Overview

Cardiovascular and cerebrovascular diseases represent the leading cause of death worldwide, creating a pressing need for novel and economically accessible therapeutic interventions. Leonurine, a key active compound derived from the traditional medicinal herb Leonurus japonicus, has long been used in Traditional Chinese Medicine for its ability to "promote blood circulation and resolve blood stasis." Recent studies have identified its significant therapeutic potential for cardiovascular and cerebrovascular diseases, and it has now entered phase II clinical trials. However, its extremely low natural abundance in the motherwort plant, coupled with inefficient extraction and high costs, severely limits its industrial application. Our project marks the first attempt in iGEM to achieve leonurine biosynthesis through synthetic biology.

To support future teams, we have re-characterized existing parts for practicality and convenience, designed and registered new basic and composite parts (enriching the Parts Registry), and documented reusable experimental protocols covering both hairy root and microbial systems. Additionally, we developed reusable educational tools and conducted policy research on drug registration pathways, providing the iGEM community with practical and systematic resources.

Looking forward, leonurine produced through these methods could serve as an Active Pharmaceutical Ingredient (API), offering a low-cost solution for the prevention and adjuvant treatment of cardiovascular and cerebrovascular diseases – fully demonstrating the potential of synthetic biology to address clinical and societal needs.

Below are our specific contributions:

Re-characterization of PSB Modules

We re-utilized the standardized plant expression toolkit BBa_K4399012, originally reported by iGEMer worldshaper-shanghai team 2022. In this system, we replaced the anthocyanin biosynthesis gene with the leonurine biosynthesis enzymes LjUGT5 and LjSCPL12. Using this framework, we successfully achieved efficient expression and stable production of leonurine in Nicotiana tabacum hairy roots.

This not only validated the robustness of BBa_K4399012 but also demonstrated its versatility for secondary metabolite biosynthesis in plants. We believe our work provides a clear example for future teams wishing to adapt this plant toolkit for different target pathways. See our detailed results in engineering success

Systematic Experience and Protocols

We documented and optimized detailed workflows for both plant and microbial systems, covering:

​● Plant hairy root system: Agrobacterium-mediated transformation, GFP-based selection, β-estradiol induction system, transcriptional validation (qRT-PCR), and metabolite detection (LC-MS).

​● Microbial system: Protein expression in E. coli BL21(DE3), IPTG induction, Ni-NTA purification, SDS-PAGE and Western blot validation, and in vitro enzymatic assays for leonurine synthesis.

These protocols are reproducible, detailed, and designed for direct reuse by other iGEM teams. By lowering the entry barrier to hairy root experiments and microbial enzyme assays, we contribute methodological resources that can accelerate future synthetic biology projects on natural products.For full experimental records, see our Notebook and Protocol page.

Reusable Education Framework

Beyond scientific research, we designed a set of reusable education tools that combine interactive learning with public engagement. These activities were successfully implemented during our project and are intentionally designed to be replicable by future iGEM teams:

​● Board Game: A strategy game themed around “Drug Synthesis and Application,” where participants learn the logic of synthetic biology through gameplay.

​● Memory Cards: A card-matching activity featuring drug molecules, metabolic pathways, and synthetic modules, lowering the learning barrier through gamified memorization.

​● Clay Cell Models: A hands-on activity designed for younger audiences to understand cell structures and functions by building them with clay.

​● Herbal Sachet Workshop: An educational workshop that integrates traditional cultural practices with modern synthetic biology concepts, offering a creative way to connect science and culture.

By documenting and standardizing these activities, we provide a reusable education framework that can be directly adapted by other iGEM teams for outreach, public engagement, or classroom teaching. These tools expand the possibilities for science communication by making synthetic biology approachable, interactive, and culturally relevant.

Drug Registration Process Summary

In advancing leonurine toward clinical and industrial application, clarifying the drug registration and compliance pathway is essential. We have reviewed the current regulatory frameworks of the National Medical Products Administration (NMPA) of China and synthesized the following process based on the context of synthetic biology drug development, serving as a reference for future iGEM teams:

(1) Preclinical Studies

Pharmacological Studies: Include mechanism of action and pharmacodynamic evaluation to validate leonurine’s pharmacological activities in cardiovascular and cerebrovascular protection.

Toxicological Studies: Cover acute toxicity, long-term toxicity, carcinogenicity, reproductive toxicity, etc.

Quality Studies: Involve purity analysis of the API (Active Pharmaceutical Ingredient), impurity profiling, and stability testing.

Upon completion, an Investigational New Drug Application (IND) must be submitted.

(2) Clinical Trials

Conducted in accordance with NMPA and international standards (ICH-GCP), clinical trials are divided into three phases:

Phase I: Small-scale (20–100 participants), focusing on safety and pharmacokinetics.

Phase II: Medium-scale (100–300 participants), exploring pharmacodynamics and recommended dosage, with preliminary efficacy validation.

Phase III: Large-scale (300–3000 participants), confirming efficacy and safety to support the New Drug Application.

All trials require step-by-step approval and oversight by an ethics committee.

(3) New Drug Application (NDA)

After completing clinical trials, the applicant must submit an NDA, including:

Comprehensive clinical data (efficacy and safety);

Full quality specifications (manufacturing process, impurity control, stability studies);

A drug risk management plan.

The NMPA conducts a technical review. If approved, a Drug Registration Certificate is issued.

(4) API Management and the DMF System

If leonurine is supplied as an Active Pharmaceutical Ingredient (API) to manufacturers, it must comply with the Drug Master File (DMF) filing system:

API producers submit complete documentation on processes and quality control to the NMPA;

Formulation manufacturers reference the DMF number in their NDAs;

The NMPA reviews both the API and the formulation for consistency and compliance.

This ensures standardization and traceability across the supply chain.

(5) International Registration & Future Applications

Given that leonurine has entered global Phase II clinical trials (e.g., for cardiovascular indications), future international marketing will require compliance with regulations from agencies such as the FDA (U.S.) and EMA (Europe). China’s IND/NDA systems align closely with ICH guidelines, making the current research and registration pathway a preparatory step for global clinical translation.

By summarizing this process, we provide not only a compliance roadmap for leonurine’s industrialization but also a general policy reference for drug-related projects within the iGEM community. Future teams developing synthetic products at the API level can directly refer to this framework for planning regulatory pathways.

New Basic Parts

We registered two new basic parts, LjUGT5 (BBa_25WVL694) and LjSCPL12 (BBa_257ZPI24), the key enzymes catalyzing the final steps of leonurine biosynthesis. Both genes were expressed in E. coli BL21(DE3), purified through Ni-NTA affinity chromatography, and functionally validated via in vitro enzymatic assays. LC-MS confirmed their ability to catalyze the formation of leonurine from syringic acid and 4-guanidinobutanol. By providing fully validated coding sequences, our contribution enables future iGEM teams to bypass the uncertainty of de novo gene mining and directly reuse these sequences in plant, microbial, or in vitro systems. These parts are especially valuable as no previous iGEM team has worked with leonurine biosynthesis, making our registration the first available standardized genetic resources for this pathway. See Parts page for more details.

New Composite Part

We designed and constructed a novel composite part that integrates GFP, LjUGT5, LjSCPL12, and XVE into one functional unit. This design was not only a technical integration but also an innovation in pathway reconstruction: GFP provided a visual screening marker for positive hairy root transformants, XVE allowed inducible regulation under β-estradiol, and LjUGT5 with LjSCPL12 completed the final biosynthetic steps of leonurine. By combining these modules, we achieved controllable and verifiable expression in tobacco hairy roots. Experimental results confirmed the stable biosynthesis of leonurine, validated by LC-MS metabolite detection. This composite part demonstrates reusability for future iGEM teams working on plant secondary metabolites, as it provides a template for integrating screening, regulation, and biosynthesis into a single construct. See Engineering Success for detailed results.