Overview

Cardiovascular and cerebrovascular diseases are the leading cause of death worldwide. Leonurine, a guanidine alkaloid from Leonurus japonicus (yi mu cao), has been used in traditional medicine for “blood-activating” functions and recently gained attention for its cardiovascular potential, now under Phase II clinical evaluation in China. However, its natural abundance is extremely low, and prior attempts in yeast and tobacco leaves confirmed enzyme activity but yielded negligible amounts.

We are the first iGEM team to implement leonurine biosynthesis in a plant chassis using synthetic biology. By engineering tobacco hairy roots, we achieved a 17-fold yield improvement compared with yeast, demonstrating the feasibility of plant-based synthetic biology for pharmaceutically relevant compounds. Although yield limitations remain, our work provided proof-of-concept, reusable plant-specific parts and protocols, and highlighted hairy roots as a promising chassis for future plant synthetic biology projects.

Introduction

Cardiovascular diseases (CVDs) account for an estimated 19.8 million deaths in 2022, with incidence and mortality still on the rise [1]. The limited efficacy and side effects of existing drugs highlight the urgent need for novel therapeutic strategies. Leonurine, a guanidine-substituted alkaloid derived from Leonurus japonicus (yi mu cao), has demonstrated multiple pharmacological effects, including antioxidative activity, vascular endothelial protection, and anti-thrombotic functions, making it a promising candidate for cardiovascular therapy [2, 3].

However, the natural content of leonurine in Leonurus japonicus is extremely low, rendering direct extraction inefficient and unsustainable [3]. Synthetic biology provides a potential alternative, yet no study had previously achieved leonurine production in a plant chassis. Building on recent multi-omics research that identified LjUGT5 and LjSCPL12 as the final two enzymes in the biosynthetic pathway, our project focused on engineering these key steps in tobacco hairy roots [4]. By reconstituting a minimal functional pathway with exogenous substrates, we aimed to demonstrate that plant chassis can support the most cost-effective leonurine biosynthesis.

Figure 1 Protective effect of LEO (leonurine) on cardio-cerebrovascular system [2, 3]

Why Tobacco Hairy Roots?

We adopted a “validate first, then optimize” logic for chassis selection. Tobacco (Nicotiana tabacum) hairy roots were chosen as our plant platform for several reasons:

​ ✓ Well-established system – Agrobacterium-mediated transformation of tobacco hairy roots is one of the most widely used systems in plant biotechnology. It offers high transformation efficiency, stable integration of transgenes, and reproducible results, which are critical for a student-driven project operating on a limited timeline.

​ ✓ Metabolic simulation – Unlike microbial hosts, hairy roots provide a metabolic environment that closely resembles native plants. This makes them particularly suitable for validating enzymes derived from medicinal plants, such as LjUGT5 and LjSCPL12. Furthermore, plant-specific factors such as membrane transporters and post-translational modifications can enhance enzyme folding and activity, advantages not readily replicated in microbial systems.

​ ✓ Experimental feasibility – Hairy roots grow rapidly on hormone-free media, maintain genetic stability, and can be continuously propagated. Their ability to be repeatedly induced from sterile seedlings ensures that sufficient biological material can be obtained for multiple rounds of testing within the scope of our project.

By leveraging these advantages, hairy roots allowed us not only to validate the catalytic activity of LjUGT5 and LjSCPL12 in a genuine plant environment but also to assess whether a plant chassis could achieve measurable leonurine production. This goes beyond a simple proof-of-function, positioning hairy roots as both a validation tool and a potential production platform in plant synthetic biology.

Engineering in Hairy Roots

3.1 Design

3.1.1 The enzymes: LjUGT5 and LjSCPL12

The biosynthetic pathway of leonurine involves several upstream steps, but its final two reactions are catalyzed by UDP-glycosyltransferase (UGT) and serine carboxypeptidase-like acyltransferase (SCPL) (Figure 3).

​ ✓ LjUGT5 activates syringic acid (SA) by glycosylation to produce SA-Glc.

​ ✓ LjSCPL12 catalyzes the condensation of SA-Glc with 4-guanidinobutanol to form leonurine.

Figure 3. Construction of the Leonurine Biosynthetic Pathway

By focusing on these two enzymes, we could bypass the complex upstream metabolic processes and directly reconstruct the key bottleneck steps. To further improve efficiency, we supplied the inexpensive substrates syringic acid (≈¥300/kg) and 4-guanidinobutanol (≈¥500/kg) exogenously, thereby reducing metabolic burden on the chassis and offering cost advantages for potential industrial scale-up.

Thus, LjUGT5 and LjSCPL12 were chosen as the core enzymes of our synthetic design, representing the minimal but sufficient pathway to achieve controllable leonurine synthesis. All the above components have been deposited in the repository (as BioBricks) with characterization data specific to plant systems.

3.1.2 Plasmid selection: pAGM4723

We selected pAGM4723 as our plant expression vector (Figure 4). This plasmid is part of the MoClo modular cloning system, optimized for Golden Gate Assembly, and allows efficient assembly of promoters, CDSs, and terminators.

Reasons for selection:

​ a) MoClo compatibility, enabling rapid modular construction.

​ b) Multi-gene assembly capacity, suitable for reconstructing the UGT-SCPL pathway.

​ c) Agrobacterium-mediated hairy root transformation, ensuring stable expression in Nicotiana tabacum.

​ d) Contains the chloramphenicol resistance gene (cat) for selection.

Thus, pAGM4723 provided an ideal platform for validating the functions of LjUGT5 and LjSCPL12 in the hairy root system.

Figure 4. Plasmid map of pAGM4723

3.1.3 Plasmid Construct Designs

Building upon the key enzymes UGT5 and SCPL12 for leonurine biosynthesis, we introduced GFP as a reporter and XVE as an inducible regulator. GFP facilitates the screening of positive hairy root transformants, providing a visual marker to confirm successful integration. Meanwhile, XVE, an artificial estrogen-responsive system, can be activated by β-estradiol. Once activated, XVE binds to the PLEXA35S promoter and recruits the transcriptional machinery, thereby driving the expression of UGT5 and SCPL12 at specific time points. This design not only reduces the metabolic burden associated with constitutive expression but also provides precise temporal control over the biosynthetic pathway.

Figure 5. Control of Protein Expression

Our plasmid design followed a stepwise assembly strategy: at Level 0, basic modules such as promoters, CDSs, and terminators were first standardized; at Level 1, these modules were combined into complete expression cassettes; and finally, at Level 2, all cassettes were integrated into one construct (Figure 6). Leveraging the pAGM4723 MoClo system, this hierarchical approach ensures high efficiency, directional fidelity, and robust success rates, making it ideal for assembling multi-gene pathways. The final Level 2 plasmid thus supports the stable expression of GFP, XVE, and UGT5/SCPL12 in hairy roots, enabling a complete workflow from selection and regulation to leonurine biosynthesis.

Figure 6. Schematic diagram of plant expression vector construction

3.2 Build

3.2.1 Recombinant vector construction – level 1

Level 0 consists of standardized basic modules such as promoters, CDSs, and terminators, with some obtained from plasmids preserved in our laboratory and others chemically synthesized by GENEWIZ (Suzhou, Jiangsu, China). The expression of the GFP gene is driven by the constitutive promoter PAtUBI5 and terminated by Tmas. Both LjUGT5 and LjSCPL12 are controlled by the inducible promoter PLEXA35S, with Thsp18.2, Tnos, and T35S serving as commonly used terminators in plant expression systems. P35S is a strong constitutive promoter that drives the overexpression of XVE, thereby enabling regulation of the target genes. The Golden Gate Assembly system and procedure for constructing Level 1 vectors from these basic modules are illustrated in Figure 7.

Figure 7. System and Procedure of Level 1 Vector Construction

After assembly, the recombinant plasmids were transformed into E. coli competent cells DH5α. Following overnight incubation, positive colonies of GFP, LjUGT5, LjSCPL12, and XVE were obtained on antibiotic-containing (Amp+) plates (Figure 8).

Figure 8. Construction of Level 1 Vectors for Four Genes (*Note: From left to right are GFP, LjUGT5, LjSCPL12, and XVE, respectively.)*

Subsequently, colony PCR verification was performed on positive clones of LjUGT5 and LjSCPL12. The results showed that all eight randomly selected clones produced bands consistent with the positive control, while no bands were observed in the negative control (Figure 9). These findings indicate that the Level 1 vectors were successfully constructed with high assembly efficiency, providing a solid foundation for the subsequent multi-gene assembly at Level 2.

Figure 9. Colony PCR Identification of the LjUGT5 (A) and LjSCPL12 (B) Level 1 Vectors (Note: PC, positive control; NC, negative control; 1-8, eight randomly selected single colonies)

3.2.2 Recombinant vector construction – level 2

After constructing the Level 1 vectors, we needed to combine the expression cassettes of the four genes into a single construct to obtain the final multi-gene expression vector, Level 2, which enables simultaneous pathway co-expression in tobacco hairy roots. The Golden Gate Assembly system and procedure for constructing Level 2 vectors are illustrated in Figure 10.

Figure 10. System and Procedure of Level 2 Vector Construction

The recombinant plasmids were transformed into E. coli DH5α and plated on LB agar containing kanamycin, followed by overnight incubation at 37 °C. Since the vector carries the chromogenic marker gene crtW aa, non-recombinant colonies appeared yellow, while successful insertion of the target fragment replaced this marker, resulting in white colonies. Thus, white colonies indicate positive recombinants, whereas yellow colonies represent empty vectors. Six parallel replicates were performed, and the results are shown in Figure 11.

Figure 11. Construction of the plant expression vector (Level 2), six repeats.

We picked 96 white single colonies for colony PCR. First, we verified the GFP gene, obtaining 74 positive clones, which served as candidates for the next step of LjSCPL12 identification (Figure 12).

Figure 12. Colony PCR verification of the GFP gene in the Level 2 vector

Based on GFP-positive clones, PCR amplification of LjSCPL12 identified 46 positive clones, which were then used as candidates for LjUGT5 identification (Figure 13).

Figure 13. Colony PCR verification of the LjSCPL12 gene in the Level 2 vector

Based on GFP and LjSCPL12 identification, PCR amplification of LjUGT5 yielded 40 positive clones, which were used as candidates for XVE identification (Figure 14).

Figure 14. Colony PCR verification of the LjUGT5 gene in the Level 2 vector

Finally, after confirming GFP, LjSCPL12, and LjUGT5, PCR amplification of XVE identified 36 positive clones, from which 3 clones were selected for sequencing (Figure 15).

Figure 15. Colony PCR verification of the XVE gene in the Level 2 vector

3.2.3 Transgenic tobacco hairy root construction

After confirming the sequence accuracy of the Level 2 plasmid, we electroporated it into the hairy root Agrobacterium rhizogenes strain Ar.1193, and plated the cells on LB agar supplemented with rifampicin (200 mg/L), kanamycin (50 mg/L), and streptomycin (100 mg/L). The plates were incubated upside down at 28 °C for 2 days. Positive colonies were identified, expanded, and subsequently used to infect tobacco leaves.

Figure 16. Process of Induction and Culture of Tobacco Hairy Roots (Note: A, 0 days after Agrobacterium infection; B, Hairy root emergence at 10 days after Agrobacterium infection; C, Individual culture of hairy roots on solid medium; D, Transfer of hairy roots to liquid culture with the addition of an inducer and substrates.)

As shown in Figure 16, hairy roots began to emerge around 10 days after infection. Once the roots grew to 1–2 cm, GFP expression was examined, and GFP-positive roots were selected (Figure 17). Genomic DNA was then extracted from these positive roots for further verification.

Figure 17. Identification of transgenic positive hairy roots based on fluorescent signals

We selected three GFP-positive hairy roots and extracted their genomic DNA for genotypic analysis. The results confirmed that the GFP, LjUGT5, LjSCPL12, and XVE genes were successfully integrated into the tobacco hairy roots, indicating a high transformation efficiency (Figure 18).

Figure 18. PCR identification of transgenic positive hairy roots (Note: A, B, C, D panels represent the GFP, LjUGT5, LjSCPL12, and XVE genes, respectively. PC, positive control; EV, empty vector; lanes 1, 2, 3 represent three independent transgenic hairy root lines.)

3.3 Test

3.3.1 Expression Analysis of LjUGT5 and LjSCPL12 Genes

After obtaining PCR-positive hairy roots, they were transferred into liquid medium and induced with β-estradiol (10 μM) for 2 days, followed by supplementation with 4-guanidinobutanol, syringic acid, and UDPG (0.5 mM). The addition of UDPG was essential because the formation of SA-Glc requires UDP-glucose as a glycosyl donor, catalyzed by LjUGT5, which promotes efficient glycosylation of syringic acid and ensures smooth downstream synthesis of leonurine. After 3 more days, total RNA was extracted for qRT-PCR analysis.

As shown in Figure 19, both genes exhibited near-background expression in the control groups (CK1–3). In contrast, in the β-estradiol–treated samples (Sample 1–3), LjUGT5 expression was significantly upregulated (approximately 10-fold), while LjSCPL12 expression also increased markedly (about 4–6 fold). These results demonstrate that the constructed multi-gene expression vector was successfully integrated into the hairy root system and that the target genes could be robustly induced by β-estradiol, confirming the feasibility of our design.

Figure 19. Expression level of LjUGT5 (A) and LjSCPL12 (B) in transgenic hairy roots (Note: Samples 1-3 were treated with β-Estradiol to a final concentration of 10 μM; CK1-3 were treated with an equal volume of DMSO.)

3.3.2 Analysis of Leonurine Yield in Tobacco Hairy Roots

Leonurine production was induced under the same conditions as described above. After 3 days of substrate supplementation, hairy roots were harvested, freeze-dried for 2 days, and ground into powder. Metabolites were extracted with 70% methanol and analyzed by LC-MS (AB SCIEX 6500+) for leonurine quantification.

In metabolite analysis, we first performed qualitative detection by LC-MS retention time. A distinct peak was observed in the transgenic samples at the same retention time as the standard, while no peak was detected in the controls, indicating successful biosynthesis of leonurine in the transgenic hairy roots (Figure 20A). We then used a standard curve to correlate peak area with concentration, enabling quantitative analysis (Figure 20B). The results showed that all three transgenic samples contained stable levels of leonurine, with an average content of 3.687 μg/g DW, while no detectable signal (ND) was observed in the controls (Figure 20C). Based on biomass yield, approximately 0.4 g of freeze-dried hairy root powder was obtained from a 200 mL culture, corresponding to about 2.0 g DW per liter. With an average leonurine content of 3.687 μg/g DW, this translates into:

​ ➢ a specific yield of 3.687 μg/g DW,

​ ➢ a volumetric titer of approximately 7.37 μg/L, and

​ ➢ a batch yield of about 1.47 μg per 200 mL culture.

These values represent apparent yields, as no correction was applied for extraction recovery during metabolite analysis.

Figure 20. Analysis of leonurine production yield in transgenic hairy roots (Note: Samples 1-3 were supplemented with β-estradiol to a final concentration of 10 μM; control groups CK1-3 were supplemented with an equal volume of DMSO.)

Together, these results demonstrate that our multi-gene expression construct not only enabled effective inducible expression of the target genes in hairy roots but also successfully drove the stable biosynthesis of leonurine, thereby validating the functional effectiveness of the reconstructed pathway.

3.4 Learn

We successfully validated the functions of LjUGT5 and LjSCPL12 in the tobacco hairy root system and detected stable biosynthesis of leonurine under substrate supplementation and β-estradiol induction. This confirmed the feasibility of our designed pathway in a plant chassis. However, the yield was only 3.687 μg/g DW (≈7.37 μg/L), which remains far below the threshold required for industrial application (≥60 mg/200 mL). Such limited productivity likely arises from intrinsic plant-specific constraints, including restricted metabolic flux, inefficient substrate transport, and suboptimal enzyme activity in the hairy root environment. These limitations highlight the unique challenges of plant synthetic biology, prompting us to seek iterative solutions in the next cycle.

Limitations & Iteration

While our work in tobacco hairy roots successfully demonstrated the functionality of LjUGT5 and LjSCPL12 and confirmed the feasibility of leonurine biosynthesis in a plant system, we also encountered important limitations that are inherent to plant synthetic biology.

First, the yield was relatively low (3.687 μg/g DW, ≈7.37 μg/L), still far below the threshold for industrial application (≥60 mg/200 mL). This can be attributed to several plant-specific bottlenecks:

​ ➢ Metabolic flux constraints – hairy roots provide limited carbon flux compared with microbial chassis, restricting precursor availability[5].

​ ➢ Substrate transport inefficiency – uptake and intracellular distribution of exogenously supplied precursors is constrained by plant membrane transport systems[6].

​ ➢ Enzyme context dependency – plant cellular environments may not always provide optimal cofactors or folding conditions, reducing catalytic efficiency[7].

By identifying these challenges, we addressed a key question in plant synthetic biology: can plant chassis serve not only for functional validation but also for scalable production? Our results suggest that while hairy roots are excellent for testing pathway feasibility and enzyme activity within a relevant metabolic context, further optimization—such as transporter engineering, flux balancing, or stable genome integration—would be required before they can become competitive production platforms.

Importantly, this limitation should not be viewed as a failure but as a valuable contribution. Our experiments produced detailed protocols, validated plant-specific parts, and quantitative benchmarks for leonurine biosynthesis in hairy roots—resources that future iGEM teams can directly reuse if they wish to further optimize plant systems.

For the next iteration, and guided by expert feedback, we complemented the hairy-root work with a microbial chassis (E. coli) and in vitro enzyme catalysis. This was not an abandonment of plants but a strategic parallel approach to test scalability. Together, our two cycles provide: (1) insights into the strengths and challenges of plant chassis, and (2) a complementary microbial strategy for production. This dual approach highlights how plant synthetic biology can benefit from cross-system comparisons, clarifying when plants are most advantageous and when hybrid solutions are needed.

How we use plant chassis's special attributes

Although the leonurine yield in hairy roots was limited, our work leveraged several unique biological attributes of plant systems that cannot be reproduced in microbial chassis.

First, membrane transport systems in plants enabled the uptake and intracellular trafficking of exogenously supplied substrates, including syringic acid, 4-guanidinobutanol, and UDP-glucose. This demonstrated how plant hairy roots can serve as a living “metabolic incubator,” capable of processing complex small molecules in ways that microbial systems cannot easily replicate.

Second, cofactor availability and enzyme folding environments in plants provided a physiologically relevant context to validate the activities of LjUGT5 and LjSCPL12. While microbial expression is often focused on enzyme yield, plant hairy roots allowed us to test whether these enzymes can function properly in a plant metabolic background, which is crucial for understanding their natural roles and potential compatibility with plant-based production.

Third, hairy roots offered visual screening and stable propagation advantages. By introducing GFP as a reporter, we could directly identify positive transformants in planta, an advantage not typically available in microbial chassis. Moreover, once established, hairy root lines can be propagated and induced repeatedly, creating a renewable testing platform.

In this way, our project highlighted not only the feasibility of leonurine biosynthesis in a plant chassis but also the unique experimental strengths that plant systems contribute to synthetic biology. These insights provide valuable guidance for future teams considering whether to choose plant systems for pathway validation, optimization, or specialized metabolite production.

Contributions to PSB

Our project contributes to the iGEM and plant synthetic biology community in three main aspects:

(1) Novel composite part for plant-based leonurine biosynthesis

We designed and assembled a new composite part (GFP–LjUGT5–LjSCPL12–XVE) in the modular cloning system BBa_253R7TKL(https://2025.igem.wiki/nais/parts). This construct integrates three functional modules: GFP as a visual marker for positive hairy root selection, XVE as an inducible regulator activated by β-estradiol, and the two pathway enzymes LjUGT5 and LjSCPL12 responsible for leonurine biosynthesis. This design not only enabled us to validate gene function in a plant metabolic context but also provided a reusable and well-characterized part for other teams interested in inducible multi-gene expression in plant systems. Its performance was confirmed by GFP fluorescence, qRT-PCR, and LC-MS analysis, as described in section 3.

(2) New basic parts for future leonurine studies

To support further work on leonurine and related natural products, we registered and characterized two key enzymes, LjUGT5 and LjSCPL12, as new basic parts. See Parts (https://2025.igem.wiki/nais/parts) for more details. These enzymes catalyze the final two steps of the leonurine pathway, converting syringic acid into syringic acid glucoside and subsequently condensing it with 4-guanidinobutanol to form leonurine. By documenting their sequences, expression conditions, and catalytic properties in both plant and microbial systems, we provide the community with foundational resources that can be directly reused or adapted in future projects involving plant alkaloids or glycosylated natural products.

(3) Detailed experimental records as reusable protocols

Beyond genetic parts, we contributed a comprehensive experimental workflow for engineering in plant hairy roots and microbial systems. This includes:

​ ✓ Agrobacterium-mediated hairy root transformation protocols, GFP-based selection, and induction system design;

​ ✓ Step-by-step records of heterologous protein expression in E. coli, purification using Ni-NTA, and activity validation by in vitro catalysis.

By sharing these protocols openly on our wiki, we provide practical resources that can reduce trial-and-error for future teams, especially those with limited experience in plant synthetic biology. Detailed methods can be found in our Notebook（https://2025.igem.wiki/nais/notebook） and Protocols（https://2025.igem.wiki/nais/protocols）.

(4) Plant chassis characterization and optimization insights

We systematically characterized tobacco hairy roots as a synthetic biology chassis, documenting:

• Transformation efficiency and stability

• Inducible expression performance in plant tissues

• Metabolic capabilities for alkaloid production

• Limitations and optimization strategies specific to plant systems

This represents a valuable resource for teams considering plant chassis for their projects.

Together, these contributions demonstrate that our work not only advanced the frontier of leonurine biosynthesis but also created generalizable tools, parts, and protocols that are useful for the broader plant synthetic biology community.

Outlook

Our project pioneered the use of synthetic biology for leonurine biosynthesis by combining tobacco hairy roots for pathway validation with E. coli for scalable in vitro production. This dual approach not only confirmed the feasibility of reconstructing late-stage biosynthetic steps in a plant chassis but also revealed the challenges of low yield, limited metabolic flux, and substrate transport in hairy roots. Looking ahead, these insights provide clear opportunities for advancing plant synthetic biology. Future directions could include optimizing hairy root metabolism through transporter engineering, multi-gene pathway balancing, or exploring stable genome integration in model plants. By documenting our protocols and providing new parts, we offer reusable resources for other teams to build upon. Ultimately, our work demonstrates how plant systems can be both a platform for functional discovery and a target for further engineering, laying the groundwork for more efficient production of leonurine and other plant-derived therapeutics.

Reference

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7. Youjun Zhang, Alisdair R. Fernie,Metabolons, enzyme–enzyme assemblies that mediate substrate channeling, and their roles in plant metabolism,Plant Communications, Volume 2, Issue 1, 2021, 100081, ISSN 2590-3462, https://doi.org/10.1016/j.xplc.2020.100081.