Metabolic Engineering of Tobacco for Heterologous Production of Rare Ginsenosides CK and Rh2

Metabolic Engineering of Tobacco for Heterologous Production of Rare Ginsenosides CK and Rh2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Metabolic Engineering of Tobacco for Heterologous Production of Rare Ginsenosides CK and Rh2 Linggai Cao, Xuemeng Li, Shanshan Lang, Yan Chen, Ping Dong, Jie Zhang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8043439/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Rare ginsenosides, such as compound K (CK) and Rh2, possess potent pharmacological activities but are present at extremely low levels in natural Panax ginseng . Here, we report the successful heterologous biosynthesis of CK and Rh2 in tobacco ( Nicotiana benthamiana and N. tabacum K326) through metabolic engineering. Three pivotal genes— DDS , CYP716A47 , and either UGTPg1 or UGTPg45 —were introduced into tobacco using a multi-gene overexpression construct. Transgenic lines were generated and screened, and ginsenoside accumulation was quantified via LC–MS. N. benthamiana exhibited superior CK production, reaching up to 47.87 µg/g DW in roots, whereas K326 showed higher Rh2 accumulation, up to 8.11 µg/g DW in roots. Across both species, roots consistently contained greater ginsenoside levels than leaves. Topping significantly enhanced yield, and callus cultures demonstrated the potential for scalable in vitro production. These results establish tobacco as a robust plant chassis for the heterologous production of rare ginsenosides. ginsenosides metabolic engineering heterologous production tobacco synthetic biology Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Ginsenosides, the principal pharmacologically active constituents of Panax ginseng , exhibit diverse biological activities that underpin their broad therapeutic potential[ 1 , 2 ]. Among them, rare ginsenosides such as compound K (CK) and Rh2 display enhanced bioactivity and improved bioavailability relative to their more abundant glycosylated precursors[ 3 – 6 ]. However, their trace natural abundance, combined with the technical and economic challenges associated with chemical synthesis, severely restricts their large-scale utilization in the pharmaceutical and nutraceutical industries[ 3 ]. Metabolic engineering presents a sustainable and scalable alternative for producing these high-value compounds[ 7 ]. Recent advances emphasize the use of plant cell, tissue, and hairy root cultures, engineered microbial hosts, and heterologous expression in plant chassis to circumvent conventional cultivation barriers[ 6 ]. Although microbial systems have been exploited for ginsenoside biosynthesis, their performance is often constrained by inefficient expression of plant-derived cytochrome P450 enzymes, elevated fermentation costs, and contamination risks[ 8 , 9 ]. In contrast, plant-based platforms—particularly tobacco—offer several advantages, including well-established genetic transformation protocols, rapid growth, high biomass accumulation, and intrinsic capabilities for complex post-translational modifications and glycosylation[ 10 – 12 ]. The progressive elucidation of the ginsenoside biosynthetic pathway has laid the groundwork for its heterologous reconstruction in alternative hosts[ 13 – 15 ]. Leveraging the 2,3-oxidosqualene precursor naturally produced through tobacco’s native MVA pathway, we introduced three key enzymes catalyzing the biosynthesis of CK and Rh2. We compared production efficiencies between N. benthamiana and N. tabacum K326, investigated tissue-specific ginsenoside accumulation, and established callus cultures to evaluate the potential for scalable in vitro production. Collectively, our study demonstrates the feasibility and efficiency of tobacco as a plant chassis for the heterologous production of rare ginsenosides, providing a strategic framework for future biotechnological and industrial applications. 2. Methods 2.1 Construction of a multi-gene overexpression vector Three key enzyme genes involved in the biosynthesis of rare ginsenosides were selected: dammarenediol synthase ( PgDDS , GenBank ID: GU183405.1), cytochrome P450 CYP716A47 (GenBank ID: JN604536.1), and the glycosyltransferases UGTPg1 (GenBank ID: KF377585.1) or UGTPg45 (GenBank ID: KM401918.1). All genes were synthesized by Biorun Co., Ltd. Each gene was placed under the control of the cauliflower mosaic virus 35S promoter at the 5′-end and terminated with the nopaline synthase (NOS) terminator sequence at the 3′-end. The final construct comprised three expression cassettes arranged in tandem as follows: 35S– PgDDS –NOS, 35S– CYP716A47 –NOS, and 35S– UGT –NOS. 2.2 Plant Transformation The multi-gene vector was introduced into Agrobacterium tumefaciens strain GV3101 via the freeze–thaw method. Transformed cells were cultured in 5 mL YEB medium containing 25 mg/L hygromycin at 28°C with shaking at 200 rpm until OD 600 ≈ 0.8. Cells were harvested by centrifugation at 5,000 rpm for 10 min at 4°C, resuspended in an equal volume of MS medium supplemented with 100 µM acetosyringone, and adjusted to OD 600 0.3–0.5. Leaf explants (0.5–1 cm²) from sterile 4–6-week-old N. benthamiana and N. tabacum K326 plants were pre-cultured on MS medium containing 2.0 mg/L 2,4-D and 0.5 mg/L 6-BA in darkness at 25°C for 2 days. Explants were immersed in the Agrobacterium suspension with gentle shaking for 10 min, blotted dry, and co-cultured on the same medium containing 100 µM acetosyringone in darkness at 20–22°C for 2–3 days. After co-cultivation, explants were transferred to decontamination medium (same as pre-culture medium plus 500 mg/L cefotaxime) and incubated in darkness at 25°C for 7 days. They were then transferred to selection medium (decontamination medium supplemented with 25 mg/L hygromycin) and subcultured every 2 weeks. After 3–4 rounds of subculture, resistant calli were transferred to differentiation medium (MS + 1.0 mg/L 6-BA + 0.2 mg/L NAA + 25 mg/L hygromycin) for 4–6 weeks to induce shoot formation. Shoots of 3–5 cm were moved to rooting medium (½ MS + 0.5 mg/L IBA + 25 mg/L hygromycin) under a 16-hour photoperiod at 25°C for 4–6 weeks. Once roots developed, plantlets were acclimatized in a greenhouse for 3–5 days and transplanted into sterilized peat soil:perlite (3:1) substrate at 25°C with 80–90% relative humidity, yielding T 2 transgenic lines through successive subculturing. 2.3 Identification of Transgenic Seedlings Genomic DNA and total RNA were extracted from tobacco seedlings using a rapid plant genomic DNA extraction kit and the GenePure polysaccharide–polyphenol plant RNA rapid extraction kit, respectively. PCR and RT–PCR were performed using primers listed in Table 1 . The reaction program was as follows: pre-denaturation at 94°C for 2 minutes; denaturation at 94°C for 30 s; annealing at 58°C; extension at 72°C for 2 minutes 40 s (34 cycles of steps 2–4); and final extension at 72°C for 5 minutes. Amplified products were analyzed by 0.8% agarose gel electrophoresis. Table 1 Primes of PCR and RT-PCR Genes Primers Sequences Nbactin Nbactin-qF 5'-AAAGACCAGCTCATCCGTGG-3' Nbactin-qR 5'-AGCAGCTTCCATTCCGATCA-3' actin actin-F 5'-CGTGATCTTACAGATAGCTTCATGA-3' actin-R 5'-AGAGAAGCTAAGATTGATCCTCC-3' PgDDS PgDDS-F 5'-ATGTGGAAGCTGAAGGTTGCTCAAGGA-3' PgDDS -R 5'-TTAAATTTTGAGCTGCTGGTGCTTAGGC-3' CYP716A47 CYP716A47-F 5'-ATGGTGTTGTTTTTCTCCCTATCT-3' CYP716A47-R 5'-TTAATTGTGGGGATGTAGATGAAT-3' UGTPg1 GT1-F 5'-ATGAAGTCAGAATTGATATTCTTGCCCG-3' GT1-R 5'-TTACATTACATAATTTCCTCAAATAGCTT-3' UGTPg45 GT2-F 5'-ATGGAGAGAGAAATGTTGAGCAAAACT-3' GT2-R 5'-TTACATCAGGAGGAAACAAGCTTTGAAA-3' 2.4 Topping Experiment T 2 -generation K326 seedlings engineered for CK and Rh2 biosynthesis were grown in 38 × 42 cm cultivation pots at the Fuquan agricultural experiment base (Latitude: 26°42′N, Longitude: 107°31′E; Fuquan City, Guizhou Province, China). At the early bud stage, the plant apex was inspected, and topping was performed by cutting at the base of the 1–2 youngest leaves above the target functional leaves (e.g., to retain 20 functional leaves, cutting was performed at the base of the 21st–22nd small leaves). The cutting height was maintained 2–3 cm above the uppermost functional leaf to avoid injury to the growth point. The apical tissue, including terminal and unopened flower buds, was excised with sterile scissors at a 45° angle to minimize wound size. The excised apex was sealed in a sterile bag and autoclaved prior to disposal to prevent the spread of transgenic material. To protect the wound from infection and hormone loss, the cut surface was covered with sterile plastic film for 1–2 days. Ten days after topping, leaves from both topped and non-topped plants were collected for quantification of CK and Rh2. 2.5 Induction and Subculture of Tobacco Callus Cells Seeds of PCR-positive T 2 transgenic tobacco were surface sterilized with 10% sodium hypochlorite for 10 min, rinsed 4–5 times with sterile water, and sown on MS medium. When seedlings developed 2–3 true leaves, they were transferred to flasks containing MS medium with 25 mg/L hygromycin. After 60 days of growth, leaves and roots were excised under sterile conditions. Leaves were cut into 0.5 × 0.5 cm pieces and cultured on callus induction medium (MS + 1.0 mg/L NAA + 1.0 mg/L 6-BA). Roots were gently scored on both sides with a sterile blade to produce 5–6 small wounds and cultured on root-derived callus induction medium (MS + 1.0 mg/L 2,4-D + 0.5 mg/L NAA + 0.5 mg/L 6-BA). Media were refreshed every two weeks. Callus tissue became visible at the wound edges after 15–25 days and was fully developed after approximately 45 days. Calli were subsequently transferred to subculture medium (MS + 1.0 mg/L NAA + 1.0 mg/L 6-BA) for maintenance and propagation. 2.6 Ginsenoside Quantification Approximately 20 g of fresh tobacco leaves were flash-frozen in liquid nitrogen and lyophilized. The dried tissue was ground into a fine powder using a high-speed grinder. Precisely 0.3 g of powder was weighed into a 15 mL centrifuge tube, and 80% methanol was added at a ratio of 1:29 (g:mL). Ultrasonic extraction was conducted for 2 hours with intermittent shaking. The extract was centrifuged at 15,000 rpm for 10 minutes, and the supernatant was collected for LC–MS analysis. The concentrations of CK and Rh2 were determined by liquid chromatography–mass spectrometry (LC–MS) under the following conditions: BEH C18 column (50 × 2.1 mm, 1.7 µm); mobile phases: (A) 0.1% formic acid in water, (B) 0.1% formic acid in methanol; gradient program: 50% B at 0 min → 100% B at 3.5 min → hold at 100% B until 5.2 min → return to 50% B at 5.3 min → hold at 50% B until 6.7 min; column temperature: 40°C; injection volume: 5 µL; flow rate: 0.4 mL/min. Mass spectrometry was conducted in positive electrospray ionization (ESI⁺) mode with an ion source temperature of 550°C, voltage of 5,500 V, curtain gas at 40 psi, and both nebulizer and auxiliary gases at 45 psi. Quantitative detection was performed using multiple reaction monitoring (MRM). 3. Results 3.1 Vector Construction and Screening of Transgenic Lines for Heterologous Synthesis of Rare Ginsenoside CK in Tobacco The multi-gene overexpression vector containing the UGTPg1 gene was successfully introduced into N. benthamiana and K326 (Fig. 1 A). Following systematic screening of 140 N. benthamiana transgenic lines, six positive lines capable of synthesizing the rare ginsenoside CK were identified: N8-7, N14-1, N14-7, N14-8, N14-9, and N14-10 (Fig. 1 B- 1 C, Table S1 ). Metabolite quantification revealed that the maximum CK content in leaves reached 24.36 µg/g DW, whereas roots accumulated up to 47.87 µg/g DW. In all lines, CK levels were consistently higher in roots than in leaves, with root-to-leaf ratios ranging from 1.53 to 2.59 (Fig. 1 F). Screening of 130 transgenic K326 lines identified nine CK-producing lines: P8-7, P8-8, P8-10, P9-8, P12-10, P13-3, P13-4, P13-5, and P13-8 (Fig. 1 D, Fig. 1 E, Table S1 ). However, CK accumulation in the K326 background was notably lower, with maximum concentrations of 0.49 µg/g DW in leaves and 1.04 µg/g DW in roots. As observed in N. benthamiana , CK levels were higher in roots than in leaves, with root-to-leaf ratios ranging from 1.52 to 3.26 (Fig. 1 F). Comparison of identical tissues across the two tobacco species showed that CK accumulation was significantly higher in transgenic N. benthamiana than in K326, indicating that N. benthamiana is a more suitable host for the heterologous biosynthesis of rare ginsenoside CK. 3.2 Vector Construction and Screening of Transgenic Lines for Heterologous Synthesis of Rare Ginsenoside Rh2 in Tobacco The multi-gene overexpression vector harboring the UGTPg45 gene was introduced into N. benthamiana and K326 (Fig. 2 A). Screening of 130 N. benthamiana transgenic lines identified eight Rh2-producing lines: N18-1, N18-8, N19-5, N26-2, N26-3, N26-4, N26-9, and N26-10 (Fig. 2 B- 2 C, Table S1 ). Metabolite analysis revealed that Rh2 levels in these lines reached 1.07 µg/g DW in leaves and 2.32 µg/g DW in roots. In productive lines, Rh2 content was consistently higher in roots than in leaves, with root-to-leaf ratios of 1.18–2.35. Notably, Rh2 was undetectable in lines N18-8, N26-4, and N26-10 (Fig. 2 F). Screening of 170 transgenic K326 lines yielded four Rh2-producing lines: P23-1, P23-3, P23-4, and P29-3 (Fig. 2 D- 2 E, Table S1 ). Rh2 accumulation in K326 was substantially higher than in N. benthamiana , with maximum concentrations of 4.08 µg/g DW in leaves and 8.11 µg/g DW in roots. As with N. benthamiana , Rh2 content in K326 roots exceeded that in leaves by 1.99–2.99 times (Fig. 2 F). When comparing equivalent tissues between the two species, transgenic K326 showed markedly higher Rh2 accumulation than N. benthamiana , suggesting that K326 is the more suitable tobacco host for heterologous Rh2 production. 3.3 Pot Experiments with Transgenic K326 Lines Producing Rare Ginsenosides To assess the phenotypic effects of CK and Rh2 biosynthesis engineering on K326 tobacco, a pot experiment was conducted with transgenic K326 lines (engineered for CK or Rh2 production) and non-transgenic K326 plants (served as the control) (Fig. 3 A- 3 B). At full flowering, CK-producing K326 lines remained phenotypically similar to the control (Fig. 3 C), while Rh2-producing lines developed more severe weather flecking (Fig. 3 D). Among these, line P23-1, which exhibited the highest Rh2 accumulation, showed extensive leaf necrosis and premature senescence. These results suggest that intracellular accumulation of heterologously synthesized Rh2 may exert cytotoxic effects on tobacco cells, compromising normal physiological functions and reducing resistance to abiotic stressors such as ozone, thereby intensifying weather flecking symptoms. After topping treatment, both CK and Rh2 levels increased markedly compared with non-topped controls. In CK-producing lines, topped plants accumulated 1.58–2.18 times higher CK content, with a maximum of 0.91 µg/g DW in line P9-8 (Fig. 3 E). Similarly, in Rh2-producing lines, topped plants exhibited 1.29–3.43 times higher Rh2 levels, with a maximum of 4.76 µg/g DW after topping (Fig. 3 F). 3.4 Acquisition and Content Detection of N. benthamiana Cells Synthesizing Rare Ginsenosides Callus cells were successfully induced from the leaves and roots of transgenic N. benthamiana lines capable of synthesizing rare ginsenosides through dedifferentiation culture (Fig. 4 A- 4 D). In callus cells derived from leaves, the contents of ginsenoside CK and Rh2 were 20.52 µg/g DW and 0.98 µg/g DW, respectively. The CK level was slightly but significantly lower than that in the leaves of transgenic N. benthamiana , whereas the Rh2 content showed no significant difference (Fig. 4 E). In contrast, root-derived callus exhibited pronounced browning, which markedly impaired cellular vitality and subsequent growth (Fig. S1 ). During culture, the callus gradually accumulated brown pigments, altering cell morphology and inhibiting proliferation. As a result, an adequate quantity of root-derived callus cells for ginsenoside detection has not yet been obtained. 4. Discussion Plant metabolic engineering has advanced rapidly, enabling the development of sustainable platforms for producing high-value phytochemicals. Compared with microbial systems, which often face challenges such as high cultivation costs, contamination risks, and the inability to replicate complex plant glycosylation patterns [ 16 ], plant chassis provide unique advantages, including robust transformation protocols, native post-translational modification capacity, and scalability for biomass-based production[ 17 ]. In this study, tobacco species ( N. benthamiana and K326) were engineered to biosynthesize CK and Rh2 by introducing only three core biosynthetic genes— DDS , CYP716A47 , and either UGTPg1 or UGTPg45 . This streamlined pathway demonstrates the capacity of plant systems to leverage endogenous metabolism, minimizing the need for extensive synthetic reconstruction[ 18 – 20 ]. Previous studies have achieved the heterologous production of rare ginsenosides in tobacco through multi-gene expression. Gwak et al . introduced PgDDS , CYP716A47 , and UGT71A28 from Panax ginseng , producing CK with the highest content of 4.65 µg/g DW in roots[ 21 ]. Similarly, Chen et al. reconstructed the biosynthetic pathway for Rh2 in tobacco using PnDDS , CYP12H , and UGTPn3 from P. notoginseng , achieving a maximum root content of 5.30 µg/g DW[ 22 ]. In the present study, CK accumulation in N. benthamiana roots reached 47.87 µg/g DW, and Rh2 content in K326 roots reached 8.11 µg/g DW—both substantially exceeding previous reports. Moreover, the successful establishment of ginsenoside-producing callus cultures highlights the feasibility of continuous, season-independent production, bridging laboratory innovation with industrial-scale application. A key finding of this study is the distinct host preference for different ginsenosides. N. benthamiana demonstrated superior CK production, with root content (47.87 µg/g DW) greatly surpassing that of K326 (1.04 µg/g DW). Conversely, K326 was more efficient for Rh2 synthesis, producing up to 8.11 µg/g DW in roots compared to only 2.32 µg/g DW in N. benthamiana . These species-specific disparities underscore the necessity of tailored chassis selection for optimal metabolite yields. Such differences likely arise from variations in metabolic flux distribution, glycosylation efficiency, and the compatibility between heterologous enzymes and endogenous host metabolism[ 23 ]. Additionally, tissue-specific transport and storage mechanisms—particularly root sequestration of triterpenoids—may further influence compound accumulation[ 24 ]. his phenomenon parallels examples in plant metabolic engineering, such as the preferential use of Artemisia annua for artemisinin production[ 25 ]. Consequently, precise host selection is a crucial determinant of efficiency in heterologous ginsenoside biosynthesis. An important physiological observation was the pronounced weather flecking in Rh2-producing K326 lines, especially P23-1, the highest-accumulating line. This phenotype, characterized by necrotic lesions and premature senescence, mirrors oxidative injury typically associated with ozone stress. The excessive accumulation of Rh2 may disrupt redox homeostasis by imposing a heavy metabolic burden, diverting ATP and NADPH from ROS-detoxifying systems and thereby increasing oxidative stress[ 26 ]. Additionally, Rh2 has known cytotoxic properties and can induce apoptosis through ROS-mediated mechanisms in mammalian cells[ 27 ]. The correlation between high Rh2 levels and leaf necrosis suggests a direct link between metabolite buildup and phytotoxicity. This oxidative damage compromises photosynthetic capacity, reducing growth and biomass[ 28 ]. Hence, a balance must be struck between metabolite accumulation and plant health. Mitigation strategies may involve tissue-specific promoters to restrict synthesis to roots or co-expression of antioxidant enzymes to enhance stress tolerance. The establishment of transgenic tobacco callus capable of ginsenoside synthesis provides a foundation for scalable, controllable production systems that may rival microbial platforms. Although engineered yeast systems have achieved impressive ginsenoside yields[ 29 – 32 ], microbial hosts often struggle to express plant cytochrome P450s functionally and reproduce complex glycosylation patterns. Tobacco cells naturally accommodate P450 activity and harbor diverse glycosyltransferases that facilitate accurate glycosylation of triterpenoid intermediates, reducing the need for extensive pathway engineering. Furthermore, plant cell suspension cultures can be expanded in bioreactors with high biomass density and are unaffected by environmental seasonality[ 33 , 34 ]. While current yields in tobacco callus (20.52 µg/g DW for CK) remain lower than top-performing microbial systems, targeted optimization—through enhanced gene expression, precursor supply tuning, elicitor application, and prevention of callus browning—could substantially increase productivity. Given their native enzymatic compatibility and scalability, tobacco cell cultures hold strong potential as sustainable, high-yielding bioplatforms for industrial-scale production of rare ginsenosides. 5. Conclusions This study successfully engineered tobacco plants and cell cultures for the heterologous production of rare and pharmaceutically valuable ginsenosides. Stable T2-generation transgenic lines of N. benthamiana and K326 were generated, each capable of synthesizing ginsenosides CK and Rh2, respectively, thereby confirming the feasibility of producing rare ginsenosides in tobacco through metabolic engineering. Root tissues served as the primary sites of ginsenoside accumulation, with concentrations consistently and significantly exceeding those in leaves, underscoring the critical role of roots in ginsenoside biosynthesis and storage. Transgenic N. benthamiana exhibited a markedly higher accumulation capacity for ginsenoside CK, whereas transgenic K326 demonstrated greater efficiency in producing ginsenoside Rh2. These findings emphasize that chassis selection is a key determinant for optimizing the yield of specific metabolites in plant-based metabolic engineering. Moreover, the application of topping, a straightforward agronomic intervention, significantly enhanced CK and Rh2 accumulation, demonstrating that field management strategies can further amplify metabolite production in engineered plants. Importantly, the successful establishment of ginsenoside-producing callus cultures provides a solid technical foundation for future large-scale, season-independent production of rare ginsenosides through cell suspension systems. Declarations Ethical approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Clinical trial number Not applicable. Funding This work was supported by the Science and Technology Project of Beijing Life Science Academy (Grant Nos. 2023000CC0080, 2025XM24) and the Guizhou Provincial Basic Research Program (Natural Science) (Grant Nos. MS [2025]027 and QN [2025]436). Author Contribution LGC, SZY and JFZ designed the experiments. LGC, XML and SSL performed the experiments, with assistance from YC, PD, JZ, JL and PZ. LGC, SZY and JFZ analyzed the data and wrote the manuscript. All authors have read and agreed to the published version of the manuscript. 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Metab Eng. 2018;48:218–32. Additional Declarations No competing interests reported. Supplementary Files TableS1.xlsx FigureS1.pptx originalagarosegelimages.rar Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Cao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYHACxgMJIIqZ+cCBDxVE6oFqYUs8OOMMsVogFI/xYd4WIpTztx8+cODhjlq7/naeDwd4Gxjk+cUO4NcicSYt4UDimePJMw7zbjgguYPBcObsBPxaDBhyDA4kth1LZgBpMTzDkGBwm5AW/jcQLfKHeR4AGcRokQDbUmNncJiH4cBBYrRI3HgG9EvbgQTDw2wGBxvOSBD2C39/8sGHP9vq7OXOH378+U+FjTy/NAEtUHA4sQFqK1HKQaDOnmilo2AUjIJRMPIAAJNaTz7G5gB2AAAAAElFTkSuQmCC","orcid":"","institution":"Beijing Life Science Academy (BLSA)","correspondingAuthor":true,"prefix":"","firstName":"Linggai","middleName":"","lastName":"Cao","suffix":""},{"id":558097688,"identity":"b910f55b-5e6a-4af1-8b8b-83f37b97a10c","order_by":1,"name":"Xuemeng Li","email":"","orcid":"","institution":"Beijing Life Science Academy (BLSA)","correspondingAuthor":false,"prefix":"","firstName":"Xuemeng","middleName":"","lastName":"Li","suffix":""},{"id":558097689,"identity":"fc08838a-ac3e-44f9-a89c-2bfc816d2951","order_by":2,"name":"Shanshan Lang","email":"","orcid":"","institution":"Beijing Life Science Academy (BLSA)","correspondingAuthor":false,"prefix":"","firstName":"Shanshan","middleName":"","lastName":"Lang","suffix":""},{"id":558097690,"identity":"b5f32b3c-eebe-4c86-8000-d7b1776bb5c3","order_by":3,"name":"Yan Chen","email":"","orcid":"","institution":"Beijing Life Science Academy (BLSA)","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Chen","suffix":""},{"id":558097691,"identity":"8d7613d0-5a7b-4d28-bd54-aace8ac32966","order_by":4,"name":"Ping Dong","email":"","orcid":"","institution":"Beijing Life Science Academy (BLSA)","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Dong","suffix":""},{"id":558097694,"identity":"25a8cac2-3e72-4c3c-a5b1-8519de591fda","order_by":5,"name":"Jie Zhang","email":"","orcid":"","institution":"Guizhou Academy of Tobacco Science","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Zhang","suffix":""},{"id":558097696,"identity":"e518acee-a84e-42b2-af4a-d52d8946174d","order_by":6,"name":"Jie Liu","email":"","orcid":"","institution":"Guizhou Academy of Tobacco Science","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Liu","suffix":""},{"id":558097698,"identity":"04c43e7f-8c79-4f0b-ae31-74ae9ce8f40d","order_by":7,"name":"Pan Zhang","email":"","orcid":"","institution":"Guizhou Academy of Tobacco Science","correspondingAuthor":false,"prefix":"","firstName":"Pan","middleName":"","lastName":"Zhang","suffix":""},{"id":558097700,"identity":"56ffd2e5-eb1c-4ba2-bc64-c8f7f44f205b","order_by":8,"name":"Shizhou Yu","email":"","orcid":"","institution":"Guizhou Academy of Tobacco 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16:34:30","extension":"html","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":103131,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8043439/v1/364d60fb2f2b40390e028364.html"},{"id":98000454,"identity":"337e2faf-62ab-4e97-ac3f-04075872cca1","added_by":"auto","created_at":"2025-12-11 16:11:04","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":162790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeterologous synthesis of rare ginsenoside CK in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eN. benthamiana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and K326\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Schematic diagram of the vector for synthesizing rare ginsenoside CK; B: Screening of positive lines synthesizing rare ginsenoside CK in \u003cem\u003eN. benthamiana\u003c/em\u003e by PCR and RT-PCR. Lane M: DNA marker, Lane 1: WT (untreated \u003cem\u003eN. benthamiana\u003c/em\u003e), Lane 2: Line N8-7, Lane 3: Line N14-1, Lane 4: Line N14-7, Lane 5: Line N14-8, Lane 6: Line N14-9, Lane 7: Line N14-10; C: Screened positive lines of\u003cem\u003e N. benthamiana\u003c/em\u003e; D: Screening of positive lines synthesizing rare ginsenoside CK in K326 by PCR and RT-PCR. Lane M: DNA marker, Lane 1: WT (untreated K326), Lane 2: Line P8-7, Lane 3: Line P8-8, Lane 4: Line P8-10, Lane 5: Line P9-8, Lane 6: Line P12-10, Lane 7: Line P13-3, Lane 8: Line P13-4, Lane 9: Line P13-5, Lane 10: Line P13-8; E: Screened positive lines of K326; F: Detection of rare ginsenoside CK content in positive lines. Note: Different letters indicate significant differences. The DNA marker used in PgDDS gene detection is DL5000 from UEland Company, which consists of 8 double-stranded DNA fragments with sizes of 5,000 bp, 3,000 bp, 2,000 bp, 1,000 bp, 750 bp, 500 bp, 250 bp, and 100 bp. For other types of gene detection, the DNA marker employed is DL2000 from UEland Company, composed of 6 double-stranded DNA fragments with lengths of 2,000 bp, 1,000 bp, 750 bp, 500 bp, 250 bp, and 100 bp.\u003c/p\u003e","description":"","filename":"Slide1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8043439/v1/67af0f73158c7a02694719fb.jpg"},{"id":98000456,"identity":"7e167947-980e-4b58-9ac8-1d8edc951dcc","added_by":"auto","created_at":"2025-12-11 16:11:04","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":156420,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeterologous synthesis of rare ginsenoside Rh2 in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eN. benthamiana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and K326\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Schematic diagram of the vector for synthesizing rare ginsenoside Rh2; B: Screening of positive lines synthesizing rare ginsenoside Rh2 in \u003cem\u003eN. benthamiana\u003c/em\u003e by PCR and RT-PCR. Lane M: DNA marker, Lane 1: WT (untreated \u003cem\u003eN. benthamiana\u003c/em\u003e), Lane 2: Line N18-1, Lane 3: Line N18-8, Lane 4: Line N19-5, Lane 5: Line N26-2, Lane 6: Line N26-3, Lane 7: Line N26-4, Lane 8: Line N26-9, Lane 9: Line N26-10; C: Screened positive lines of\u003cem\u003e N. benthamiana\u003c/em\u003e; D: Screening of positive lines synthesizing rare ginsenoside Rh2 in K326 by PCR and RT-PCR. Lane M: DNA marker, Lane 1: WT (untreated K326), Lane 2: Line P23-1; Lane 3: Line P23-3; Lane 4: Line P23-4; Lane 5: Line P29-3; E: Screened positive lines of K326; F: Detection of rare ginsenoside Rh2 content in positive lines. Note: Different letters indicate significant differences. The usage of the DNA marker is the same as that shown in Fig. 1.\u003c/p\u003e","description":"","filename":"Slide2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8043439/v1/5e01859e98cc68fcfaa74801.jpg"},{"id":98000464,"identity":"ca6e1e24-f400-4230-9fea-3fd87b476a33","added_by":"auto","created_at":"2025-12-11 16:11:04","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":206285,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePot experiment of transgenic K326 lines synthesizing rare ginsenosides CK and Rh2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Pot-cultured seedling stage; B: Pot-cultured full flowering stage; C: Upper-middle leaves of transgenic K326 lines synthesizing rare ginsenoside CK. In the first row, from left to right: P8-7, P8-8, P8-10, P9-8, P12-10; In the second row, from left to right: P13-3, P13-4, P13-5, P13-8, K326 (control); D: Upper-middle leaves of transgenic K326 lines synthesizing rare ginsenoside Rh2. From left to right: P23-1, P23-3, P23-4, P29-3, K326 (control); E: Comparison of rare ginsenoside CK content between topped and non-topped plants; F: Comparison of rare ginsenoside Rh2 content between topped and non-topped plants. Note: Different letters indicate significant differences.\u003c/p\u003e","description":"","filename":"Slide3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8043439/v1/0fafbd3116e07d2c606f1d93.jpg"},{"id":98425612,"identity":"ca277d53-7aea-4835-9c5d-93f793325d17","added_by":"auto","created_at":"2025-12-17 16:34:58","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":79908,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeterologous synthesis of rare ginsenosides CK and Rh2 in tobacco cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Dedifferentiation treatment of leaves; B: Induction of callus cells; C: Acquisition of callus cells; D: Culture of callus cells; E: Detection of saponin content in cells. Note: Different letters indicate significant differences.\u003c/p\u003e","description":"","filename":"Slide4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8043439/v1/66daba99c4701438363803dd.jpg"},{"id":98622691,"identity":"6e174dc7-0f2b-4752-8639-01bdf5b43bd6","added_by":"auto","created_at":"2025-12-19 17:00:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1529419,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8043439/v1/8c414ad4-88e2-49c0-a455-8b345608a9f1.pdf"},{"id":98424190,"identity":"ae686071-ce93-469e-aae7-d32a57047e96","added_by":"auto","created_at":"2025-12-17 16:33:03","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":9843,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8043439/v1/b81c541a3acd4fa731e38a3b.xlsx"},{"id":98000472,"identity":"2fa7680c-72b4-4675-8fdb-bbda3ab0c6e7","added_by":"auto","created_at":"2025-12-11 16:11:04","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19790495,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8043439/v1/a9f0f6f2d5d5f54c35f5f325.pptx"},{"id":98000475,"identity":"fc86f3ab-76a6-4057-9997-e837a6f7f319","added_by":"auto","created_at":"2025-12-11 16:11:05","extension":"rar","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":22099002,"visible":true,"origin":"","legend":"","description":"","filename":"originalagarosegelimages.rar","url":"https://assets-eu.researchsquare.com/files/rs-8043439/v1/136e2c2f2abdf50f82b61d76.rar"}],"financialInterests":"No competing interests reported.","formattedTitle":"Metabolic Engineering of Tobacco for Heterologous Production of Rare Ginsenosides CK and Rh2","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGinsenosides, the principal pharmacologically active constituents of \u003cem\u003ePanax ginseng\u003c/em\u003e, exhibit diverse biological activities that underpin their broad therapeutic potential[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Among them, rare ginsenosides such as compound K (CK) and Rh2 display enhanced bioactivity and improved bioavailability relative to their more abundant glycosylated precursors[\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, their trace natural abundance, combined with the technical and economic challenges associated with chemical synthesis, severely restricts their large-scale utilization in the pharmaceutical and nutraceutical industries[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMetabolic engineering presents a sustainable and scalable alternative for producing these high-value compounds[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Recent advances emphasize the use of plant cell, tissue, and hairy root cultures, engineered microbial hosts, and heterologous expression in plant chassis to circumvent conventional cultivation barriers[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Although microbial systems have been exploited for ginsenoside biosynthesis, their performance is often constrained by inefficient expression of plant-derived cytochrome P450 enzymes, elevated fermentation costs, and contamination risks[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In contrast, plant-based platforms\u0026mdash;particularly tobacco\u0026mdash;offer several advantages, including well-established genetic transformation protocols, rapid growth, high biomass accumulation, and intrinsic capabilities for complex post-translational modifications and glycosylation[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe progressive elucidation of the ginsenoside biosynthetic pathway has laid the groundwork for its heterologous reconstruction in alternative hosts[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Leveraging the 2,3-oxidosqualene precursor naturally produced through tobacco\u0026rsquo;s native MVA pathway, we introduced three key enzymes catalyzing the biosynthesis of CK and Rh2. We compared production efficiencies between \u003cem\u003eN. benthamiana\u003c/em\u003e and \u003cem\u003eN. tabacum\u003c/em\u003e K326, investigated tissue-specific ginsenoside accumulation, and established callus cultures to evaluate the potential for scalable \u003cem\u003ein vitro\u003c/em\u003e production. Collectively, our study demonstrates the feasibility and efficiency of tobacco as a plant chassis for the heterologous production of rare ginsenosides, providing a strategic framework for future biotechnological and industrial applications.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Construction of a multi-gene overexpression vector\u003c/h2\u003e\u003cp\u003eThree key enzyme genes involved in the biosynthesis of rare ginsenosides were selected: \u003cem\u003edammarenediol synthase\u003c/em\u003e (\u003cem\u003ePgDDS\u003c/em\u003e, GenBank ID: GU183405.1), \u003cem\u003ecytochrome P450 CYP716A47\u003c/em\u003e (GenBank ID: JN604536.1), and the \u003cem\u003eglycosyltransferases UGTPg1\u003c/em\u003e (GenBank ID: KF377585.1) or \u003cem\u003eUGTPg45\u003c/em\u003e (GenBank ID: KM401918.1). All genes were synthesized by Biorun Co., Ltd. Each gene was placed under the control of the cauliflower mosaic virus 35S promoter at the 5\u0026prime;-end and terminated with the \u003cem\u003enopaline synthase\u003c/em\u003e (NOS) terminator sequence at the 3\u0026prime;-end. The final construct comprised three expression cassettes arranged in tandem as follows: 35S\u0026ndash;\u003cem\u003ePgDDS\u003c/em\u003e\u0026ndash;NOS, 35S\u0026ndash;\u003cem\u003eCYP716A47\u003c/em\u003e\u0026ndash;NOS, and 35S\u0026ndash;\u003cem\u003eUGT\u003c/em\u003e\u0026ndash;NOS.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Plant Transformation\u003c/h2\u003e\u003cp\u003eThe multi-gene vector was introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101 via the freeze\u0026ndash;thaw method. Transformed cells were cultured in 5 mL YEB medium containing 25 mg/L hygromycin at 28\u0026deg;C with shaking at 200 rpm until OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;\u0026asymp;\u0026thinsp;0.8. Cells were harvested by centrifugation at 5,000 rpm for 10 min at 4\u0026deg;C, resuspended in an equal volume of MS medium supplemented with 100 \u0026micro;M acetosyringone, and adjusted to OD\u003csub\u003e600\u003c/sub\u003e 0.3\u0026ndash;0.5. Leaf explants (0.5\u0026ndash;1 cm\u0026sup2;) from sterile 4\u0026ndash;6-week-old \u003cem\u003eN. benthamiana\u003c/em\u003e and \u003cem\u003eN. tabacum\u003c/em\u003e K326 plants were pre-cultured on MS medium containing 2.0 mg/L 2,4-D and 0.5 mg/L 6-BA in darkness at 25\u0026deg;C for 2 days. Explants were immersed in the \u003cem\u003eAgrobacterium\u003c/em\u003e suspension with gentle shaking for 10 min, blotted dry, and co-cultured on the same medium containing 100 \u0026micro;M acetosyringone in darkness at 20\u0026ndash;22\u0026deg;C for 2\u0026ndash;3 days.\u003c/p\u003e\u003cp\u003eAfter co-cultivation, explants were transferred to decontamination medium (same as pre-culture medium plus 500 mg/L cefotaxime) and incubated in darkness at 25\u0026deg;C for 7 days. They were then transferred to selection medium (decontamination medium supplemented with 25 mg/L hygromycin) and subcultured every 2 weeks. After 3\u0026ndash;4 rounds of subculture, resistant calli were transferred to differentiation medium (MS\u0026thinsp;+\u0026thinsp;1.0 mg/L 6-BA\u0026thinsp;+\u0026thinsp;0.2 mg/L NAA\u0026thinsp;+\u0026thinsp;25 mg/L hygromycin) for 4\u0026ndash;6 weeks to induce shoot formation. Shoots of 3\u0026ndash;5 cm were moved to rooting medium (\u0026frac12; MS\u0026thinsp;+\u0026thinsp;0.5 mg/L IBA\u0026thinsp;+\u0026thinsp;25 mg/L hygromycin) under a 16-hour photoperiod at 25\u0026deg;C for 4\u0026ndash;6 weeks. Once roots developed, plantlets were acclimatized in a greenhouse for 3\u0026ndash;5 days and transplanted into sterilized peat soil:perlite (3:1) substrate at 25\u0026deg;C with 80\u0026ndash;90% relative humidity, yielding T\u003csub\u003e2\u003c/sub\u003e transgenic lines through successive subculturing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Identification of Transgenic Seedlings\u003c/h2\u003e\u003cp\u003eGenomic DNA and total RNA were extracted from tobacco seedlings using a rapid plant genomic DNA extraction kit and the GenePure polysaccharide\u0026ndash;polyphenol plant RNA rapid extraction kit, respectively. PCR and RT\u0026ndash;PCR were performed using primers listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The reaction program was as follows: pre-denaturation at 94\u0026deg;C for 2 minutes; denaturation at 94\u0026deg;C for 30 s; annealing at 58\u0026deg;C; extension at 72\u0026deg;C for 2 minutes 40 s (34 cycles of steps 2\u0026ndash;4); and final extension at 72\u0026deg;C for 5 minutes. Amplified products were analyzed by 0.8% agarose gel electrophoresis.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimes of PCR and RT-PCR\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGenes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimers\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSequences\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eNbactin\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eNbactin-qF\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-AAAGACCAGCTCATCCGTGG-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eNbactin-qR\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-AGCAGCTTCCATTCCGATCA-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eactin\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eactin-F\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-CGTGATCTTACAGATAGCTTCATGA-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eactin-R\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-AGAGAAGCTAAGATTGATCCTCC-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003ePgDDS\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePgDDS-F\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-ATGTGGAAGCTGAAGGTTGCTCAAGGA-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePgDDS -R\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-TTAAATTTTGAGCTGCTGGTGCTTAGGC-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eCYP716A47\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eCYP716A47-F\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-ATGGTGTTGTTTTTCTCCCTATCT-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eCYP716A47-R\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-TTAATTGTGGGGATGTAGATGAAT-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eUGTPg1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGT1-F\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-ATGAAGTCAGAATTGATATTCTTGCCCG-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGT1-R\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-TTACATTACATAATTTCCTCAAATAGCTT-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eUGTPg45\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGT2-F\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-ATGGAGAGAGAAATGTTGAGCAAAACT-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGT2-R\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-TTACATCAGGAGGAAACAAGCTTTGAAA-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Topping Experiment\u003c/h2\u003e\u003cp\u003eT\u003csub\u003e2\u003c/sub\u003e-generation K326 seedlings engineered for CK and Rh2 biosynthesis were grown in 38 \u0026times; 42 cm cultivation pots at the Fuquan agricultural experiment base (Latitude: 26\u0026deg;42\u0026prime;N, Longitude: 107\u0026deg;31\u0026prime;E; Fuquan City, Guizhou Province, China). At the early bud stage, the plant apex was inspected, and topping was performed by cutting at the base of the 1\u0026ndash;2 youngest leaves above the target functional leaves (e.g., to retain 20 functional leaves, cutting was performed at the base of the 21st\u0026ndash;22nd small leaves). The cutting height was maintained 2\u0026ndash;3 cm above the uppermost functional leaf to avoid injury to the growth point. The apical tissue, including terminal and unopened flower buds, was excised with sterile scissors at a 45\u0026deg; angle to minimize wound size. The excised apex was sealed in a sterile bag and autoclaved prior to disposal to prevent the spread of transgenic material. To protect the wound from infection and hormone loss, the cut surface was covered with sterile plastic film for 1\u0026ndash;2 days. Ten days after topping, leaves from both topped and non-topped plants were collected for quantification of CK and Rh2.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Induction and Subculture of Tobacco Callus Cells\u003c/h2\u003e\u003cp\u003eSeeds of PCR-positive T\u003csub\u003e2\u003c/sub\u003e transgenic tobacco were surface sterilized with 10% sodium hypochlorite for 10 min, rinsed 4\u0026ndash;5 times with sterile water, and sown on MS medium. When seedlings developed 2\u0026ndash;3 true leaves, they were transferred to flasks containing MS medium with 25 mg/L hygromycin. After 60 days of growth, leaves and roots were excised under sterile conditions. Leaves were cut into 0.5 \u0026times; 0.5 cm pieces and cultured on callus induction medium (MS\u0026thinsp;+\u0026thinsp;1.0 mg/L NAA\u0026thinsp;+\u0026thinsp;1.0 mg/L 6-BA). Roots were gently scored on both sides with a sterile blade to produce 5\u0026ndash;6 small wounds and cultured on root-derived callus induction medium (MS\u0026thinsp;+\u0026thinsp;1.0 mg/L 2,4-D\u0026thinsp;+\u0026thinsp;0.5 mg/L NAA\u0026thinsp;+\u0026thinsp;0.5 mg/L 6-BA). Media were refreshed every two weeks. Callus tissue became visible at the wound edges after 15\u0026ndash;25 days and was fully developed after approximately 45 days. Calli were subsequently transferred to subculture medium (MS\u0026thinsp;+\u0026thinsp;1.0 mg/L NAA\u0026thinsp;+\u0026thinsp;1.0 mg/L 6-BA) for maintenance and propagation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Ginsenoside Quantification\u003c/h2\u003e\u003cp\u003eApproximately 20 g of fresh tobacco leaves were flash-frozen in liquid nitrogen and lyophilized. The dried tissue was ground into a fine powder using a high-speed grinder. Precisely 0.3 g of powder was weighed into a 15 mL centrifuge tube, and 80% methanol was added at a ratio of 1:29 (g:mL). Ultrasonic extraction was conducted for 2 hours with intermittent shaking. The extract was centrifuged at 15,000 rpm for 10 minutes, and the supernatant was collected for LC\u0026ndash;MS analysis.\u003c/p\u003e\u003cp\u003eThe concentrations of CK and Rh2 were determined by liquid chromatography\u0026ndash;mass spectrometry (LC\u0026ndash;MS) under the following conditions: BEH C18 column (50 \u0026times; 2.1 mm, 1.7 \u0026micro;m); mobile phases: (A) 0.1% formic acid in water, (B) 0.1% formic acid in methanol; gradient program: 50% B at 0 min \u0026rarr; 100% B at 3.5 min \u0026rarr; hold at 100% B until 5.2 min \u0026rarr; return to 50% B at 5.3 min \u0026rarr; hold at 50% B until 6.7 min; column temperature: 40\u0026deg;C; injection volume: 5 \u0026micro;L; flow rate: 0.4 mL/min. Mass spectrometry was conducted in positive electrospray ionization (ESI⁺) mode with an ion source temperature of 550\u0026deg;C, voltage of 5,500 V, curtain gas at 40 psi, and both nebulizer and auxiliary gases at 45 psi. Quantitative detection was performed using multiple reaction monitoring (MRM).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cb\u003e3.1 Vector Construction and Screening of Transgenic Lines for Heterologous Synthesis of Rare Ginsenoside CK in Tobacco\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe multi-gene overexpression vector containing the \u003cem\u003eUGTPg1\u003c/em\u003e gene was successfully introduced into \u003cem\u003eN. benthamiana\u003c/em\u003e and K326 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Following systematic screening of 140 \u003cem\u003eN. benthamiana\u003c/em\u003e transgenic lines, six positive lines capable of synthesizing the rare ginsenoside CK were identified: N8-7, N14-1, N14-7, N14-8, N14-9, and N14-10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Metabolite quantification revealed that the maximum CK content in leaves reached 24.36 \u0026micro;g/g DW, whereas roots accumulated up to 47.87 \u0026micro;g/g DW. In all lines, CK levels were consistently higher in roots than in leaves, with root-to-leaf ratios ranging from 1.53 to 2.59 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eScreening of 130 transgenic K326 lines identified nine CK-producing lines: P8-7, P8-8, P8-10, P9-8, P12-10, P13-3, P13-4, P13-5, and P13-8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). However, CK accumulation in the K326 background was notably lower, with maximum concentrations of 0.49 \u0026micro;g/g DW in leaves and 1.04 \u0026micro;g/g DW in roots. As observed in \u003cem\u003eN. benthamiana\u003c/em\u003e, CK levels were higher in roots than in leaves, with root-to-leaf ratios ranging from 1.52 to 3.26 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eComparison of identical tissues across the two tobacco species showed that CK accumulation was significantly higher in transgenic \u003cem\u003eN. benthamiana\u003c/em\u003e than in K326, indicating that \u003cem\u003eN. benthamiana\u003c/em\u003e is a more suitable host for the heterologous biosynthesis of rare ginsenoside CK.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.2 Vector Construction and Screening of Transgenic Lines for Heterologous Synthesis of Rare Ginsenoside Rh2 in Tobacco\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe multi-gene overexpression vector harboring the \u003cem\u003eUGTPg45\u003c/em\u003e gene was introduced into \u003cem\u003eN. benthamiana\u003c/em\u003e and K326 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Screening of 130 \u003cem\u003eN. benthamiana\u003c/em\u003e transgenic lines identified eight Rh2-producing lines: N18-1, N18-8, N19-5, N26-2, N26-3, N26-4, N26-9, and N26-10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Metabolite analysis revealed that Rh2 levels in these lines reached 1.07 \u0026micro;g/g DW in leaves and 2.32 \u0026micro;g/g DW in roots. In productive lines, Rh2 content was consistently higher in roots than in leaves, with root-to-leaf ratios of 1.18\u0026ndash;2.35. Notably, Rh2 was undetectable in lines N18-8, N26-4, and N26-10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eScreening of 170 transgenic K326 lines yielded four Rh2-producing lines: P23-1, P23-3, P23-4, and P29-3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Rh2 accumulation in K326 was substantially higher than in \u003cem\u003eN. benthamiana\u003c/em\u003e, with maximum concentrations of 4.08 \u0026micro;g/g DW in leaves and 8.11 \u0026micro;g/g DW in roots. As with \u003cem\u003eN. benthamiana\u003c/em\u003e, Rh2 content in K326 roots exceeded that in leaves by 1.99\u0026ndash;2.99 times (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eWhen comparing equivalent tissues between the two species, transgenic K326 showed markedly higher Rh2 accumulation than \u003cem\u003eN. benthamiana\u003c/em\u003e, suggesting that K326 is the more suitable tobacco host for heterologous Rh2 production.\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Pot Experiments with Transgenic K326 Lines Producing Rare Ginsenosides\u003c/h2\u003e\u003cp\u003eTo assess the phenotypic effects of CK and Rh2 biosynthesis engineering on K326 tobacco, a pot experiment was conducted with transgenic K326 lines (engineered for CK or Rh2 production) and non-transgenic K326 plants (served as the control) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). At full flowering, CK-producing K326 lines remained phenotypically similar to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), while Rh2-producing lines developed more severe weather flecking (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Among these, line P23-1, which exhibited the highest Rh2 accumulation, showed extensive leaf necrosis and premature senescence. These results suggest that intracellular accumulation of heterologously synthesized Rh2 may exert cytotoxic effects on tobacco cells, compromising normal physiological functions and reducing resistance to abiotic stressors such as ozone, thereby intensifying weather flecking symptoms.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAfter topping treatment, both CK and Rh2 levels increased markedly compared with non-topped controls. In CK-producing lines, topped plants accumulated 1.58\u0026ndash;2.18 times higher CK content, with a maximum of 0.91 \u0026micro;g/g DW in line P9-8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Similarly, in Rh2-producing lines, topped plants exhibited 1.29\u0026ndash;3.43 times higher Rh2 levels, with a maximum of 4.76 \u0026micro;g/g DW after topping (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Acquisition and Content Detection of \u003cem\u003eN. benthamiana\u003c/em\u003e Cells Synthesizing Rare Ginsenosides\u003c/h2\u003e\u003cp\u003eCallus cells were successfully induced from the leaves and roots of transgenic \u003cem\u003eN. benthamiana\u003c/em\u003e lines capable of synthesizing rare ginsenosides through dedifferentiation culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In callus cells derived from leaves, the contents of ginsenoside CK and Rh2 were 20.52 \u0026micro;g/g DW and 0.98 \u0026micro;g/g DW, respectively. The CK level was slightly but significantly lower than that in the leaves of transgenic \u003cem\u003eN. benthamiana\u003c/em\u003e, whereas the Rh2 content showed no significant difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn contrast, root-derived callus exhibited pronounced browning, which markedly impaired cellular vitality and subsequent growth (Fig.\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). During culture, the callus gradually accumulated brown pigments, altering cell morphology and inhibiting proliferation. As a result, an adequate quantity of root-derived callus cells for ginsenoside detection has not yet been obtained.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003ePlant metabolic engineering has advanced rapidly, enabling the development of sustainable platforms for producing high-value phytochemicals. Compared with microbial systems, which often face challenges such as high cultivation costs, contamination risks, and the inability to replicate complex plant glycosylation patterns [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], plant chassis provide unique advantages, including robust transformation protocols, native post-translational modification capacity, and scalability for biomass-based production[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In this study, tobacco species (\u003cem\u003eN. benthamiana\u003c/em\u003e and K326) were engineered to biosynthesize CK and Rh2 by introducing only three core biosynthetic genes\u0026mdash;\u003cem\u003eDDS\u003c/em\u003e, \u003cem\u003eCYP716A47\u003c/em\u003e, and either \u003cem\u003eUGTPg1\u003c/em\u003e or \u003cem\u003eUGTPg45\u003c/em\u003e. This streamlined pathway demonstrates the capacity of plant systems to leverage endogenous metabolism, minimizing the need for extensive synthetic reconstruction[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePrevious studies have achieved the heterologous production of rare ginsenosides in tobacco through multi-gene expression. Gwak \u003cem\u003eet al\u003c/em\u003e. introduced \u003cem\u003ePgDDS\u003c/em\u003e, \u003cem\u003eCYP716A47\u003c/em\u003e, and \u003cem\u003eUGT71A28\u003c/em\u003e from \u003cem\u003ePanax ginseng\u003c/em\u003e, producing CK with the highest content of 4.65 \u0026micro;g/g DW in roots[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Similarly, Chen et al. reconstructed the biosynthetic pathway for Rh2 in tobacco using \u003cem\u003ePnDDS\u003c/em\u003e, \u003cem\u003eCYP12H\u003c/em\u003e, and \u003cem\u003eUGTPn3\u003c/em\u003e from \u003cem\u003eP. notoginseng\u003c/em\u003e, achieving a maximum root content of 5.30 \u0026micro;g/g DW[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In the present study, CK accumulation in \u003cem\u003eN. benthamiana\u003c/em\u003e roots reached 47.87 \u0026micro;g/g DW, and Rh2 content in K326 roots reached 8.11 \u0026micro;g/g DW\u0026mdash;both substantially exceeding previous reports. Moreover, the successful establishment of ginsenoside-producing callus cultures highlights the feasibility of continuous, season-independent production, bridging laboratory innovation with industrial-scale application.\u003c/p\u003e\u003cp\u003eA key finding of this study is the distinct host preference for different ginsenosides. \u003cem\u003eN. benthamiana\u003c/em\u003e demonstrated superior CK production, with root content (47.87 \u0026micro;g/g DW) greatly surpassing that of K326 (1.04 \u0026micro;g/g DW). Conversely, K326 was more efficient for Rh2 synthesis, producing up to 8.11 \u0026micro;g/g DW in roots compared to only 2.32 \u0026micro;g/g DW in \u003cem\u003eN. benthamiana\u003c/em\u003e. These species-specific disparities underscore the necessity of tailored chassis selection for optimal metabolite yields. Such differences likely arise from variations in metabolic flux distribution, glycosylation efficiency, and the compatibility between heterologous enzymes and endogenous host metabolism[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Additionally, tissue-specific transport and storage mechanisms\u0026mdash;particularly root sequestration of triterpenoids\u0026mdash;may further influence compound accumulation[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. his phenomenon parallels examples in plant metabolic engineering, such as the preferential use of \u003cem\u003eArtemisia annua\u003c/em\u003e for artemisinin production[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Consequently, precise host selection is a crucial determinant of efficiency in heterologous ginsenoside biosynthesis.\u003c/p\u003e\u003cp\u003eAn important physiological observation was the pronounced weather flecking in Rh2-producing K326 lines, especially P23-1, the highest-accumulating line. This phenotype, characterized by necrotic lesions and premature senescence, mirrors oxidative injury typically associated with ozone stress. The excessive accumulation of Rh2 may disrupt redox homeostasis by imposing a heavy metabolic burden, diverting ATP and NADPH from ROS-detoxifying systems and thereby increasing oxidative stress[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Additionally, Rh2 has known cytotoxic properties and can induce apoptosis through ROS-mediated mechanisms in mammalian cells[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The correlation between high Rh2 levels and leaf necrosis suggests a direct link between metabolite buildup and phytotoxicity. This oxidative damage compromises photosynthetic capacity, reducing growth and biomass[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Hence, a balance must be struck between metabolite accumulation and plant health. Mitigation strategies may involve tissue-specific promoters to restrict synthesis to roots or co-expression of antioxidant enzymes to enhance stress tolerance.\u003c/p\u003e\u003cp\u003eThe establishment of transgenic tobacco callus capable of ginsenoside synthesis provides a foundation for scalable, controllable production systems that may rival microbial platforms. Although engineered yeast systems have achieved impressive ginsenoside yields[\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], microbial hosts often struggle to express plant cytochrome P450s functionally and reproduce complex glycosylation patterns. Tobacco cells naturally accommodate P450 activity and harbor diverse glycosyltransferases that facilitate accurate glycosylation of triterpenoid intermediates, reducing the need for extensive pathway engineering. Furthermore, plant cell suspension cultures can be expanded in bioreactors with high biomass density and are unaffected by environmental seasonality[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. While current yields in tobacco callus (20.52 \u0026micro;g/g DW for CK) remain lower than top-performing microbial systems, targeted optimization\u0026mdash;through enhanced gene expression, precursor supply tuning, elicitor application, and prevention of callus browning\u0026mdash;could substantially increase productivity. Given their native enzymatic compatibility and scalability, tobacco cell cultures hold strong potential as sustainable, high-yielding bioplatforms for industrial-scale production of rare ginsenosides.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThis study successfully engineered tobacco plants and cell cultures for the heterologous production of rare and pharmaceutically valuable ginsenosides. Stable T2-generation transgenic lines of \u003cem\u003eN. benthamiana\u003c/em\u003e and K326 were generated, each capable of synthesizing ginsenosides CK and Rh2, respectively, thereby confirming the feasibility of producing rare ginsenosides in tobacco through metabolic engineering. Root tissues served as the primary sites of ginsenoside accumulation, with concentrations consistently and significantly exceeding those in leaves, underscoring the critical role of roots in ginsenoside biosynthesis and storage.\u003c/p\u003e\u003cp\u003eTransgenic \u003cem\u003eN. benthamiana\u003c/em\u003e exhibited a markedly higher accumulation capacity for ginsenoside CK, whereas transgenic K326 demonstrated greater efficiency in producing ginsenoside Rh2. These findings emphasize that chassis selection is a key determinant for optimizing the yield of specific metabolites in plant-based metabolic engineering. Moreover, the application of topping, a straightforward agronomic intervention, significantly enhanced CK and Rh2 accumulation, demonstrating that field management strategies can further amplify metabolite production in engineered plants. Importantly, the successful establishment of ginsenoside-producing callus cultures provides a solid technical foundation for future large-scale, season-independent production of rare ginsenosides through cell suspension systems.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eConsent for publication\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eClinical trial number\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the Science and Technology Project of Beijing Life Science Academy (Grant Nos. 2023000CC0080, 2025XM24) and the Guizhou Provincial Basic Research Program (Natural Science) (Grant Nos. MS [2025]027 and QN [2025]436).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLGC, SZY and JFZ designed the experiments. LGC, XML and SSL performed the experiments, with assistance from YC, PD, JZ, JL and PZ. LGC, SZY and JFZ analyzed the data and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data and materials supporting the findings of this study are available upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCao L, Wu H, Zhang H, Zhao Q, Yin X, Zheng D, et al. Highly efficient production of diverse rare ginsenosides using combinatorial biotechnology. Biotechnol Bioeng. 2020;117:1615\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee DY, Noren HN, O'Connell JF, Lee BY, Kim Y. 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Plant Biotechnol J. 2007;5:579\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAppelhagen I, Wulff-Vester AK, Wendell M, Hvoslef-Eide AK, Russell J, Oertel A, et al. Colour bio-factories: Towards scale-up production of anthocyanins in plant cell cultures. Metab Eng. 2018;48:218\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ginsenosides, metabolic engineering, heterologous production, tobacco, synthetic biology","lastPublishedDoi":"10.21203/rs.3.rs-8043439/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8043439/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRare ginsenosides, such as compound K (CK) and Rh2, possess potent pharmacological activities but are present at extremely low levels in natural \u003cem\u003ePanax ginseng\u003c/em\u003e. Here, we report the successful heterologous biosynthesis of CK and Rh2 in tobacco (\u003cem\u003eNicotiana benthamiana\u003c/em\u003e and \u003cem\u003eN. tabacum\u003c/em\u003e K326) through metabolic engineering. Three pivotal genes\u0026mdash;\u003cem\u003eDDS\u003c/em\u003e, \u003cem\u003eCYP716A47\u003c/em\u003e, and either \u003cem\u003eUGTPg1\u003c/em\u003e or \u003cem\u003eUGTPg45\u003c/em\u003e\u0026mdash;were introduced into tobacco using a multi-gene overexpression construct. Transgenic lines were generated and screened, and ginsenoside accumulation was quantified via LC\u0026ndash;MS. \u003cem\u003eN. benthamiana\u003c/em\u003e exhibited superior CK production, reaching up to 47.87 \u0026micro;g/g DW in roots, whereas K326 showed higher Rh2 accumulation, up to 8.11 \u0026micro;g/g DW in roots. Across both species, roots consistently contained greater ginsenoside levels than leaves. Topping significantly enhanced yield, and callus cultures demonstrated the potential for scalable \u003cem\u003ein vitro\u003c/em\u003e production. These results establish tobacco as a robust plant chassis for the heterologous production of rare ginsenosides.\u003c/p\u003e","manuscriptTitle":"Metabolic Engineering of Tobacco for Heterologous Production of Rare Ginsenosides CK and Rh2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-11 16:10:59","doi":"10.21203/rs.3.rs-8043439/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d94fe961-0485-4185-911d-6ac135e70efd","owner":[],"postedDate":"December 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-17T07:09:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-11 16:10:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8043439","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8043439","identity":"rs-8043439","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}