Bt Research 2025, Vol.16, No.4, 157-167 http://microbescipublisher.com/index.php/bt 161 4.2 Modular assembly and expression of multigene clusters In traditional Bt preparations, single toxins are often relied on to control pests, but single toxins may not only have limited insecticide spectrum, but also easily fail due to the resistance of target pests. Gene combinations can expand the insecticidal spectrum. Different Cry or Vip toxins are good at each of the insect categories. For example, Cry1 is targeted against Lepidoptera, Cry3 is targeted against Coleoptera, and Vip3A is effective against some pests that are resistant to Cry. Combining multiple toxin genes into a Bt strain can enable them to simultaneously target multi-target pests (Wang et al., 2020). An international study reported that Bt strains containing up to eight different cry genes showed broad-spectrum high virulence to both Lepidoptera and Coleoptera pests. China already has an engineering strain G033A, which carries a variety of Cry proteins, and has become the first Bt engineering strain registered for the prevention and treatment of Coleoptera and Lepidoptera in China. Multigene combinations can delay the formation of pest resistance. When the pest is exposed to several toxins with different mechanisms of action at the same time, multi-site mutations are required to obtain resistance, which greatly reduces the probability of resistance evolution. Modular assembly facilitates regulating the expression ratio of different toxins. Using synthetic biology, appropriate promoters and replicons can be configured for each toxin gene to balance their expression. For example, Chen et al. adjust the plasmid copy number and promoter strength to make engineering Bt produce both Cry toxin and a lysozyme, thus both killing insects and accelerating decomposition in insect corpses and reducing environmental residues (Chen et al., 2022). Multigenetic engineering can also combine insecticidal proteins with pro-efficiency factors, such as cooperating with insect intestinal peptidase inhibitors, insecticidal toxin antibodies, etc., to attack pests from different angles. 4.3 Metabolic pathway optimization to enhance the stability and yield of Bt In addition to directly modifying the toxin gene itself, improving the field efficacy of Bt strains also requires consideration of the strain's environmental adaptability and toxin production ability. The mature experience in cell plant metabolic engineering is also applicable to Bt strain optimization. Bt preparations are often affected by adverse conditions such as ultraviolet rays and temperature in the field, resulting in a decrease in spore and toxin activity. Through metabolic engineering, Bt strains can be conferred on environmental stress resistance. Avetisyan et al. (2024) used synthetic biology methods to introduce the melanin synthesis pathway into the Bt strain, allowing it to synthesize biomelanin particles. Melanin has strong UV absorption capacity, and the modified Bt strain has significantly improved survival rate and toxin activity in sunlight (Avetisyan et al., 2024). Bt strains need to allocate resources between cell growth and toxin synthesis during growth. Synthetic biology can improve toxin yield per unit cell by reprogramming the strain metabolic pathway (Figure 2). Resources can be saved for toxic protein synthesis by knocking out or downregulating secondary metabolic pathways that are not associated with budding formation. A series of genes that synthesize competitive by-products in Bt strains were deleted, and the results showed that the Cry toxin yield of the engineered strains increased by more than 20% compared with the wild type (Zhu et al., 2022). In addition, regulating the flow of key precursor substances is also an effective means: the large-scale expression of Cry toxin requires sufficient amino acid supply and energy support. By enhancing the enzyme activity of Bt central metabolism, the supply of toxin synthesis precursors can be increased, thereby improving yield. 5 Case Analysis: Successful Practice of Synthetic Biology Transformation of Bt 5.1 Design and synthesis examples of newCry toxin genes One of the typical cases of creating new toxins through synthetic biological strategies, from Chae et al. (2022) research on chimeric Cry toxin. The study designed and synthesized a brand new chimeric Cry toxin gene to address the problem of the global resistant pest Spodoptera frugiperda's resistance to multiple Bt toxins. The researchers first compared the evolutionary sequences of multiple Cry toxins, and selected Cry1Gb and Cry1Ie, two toxins with low natural virulence but large sequence differences as templates. They used DNA synthesis technology to splice their different domains and constructed a series of candidate chimeric toxin genes. These genes were artificially synthesized and expressed and purified in E. coli, and the virulence of different strains of Fallia meadow was determined separately. It was found that one of the chimera (eCry1Gb.1Ig) had high-efficiency
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