Journal of Energy Bioscience 2024, Vol.15, No.6, 378-387 http://bioscipublisher.com/index.php/jeb 379 shown that by regulating key genes in the biosynthetic pathway, soybean disease resistance can be enhanced and its nutritional value can be improved (Yu et al., 2003; Yu and McGonigle, 2005). This study will systematically review the latest research progress in soybean isoflavone biosynthesis from the perspective of metabolic regulation, focusing on the key enzyme functions in the synthesis pathway, transcriptional regulation mechanisms, and the development of metabolic engineering technology. It is hoped that it will provide theoretical support for molecular breeding and functional food research and development to increase the content of soybean isoflavones, and analyze the potential application value of isoflavones in soybean agricultural production and human health. 2. Biochemical Pathways of Isoflavone Biosynthesis 2.1 Key precursors and initial steps for isoflavone formation The biosynthesis of soy isoflavones begins with the phenylpropanoid metabolic pathway, which provides the necessary precursor molecules for the synthesis of flavonoids. The initial step of biosynthesis is catalyzed by phenylalanine ammonia lyase (PAL), which converts phenylalanine from ammonia to cinnamic acid. Subsequently, under the catalysis of cinnamate 4-hydroxylase (C4H), cinnamic acid was further hydroxylated to form p-coumaric acid (Dastmalchi et al., 2016). On this basis, Chalcone Synthase (CHS) catalyzes the condensation reaction of multiple precursor molecules to form naringenin chalcone, which is considered a key rate limiting step in the synthesis of flavonoids and marks the official start of flavonoid biosynthesis (Imaizumi et al., 2020). 2.2 Functions of chalcone synthase (CHS) and isoflavone synthase (IFS) Chalcone Synthase (CHS) is a key rate limiting enzyme in the biosynthesis pathway of isoflavones, catalyzing the condensation of p-coumaroyl-CoA and malonyl CoA to produce naringenin chalcone (Imaizumi et al., 2020). CHS not only serves as the core catalytic enzyme of this pathway, but also forms metabolic complexes with other flavonoid biosynthetic enzymes, known as isoflavone metabolome. Its function is to improve metabolic efficiency through substrate channel effects and optimize the synthesis rate of isoflavones (Dastmalchi et al., 2016; Waki et al., 2016). Isoflavone Synthase (IFS) is a cytochrome P450 monooxygenase that catalyzes the conversion of naringenin to 2-hydroxyisoflavones, which then undergo spontaneous dehydration to produce daidzein and genistein (Waki et al., 2016; Mameda et al., 2018). The activity of IFS largely determines the flow of isoflavone synthesis, therefore, its expression regulation is crucial for controlling the accumulation of isoflavone compounds. 2.3 Intermediate pathways involved in the synthesis of daidzein and genistein The biosynthesis of daidzein and genistein involves multiple intermediate metabolites, among which naringenin chalcone is a key precursor that undergoes isomerization under the catalytic action of chalcone isomerase (CHI) to naringenin (Waki et al., 2016). Subsequently, naringin was catalyzed by IFS to undergo hydroxylation, forming 2-hydroxyisoflavones, which were then dehydrated to produce daidzein and genistein (Waki et al., 2016; Mameda et al., 2018). This metabolic pathway is co regulated by multiple enzymes to ensure stable and efficient metabolic flux (Dastmalchi et al., 2016). In addition, Chalcone Reductase (CHR) plays an important role in the biosynthesis of 5-deoxyisoflavones (such as daidzein), catalyzing the reduction of coumaroyl CoA to produce 6 '- deoxychalcone. This reaction provides CHS specific substrates and further promotes the branching metabolic pathways of isoflavone biosynthesis (Mameda et al., 2018). 3 Regulatory Genes of Isoflavone Biosynthesis 3.1 Transcription factors regulating the isoflavone biosynthesis pathway Transcription factors play a vital role in the transcriptional regulatory network of soybean isoflavone biosynthesis. GmMYB29, a member of the R2R3-MYB family, is one of the core regulatory factors of isoflavone biosynthesis. It can directly activate the promoters of key enzyme genes (such as IFS2 (isoflavone synthase 2) and CHS8 (chalcone synthase 8)). In their 2017 study, Chu et al. found that overexpression of GmMYB29 significantly increased the accumulation of isoflavones, while gene silencing led to a decrease in its synthesis level. The C2H2-type zinc finger protein GmZFP7 is also a key regulator of isoflavone metabolic flux. By upregulating the
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