Journal of Energy Bioscience 2024, Vol.15, No.6, 378-387 http://bioscipublisher.com/index.php/jeb 381 that is consistent with the metabolic needs of plants throughout the day (Matsuda et al., 2020). This co-regulation ensures that metabolic flux is effectively used for the production of isoflavones and other essential secondary metabolites. 3.3 Genetic modification to increase isoflavone content Genetic modification technology is an important strategy to increase the content of soybean isoflavones, in which the regulation of transcription factors plays an important role in this technology. Studies by Chu et al. (2017) and Feng et al. (2022) both showed that overexpression of GmMYB29 and GmZFP7 can significantly promote gene expression in the isoflavone synthesis pathway, thereby increasing its accumulation level. In addition to directly regulating transcription factors, metabolic engineering strategies are also widely used. Combined inhibition of flavanone 3-hydroxylase (F3H) can effectively block the anthocyanin synthesis branch in the phenylpropanoid metabolic pathway, so that the metabolic flux can be more biased towards the isoflavone synthesis pathway. In a study in 2003, Yu et al. found that when genetic modification technology was co-expressed with transcription factors such as C1 and R that regulate anthocyanin synthesis, the accumulation level of isoflavones would be significantly increased. In a study in 2019, Gupta et al. found that increased cytosine methylation in the coding regions of IFS1 and IFS2 genes was positively correlated with enhanced isoflavone biosynthesis, which provided a new molecular regulation pathway for genetic improvement. 4 Enzymatic Mechanism of Isoflavone Metabolism 4.1 Isoflavone synthase and related enzymes Isoflavone synthase (IFS) is a key catalytic enzyme in the biosynthesis pathway of soybean isoflavones. Its function is to catalyze the specific conversion of flavonoids to isoflavones and open the synthesis pathway of isoflavone compounds. As a type of cytochrome P450 monooxygenase, IFS is a member of the soybean CYP93C subfamily, and its activity determines the metabolic flux of isoflavone synthesis (Jung et al., 2000; Waki et al., 2016). IFS does not act alone, but forms a metabolic complex (metabolon) with other key enzymes in the phenylpropanoid metabolic pathway, including chalcone synthase (CHS) and chalcone isomerase (CHI). The subcellular localization of IFS and its related enzymes is also important in metabolic regulation. These enzymes are anchored on the endoplasmic reticulum (ER) membrane to form a stable enzyme complex to optimize substrate transport and metabolite synthesis efficiency. This spatial organization structure not only helps to enhance the coordination of metabolic pathways, but also avoids competition from bypass metabolism and ensures the robustness of isoflavone synthesis (Dastmalchi et al., 2016; Waki et al., 2016). 4.2 Enzymatic specificity and reaction kinetics In the biosynthesis of isoflavones, the substrate specificity of key enzymes determines the synthesis efficiency and product distribution of specific isoflavones (genistein, soy flavonoids, etc.). Isoflavone synthase (IFS) shows high selectivity for its flavanone substrates, and its catalytic activity is not only regulated by its own enzyme structure, but also by the interaction with other metabolic enzymes. Studies by Dastmalchi et al. (2016) and Waki et al. (2016) showed that IFS forms a functional complex with chalcone synthase (CHS) or chalcone isomerase (CHI) to regulate the catalytic efficiency and substrate flow direction of the isoflavone synthesis pathway. The formed complex achieves efficient transfer of metabolic intermediates between enzymes through tightly coupled enzyme-enzyme interactions in space, avoiding diffusion losses. This organizational pattern can significantly improve the overall flux of the synthesis pathway and reduce the possibility of by-product generation. 4.3 Research progress on enzyme engineering for isoflavone production In recent years, the main research direction of metabolic engineering is to achieve precise regulation of isoflavone metabolic flow by regulating the expression of key enzymes and transcription factors involved in biosynthesis. Studies by Chu et al. (2017) and Feng et al. (2022) both found that overexpression of transcription factors GmMYB29 and GmZFP7 can significantly enhance the expression of IFS and its related enzymes, and improve the overall synthesis efficiency of isoflavones. In order to further optimize the metabolic flow, Yu et al. (2003) also adopted a competitive pathway inhibition strategy (such as combined inhibition of key enzymes in the
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