Abstract

Our project addresses the urgent need for scalable production of leonurine, a bioactive alkaloid from Leonurus japonicus strong cardiovascular protective effects. Natural extraction is limited by extremely low content, and previous heterologous systems like yeast achieved only trace yields. We are the first team to establish a synthetic biology–based route to leonurine biosynthesis. To achieve this, we designed and constructed multiple new parts. These include two basic parts, LjUGT5 (BBa_25WVL694) and LjSCPL12 (BBa_257ZPI24), which catalyze the glycosylation of syringic acid and the subsequent condensation reaction with 4-guanidinobutanol, respectively, completing the terminal two steps of the pathway. On this basis, we developed composite parts, including a multi-gene construct GFP-LjUGT5-LjSCPL12-XVE (BBa_253R7TKL) for inducible expression in hairy roots, and expression cassettes SUMO-LjUGT5 (BBa_250OPNL1) and MBP-LjSCPL12 (BBa_25UTQ8JW) optimized for soluble protein production and purification in E. coli.

Functionally, these parts enabled us to reconstruct the pathway in tobacco hairy roots, yielding a 17-fold improvement over yeast-based systems, and further achieve in vitro cascade catalysis in E. coli with a 5.9-fold higher yield than hairy roots. Together, these parts provide a versatile toolkit that integrates plant validation and microbial production, demonstrating both innovation and practical potential. In the future, our constructs lay the foundation for supplying leonurine as an active pharmaceutical ingredient (API) to pharmaceutical companies, enabling low-cost cardiovascular protection and adjunctive therapy.

Table 1 Part list

No.	Name	Type	Description	Length
1	BBa_25WVL694	Basic	LjUGT5	1392bp
2	BBa_257ZPI24	Basic	LjSCPL12	1347bp
3	BBa_253R7TKL	Composite	GFP-LjUGT5-LjSCPL12-XVE	9223bp
4	BBa_250OPNL1	Composite	SUMO-LjUGT5	1852bp
5	BBa_25UTQ8JW	Composite	MBP-LjSCPL12	2746bp

Best New Composite Parts

GFP-LjUGT5-LjSCPL12-XVE Composite part

3.1 Description

This composite part integrates GFP, LjUGT5, LjSCPL12, and XVE expression cassettes into a single construct, enabling inducible leonurine biosynthesis in plant hairy roots while providing a fluorescent reporter for screening.

3.2 Source of the Part

The part is a Composite part.

3.3 Design Considerations

The construct was designed with three functional modules: Reporter module (GFP) driven by the constitutive promoter PAtUBI5 and terminated by Tmas, allowing easy visualization and screening of positive transformants. Biosynthesis module (LjUGT5 + LjSCPL12) under the control of inducible promoters PLEXA35S, ensuring temporal regulation of the two key enzymes responsible for the final steps of leonurine biosynthesis. LjUGT5 catalyzes glycosylation of syringic acid to SA-Glc, while LjSCPL12 condenses SA-Glc with 4-guanidinobutanol to form leonurine. Inducible regulator module (XVE) driven by the strong constitutive P35S promoter. Upon β-estradiol induction, XVE activates the PLEXA35S promoters, recruiting transcriptional machinery to drive expression of LjUGT5 and LjSCPL12. This design ensures precise temporal control, reduces metabolic burden during root growth, and allows induction of leonurine synthesis at specific time points. By combining reporter selection, enzyme expression, and inducible regulation into a single plasmid, the composite part establishes a robust and controllable plant chassis for studying and producing leonurine.

3.4 Result

1. Construct the composite part

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 1). 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 1. Schematic diagram of plant expression vector construction

2. 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 2.

Figure 2. 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 3).

Figure 3. 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 4). 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 4. 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. 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 5.

Figure 5. 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 6.

Figure 6. 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 7).

Figure 7. 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 8).

Figure 8. 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 9).

Figure 9. 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 10).

Figure 10. Colony PCR verification of the XVE gene in the Level 2 vector

4. 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 11. 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 11, 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 12). Genomic DNA was then extracted from these positive roots for further verification.

Figure 12. 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 13).

Figure 13. 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.)

5. 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 14, 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 14. 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.)

6. 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 15A). We then used a standard curve to correlate peak area with concentration, enabling quantitative analysis (Figure 15B). 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 15C). 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:

Øa specific yield of 3.687 μg/g DW,

Øa volumetric titer of approximately 7.37 μg/L, and

Øa 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 15. 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.

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. The low yield may be attributed to limited overall metabolic flux in hairy roots, inefficient substrate transport, or restricted enzyme activity in the plant environment. These findings suggest that hairy roots provide a reliable platform for functional validation.