Plant Gene and Trait 2025, Vol.16, No.5, 225-233 http://genbreedpublisher.com/index.php/pgt 228 pathways (Kim et al., 2018; Yoon et al., 2022). Combined with metabolic flux analysis, the bottleneck links in the pathway can be located, and the accumulation of target metabolites can be increased by overexpression or knockout of key enzyme genes (Yao et al., 2022; Yuan et al., 2023). Furthermore, metabolomics can also compare metabolic differences under different environments, varieties and physiological states, providing data support for precise metabolic engineering design (Yoon et al., 2022). 5 Synthetic Pathway Design and Optimization 5.1 Reconstruction of ginsenoside biosynthetic pathways in microbial systems In recent years, synthetic biology has promoted the efficient synthesis of ginsenosides in microorganisms. Researchers analyzed and reconstructed the complete pathways of ginsenosides (such as Rg2, Re, Ro, etc.) and achieved de novo synthesis starting from glucose in Saccharomyces cerevisiae. For instance, they identified and validated glycosyltransferases PgURT94, PgCSyGT1, PgUGT18, and PgUGT8, and modularly integrated them into Saccharomyces cerevisiae genome, resulting in the yields of Rg2 and Re reaching 1.3 g/L and 3.6 g/L, respectively (Li et al., 2022a; Zhang et al., 2022). This microbial factory provides a sustainable platform for the industrial production of saponins (Son et al., 2024). 5.2 Engineering rate-limiting enzymes for improved productivity In the ginsenoside pathway, rate-limiting enzymes such as squalene synthase (SS), squalene epoxidase (SQE), β-amyrin synthase (bAS), CYP450 family and UDP-glycosyltransferase (UGT) play a key role in product accumulation. The content of target saponins can be significantly increased through methods such as gene overexpression, site-directed mutagenesis or CRISPR/Cas9 knockout of bypass enzymes. For example, overexpression of SS and downstream enzymes can increase triterpene saponins and phytosterols (Yao et al., 2022). In the semi-rational design and mutation experiments of glycosyltransferase Pq3-O-UGT2, its catalytic efficiency was enhanced. Combined with CRISPR knockout of the bypass enzyme CYP716A53v2, the yield of Rg3 was increased to 21 times that of the wild type (Yang et al., 2018; Yao et al., 2022). These methods provide feasible paths for the targeted production of specific saponins. 5.3 Systems biology approaches for balancing precursor supply and metabolite output Systems biology integrates genomic, transcriptomic and metabolomic data to construct a global network for saponin synthesis. Studies have found that the distribution of precursor molecules (such as 2,3-oxidosqualene) in both the MVA and MEP pathways determines the accumulation of downstream products (Kim et al., 2018; Yu et al., 2024). Through metabolic flow analysis and model prediction, key genes (such as DDS, SQE) can be targeted and regulated to balance the supply of precursors and the output of downstream products, and reduce bottlenecks and by-products (Xu et al., 2023; Yu et al., 2024). These data also provide a basis for chassis cell design and multi-enzyme synergy, accelerating the development of high-yield strains (Xu et al., 2023; Son et al., 2024). 6 Case Study: Application of Synthetic Biology for Ginsenoside Enhancement 6.1 Research background and goals Ginsenosides are the most important active components in ginseng and have functions such as anti-cancer, anti-oxidation and immune regulation. However, the traditional method of extracting saponins from ginseng is highly restricted. This is because ginseng has a long growth cycle, low yield, and is easily affected by the environment, making it difficult to meet the increasing market demand. The development of synthetic biology has provided a new approach for the efficient and sustainable production of saponins. The research objective is to achieve high-yield and diversified production of saponins and their derivatives by modifying metabolic pathways and optimizing host systems, and to promote their application in the pharmaceutical and health industry (Li et al., 2022b; Li et al., 2023; Qiu and Blank, 2023; Son et al., 2024). 6.2 Methodology: pathway engineering, host system selection, and validation In synthetic biology, there are mainly three strategies for the production of ginsenosides. First, metabolic pathway engineering. Researchers reconstructed the saponin synthesis pathway by cloning and optimizing key enzyme genes (such as DDS, CYP450, UGT), and increased the accumulation of precursor substances (such as dammarenediol-II, protopanaxadiol) and target products (Wang et al., 2019; Yao et al., 2022). Second, host system
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