Journal of Energy Bioscience 2024, Vol.15, No.6, 378-387 http://bioscipublisher.com/index.php/jeb 385 of the pathway, combined with C1/R expression can further increase the content of isoflavones (Yu et al., 2003). The R2R3-type MYB transcription factor GmMYB29 can regulate isoflavone biosynthesis by activating the promoters of key genes such as IFS2 and CHS8. As early as 2017, Chu et al. found that overexpression of GmMYB29 in soybean hairy roots can increase isoflavone content, while RNAi-mediated silencing leads to a decrease in isoflavone content. Feng et al. (2022) found that the C2H2-type zinc finger transcription factor GmZFP7 can affect the expression of gateway enzymes such as GmIFS2 and GmF3H1, and is a regulator of isoflavone accumulation. 8.2 CRISPR-based modifications to enhance isoflavone content CRISPR/Cas9 technology provides a more precise method for modifying genes involved in isoflavone biosynthesis. Although specific studies on CRISPR-based modification in soybean to increase isoflavone content are limited, the technology has great potential. By targeting key regulatory genes (GmMYB29 and GmZFP7), CRISPR can be used to create knockouts or overexpressions to study the effects of key genes on isoflavone content. Chu et al. (2017) identified SNPs associated with isoflavone concentrations through genome-wide association studies (GWAS), providing valuable targets for CRISPR editing. In addition, transcriptome analysis can observe the dynamics of isoflavone biosynthesis genes during seed development, which can help researchers further understand potential CRISPR targets for increasing isoflavone content (Chen et al., 2023). 8.3 Synthetic biology for isoflavone production Synthetic biology approaches have been widely used to reconstruct and optimize isoflavone biosynthesis in heterologous systems. In 2000, Jung et al. found that soybean isoflavone synthase could be successfully expressed in Arabidopsis to produce the isoflavone genistein, demonstrating the feasibility of transferring the pathway to other species. Researchers have also explored the concept of metabolons, protein complexes that promote efficient metabolic flux. In 2016, Dastmalchi et al. found that isoflavone synthase (IFS) and cinnamate 4-hydroxylase (C4H) can be anchored to the endoplasmic reticulum and interact with other enzymes in the pathway to form metabolons. This organization can improve the efficiency of isoflavone biosynthesis and provide a framework for synthetic biology applications. In addition, the identification of protein-protein interactions between isoflavone biosynthetic enzymes, such as GmIFS1 with GmCHS1 and GmCHIs, also further showed the potential to design these interactions to promote isoflavone production (Waki et al., 2016). 9 Conclusion and Future Directions Isoflavones, plant secondary metabolites, have dual value in crop stress resistance and nutritional function. In recent years, important breakthroughs have been made in the biosynthesis of soybean isoflavones, and researchers have systematically analyzed the regulatory mechanism of its metabolic network. The dynamic assembly pattern of key enzyme complexes on the endoplasmic reticulum membrane has been revealed, and the synergistic mechanism of core catalytic elements such as isoflavone synthase (IFS) and chalcone synthase (CHS) has been elucidated. Genetic studies have identified key transcriptional regulatory factors such as GmMYB29 through genome-wide association analysis, confirming that they affect the activity of metabolic pathways through cascade regulatory networks. Gene editing strategies based on metabolic flux analysis have been successfully applied to seed isoflavone enrichment breeding, and targeted accumulation of target components has been achieved through competitive pathway inhibition and transcription factor co-expression technology. Current research still faces bottlenecks in field transformation: First, isoflavone biosynthesis is regulated by the photoperiod and has developmental stage specificity, and field environmental fluctuations can easily lead to imbalances in the metabolic network; second, there are multi-level feedback regulations in the phenylpropanoid metabolic network, and genetic improvement of a single target may cause unexpected metabolic deviations. For example, although overexpression of IFS can increase the isoflavone content, it may change the distribution of lignin precursors, thereby affecting the mechanical strength of the plant. These complexities require researchers to establish a systems biology perspective and take into account both network homeostasis and target product output in metabolic engineering design.
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