Abstract

Cardiovascular and cerebrovascular diseases continue to be a major global health challenge due to their high rates of incidence, disability, and death. Leonurine, the main active compound in Leonurus japonicus, has shown promising pharmacological effects, including protecting the heart, reducing oxidative stress, and repairing blood vessels.

However, its clinical use has been severely limited by its very low natural abundance and the inefficiency of traditional chemical synthesis. To date, no production of leonurine using synthetic biology has been reported, with the recent 2024 multi-omics study being the only research to suggest its potential biosynthetic pathway.

Building on these findings, our project develops the first industry-focused synthetic biology approach for producing leonurine. We reconstructed the final biosynthetic steps (UGT and SCPL) in both tobacco hairy root and E. coli BL21 (DE3) systems, while adding inexpensive substrates (syringic acid and 4-guanidinobutanol) to improve yield and reduce costs. By comparing and refining these two platforms, we aim to create a scalable and sustainable method for producing leonurine, which could meet urgent clinical needs in cardiovascular health and help modernize traditional Chinese medicine through synthetic biology.

Inspiration

From the very beginning, since our team was formed at the end of 2024, the development of novel therapeutics for cardiovascular and cerebrovascular diseases has remained at the forefront of our minds. Many of our team members had witnessed elderly family members regularly taking multiple medications or undergoing rehabilitation due to cardiovascular conditions. These experiences made us acutely aware of the growing burden of cardiovascular disease and the limitations of current treatments, including high costs, side effects, and the difficulty of long-term adherence.

In our search for potential alternatives, we noticed that natural herbal products have long been valued in healthcare, but their clinical use is often restricted by inconsistent yields, fluctuating quality, and high prices. This gap between traditional remedies and modern therapeutic needs drew our attention to the possibility of exploring active compounds from traditional Chinese medicine using synthetic biology.

A key turning point came in April 2025, when our school organized a study tour to Chenshan Botanical Garden—an institution that had also served as a guiding partner for our school’s NAIS team the previous year. During the visit, researchers in charge of germplasm conservation and medicinal plant development introduced us to the remarkable biodiversity of the garden and to many traditional medicinal herbs. It was here that we first encountered Leonurus japonicus, known in Chinese as yi mu cao. For over two thousand years, this herb has been widely used in gynecology for its well-recognized efficacy in promoting blood circulation and removing blood stasis.

Learning about this history and pharmacological property immediately sparked our attention toward its major bioactive component—leonurine. Inspired by its therapeutic potential and the possibility of bridging traditional medicine with modern biotechnology, we decided to explore the biosynthesis of leonurine and evaluate its potential as a new therapeutic strategy for cardiovascular and cerebrovascular diseases.

Background

Cardiovascular diseases (CVDs) are a group of disorders characterized primarily by pathological changes in the heart and blood vessels, including coronary heart disease, heart failure, hypertension, arrhythmias, and stroke [1]. They share common features of insidious onset and slow progression; however, once they enter the acute stage, they often become life-threatening, showing epidemiological characteristics of “high prevalence, high mortality, and high disability rates” [2].

According to the World Health Organization (WHO, 2025), CVDs have become the leading cause of death worldwide, responsible for approximately 19.8 million deaths in 2022, accounting for 32% of total global mortality [3]. The situation in China is equally alarming. The China Cardiovascular Health and Disease Report 2025 indicates that from 1990 to 2019, the number of CVD cases in the Chinese population increased from 5.30 million to 12.34 million, a rise of 132.82%; meanwhile, deaths rose from 2.42 million to 4.58 million, an increase of 89.12% [4]. Deaths related to cardiovascular disease now account for over 47% of total mortality in China, far exceeding cancer and other chronic diseases [4].

Figure 1. Comparison of Cardiovascular Disease Profiles in Chinese Residents: 1990 vs. 2019 (From Chinese Circulation Journal, June 2025, Vol. 40 No.6(Serial No.324)

Figure 2. Proportion of major causes of death among rural and urban residents in China, 2021 (Source: Chinese Circulation Journal, June 2025, Vol. 40, No. 6, Serial No. 324).

The development of cardiovascular diseases (CVDs) is often closely linked to pathological processes such as atherosclerosis, vascular endothelial dysfunction, and thrombosis. Common risk factors include hypertension, hyperlipidemia, diabetes, smoking, obesity, lack of physical activity, and unhealthy eating habits [5]. Notably, these conditions often progress without symptoms in their early stages. Many individuals accumulate risk without noticeable discomfort, only to be diagnosed after a severe event such as an acute myocardial infarction or stroke [5]. This hidden progression complicates prevention and control efforts and contributes to the growing burden of CVDs.

At the individual level, CVDs significantly reduce patients’ quality of life. For example, stroke is a leading cause of long-term disability in adults; about 75% of survivors experience some degree of functional impairment, such as hemiplegia, speech difficulties, or cognitive decline [6]. Patients with heart failure, on the other hand, often require lifelong medication and rehabilitation, with severely limited ability to perform daily activities.

At the societal level, CVDs place a considerable economic strain on healthcare systems. According to HQMS data, the total hospitalization costs for cases primarily diagnosed with CVDs reached 283.43 billion RMB in 2023. This cost is projected to increase further with the aging population and ongoing exposure to risk factors [4].

In summary, the high prevalence, mortality, and disability rates associated with CVDs—along with their substantial impact on individual well-being and socioeconomic development—highlight their role as a critical public health challenge both in China and worldwide. This reality underscores the urgent need for innovative strategies in prevention and treatment and reinforces the relevance and importance of our research project.

Current solution

Currently, the treatment of cardiovascular and cerebrovascular diseases (CVDs) mainly includes pharmacological therapy, interventional and surgical procedures, rehabilitation interventions, and complementary approaches such as traditional Chinese medicine (TCM).

Pharmacological therapy is the most common strategy. Antiplatelet drugs (e.g., aspirin, clopidogrel) are effective in preventing thrombosis; statins are widely prescribed for lipid-lowering; while ACE inhibitors (ACEIs), angiotensin receptor blockers (ARBs), and β-blockers are commonly used to control blood pressure and reduce cardiac load [7,8]. However, through our human practice discussions with Dr. Huanchun Ni, we learned that long-term use of these drugs often entails challenges such as increased bleeding risk, hepatic and renal impairment, and poor patient compliance.

Interventional and surgical procedures play a critical role in patients with coronary artery disease or severe atherosclerosis. Percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG) can significantly improve blood supply and patient outcomes [9]. Nevertheless, these procedures are costly, invasive, and carry risks of postoperative complications and restenosis.

Lifestyle modification and rehabilitation interventions—including dietary control, regular exercise, smoking cessation, and alcohol restriction—are regarded as the cornerstone of secondary prevention in clinical guidelines [3]. Yet in practice, their effectiveness is often limited due to the difficulty of adherence over the long term.

Traditional Chinese medicine (TCM) and adjuvant therapies are also widely applied. For example, active compounds such as tanshinones and leonurine have been reported to improve hemodynamics and inhibit platelet aggregation in certain studies [10]. However, due to the complexity of herbal constituents and the lack of standardized efficacy, they are difficult to directly translate into modern drug development.

Overall, while existing treatments have significantly reduced mortality and recurrence rates of CVDs, they remain limited by prominent side effects, high costs, heterogeneous efficacy, and insufficient compliance. Against this backdrop, our project aims to address these limitations by exploring a synthetic biology-based production route for leonurine. Unlike conventional extraction from plants or direct chemical synthesis, our approach reconstructs key biosynthetic steps in both plant and microbial systems, thereby offering the potential for a safer, more controllable, and cost-effective therapeutic option that could meet future industrial needs.

Our solution

Part I. The Value of Traditional Chinese Medicine and Natural Active Compounds

In the framework of traditional Chinese medicine (TCM), herbal remedies have long played a vital role in the prevention and treatment of cardiovascular and cerebrovascular diseases, with typical representatives including Salvia miltiorrhiza (Danshen), Panax notoginseng (Sanqi), and Leonurus japonicus (Motherwort) [10, 11]. During our human practice, Professor Yizhun Zhu highlighted that leonurine—the representative active compound from Leonurus japonicus—has recently gained increasing pharmacological attention for its mechanisms in cardiovascular protection (Figure 3).

Extensive experimental evidence has demonstrated that leonurine exerts significant antioxidant, anti-apoptotic, cardioprotective, and vascular endothelium–repairing effects [12, 13] (Figure 4). Preclinical animal studies have further shown that leonurine can reduce myocardial infarct size and improve neurological function, suggesting promising potential for its application in cardiovascular and cerebrovascular diseases [13].

Leonurine has already completed a Phase I clinical trial (safety evaluation) without reports of severe adverse events (data not fully disclosed) (https://www.chictr.org.cn/index.html, Registration number: ChiCTR1800019071). A Phase II clinical trial was launched in 2023.

Currently, leonurine has progressed beyond preclinical evaluation and entered registered clinical trials, making it one of the relatively few natural compounds derived from TCM to reach this stage, and thus demonstrating its translational potential from bench to bedside.

Figure 3 The photograph of Leonurus japonicus Houtt. and the structure of leonurine [12]

Figure 4 Protective effect of LEO (leonurine) on cardio-cerebrovascular system [12, 13]

Part II Current Challenges: Source and Industrialization Issues

Despite its promising pharmacological activities, the natural availability of leonurine is extremely limited. Its content in Leonurus japonicus is only about 0.02%–0.12% of dry weight, resulting in inefficient extraction and high production costs [13]. As revealed through human practice, the yield and quality of herbal materials are highly susceptible to factors such as seasonality, geographic origin, and processing methods, making it difficult to achieve the standardization and batch-to-batch consistency required for drug development. Meanwhile, the existing chemical synthesis routes are complex, involve multiple steps, suffer from low yields, and generate significant environmental pollution, which further hinders large-scale industrial application.

Therefore, it is urgent to establish a novel, sustainable, and controllable approach for the large-scale production of leonurine. Synthetic biology has emerged as the most promising solution in this context.

Part III Biosynthetic Pathway of Leonurine

Recent metabolomic and transcriptomic studies have progressively revealed the biosynthetic pathway of leonurine in Leonurus japonicus. Key intermediates, including syringic acid (SA), arginine, and its derivatives (agmatine, 4-guanidinobutanal, 4-guanidinobutanol), are present at high levels in L. japonicus, whereas their amounts in L. sibiricusare markedly lower, with 4-guanidinobutanol and leonurine nearly absent [14-16](Figure 5).

Association analyses between metabolome and transcriptome data have identified candidate genes encoding enzymes involved in this pathway, including those responsible for syringic acid biosynthesis (F5H), arginine metabolism (NAGK, NAOD), and the conversion of arginine derivatives to 4-guanidinobutanol (ADC, AO, GLYR) (Figure 5). Importantly, UGTs (UDP-glycosyltransferases) and SCPL/BAHD acyltransferases play key roles in the final condensation step [17].

The proposed pathway proceeds as follows: SA is first activated by UGTs to produce SA-Glc, while arginine undergoes multiple reactions to generate 4-guanidinobutanol. In the final step, SA-Glc and 4-guanidinobutanol are condensed by SCPL enzymes to form leonurine [17] (Figure 5). This comprehensive pathway elucidation not only provides the molecular basis for the pharmacological effects of leonurine, but also serves as a theoretical foundation for heterologous synthesis in plant or microbial systems.

Figure 5 Construction of the leonurine biosynthesis pathway through integrated multiomics analyses [17]

Part IV: Our Solution Design

To achieve the controllable synthesis of leonurine, our technical design focuses on the final two steps of its biosynthetic pathway, which are jointly catalyzed by UGT (UDP-glycosyltransferase) and SCPL (Serine Carboxypeptidase-like acyltransferase) to produce leonurine. Within the system, we exogenously supply two key substrates—syringic acid (SA) and 4-guanidinobutanol—to bypass upstream complex metabolic steps (Figure 6). This strategy not only significantly reduces the metabolic burden on the chassis cells but also offers cost advantages for industrial-scale production, as both substrates are inexpensive (syringic acid costs approximately ¥300/kg, and 4-guanidinobutanol costs about ¥500/kg).

Figure 6 Construction of the Leonurine Biosynthetic Pathway

In terms of vector design, we adopted tailored strategies for different chassis systems:

​ ● For the plant system, we used the pK7WG2R plant expression vector, constructed via the Gateway® system.

​ ● For the prokaryotic system, the UGT gene was cloned into pRSF-Duet-M6, and the SCPL12 gene was cloned into pETDuet-MBP, both assembled using double digestion and homologous recombination.This design ensures highly efficient and stable expression of the target enzymes in both types of hosts.

Regarding chassis selection, we followed a “validate first, then iterate” logic. We first selected the tobacco (Nicotiana tabacum) hairy root system as the plant chassis. This platform is well-established for plant transformation and provides a relevant metabolic environment, making it suitable not only for enzyme validation but also for testing whether a plant chassis could support meaningful production. Indeed, our hairy root system achieved a 17-fold higher yield compared with yeast, highlighting the potential of plants as a chassis for secondary metabolite biosynthesis. However, the absolute yield was still insufficient for industrial standards (≥60 mg/200 mL), reflecting plant-specific bottlenecks such as limited metabolic flux, substrate transport barriers, and enzyme context dependency [18, 19, 20].

To complement this, and informed by expert feedback, we further transferred the pathway into Escherichia coli BL21 (DE3). This was not an abandonment of plant systems but a strategic parallel approach to test scalability. Compared with plants, BL21 (DE3) offers reduced metabolic burden, lower cultivation costs, and mature fermentation processes, making it more suitable for industrial-scale enzyme production and in vitro catalysis.

Taken together, our dual-chassis strategy highlights both the strengths and limitations of plant systems, while also providing an alternative microbial pathway for industrial application (Figure 7). This not only ensures reliable scientific validation but also expands the toolbox of plant synthetic biology by clarifying when plant systems are advantageous and when hybrid strategies are needed.

Figure 7 Experimental Design Diagram

Overall, our solution achieves a “threefold balance” in route design:

​ ● By concentrating on the last two steps of the pathway, we ensure simplicity and efficiency in system construction.

​ ● By supplementing with inexpensive exogenous substrates, we maintain a cost advantage.

​ ● By leveraging a dual-chassis system—tobacco hairy roots for functional validation and E. coli for scalable enzyme production—we balance pathway feasibility with industrial potential.

This strategy not only provides a solid technical foundation for the controllable synthesis of leonurine but also demonstrates strong potential for industrial application.

Summary

In conclusion, cardiovascular and cerebrovascular diseases pose a critical global health challenge due to their high incidence, disability, and mortality rates. Although current therapeutic approaches continue to evolve, they are often limited by side effects, long-term costs, and insufficient efficacy. Leonurine, a guanidine alkaloid from Leonurus japonicus, has long been used in traditional Chinese medicine for “blood-activating” functions and has recently shown promising preclinical effects in cardiovascular protection. It is currently under Phase II clinical evaluation in China. However, its clinical translation has long been hindered by its extremely low natural abundance, inefficient extraction, and the challenges of achieving sustainable chemical synthesis.

To overcome these barriers, we designed a synthetic biology–based strategy that reconstructs the final steps of leonurine biosynthesis using UGT and SCPL enzymes, coupled with the supplementation of low-cost substrates. By implementing this pathway in both tobacco hairy root and E. coli BL21 (DE3) systems, we established a dual-platform approach balancing scientific validation and industrial scalability. This design lays the foundation for future routes toward stable, cost-effective production of high-purity leonurine, while also reflecting our commitment to aligning synthetic biology with pressing clinical and societal needs, highlighting its potential to advance cardiovascular health in the future.

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