Wet Lab

Converting Low-Value Waste Oils into High-Value Products

In this project, we designed the key enzymes of the mevalonate (MVA) pathway—Acetyl-CoA thiolase (Erg10), HMG-CoA synthase (Erg13), and HMG-CoA reductase (HMG1)—to be targeted to the peroxisomes of Saccharomyces cerevisiae. This allows acetyl-CoA generated from fatty acid β-oxidation to be immediately utilized in the biosynthesis of ginsenoside Ro.

Unlike conventional approaches that rely on glucose fermentation or extraction from ginseng plants, we are the first to propose using glycerol and free fatty acids from waste oil byproducts as carbon sources. Through engineered yeast, these low-value substrates can be efficiently converted into the rare ginsenoside Ro.

This strategy not only reduces substrate costs but also provides a high-value use for crude glycerol and free fatty acids, avoiding environmental pollution caused by their direct disposal. By transforming industrial waste streams into bioactive natural products, our work demonstrates a novel circular utilization of waste oils and provides a practical example of green pharmaceutical production and the circular bioeconomy.

Subcellular Compartmentalization Strategy in Metabolic Engineering

We strategically exploit peroxisomes as subcellular compartments to directly link fatty acid β-oxidation with the ginsenoside Ro biosynthetic pathway. In conventional yeast metabolism, acetyl-CoA generated from fatty acids is largely confined within peroxisomes and cannot efficiently enter the cytosolic MVA pathway, resulting in low carbon utilization efficiency.

To overcome this limitation, we targeted key MVA pathway enzymes—including Erg10, Erg13, and HMG1—to the peroxisomes by fusing them with PTS1 signals. This allows acetyl-CoA derived from fatty acids to be directly channeled into Ro biosynthesis, avoiding loss due to membrane compartmentalization.

This design not only reduces competition with endogenous sterol synthesis but also fully leverages fatty acids as a low-cost carbon source. By creating a “microfactory”-style metabolic reconstruction, we significantly enhance the supply of precursors for Ro production and provide a transferable engineering strategy for the industrial-scale biosynthesis of other complex triterpenoids.

Efficient Yeast-Based Production of Rare Ginsenoside Ro

Unlike traditional methods that rely on years of cultivation and low-yield extraction, our project decomposes ginsenoside Ro biosynthesis into four independent modules. The relevant genes for each module are introduced into Saccharomyces cerevisiae, creating engineered yeast capable of rapidly producing the rare ginsenoside Ro.

This approach addresses the scarcity of Ro and the imbalance between supply and market demand, while avoiding the high costs and environmental pollution associated with organic solvents. By integrating heterologous genes, optimizing UDP-glycosyltransferases, and expanding sugar donor pools, we established a scalable synthetic system for the sustainable production of this rare medicinal compound.

For more information on the design of the yeast chassis for ginsenoside Ro production, please see Design

Part

JLU-NBBMS 2025 has designed and characterized a large collection of basic and composite parts, which have been uploaded to Parts.

These components cover the complete pathway from precursor generation to final product modification. Promoters (e.g., ADH1, TEF1) and terminators regulate gene expression, while (GGGGS)₂ linkers and Myc tags enable protein fusion and detection. PTS1 signal peptides target key enzymes to peroxisomes, allowing efficient utilization of acetyl-CoA derived from fatty acids. Core enzymes of the mevalonate (MVA) pathway, including ERG10, ERG13, and HMG1, ensure sufficient supply of triterpene precursors. β-amyrin synthase catalyzes the formation of the triterpene backbone from 2,3-oxidosqualene, and cytochrome P450 monooxygenases with their reductases perform further oxidative modifications. Finally, glycosyltransferases such as UGT73P40 attach specific sugar moieties to the triterpene backbone, producing the bioactive rare ginsenoside Ro.

Model

Portable end-to-end design pipeline

Discovery → Pathway → Control → Protein Engineering → Validation, all in one flow:

Discovery: Start from reference sequences → generate BLAST/HMM candidates → motif/family identification → multiple sequence alignment & phylogeny → structure prediction → ligand docking prescreen, to build a cross-species candidate enzyme library.

Pathway: Use ODEs + Michaelis-Menten to model the CE→IVa/R1→Ro main/branch network; collect/estimate (kcat, Km); apply a genetic algorithm (GA) to search the large enzyme space for optimal combinations, avoiding exhaustive enumeration.

Control: Compute flux control coefficients with MCA to answer, quantitatively, “which enzyme should we engineer first?” instead of deciding by intuition.

Protein Engineering: Score all single-site substitutions with dESM (differential embeddings from protein language models) + structural neighborhood graphs; triage the Top-N by docking; run GROMACS MD (CHARMM36/TIP3P) and MM/PBSA on representative variants to provide mechanistic evidence.

Validation: Align three classes of readouts—flux, structural stability, and binding energy—to form a “model-as-evidence” loop.

A general rule for deciding “what to optimize first” at the pathway level

In the ginsenoside Ro case, MCA + sensitivity analysis point to UGT73F3 as the primary bottleneck. A GA+ODE search over a large candidate space yields an optimal combination (e.g., E1 = E4 = UGT73P40; E2 = E3 = UGT73F3). From this we propose a generalizable order of operations: increase the rate-limiting step's kcat first, then refine Km; prioritize resolving branch competition.

An interpretable AI-assisted protein-engineering paradigm

We scored 9,443 single-site mutants across the full UGT73F3 sequence and identified high-potential variants such as S175P. In 100-ns MD runs, these variants show more stable RMSD/RMSF, an optimized hydrogen-bond network, and more favorable binding free energy, consistent with docking results and the observed flux gains—shifting from mere recommendation to evidence.

Portability and reuse—what other teams can adopt

he six-step enzyme discovery procedure can be directly reused to seed starting libraries for other triterpenoid/steroidal glycosylation steps.

GA+ODE is well-suited for scenarios with multiple sites, multiple isoenzymes, and strong branch competition when selecting enzyme combinations.

MCA helps decide what to optimize first before committing experimental resources.

The combined dESM + graph-neural-network ML + docking + MD workflow is ideal when the active site is well characterized and task performance hinges on substrate specificity and stability.

Human Practices

Decentralized Stakeholder Planetary Network

We proposed the concept of a “Decentralized Stakeholder Planetary Network” as the overarching framework for our Human Practices. Inspired by planetary metaphysics and Confucian philosophy, this model rejects a single dominant perspective and instead emphasizes equal dialogue and collaboration among six “planets”: government, enterprises, consumers, farmers, scholars, and iGEMers. Through this approach, we were able to capture the genuine needs and contributions of diverse groups within the complex socio-technical ecosystem of the ginseng industry. This framework not only guided our own practices but also offers a replicable methodology for future iGEM teams to construct stakeholder networks and foster more balanced, inclusive interactions between synthetic biology and society.

Applying the Double Diamond Model in Human Practices

In the iGEMers' planet, we adopted the Double Diamond model developed by the British Design Council to structure and analyze our Human Practices activities. The model's dual cycles of divergence and convergence—Discover, Define, Develop, Deliver—helped us systematically show how stakeholders contributed to project development, from the exploration of possibilities (Could be) to the refinement of practical solutions (Should be). By applying this model, we enhanced the coherence and transparency of our activities, while also providing future iGEM teams with a transferable tool to organize stakeholder engagement, integrate feedback, and optimize project direction.

An Illustrated White Paper on Chassis Organisms

Our team produced An Illustrated White Paper on Chassis Organisms. Based on literature review and expert interviews, it systematically summarizes the characteristics, safety aspects, application potential, and ethical and regulatory considerations of commonly used chassis organisms, while also including case studies from previous iGEM teams to showcase their practical value in diverse projects. By using illustrations and infographics, we transformed complex concepts into accessible content for the public and young students. This resource not only provides future iGEM teams with ready-to-use references for project design, risk assessment, and application exploration, but also serves as a versatile tool for outreach and education, ensuring strong reusability and impact.

Education

Educational Philosophy and the ROOT Model

We proposed an educational philosophy centered on “teaching according to aptitude and timing”, using the growth stages of ginseng as a metaphor to design a progressive, layered framework for public engagement. In addition, we developed the ROOT model (Raise-Organize-Operate-Test), which provides a systematic and cyclical structure for planning and evaluating educational activities. This philosophy and model not only guided our own practice but also serve as a reusable reference for future iGEM teams aiming to design multi-level and sustainable education programs.

Fertile Ground Initiative: Growth Log for Team Members

We created the “Fertile Ground Initiative”, encouraging members to keep a Growth Log throughout the project to promote continuous reflection, self-correction, and improvement. This practice strengthened both personal understanding of synthetic biology and commitment to education, while forming a replicable model of self-education within teams. Future iGEM groups can adopt similar log-based reflection mechanisms to enhance knowledge retention, foster team growth, and ensure iterative improvement in their projects.

Entrepreneurship

Industrial Value Contributions

We analyzed the current state and challenges of the ginseng industry, highlighting the limitations of low Ro yield, high production costs, and traditional extraction methods. We proposed a framework for integrating synthetic biology to complement conventional industry practices. Utilizing value proposition canvases, stakeholder matrices, and market size analyses, we demonstrated a viable industrialization pathway for synthetic biology products. This work not only aids in translating scientific achievements into commercial value but also offers a reference framework for downstream applications in health products, cosmetics, and related fields.

Contributions to Circular Resource Utilization

We introduced an innovative approach to repurpose glycerol and fatty acids from waste oils as low-cost carbon sources for the synthesis of high-value ginsenoside Ro, establishing a sustainable "waste-to-resource" pipeline. This solution reduces reliance on land-intensive and long-cycle cultivation while minimizing environmental risks associated with organic solvent usage, thereby balancing economic and ecological benefits. We hope this exploration inspires further integration of synthetic biology with circular resource strategies to advance sustainable development goals.