Metabolic Subsystem

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Overview

In natural cells, continuous energy generation and recycling are essential for supporting metabolic and synthetic functions. Various strategies that have been explored to supply synthetic cells with energy so that genetic circuits and protein expression can be sustained over extended periods. These methods focus on mimicking these energy management strategies by integrating one or more energy‐supplying modules.

Cell-Free Metabolism

We start by focusing on techniques for extending the operation cell-free systems (without encapsulation).

Continuous External Feeding and Substrate Supply

One fundamental strategy involves continuously replenishing the synthetic cell's interior with fresh energy substrates and nutrients. In many cell‐free systems encapsulated in liposomes or giant unilamellar vesicles (GUVs), limited supply of substrates (e.g., ATP, nucleotides, amino acids) leads to eventual depletion that stops protein expression. To overcome this, external feeding protocols have been established such as microfluidic continuous exchange of reaction components. For example, microfluidic chemostats have been used to periodically replace part of the reaction volume with an energy solution that contains chemical substrates (e.g., creatine phosphate, nucleoside triphosphates) and replenishes lost amino acids and cofactors, thereby extending the time over which genetic circuits operate and proteins are synthesized . In these systems, an external apparatus continuously feeds energy-rich substrates into synthetic compartments, offsetting the stoichiometric consumption that occurs during transcription and translation. This approach partially mimics the nutrient uptake and waste removal seen in living cells and is particularly useful in cell-free environments where metabolic regeneration is not intrinsic .

Reconstituted ATP Regeneration Systems

Cell-free protein synthesis systems that traditionally rely on high-energy phosphate compounds such as phosphoenolpyruvate (PEP) or 3-phosphoglycerate (3-PGA) can be optimized by coupling with engineered metabolic enzymes to recycle phosphate and regenerate ATP . These systems take advantage of enzymatic cascades in which one enzyme's product becomes the substrate for the next, effectively maintaining a pool of high-energy molecules to sustain protein synthesis. Although these methods can extend the duration of cell-free expression, challenges remain regarding phosphate bond instability and catalyst poisoning, which can lead to eventual cessation of activity.

Enzymatic Cofactor and Metabolite Recycling

Efficient energy supply within synthetic cells not only depends on ATP regeneration but also on the reconstitution and continuous recycling of cofactors such as NADH and NADPH. Synthetic compartments have been developed that incorporate enzymatic cascades able to regenerate essential cofactors, thereby maintaining redox balance and sustaining metabolic reactions necessary for protein expression , . For instance, specific enzyme and electron donor systems have been demonstrated in polymersomes to continuously recycle NADPH, which in turn supports downstream biosynthetic reactions and energizes genetic circuits. These enzymatic recycling modules help sustain the out-of-equilibrium conditions required for extended operation of synthetic biological processes.

Integration of Microfluidic Systems for Continuous Energy Renewal

Many synthetic cell platforms operate in a closed, batch-style environment, which limits the duration of protein expression because energy substrates are eventually depleted and inhibitory accumulations occur. Microfluidic platforms have been employed to overcome these limitations by creating a continuous exchange system, where fresh reaction solutions are fed into the synthetic cell environment at regular intervals . In these microfluidic chemostats, a portion of the reaction volume is periodically replaced with a nutrient-rich feed that contains all the necessary components for energy generation and gene expression. This approach not only sustains ATP levels but also buffers against waste accumulation, thereby extending the operational lifespan of the synthetic cells. The integration of such continuous-flow systems bridges the gap between static, closed-cell assays and the dynamic conditions that living cells experience, offering a promising route for long-term operation of artificial cells.

Encapsulation-Based Approaches

For use in synthetic cells, the energy regeneration and waste processing systems must operate in an encapsulated environment. Several approaches have been explored in the literature.

Integration of Artificial Organelles

Another promising approach is the design of modular artificial organelles—compartmentalized subunits embedded within synthetic cells that mimic the energy conversion functions of mitochondria or chloroplasts. Such artificial organelles typically integrate a photoconverter (e.g., bacteriorhodopsin or photosystem II), an ATP synthase, and a compartment that maintains the proton motive force , . By partitioning the energy-generating reactions into discrete subcompartments, synthetic cells can achieve spatial organization similar to eukaryotic cells, which in turn helps protect sensitive reactions from interference and allows for regulated energy supply. These enzyme-coupled systems have been further optimized by modulating the membrane composition and protein orientation to maximize the efficiency of ATP synthesis and reduce leakiness .

Light-Driven Energy Systems

A common goal is to establish internal modules within synthetic cells that can cyclically regenerate ATP, the universal energy currency. One successful approach has been to incorporate membrane-bound ATP synthase together with proton pumps into vesicles, thereby recreating a minimal version of natural bioenergetics. Light-driven systems are a prominent example. In such systems, proteins such as bacteriorhodopsin or proteorhodopsin are co-reconstituted with ATP synthase in lipid bilayers or polymersomes; upon illumination, the light-sensitive proton pump establishes a proton gradient across the membrane, which the ATP synthase then harnesses to convert ADP into ATP , . This strategy has been validated by early work showing that light-induced proton gradients can drive ATP production, drawing analogies to natural photosynthesis, and it is now under active refinement to achieve higher synthesis rates and longer operation times , .

Light-driven energy generation stands out as one of the most attractive strategies for powering synthetic cells, primarily because it allows for energy input in a renewable and externally controllable manner. The reconstitution of light-activated proton pumps such as bacteriorhodopsin (or its variants) in combination with ATP synthase enables synthetic cells to utilize light as a free energy source. Not only is this strategy renewable, but it also allows for precise external control over energy production, which is advantageous in systems where timing and spatial regulation of genetic circuits are crucial.

Membrane Permeabilization and Nutrient Uptake

Another necessary element for long-term operation is ensuring that the synthetic cell membrane can both retain key biomacromolecules while allowing the controlled exchange of small energy substrates and waste products. Several approaches have been developed to modify vesicle permeability. One effective strategy is the incorporation of pore-forming proteins such as α-hemolysin into liposomal membranes, thereby permitting passive diffusion of small molecules including nutrients, ATP, and cofactors , . The presence of these pores allows for a continuous supply of vital substrates and removal of inhibitory products from within the synthetic cell, enabling sustained protein expression and circuit operation. Importantly, the selective permeability of these membranes can be engineered by tuning the composition of lipid mixtures to favor the necessary pore formation while maintaining compartment integrity .

Metabolic Pathway Engineering and Substrate-Level Phosphorylation

Beyond the reconstitution of classical energy modules involving ATP synthase, synthetic cells have been designed to include minimal metabolic pathways that directly generate ATP through substrate-level phosphorylation. One example is the arginine breakdown pathway, which has been reconstituted in liposomes to drive ATP production from energy-rich substrates . In such systems, the conversion of arginine to ornithine is coupled to ATP generation via carbamate kinase, and the process is facilitated by membrane transporters that exchange substrates and products. These pathways, although simpler than full respiratory chains, can provide a bona fide ATP supply to support energetically demanding processes such as translation and genetic circuit operation . By designing these pathways carefully, researchers can mimic the efficiency of natural mitochondrial ATP production in a much more simplified and controlled environment.

Use of Synthetic Membrane Materials and Compartmentalization Strategies

The choice of membrane material is critical not only for providing structural integrity but also for functional support of embedded energy-conversion modules. Synthetic cells have been constructed using lipid vesicles, polymersomes, or hybrid membranes that can be tailored to optimize both permeability and stability , . Hybrid membranes, particularly those incorporating block-copolymers with phospholipids, offer enhanced stability and controlled permeability, which is necessary when integrating sensitive proteins such as ATP synthase and proton pumps. In addition, compartmentalization via the creation of internal subcompartments (artificial organelles) enables spatial separation of incompatible reactions while concentrating key enzymes and substrates. This design mimics the organelle organization found in natural eukaryotic cells and facilitates higher local concentrations of metabolic components, thereby increasing ATP synthesis efficiency , .

Additional Topics

Nucleotide Feeding and Waste Management

In addition to feeding energy, it is also possible to feed additional components necessary for synthetic cell operation into a synthetic cell.

Incorporation of Energy Sensors and Feedback Regulation

A further refinement in the design of energy-supplying synthetic cells is the integration of sensors that monitor intracellular parameters such as ATP levels, pH, and redox states. Genetically encoded or chemically based sensors can provide real-time information about the energetic state of the cell and trigger feedback loops to regulate substrate uptake or enzyme activity . With such sensors in place, synthetic cells can be engineered to dynamically adjust their metabolic pathways or to upregulate transport mechanisms when energy levels fall below a certain threshold. This self-regulating capability contributes significantly to the sustained operation of genetic circuits and protein expression, as the cell is continuously maintained in an optimal energetic state .

Integration with Native or Engineered Metabolic Systems

In some approaches, synthetic cells are designed to incorporate elements of natural metabolism, borrowing components from living cells to jumpstart robust energy production. For example, cell-free protein synthesis systems that reconstitute elements of the E. coli cytoplasm have been used to support long-term protein production. Such systems include not only the biochemical machinery for transcription and translation but also enzymes for ATP and cofactor regeneration , . By adopting metabolic modules from natural organisms, synthetic cell designs can leverage billions of years of evolutionary optimization to maintain high energetic efficiency and resilience against metabolic imbalance.

Combining Multiple Energy-Supplying Strategies

Because no single approach perfectly recapitulates the energy supply mechanisms of living cells, many recent studies have embraced hybrid strategies that combine multiple methods. For instance, an artificial cell might include both a light-driven ATP generation module and a chemical ATP regeneration pathway, with membrane pores ensuring continuous exchange of substrates. This redundancy not only prolongs the duration of protein expression and genetic circuit operation but also increases system robustness under varying environmental conditions , . In some designs, modular assembly allows synthetic cells to switch between energy sources depending on the availability of light or nutrients, which closely mimics metabolic flexibility observed in living organisms , .

Future Perspectives and Remaining Challenges

Although significant progress has been made, several challenges remain in fully realizing autonomous energy supply within synthetic cells. One key challenge is matching the efficiency and dynamic range of natural metabolic networks. For long-term operation, the synthetic energy modules must not only produce sufficient ATP at high rates but also recycle all necessary cofactors and remove inhibitory byproducts. Ensuring membrane integrity while embedding multiple active proteins also remains a technical hurdle, as does the precise calibration of substrate and enzyme concentrations to avoid imbalances that could shut down energy production , .

Furthermore, while continuous feeding through microfluidic systems has shown promise in maintaining steady-state conditions, integration of such systems into fully autonomous or implantable synthetic cells is still in its infancy , . The eventual goal is to develop synthetic cells that are capable of self-sustained energy production over long periods without the need for external intervention—a milestone that will require further optimization of membrane materials, metabolic pathway integration, and feedback control mechanisms , .

Consequently, continued research in reconstituting natural energy-converting enzyme complexes, designing modular artificial organelles, and optimizing microfluidic continuous replacement strategies is essential. Advances in synthetic biology techniques, combined with insights from natural cellular bioenergetics, will undoubtedly propel the field closer to creating fully autonomous synthetic cells. Future designs may also integrate environmentally responsive elements that allow synthetic cells to adaptively alter their energy regimes in response to changing external conditions , .

In summary, the current approaches to supplying synthetic cells with energy include: continuous external supply of energy substrates via microfluidic feeding, reconstitution of ATP regeneration systems that harness light-driven or chemical energy, enzymatic recycling of cofactors such as NADPH and NADH, incorporation of artificial organelles that mimic natural bioenergetic organelles, and the development of membranes with tunable permeability to allow selective nutrient influx and waste efflux. These strategies are often combined in hybrid systems to maximize energy production efficiency, improve robustness, and enable extended operation of genetic circuits and protein expression. Advances in material science, enzyme reconstitution, and system integration are critical to overcoming current limitations and achieving self-sustaining synthetic cells that can operate for prolonged periods with minimal external intervention , , , , .

This multi-pronged approach to energy supply is essential not only for sustaining protein synthesis and gene expression but also for enabling more complex cell-like behaviors such as growth, division, and response to environmental cues. As researchers continue to refine these techniques, the integration of energy regeneration modules will remain one of the central challenges and opportunities for the field of artificial cells.

Overall, the field has evolved from relying on simple, batch-fed cell-free protein expression systems to developing sophisticated, compartmentalized energy regeneration strategies that recapitulate natural metabolic and bioenergetic processes. This progress paves the way for the development of synthetic cells that can autonomously sustain complex genetic circuits and perform prolonged, life-like functions in both in vitro settings and, eventually, in vivo applications , , .

By combining continuous nutrient supply, in situ ATP and cofactor regeneration, selective membrane permeability via channel proteins, and integration of artificial organelles, researchers are steadily advancing toward the creation of a fully autonomous synthetic cell with robust energy management. Future research will need to address remaining challenges such as protein insertion efficiency, control of reaction byproducts, and fine-tuning biophysical properties of synthetic membranes to further bridge the gap between engineered systems and natural cells , , .

The cumulative progress in these areas represents a significant step forward in synthetic biology and brings us closer to the ultimate goal of constructing artificial cells that are capable of sustained, self-regulated operation, thereby providing a viable platform for applications ranging from drug delivery to biosensing and beyond , .