Plant Gene and Trait 2025, Vol.16, No.5, 225-233 http://genbreedpublisher.com/index.php/pgt 226 et al., 2024). There are mainly two synthetic pathways for them: one is the mevalonic acid (MVA) pathway in the cytoplasm, and the other is the methylerythritol phosphate (MEP) pathway in the plastid. The entire process involves more than twenty enzymatic reactions (Liu et al., 2023). The pharmacological effects of ginsenosides are very extensive, including anti-inflammatory, anti-tumor, cardiovascular protection, anti-fatigue and immune regulation, etc. (Jiang et al., 2024). For example, PPT-type saponins can significantly inhibit the activation of NLRP3 inflammasomes, thereby exerting anti-inflammatory effects (Jiang et al., 2020). 2.2 Other metabolites: polysaccharides, flavonoids, and minor compounds In addition to saponins, ginseng also contains polysaccharides, flavonoids and other components. Ginseng polysaccharides can regulate immunity and have antioxidant effects. Enzymatic extraction can also enhance the immune stimulation effect (Song et al., 2018). Flavonoids and some other minor components, such as alkaloids, volatile oils, fatty acids, etc., also play auxiliary roles in anti-inflammation and antioxidation (Morshed et al., 2023). In addition, researchers also discovered novel indole alkaloids in it, which made the chemical composition of ginseng more diverse (Vu et al., 2023). 2.3 Current limitations in natural metabolite yield and stability Although there are many types of ginseng metabolites, the problems of yield and stability remain prominent. Firstly, ginseng has a long growth cycle and high requirements for climate and soil. Therefore, the output of active components such as saponins is limited and fluctuates greatly (Xu et al., 2023; Jiang et al., 2024). Secondly, the environment, varieties and cultivation methods all affect the composition and content of metabolites (Yoon et al., 2022). Furthermore, saponins have a low absorption rate in the human body and are easily decomposed by intestinal microorganisms, which will reduce their bioavailability and efficacy (Mi et al., 2023; Wang et al., 2023). The extraction and purification processes of polysaccharides can also affect their structure and activity (Song et al., 2018). These issues have made synthetic biology the main approach to enhancing the yield and stability of functional components in ginseng. 3 Synthetic Biology Tools for Metabolite Engineering 3.1 Genome editing technologies: CRISPR/Cas, TALENs, ZFNs Genome editing technology has brought new opportunities to ginseng metabolite engineering. The CRISPR/Cas9 system has been applied in ginseng to knock out or regulate key metabolic genes, thereby significantly increasing the production of some saponins, such as Rg3. Studies have shown that after knocking down the CYP716A53v2 gene, the synthetic flux of the original ginsenoside Rg3 was significantly enhanced. Combined with other metabolic engineering methods, the yield of Rg3 was more than 21 times higher than that of the wild type (Yao et al., 2022). Although TALENs and ZFNs are also useful in other plants, research on ginseng is still scarce and may be expanded in the future. 3.2 Synthetic promoters, transcriptional regulation, and metabolic switches The design of synthetic promoters and transcription factors also provides new approaches for regulating the metabolic genes of ginseng. For instance, WRKY-like transcription factors (PgWRKY4X) can activate the transcription of squalene epoxidase (PgSE), thereby significantly promoting the synthesis of saponins (Yao et al., 2020). In addition, researchers have designed metabolic switches and artificial regulatory elements, allowing the metabolic flow to be regulated according to external signals or inducers, which enables the accumulation of more target metabolites. These methods provide a molecular basis for directed synthesis and increasing the yield of ginsenosides. 3.3 Heterologous expression systems for metabolite biosynthesis (yeast, E. coli, plants) Heterologous expression systems are the key for synthetic biology to achieve efficient production of ginseng metabolites. Saccharomyces cerevisiae is widely used to reconstruct the synthetic pathway of ginsenosides. By introducing multiple glycosyltransferases and key enzyme genes, the research has achieved the de novo synthesis of various saponins such as Rg2, Re, F1, Rh1, etc., and the yield can even reach the gram level (Figure 1) (Wang et al., 2020; Li et al., 2022a; Zhang et al., 2022). In addition, E. coli and plant cell culture systems are also
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