lactic acid concentration. When lactic acid accumulates, LtmR will feedback inhibit certain metabolic genes, prompting the bacteria to temporarily suspend energy metabolism and strengthen the alkali resistance mechanism. Bt may also use these metabolite concentration ratios to determine whether the host environment is nutritious or poor, thereby deciding whether to maintain growth or initiate sporotoxin synthesis. 5.2 Regulation of metabolic pathways by global transcription factors There are a variety of global transcription factors in Bt, which can convert metabolic status into gene expression regulation, and have an important impact on bacterial virility. One of these is nutritionally responsive transcription factors, such as CodY, CcpA, AbrB, etc. CodY senses the levels of GTP and branched chain amino acids in cells, binds these molecules when nutrients are abundant and acts as transcriptional repressors, shutting down many spore production and virulence-related genes; when nutrients decrease, CodY loses the effect and the related genes are expressed. This mechanism has been demonstrated in Bacillus subtilis and is also believed to exist and regulate the production of crystal toxins and enzymes in Bt (Mei et al., 2016). CcpA is responsible for carbon metabolism repression. When there is sufficient glucose, CcpA actively inhibits the genes of secondary metabolic enzymes. AbrB is a transition phase regulatory protein that is abundant in the exponential growth phase and
Bt Research 2025, Vol.16, No.5, 182-193 http://microbescipublisher.com/index.php/bt 188 usually acts as a transcriptional repressor, suppressing numerous targets including virulence genes. In the late stage of growth, the rise of Spo0A inhibits AbrB, causing many previously repressed genes to begin expression (Lin and Xu, 2024). Another type of global regulatory factor is stress-responsive. For example, the σ^B factor is activated under stress such as oxidation and osmosis. σ^B will induce the expression of some protective genes (such as oocyte-laying, repair enzymes, etc.), and may also affect the time of spores and toxin production. When the insect's internal environment poses a stress on Bt, the activation of these global factors helps Bt overcome the difficulties and complete the infection process. 5.3 Dynamic response of environmental adaptation and metabolic pathways The environment in which Bt is located is changing rapidly, from laboratory culture medium to field leaves surfaces to insect intestines, the physical and chemical conditions vary greatly. The ability of Bt to successfully cause disease is inseparable from its dynamic response to environmental changes in metabolic pathways. In the insect gut, Bt encounters environmental challenges of alkaline, high concentrations of enzymes and complex microbiota. A study on corn borer larvae showed that after Bt enters the intestine, it triggers dynamic changes in the composition of the insect's midgut microbiome and increases the total bacterial volume. When insects lose gut symbionts, their sensitivity to Bt toxins is actually significantly improved. This shows that the metabolic activity of Bt in the intestine may interact with the intestinal microbiota (Li et al., 2020). When Bt is metabolized vigorously, it can suppress certain beneficial bacteria, thereby weakening insect resistance to Bt. This is an environmental-metabolic-virulence chain reaction. In addition, Bt also has a metabolic response mechanism for environmental parameters such as temperature. Different Bt strains have different adaptations to temperature changes. An experiment compared the virulence of Bt israelensis (mosquitoicidal strain) to Aedes larvae at different temperatures, and found that high-temperature culture can induce Bt to produce more melanin, thereby enhancing the spores' anti-ultraviolet and high-temperature ability and indirectly improving the mosquito-killing effectiveness in fields (Cao et al., 2018). This shows that Bt can adapt to fluctuations in ambient temperature and other factors by changing secondary metabolism (such as pigment synthesis). 6 Metabolic Pathways Interact with Insect Intestinal Environment 6.1 The survival and adaptation mechanism of Bt in the intestines of insects The insect midgut is the main place where Bt works and is also a challenging living environment. After Bt spores and crystals enter the insect's midgut with feeding, they first face the effects of strong alkaline pH and digestive enzymes. For Bt vegetative cells, the environment around pH 10 is not friendly. Bt needs to adapt to high pH as soon as possible in the intestines of insects to germinate spores and reproduce vegetative cells, thereby assisting in infection (Peng et al., 2024). In addition to pH, there are also a variety of antibacterial substances (such as antibacterial peptides, lysozymes) and complex symbiotic bacteria in the intestines, which put pressure on Bt colonization. When Bt enters the insect intestine, it uses metabolic pathways to enhance its own defense and competitiveness (Grizanova et al., 2022). In the insect intestine, the metabolic activity of Bt is also reflected in chemotaxis and colonization. Bt may use flagellar movement and chemotaxis to gather in areas that are prone to colonization in the intestine (such as the posterior part of the midgut). In addition, Bt-germinated spore cells adhere to the midgut surface, which requires the formation of adhesion factors and biofilm. 6.2 Interaction between intestinal pH, enzyme and Bt metabolism The high alkalinity of the insect midgut environment and various digestive enzymes are not only a necessary condition for the Bt toxin to play a role, but also a severe test for Bt bacteria. There is actually an interaction and balance between Bt and this environmental factor. The midgut pH ~10 is conducive to the rapid dissolution of Bt-spore crystals to explain the release of protoxins, but excessive pH may also affect the stability and receptor binding of toxins (Liu et al., 2020). Bt adopts two strategies in evolution, on the one hand, its Cry toxin structure is required for activation at high pH, but if the pH continues to rise to extremes (>11), the toxin activity may be reduced. Secondly, midgut digestive enzymes (such as trypsin, chymotrypsin, etc.) have both activation and potential destructive effects on Bt toxins.
Bt Research 2025, Vol.16, No.5, 182-193 http://microbescipublisher.com/index.php/bt 189 Bt prototoxin requires these proteases to remove nontoxic fragments at the N- and C-terminal ends to be converted into activated toxins (Guo et al., 2020). Therefore, an appropriate amount of enzyme activity is necessary. Other chemical components in the gut environment may also affect Bt metabolism and virulence. For example, certain secondary compounds in plants fed by plant-eating insects may inhibit Bt metabolic enzymes, thereby reducing the effect of Bt infection. 6.3 Feedback from insect immune response on Bt metabolism When Bt successfully breaks through the insect midgut barrier and enters the blood body cavity, the host's humoral and cellular immunity will be quickly activated to fight the invading pathogen. The immune response of insects includes the production of antimicrobial peptides, activation of phenol oxidase cascades to cause blood aggregation, phagocytosis of bacteria, etc. These immune effects also have feedback effects on the metabolic state of Bt (Sun et al., 2025). Antimicrobial peptides produced by insects can act directly on the Bt cell membrane, causing pores and metabolic disorders. Faced with this attack, Bt may induce membrane repair mechanisms and stress metabolic pathways, such as enhancing lipid metabolism to increase membrane steroids against antimicrobial peptide inserts. Some studies have pointed out that when Bt infects rhododendron moth, it triggers a strong immune response in the intestine, including activation of a series of antibacterial genes, but these immune genes are downregulated after Bt destroys the intestinal flora. This suggests that Bt weakens host immunity by destroying insect intestinal microbial balance, thereby reducing its killing to itself. Oxidative stress in insect immune responses can put stress on Bt (Wu et al., 2022). Insect blood cells can kill bacteria by producing reactive oxygen species such as hydrogen peroxide (ROS) through DUOX enzyme. 7 Case Analysis: Metabolic Mechanism of Bt in Corn Bordy Control 7.1 Bt is the key metabolic link in the pathogenesis of corn borer Corn borer (Osrinia furnacalis) is an important feeding pest on corn, and Bt and its toxins play a central role in the biological control of corn borer. During the Bt-corn borer interaction, the development of Bt pathogenicity involves a series of key metabolic links. First, the survival of Bt in the midgut of corn borer and the release of toxins. The midgut of corn borer larvae is highly alkaline and rich in proteases, which provides conditions for the dissolution and activation of Bt crystal toxins. Studies have shown that Bt in the intestine of corn borer will enhance glycolysis and organic acid output to neutralize the local environment to help itself survive (Peng et al., 2024). This metabolic adjustment ensures that Bt successfully enters the spore production and toxin expression stages in the host. The proliferation and colonization of Bt in the intestines of corn borer is a pathogenic amplification step. Bt proliferation requires nutrients from the host. Corn borer feeds on plant tissues, and its intestinal contents are relatively large and free amino acids are limited. The immune response of corn borer larvae can affect the Bt infection process. Recent studies have found that the intestinal flora of corn borer undergoes drastic changes under the action of Bt toxin, and many symbiotic bacteria die or escape from the intestine, resulting in downregulation of host immune gene expression (Li et al., 2020; Xu et al., 2023). During the spread of Bt in corn borer and host death stage, Bt reproduces in large quantities and uses host carcasses to form new spores in preparation for the next round of transmission (Figure 3). In field prevention and control, the pathogenic efficiency of Bt on corn borer is affected by many factors, such as the dosage form of the application, the target insect age, the ambient temperature and humidity, etc. 7.2 Metabolic support mechanism of Cry1Ab toxin Cry1Ab is one of the main insecticidal crystal proteins used by Bt to prevent and control corn borer, and is widely used in both natural Bt strains and trans-Bt gene corn. The successful play of Cry1Ab toxin is closely related to metabolic optimization in Bt strains and host plants. In terms of Bt strains, producing high levels of Cry1Ab requires a strong metabolic supply. Many industrialized Bt preparation strains have obtained higher Cry1Ab expression through mutation and domestication, which often involve changes in metabolic pathways. In transgenic Bt corn, the expression of Cry1Ab toxin also depends on metabolic optimization. As a "factory" for the production of Cry1Ab, plants must contain the synthesis of large amounts of exogenous proteins without
Bt Research 2025, Vol.16, No.5, 182-193 http://microbescipublisher.com/index.php/bt 190 damaging their own growth. To this end, researchers have codon optimization and fragment modification of the Cry1Ab gene sequence to make it more suitable for the transcriptional translation system of plants (Huang, 2011). This gene optimization is essentially to better utilize the metabolic pathway resources of plant cells, improve mRNA stability and translation efficiency, and avoid unnecessary energy waste. Figure 3 Insect gut immunity protects against infections and maintains gut microbiota homeostasis (Adopted from Li et al., 2020) 7.3 Metabolic optimization examples of genetically engineered Bt corn Genetically engineered insect-resistant corn (often called Bt corn) resists insect pests such as corn borer and sticky insects by expressing the Bt toxin gene in the plant body. In the process of cultivating Bt corn, researchers have optimized the Bt gene and corn's own metabolism in many aspects to achieve efficient and stable yield and anti-worm effect. One classic example is the MON810 corn incident of Mengsandu. The modified cry1Ab gene it carries is codon-optimized, removes unstable sequences, and uses strong promoter drivers to continuously express Cry1Ab toxic protein at a high level in corn leaves. This optimization fully takes into account the transcriptional translational metabolic characteristics of maize cells, so that the transferred Bt gene is integrated into the plant metabolic network without causing metabolic burden. Experiments have shown that MON810 corn has lasting resistance to European corn borers, but has no significant adverse effects on corn yield and physiology, which indicates that plant metabolism has reached a new balance on the expression of exogenous insect-resistant proteins (AL-Harbi et al., 2019). Another example is the insect-resistant corn strain cultivated in China in recent years, such as superposition of multiple Bt toxin genes (gold brick combinations such as Cry1Ac/Cry2Ab, etc.). In order to avoid the stress on plant metabolism of multiple exogenous protein expression, the researchers adopted strategies such as tissue-specific promoter and content optimization to enable different Bt toxins to be highly expressed in different parts of the plant or at different growth periods (Huang, 2011). 8 Future Outlook and Challenges 8.1 Application prospects of metabolomics and systems biology in Bt research With the development of high-throughput omics technology, we have the opportunity to deeply analyze the metabolic network of Bt and its association with pathogenicity from the overall level. Metabolomics can comprehensively capture the metabolic profile changes of Bt at different growth conditions and in different infection stages, thereby helping to identify key metabolic pathways and signaling molecules. By comparing Bt's
Bt Research 2025, Vol.16, No.5, 182-193 http://microbescipublisher.com/index.php/bt 191 metabolites in culture medium and host insects, it can be found which metabolites change significantly during infection and their function can be inferred. These may include secondary metabolites or signaling molecules that have not received previous attention and are of great value for elucidating the pathogenic mechanism of Bt. At the same time, the combination of transcriptomics and proteomics will improve the perspective of metabolic research. Systematic biological methods integrate multi-omics data and can reconstruct Bt's metabolic regulation network model. Using these models, we can simulate metabolic flow allocation and virulence factor expression of Bt in different environments, thereby predicting optimization strategies. 8.2 Transformation and improvement of metabolic pathway Bt pathogenicity and stability Based on an in-depth understanding of the relationship between Bt metabolism and virulence, we can try to further improve the pathogenic performance and environmental adaptability of Bt strains through metabolic engineering. A direct idea is to remove metabolic inhibition and improve toxin synthesis. By knocking out the global inhibitor gene in the Bt strain, it can continue to express toxins under nutritious conditions, rather than waiting for the nutrients to be exhausted before producing toxins. This type of transformation may allow Bt to release insecticide factors earlier when entering the insect body, speeding up the lethal process. Of course, attention should be paid to avoid the strains that have premature spore production during the fermentation period affects growth, and regulatory elements should be used to carefully control the knockout effect. Another idea is to introduce or strengthen specific metabolic pathways to improve the environmental stress resistance and survival rate of Bt. For example, Bt can be overexpressed by genetic engineering to overexpress key enzymes of the melanin synthesis pathway, thereby accumulating more melanin particles on the spore surface area and improving the anti-UV capability of the bacteria agent. 8.3 Application direction of green agriculture and sustainable development In the context of pursuing green agriculture and sustainable development, the metabolic research and application of Bt have ushered in new opportunities and challenges. On the one hand, it is expected to develop more efficient, environmentally friendly biopesticides through in-depth understanding of Bt metabolism to reduce dependence on chemical pesticides. On the other hand, the application of Bt is expanding from a single pesticide to a broader biotechnology field such as soil improvement and plant health. Studies have found that metabolites of some Bt strains can promote plant growth or inhibit plant pathogens. For example, certain volatile substances produced by Bt metabolism may induce insect resistance and improve crop self-resistance; for example, Bt is applied in combination with crop endophytes, and its metabolic activity can improve rhizosphere microbial community and thereby enhance plant nutrient absorption. These new application directions require us to break out of the traditional framework of "Bt is insecticide" and re-examine the function of Bt from the perspective of metabolic interaction. Acknowledgments We would like to thank Cuixi Biotechnology Institute for its funding and technical platform support for this research, and we would also like to thank the peer reviewer for its review opinions. Conflict of Interest Disclosure The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest. References Al-Harbi A., Lary S., Edwards M., Qusti S., Cockburn A., Poulsen M., and Gatehouse A., 2019, A proteomic-based approach to study underlying molecular responses of the small intestine of Wistar rats to genetically modified corn (MON810), Transgenic Research, 28: 479-498. https://doi.org/10.1007/s11248-019-00157-y Cao Z., Tan T., Jiang K., Mei S., Hou X., and Cai J., 2018, Complete genome sequence of Bacillus thuringiensis L-7601 a wild strain with high production of melanin, Journal of Biotechnology, 275: 40-43. https://doi.org/10.1016/j.jbiotec.2018.03.020 Chen H., Verplaetse E., Slamti L., and Lereclus D., 2022, Expression of the Bacillus thuringiensis vip3A insecticidal toxin gene is activated at the onset of stationary phase by VipR an autoregulated transcription factor, Microbiology Spectrum, 10(4): e01205-22. https://doi.org/10.1128/spectrum.01205-22
Bt Research 2025, Vol.16, No.5, 182-193 http://microbescipublisher.com/index.php/bt 192 Gong Y., Li M., Xu D., Wang H., He J., Wu D., Chen D., Qiu N., Bao Q., Sun M., and Yu Z., 2012, Comparative proteomic analysis revealed metabolic changes and the translational regulation of Cry protein synthesis in Bacillus thuringiensis, Journal of Proteomics, 75(4): 1235-1246. https://doi.org/10.1016/j.jprot.2011.10.037 Grizanova E., Krytsyna T., Kalmykova G., Sokolova E., Alikina T., Kabilov M., Coates C., and Dubovskiy I., 2022, Virulent and necrotrophic strategies of Bacillus thuringiensis in susceptible and resistant insects Galleria mellonella, Microbial Pathogenesis, 175: 105958. https://doi.org/10.1016/j.micpath.2022.105958 Guo Z., Gong L., Kang S., Zhou J., Sun D., Qin J., Guo L., Zhu L., Bai Y., Bravo A., Soberón M., and Zhang Y., 2020, Comprehensive analysis of Cry1Ac protoxin activation mediated by midgut proteases in susceptible and resistant Plutella xylostella (L.), Pesticide Biochemistry and Physiology, 163: 23-30. https://doi.org/10.1016/j.pestbp.2019.10.006 Hrithik M.T.H., Park Y., Park H., and Kim Y., 2022, Integrated biological control using a mixture of two entomopathogenic bacteria Bacillus thuringiensis and Xenorhabdus hominickii against Spodoptera exigua and other congeners, Insects, 13(10): 860. https://doi.org/10.3390/insects13100860 Huang D.F., 2011, Improvement of Bt cry1Ah gene expression in transgenic maize (Zea mays L.) through codon optimization, Journal of Agricultural Science and Technology, 13(6): 20-26. Li S., De Mandal S., Xu X., and Jin F., 2020, The tripartite interaction of host immunity–Bacillus thuringiensis infection–gut microbiota, Toxins, 12(8): 514. https://doi.org/10.3390/toxins12080514 Lin Y., and Xu Y., 2024, Potential effects of Rhodococcus erythropolis on other insects in mosquito control, Journal of Mosquito Research, 14(1): 49-60. Liu S., Wang S., Wu S., Wu Y., and Yang Y., 2020, Proteolysis activation of Cry1Ac and Cry2Ab protoxins by larval midgut juice proteases fromHelicoverpa armigera, PLoS ONE, 15(1): e0228159. https://doi.org/10.1371/journal.pone.0228159 López V.E.L., Tolibia S.E.M., Pacheco A.D., Girón J.D.T., and Martínez P., 2023, Influence of nutrient feeding variations on AbrB accumulation sporulation and cry1Ac expression during fed-batch cultures of Bacillus thuringiensis, Mexican Journal of Biotechnology, 8(4): 46-67. https://doi.org/10.29267/mxjb.2023.8.4.46 Martínez-Zavala S.A., Barboza-Pérez U.E., Hernandez-Guzman G., Bideshi D.K., and Barboza‐Corona J., 2020, Chitinases of Bacillus thuringiensis: phylogeny modular structure and applied potentials, Frontiers in Microbiology, 10: 3032. https://doi.org/10.3389/fmicb.2019.03032 Mei F., Qi M., and Li M., 2016, Genome-wide identification of CodY target genes in Bacillus thuringiensis by in vitro binding analysis, Acta Microbiologica Sinica, 56(7): 1178-1185. Palma L., Muñoz D., Berry C., Murillo J., and Caballero P., 2014, Bacillus thuringiensis toxins: an overview of their biocidal activity, Toxins, 6: 3296-3325. https://doi.org/10.3390/toxins6123296 Peng Q., Qin J., Xu H., Kao G., Yang F., Sun Z., Zhang X., Slamti L., Guo S., and Song F., 2024, Rapid adaptation of Bacillus thuringiensis to alkaline environments via the L-lactate metabolism pathway regulated by the CRP/FNR family regulator LtmR, Pesticide Biochemistry and Physiology, 208: 106255. https://doi.org/10.1016/j.pestbp.2024.106255 Peralta C., Sauka D.H., Pérez M., Onco M., Fiodor A., Caballero J., Caballero P., Berry C., Valle E., and Palma L., 2021, Genome sequence analysis and insecticidal characterization of Bacillus thuringiensis Bt-UNVM_94 a strain showing dual insecticidal activity against lepidopteran and coleopteran pests, Proceedings of 1st International Electronic Conference on Toxins, 2021: 16-31. https://doi.org/10.3390/iect2021-09139 Qi M., Mei F., Wang H., Sun M., Wang G., Yu Z., Je Y., and Li M., 2015, Function of global regulator CodY in Bacillus thuringiensis BMB171 by comparative proteomic analysis, Journal of Microbiology and Biotechnology, 25(2): 152-161. https://doi.org/10.4014/JMB.1406.06036 Rajadurai G., Anandakumar S., and Raghu R., 2023, Bacillus thuringiensis in pest management, Plant Health Archives, 29(5): 641-653. https://doi.org/10.54083/pha/1.1.2023/11-13 Šolinc G., Anderluh G., and Podobnik M., 2023, Bacillus thuringiensis toxin Cyt2Aa forms filamentous oligomers when exposed to lipid membranes or detergents, Biochemical and Biophysical Research Communications, 674: 44-52. https://doi.org/10.1016/j.bbrc.2023.06.078 Sun Y., Wen H., Xue W., and Xia X., 2025, PxDorsal regulates the expression of antimicrobial peptides and affects the Bt susceptibility of Plutella xylostella, Insects, 16(2): 163. https://doi.org/10.3390/insects16020163 Wang J., Mei H., Zheng C., Qian H., Cui C., Fu Y., Su J., Liu Z., Yu Z., and He J., 2013, The metabolic regulation of sporulation and parasporal crystal formation in Bacillus thuringiensis revealed by transcriptomics and proteomics, Molecular and Cellular Proteomics, 12: 1363-1376. https://doi.org/10.1074/mcp.M112.023986 Wu G., Liu J., Li M., Xiao Y., and Yi Y., 2022, Prior infection of Galleria mellonella with sublethal dose of Bt elicits immune priming responses but incurs metabolic changes, Journal of Insect Physiology, 139: 104401. https://doi.org/10.1016/j.jinsphys.2022.104401
Bt Research 2025, Vol.16, No.5, 182-193 http://microbescipublisher.com/index.php/bt 193 Xie J., Peng J., Yi Z., Zhao X., Li S., Zhang T., Quan M., Yang S., Lu J., Zhou P., Xia L., and Ding X., 2019, Role of hsp20 in the production of spores and insecticidal crystal proteins in Bacillus thuringiensis, Frontiers in Microbiology, 10: 2059. https://doi.org/10.3389/fmicb.2019.02059 Xu C., Luo J., Wang L., Zhu X., Xue H., Huangfu N., Gao X., Li D., Zhang K., Chen R., Ji J., Niu C., and Cui J., 2023, Gut bacterial community and gene expression alterations induced by transgenic Bt maize contribute to insecticidal activity against Mythimna separata, Journal of Pest Science, 97(2): 685-700. https://doi.org/10.1007/s10340-023-01671-z Xu F.Q., 2024, Research on insect pathogen resistance based on GWAS: methods challenges and prospects, Molecular Entomology, 15(1): 8-17. https://doi.org/10.5376/me.2024.15.0002 Yi Z., Zhang T., Xie J., Zhu Z., Luo S., Zhou K., Zhou P., Chen W., Zhao X., Sun Y., Xia L., and Ding X., 2020, iTRAQ analysis reveals the effect of gabDand sucAgene knockouts on lysine metabolism and crystal protein formation in Bacillus thuringiensis, Environmental Microbiology, 23(4): 2230-2243. https://doi.org/10.1111/1462-2920.15359 Yılmaz S., Idris A., Ayvaz A., Temizgül R., Çetin A., and Hassan M., 2024, Genome mining of Bacillus thuringiensis strain SY49.1 reveals novel candidate pesticidal and bioactive compounds, Pest Management Science, 81: 298-307. https://doi.org/10.1002/ps.8433
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