V-CHARGEs provides continuous and stable energy support for small molecule drug synthesis, in vitro protein expression, biochemical pathway studies, cell-free detection and biosensing, as well as cell-free metabolic engineering. Future performance enhancement includes optimization of P22-VLP surface charge, refinement of enzyme encapsulation methods, and expansion of substrate range, which will further strengthens its technical support for life science research and biomanufacturing.

1. Small Molecule Drugs Production

With the development of synthetic biology and cell-free systems, an increasing number of small molecule drugs and natural products are being synthesized through in vitro multi-enzyme cascade reactions. This approach not only offers mild reaction conditions and high selectivity but also avoids the complex regulatory mechanisms of cellular metabolism, demonstrating significant application potential. However, one of the major bottlenecks hindering the practical application of in vitro synthesis is the high cost and rapid consumption of ATP. In many drug synthesis pathways, numerous kinases and energy-dependent enzymes continuously consume ATP. When energy is insufficient, the efficiency of synthetic reactions drops sharply, and the accumulation of byproducts such as ADP or AMP further inhibits enzyme activity, making it difficult for the system to maintain high efficiency over long periods^[1]. To address this challenge, we chose a novel approach distinct from traditional methods. V-CHARGEs is a new in vitro ATP regeneration system that uses low-cost polyphosphate as the donor material, achieving continuous ATP regeneration through a multi-enzyme synergistic cycle, significantly reducing the cost of exogenous ATP usage. In specific applications, such as limonene synthesis (the pathway from mevalonate to IPP/DMAPP, then to GPP, and finally to limonene), involves at least three key reactions that consume ATP. Without an ATP regeneration cycle, the reaction stalls due to rapid ATP depletion. By introducing our system, ADP/AMP can be recycled back into ATP, ensuring the continuity of the synthetic reaction and significantly improving product yield.

More importantly, our system incorporates unique functionalities through the design of P22 Virus-Like Particle (P22-VLP). The P22-VLP structure protects the activity of internal enzymes under high temperatures or in the presence of proteases, enabling stable ATP regeneration under more demanding conditions. Additionally, multiple enzymes are immobilized on the same P22-VLP, shortening substrate transfer distances and significantly enhancing catalytic efficiency. Furthermore, specific linkers on the P22-VLP surface enable platform-based and modular designs, allowing flexible assembly of different enzymes to quickly construct customized multi-enzyme modules tailored to various synthetic pathways. This system, combining stability, efficiency, and scalability , transforms ATP regeneration from merely an energy cycling tool into a critical platform for advancing the efficiency and industrialization of in vitro drug synthesis.

Therefore, whether for monoterpenoids like limonene, or high-value small molecule drugs such as linalool, nucleoside analogs, peptides, and coenzyme derivatives, our ATP regeneration system provides stable, cost-effective, and efficient energy support, offering new possibilities for the scalability and practical application of in vitro drug synthesis.

2. In Vitro Protein Synthesis

In vitro protein synthesis refers to the process of synthesizing specific proteins in a cell-free environment using cell extracts, multi-enzyme complexes, or other artificial systems by providing template nucleic acids (such as DNA or mRNA) and necessary biochemical components. Compared to traditional in vivo protein synthesis, in vitro protein synthesis can be precisely controlled, avoiding interference from complex metabolic processes in living cells, and enables the production of proteins toxic to living cells, representing a new pathway for protein synthesis. From using cell extracts to synthesize proteins to deciphering the genetic code with artificial in vitro translation systems, in vitro protein synthesis has continued to evolve and is now becoming a new direction for large-scale protein production^[2].

The emergence of V-CHARGEs provides a new supportive platform for in vitro protein synthesis, promising to promote the technological development and large-scale application of in vitro protein synthesis.

3. In Vitro Studies of Biochemical Mechanisms

In life science research, the in vitro reconstruction of biochemical pathways is a core approach for deciphering molecular mechanisms, such as simulating MAPK phosphorylation signaling cascade reactions within cells. This type of reaction requires continuous ATP consumption by kinases to achieve signal transmission; it also involves the accurate determination of catalytic kinetic parameters of energy-dependent enzymes.

Traditional experiments rely on the one-time addition of high-concentration ATP, which is not only costly but also leads to reaction stagnation midway due to the rapid degradation of ATP. This fails to simulate the real environment of continuous energy supply in vivo, thereby compromising the accuracy of experimental data^[3].

V-CHARGEs can address these issues in a targeted manner. First, it regenerates ATP through multi-enzyme cycling, providing a stable energy source for in vitro pathways. This extends the duration of phosphorylation cascade reactions and enzyme-catalyzed reactions from the traditional tens of minutes to several hours, facilitating the acquisition of experimental data that more closely reflects physiological states. Second, it eliminates the need for frequent addition of expensive ATP, significantly reducing the cost of high-throughput experiments such as enzyme kinetics studies and making it suitable for laboratory-scale mechanism exploration. Third, the P22-VLP structure protects the enzymes encapsulated within from degradation by proteases in the experimental environment, avoiding experimental errors caused by enzyme activity attenuation and significantly improving data reproducibility.

4. Cell-Free Detection and Biosensing

Cell-free detection systems have emerged as powerful tools for rapid, sensitive, and programmable biosensing, capable of detecting nucleic acids, metabolites, or environmental toxins outside living cells.^[4] However, many cell-free sensors rely on ATP-dependent enzymes, transcription–translation modules, or phosphorylation cascades, which require a continuous and stable energy supply to sustain accurate signal output. V-CHARGEs provides a low-cost, robust ATP regeneration platform that ensures long-lasting activity of ATP-consuming enzymes in detection assays. The encapsulated design further protects the regeneration machinery from protease degradation or harsh environmental conditions, allowing biosensors to function reliably even in field applications. By coupling V-CHARGEs with programmable cell-free sensors, it becomes possible to develop portable, durable, and highly sensitive detection devices for medical diagnostics, food safety monitoring, and environmental surveillance.

5. Cell-Free Metabolic Engineering

Cell-free metabolic engineering aims to reconstruct and optimize entire biosynthetic pathways in vitro, bypassing the constraints of living organisms such as competing pathways, complex regulation, or cytotoxic intermediates. These reconstructed systems often require large and continuous amounts of ATP to drive key phosphorylation and ligation reactions. Traditional approaches relying on bulk ATP supplementation significantly increase costs and limit scalability. V-CHARGEs addresses this challenge by establishing an efficient, modular energy supply that couples directly to diverse enzymatic pathways. Its Virus-Like Particle architecture enables co-localization of ATP regeneration and pathway enzymes, enhancing flux through proximity effects and reducing intermediate diffusion loss. This makes V-CHARGEs not only a sustainable ATP supply solution but also a generalizable platform for designing and scaling complex metabolic modules in cell-free biomanufacturing.

Improvement Directions

The V-CHARGEs platform is highly versatile and easily modifiable. In future research, we aim to further improve this platform technology to expand its application scope and effectiveness, addressing more challenges in production and daily life.

(1) Charge Modification

(1) Modification of P22-VLP Surface Charge

The surface charge of P22-VLP significantly affects the efficiency of the ATP-ADP cycle and the ingress and egress of polyphosphate and phosphate ions. This is because ATP, ADP, polyphosphate, and phosphate ions all carry negative charges, making them attracted to positive charges and repelled by negative charges, thus influencing their movement^[5]. In future research, we plan to modify the surface charge distribution of P22-VLP through protein design and engineering to further enhance ATP regeneration efficiency.

(2) Novel Encapsulation

(2) Modification of Enzyme Encapsulation Methods

To further improve the production efficiency of V-CHARGEs and withstand more complex environmental changes, we aim to modify the method of enzyme immobilization from the outer surface of P22-VLP to the internal cavity of P22-VLP. This approach would protect SlPPK while also safeguarding enzymes that synthesize high-value products, enabling the entire system to effectively resist environmental factors such as pH and phosphate.

(3)Choose New PPKs

(3) Encapsulation of Other Regeneration Enzymes in P22-VLP for Regenerating GTP and Other Substances

To further expand the applications of V-CHARGEs, we plan to explore encapsulating other regeneration enzymes, such as PPK2 from Pseudomonas aeruginosa, within the P22-VLP cavity in future research. This could enable V-CHARGEs to efficiently regenerate GTP or other substances.