Problems We Face

Natural products are fundamental resources for the development of biopharmaceuticals, novel agrochemicals, and other high-value chemicals. However, isolating natural products directly from their original producers (e.g., bacteria, fungi, plants) remains challenging. In recent years, cell-free synthetic biology has been increasingly applied to the synthesis of natural products^[1].

ATP is the most important energy currency in living organisms and it is the essential substrate for many industrial-scale biosynthetic reactions. However, in industrial-scale cell-free biosynthesis, the direct use of expensive ATP as a substrate greatly increases production costs, which is a core bottleneck limiting the large-scale application of this technology^[2].

Researchers have attempted various ATP regeneration systems, including acetate kinase-acetyl phosphate system^[3], pyruvate kinase-phosphoenolpyruvate system^[4], and creatine kinase-phosphocreatine system^[5] . However, these systems still rely on costly or unstable substrates, limiting their industrial scalability.

In contrast, Polyphosphate Kinases (PPKs), particularly the PPK2 family, use inexpensive, stable, and readily available Polyphosphate (PolyP) as the substrate for ATP regeneration. PPK2-mediated ATP regeneration systems can be directly coupled with product-forming enzymes^[6,7], offering unrivaled cost advantages and application potential.

Literature surveys revealed that the application of PPK2 enzymes under industrial conditions continues to face two persistent bottlenecks:

1. Phosphate inhibition: During ATP consumption, large amounts of inorganic Phosphate (Pi) are released. Beyond altering system pH, phosphate itself inhibits PPK2 activity. Studies have shown that polyphosphate resulted in activity loss at concentrations higher than 5 mM, with the enzyme reaching only 50% of maximal activity at 50 mM polyphosphate^[8]. This significantly reduces ATP regeneration efficiency.

2. Poor environmental tolerance: Multiple PPK2 family enzymes are highly sensitive to temperature, pH, and proteases^[9]. Previous attempts using site-directed mutagenesis or directed evolution to improve kinase activity have yielded only limited improvements in overall stability^[10].

To further investigate the current status and bottlenecks of industrial-scale cell-free synthesis, we interviewed Dr. Yong Tao, Chief Scientist at MicroSyn Biotech and a leading figure in enzyme engineering and synthetic biology. Dr. Tao highlighted that many companies, including MicroSyn, are using PPK2 systems, but phosphate inhibition significantly reduces yield. There is an urgent industrial need for cost-effective, pH-tolerant, phosphate-resistant, and highly efficient enzyme systems. Solving the stability problem of PPK2 would make it the most suitable ATP regeneration system for industrial application.

This interview provided us with key practical insights and strengthened our determination to overcome technical barriers and advance the industrial application of cell-free biosynthesis.

Solutions We Propose

Based on these considerations, we focused on a specific PPK2 this year——SlPPK, isolated from Sulfurovum lithotrophicum. Compared with other PPK2s, SlPPK exhibits higher K_cat , V_max and K_m , enabling highly efficient ATP regeneration. However, studies have shown that at 60 ℃, SlPPK has a half-life of only ~3 h^[11], limiting its long-term stability at elevated temperatures. Therefore, improving the thermostability and environmental tolerance of SlPPK is crucial.

To this end, we integrated Virus-Like Particle (VLP) encapsulation technology to develop a novel ATP regeneration platform——V-CHARGEs (VLP-Coupled High-efficiency ATP Re-Generating Enzyme system).

Instead of conventional enzyme engineering strategies, we employed a novel approach to enhance SlPPK stability and performance. The P22-VLP (Virus-Like Particle derived from P22 bacteriophage) we selected consists of Coat Protein (CP) and Scaffold Protein (SP). CP can self-assemble into a protein nanocage, while SP is fixed to the inner surface of the cage through non-covalent interactions. We wrapped SlPPK in P22-VLP and use the cage-like structure of the VLP shell to protect it. By anchoring ATP-consuming enzymes to the P22-VLP exterior, we created an artificial multi-enzyme complex that boosts production efficiency—essentially a nanoscale 'charging station' to power reactions.

Our system consists of three components:

SlPPK-SP fusion protein: SlPPK fused to a P22-VLP Scaffold Protein (SP) to anchor it inside P22-VLP, where encapsulation enhances enzyme activity and stability.

CP-SpyTag fusion protein: The P22-VLP Coat Protein (CP) co-assembles with SP to form a nanocage, displaying SpyTag on the outer surface.

SpyCatcher-Enzyme fusion protein: Target biosynthetic enzymes are fixed to the P22-VLP exterior via high-affinity SpyTag/SpyCatcher interaction.

In our design, ADP and PolyP diffuse freely through the ~2 nm pores of the P22-VLP^[12]. Inside, SlPPK catalyzes ATP regeneration, and newly synthesized ATP diffuses outward to be immediately consumed by the immobilized enzymes. This continuous ATP ↔ ADP cycle ensures a sustained energy supply, with only inexpensive PolyP supplementation required.

Validation of Effectiveness

We verified the stability and effectiveness of V-CHARGEs through a series of experiments, providing experimental data to support their potential industrial applications. For more details, please visit our Results page.

1. Stability Validation

We evaluated the system under conditions of temperature, pH, phosphate, and protease stress. Results showed that V-CHARGEs significantly enhanced SlPPK tolerance toward pH, phosphate, and proteases.

2. Product Validation

We designed three ATP-consuming systems to demonstrate the industrial applicability of V-CHARGEs. Using SpyCatcher linkages, we attached two enzymes, Firefly Luciferase (FLuc) and Uridine-Cytidine Kinase (UCK), as well as a dual-enzyme system comprising γ-Glutamylcysteine Synthetase (γ-GCS) and Glutathione Synthetase (GS).

FLuc is capable of bioluminescence in the presence of ATP. We anchored FLuc on the outer surface of V-CHARGEs to verify whether the system can operate normally.

UCK catalyzes the conversion of Cytidine (CR) to Cytidine 5'-Monophosphate (5'-CMP). 5'-CMP serves as an important precursor for nucleotide derivatives and is widely applied in food, agriculture, and pharmaceuticals. For example, infant formula supplemented with 5'-CMP more closely resembles breast milk and significantly enhances infant immunity^[13].

γ-GCS and GS catalyze glutathione (GSH) biosynthesis. Glutathione exhibits strong reducing capacity and serves as a key antioxidant. Studies have shown that GSH plays critical roles in combating pulmonary infections, suppressing chronic disease progression, and slowing aging^[14,15].

Experimental results confirmed successful bioluminescence and synthesis of both 5'-CMP and GSH, demonstrating the practical value and future potential of our system. These results also verified that V-CHARGEs can power diverse reactions, highlighting its platform characteristics.

Highlights

We are revolutionizing the industrialization pathway of cell-free biosynthesis.

1. Enhancing Enzyme Stability

1. Simultaneously Enhancing Enzyme Stability and Industrial Applicability

The PPK2 family regenerates ATP using inexpensive, stable polyphosphate, providing distinct cost and application advantages. SlPPK is a preferred enzyme with strong catalytic activity and high substrate affinity. V-CHARGEs can self-assemble in Escherichia coli, offering simplicity, high product uniformity, low cost, and industrial adaptability.

2. Pioneering a New Approach

2. Pioneering a Brand-New Approach to Enzyme Engineering Technology

V-CHARGEs break free from the limitations of site-directed mutagenesis and directed evolution by shifting the focus from optimizing single enzyme molecules to constructing protective microenvironments. This addresses key industrial challenges such as phosphate inhibition and poor environmental tolerance, offering a transferable design framework for other industrial enzymes.

3. Establishing a Platform

3. Establishing a Universal Energy Supply Platform for Cell-Free Biosynthesis

V-CHARGEs can link with different ATP-consuming enzymes on the surface to power diverse reactions, enabling synthesis of a wide range of products. With a flexible linker design platform, different enzymes can be conveniently and precisely coupled. Furthermore, multiple enzymes can be co-anchored to form multi-enzyme complexes, leveraging proximity effects to boost industrial efficiency and highlighting the platform's universality and effectiveness.

Dry Lab

Dry Lab: Construction from Micro to Macro Scale

The successful development and optimization of wet lab experiments fundamentally rely on robust models. Our modeling work focuses on a comprehensive, multi-scale construction of the complex V-CHARGEs system, spanning from the microscopic to the macroscopic level. The goal of this work is to enable predictive design of the system, precise parameter simulation, and forecasting of industrial application benefits. This effort aims to maximize the optimization of costly and laborious wet lab experiments, thereby accelerating the translation of V-CHARGES from concept to application. For more details about our dry lab, please visit our Dry Lab page.

1. Construction of Molecular Containers

1. Construction of Molecular Containers

The entire framework commences at the most minute, atomic level. We successfully constructed high-resolution three-dimensional structural models of the P22-VLP Coat Protein and the key catalytic enzyme SlPPK using tools such as SWISS-MODEL and AlphaFold. Through structural analysis, we identified the key electrostatic interfaces, such as hydrogen bonds and salt bridges, required to form the stable T=7 icosahedral capsid. Furthermore, using Z-DOCK molecular docking, we predicted various binding modes between SlPPK and P22-VLP, confirming that the enzyme's critical catalytic domain remains exposed within the complex, thus ensuring the retention of enzyme activity. Additionally, to design the function and stability of the linker protein responsible for integrating the external enzyme onto the P22-VLP, we developed the Linker Designer Tool. This tool performs a multi-level computational assessment of the physicochemical properties of the proteins flanking the linker and the stability of the connecting peptide.

2. Substrate Diffusion Simulation

2. Substrate Diffusion Simulation

Building upon this robust molecular structure blueprint, we delved into the fields of micro- and meso-scale diffusion-reaction kinetics to provide parameter simulations and design recommendations for the experimental team. For structural modeling, we constructed the fullerene-like spherical shell using the Goldberg-Coxeter parameters. We employed methods such as the greedy algorithm to determine the bounding range for the number of internalized enzymes, offering a viable method to indirectly estimate enzyme concentration from protein concentration. For kinetic analysis, we simultaneously established and cross-validated discrete and continuous models. The discrete model simulated substrate diffusion via 3D meshing and random walk, coupling it with the single-molecule probability density integration method based on Michaelis-Menten kinetics for enzyme-catalyzed reactions. The continuous model, based on a modified Fick's First Law and the Renkin equation, characterized the dynamic evolution of substrate and product concentrations within the cage. The comparison of these two models showed consistency in trends, and both models collectively verified the key finding that the diffusion process is significantly faster than the reaction process, providing direct and reliable evidence for experimental design.

With a clear understanding of the microscopic kinetic mechanism, we further extended our research into electric field optimization and industrial application. By incorporating the potential distribution from the self-assembly model, we simulated the Debye-shielded electric potential field in the solution using the linear Poisson-Boltzmann equation. Simulation results indicated that the existing spherical shell causes approximately 23.3% inhibition of diffusion flux. Therefore, we proposed optimization strategies: designing positive charges inside the shell to mitigate diffusion hindrance, and introducing positive charges outside the shell to enhance substrate capture efficiency.

3. Industrial Application

3. Industrial Application

All these microscopic mechanisms, including the effects of pore confinement and the electric field on substrate and product transmembrane migration, were ultimately integrated into a continuous reaction-diffusion-electric field-economic integrated model based on the spherical shell structure. In the macroscopic economic assessment section, we mapped the cumulative production of GSH, ATP consumption, and enzyme usage to economic variables. We established a comprehensive mathematical model including production cost, cost per unit product, annual profit, and customer payback period. Through scenario analysis, the model clearly demonstrated that the P22-VLP compartmentalization structure significantly increases GSH production per unit volume while reducing net ATP consumption, leading to a lower unit cost and a faster payback period. This provides a feasible engineering pathway for the future low-cost and high-efficiency industrial production of GSH.

Human Practices

Integrated Human Practices: Together For a Shared STEAM Educational and Sustainable Future

Our project carried out extensive and comprehensive Human Practices, covering multiple dimensions such as education and entrepreneurship. For more details about our Human Practices, please visit our Human Practices page, Education page and Entrepreneurship Page.

1. V-CHARGEs:EDU Catalyst

1. V-CHARGEs:EDU Catalyst for Regional Transformation

At the beginning of the project, the 2025 BNU-China team conducted surveys, teacher interviews, and field visits to assess the current state of STEAM education in China and ASEAN regions. Despite growing policy-level support, significant inequalities persist across gender, urban-rural, and regional lines: girls lack early exposure to science, rural schools face severe resource shortages, and iGEM education opportunities are unevenly distributed. As a youth-led scientific team, we saw a responsibility to help build a more equitable, inclusive, and forward-looking regional STEAM education ecosystem.

2. ICD: From Ideas to Action

2. ICD: From Ideas to Action

To address these disparities, BNU-China partnered with seven project teams and launched initiatives grounded in the ICD (Inspiration-Cultivation-Dissemination) model . We promoted digital education platforms, developed diverse teaching activities, and established a systematic support framework for both teachers and students (Refer to the Education section for more details). To ensure the long-term sustainability and scalability of these efforts, we founded a nonprofit organization-UDGIC (United Digital and Green Innovation Center) -in the most exemplary region of our DAYSTAR initiative.

3. UDGIC: Voices on Global Stages

3. UDGIC: Voices on Global Stages

With the support of local government, UDGIC was provided with an office space and a startup grant of 30,000 RMB. UDGIC serves as a demonstration base focused on integrating educational resources, promoting equity, and cultivating sustainable development talents.

Beyond managing projects and coordinating resources, UDGIC actively promotes its outcomes on global platforms. We were invited to present at both the UNESCO Earth Charter Education Chair's 25th Anniversary Conference and the China-ASEAN Education Exchange Week . At the main forum of the Earth Charter 25+ conference held at the Peace Palace in The Hague, the representative of iGEM BNU-China delivered a speech sharing the team's vision for STEAM education. Through partnerships with ECI offices in Indonesia and Laos, UDGIC established collaborations with their national ESD networks to facilitate cross-border educational resource sharing. We also launched the iGEMers STEAM Alliance , fostering collaboration among young scientists and educators from China and ASEAN to advance regional STEAM education.

4. Sustainable Loops, Lasting Change

4. Sustainable Loops, Lasting Change

Through ongoing DBTL (Design-Build-Test-Learn) cycles, we came to recognize the dual value of our core technical platform, V-CHARGEs: it delivers measurable cost reduction and efficiency improvements for enterprises via a ToB model, while also bridging educational efforts with real industrial needs. As a result, UDGIC has redefined its strategic mission-not only as a coordinator of education alliances, but as a regional innovation hub integrating ESG consulting, industrial upgrading services, and talent development.

This vision is embodied in our closed-loop model linking education, technology, industry, and talent:

On the education side: we cultivate youth with cross-disciplinary skills and a deep awareness of sustainability through STEAM and global citizenship education.

On the technology side: we deploy green process innovations like V-CHARGEs into industrial settings.

On the industry side: we help enterprises adopt new technologies and fulfill ESG goals.

On the talent side: we provide young people with meaningful work opportunities and career growth, maximizing the value of their training.

This circular ecosystem not only transforms educational achievements into real-world industrial outcomes, but also injects lasting talent momentum into regional green transformation-driving China-ASEAN toward a more sustainable, inclusive, and resilient future.