Yiyang Lin

Cell‐free protein synthesis (CFPS) has emerged as a versatile platform for producing proteins in vitro with exceptional programmability, rapid prototyping capability, and an open biochemical environment. However, conventional CFPS is typically performed in homogeneous solutions that lack the spatial organization, molecular confinement, and dynamic regulation characteristic of living cells. Integrating CFPS with protocells overcomes these limitations by recreating cell‐like microenvironments that enable compartmentalized gene expression and increasingly life‐like biological functions. In this review, we summarize recent advances in protocell platforms for CFPS and discuss how compartmentalization influences the efficiency, regulation, and functionality of protein synthesis. We compare the major protocell platforms and highlight the physicochemical principles governing confined protein synthesis, such as molecular crowding, selective permeability, molecular partitioning, and phase separation. We then review strategies for enhancing protein production and constructing life‐like behaviors, including programmable gene expression, membrane protein synthesis, DNA replication, membrane growth, morphogenesis, division, and intercellular communication. Finally, we discuss emerging applications in biosensing, protein engineering, and therapeutics, and outline future challenges and opportunities for developing protein‐producing protocells toward living systems.

Biomolecular condensates undergo dynamic maturation, transitioning from liquid-like droplets to gel-like or solid-like assemblies, exhibiting structural heterogeneity in response to biochemical cues. Synthetic coacervate droplets formed via liquid-liquid phase separation (LLPS) have emerged as simplified models of these condensates and of protocells. Yet, the understanding of complex phase behaviors of liquid-like droplets in both biological and chemical contexts remains less explored, limiting our understanding of biological function and the design of droplet-based soft materials and protocells. Here, we introduce a chemically programmable strategy to dynamically modulate droplet phase transitions through controlled polymer-network cross-linking. Reactive cross-linkers selectively engage polymers within liquid-like droplets, progressively increasing internal microviscosity and inducing a liquid-to-solid transition. Beyond uniform phase regulation, network cross-linking drives spatially heterogeneous phase separation via polymer demixing and Ostwald ripening, with reaction-diffusion dynamics critically shaping both thermodynamically stable and metastable droplets. Using orthogonal cleavable cross-linkers, we further demonstrate chemical control over droplet liquefaction and the generation of multiphasic structures with pathway-dependent configurations. Integration of photoresponsive cross-linkers with digital-micromirror device (DMD) technology enables precise spatial photopatterning of droplet networks. This work establishes a versatile framework for elucidating the structural principles of microphase separation within coacervates and provides a blueprint for designing dynamic soft materials and synthetic protocells.

Liquid crystalline droplets with molecularly crowded interiors capable of selective biomolecular sequestration and interfacial wetting have been recently developed for the construction of artificial cells (protocells) and chain-like protocell networks^1-3. The controlled division of these synthetic protocells^4-16, however, remains a challenge. Symmetric fission of vesicles and droplets has been shown^17-26 using thermal gradients, dissipative self-assembly, wetting energies and chemical reactions^27-31, but asymmetric division is rare. For example, osmotic pressure has been used to induce the asymmetric division of lipid vesicles containing a polyethylene glycol-dextran aqueous two-phase system³² as well as giant unilamellar vesicles prepared with lipid-phase-separated bilayers³³. Here we show that structured liquid droplets exhibit asymmetric division in the absence of reconstituted protein machinery. In the presence of alkaline phosphatase or multivalent metal cations, individual multilamellar droplets split to produce two morphologically distinct progeny (droplet and vesicle). We show that heteromorphic division occurs by circumferential growth of a single surface caveola along a latent core-shell domain boundary because of induced changes in lipid headgroup-nucleotide counterion interactions and demonstrate that functional biomolecules are transferred between the different protocell generations. Taken together, our results provide a step towards the bottom-up assembly of proliferating artificial cells.

Nature has evolved sophisticated light‐harvesting systems and sensory mechanisms that detect light, convert it into biochemical energy, regulate biological processes, and synthesize organic compounds and oxygen. Extensive studies have explored the chemical and physical principles underlying the structure and function of photoreceptors and chromophore‐embedded proteins, providing insights into light‐matter interactions in biological systems. Artificial cells, designed to replicate natural cells, offer a unique platform for investigating biological processes and the origins of life. The development of hierarchical structures capable of light harvesting, sensory detection, and photoenergy conversion is a critical step toward creating effective cell mimics. This review highlights the role of photosensation and photoenergy transduction in biology, summarizes the bottom‐up construction of artificial cells, and examines recent advancements in light‐harvesting, phototransduction, and energy conversion within these systems, along with light‐controlled cytomimetic behaviors.

The bottom-up construction of cell-like entities or protocells is essential for emulating cytomimetic behaviours within artificial cell consortia. Complex coacervate microdroplets are promising candidates for primordial cells; however, replicating the complex cellular organization and cell–cell interactions using membraneless coacervates remains a major challenge. To address this, we developed membrane-bound coacervate protocells by interfacial assembly of metal–organic framework nanoparticles around coacervate microdroplets. By leveraging the inherently porous structure and surface chemistry of metal–organic frameworks, we demonstrated the ability to regulate biomolecular organization within the protocells and integrate proteins into the membrane, thereby imitating both integral and peripheral membrane proteins. These membranized coacervates were further engineered into artificial-organelle-incorporated protocells and tissue-like assemblies capable of signal processing and protocell-to-protocell communication. Our findings highlight the potential of designing artificial systems with spatially controlled biomolecular organization to mimic natural cellular functions, paving the way for the assembly of membranized coacervates into prototissues.

The assembly of protocells into prototissues represents a significant step forward in mimicking the hierarchical structure and complex functions of biological tissues. This review explores the construction of various protocell models, including lipid vesicles, polymer vesicles, proteinosomes, membraneless coacervates, and emulsion droplets. It examines the range of chemical and physical methods employed to spatially organize these protocells into structured, functional prototissues. Furthermore, the review highlights the distinctive properties of prototissues, such as their ability to facilitate intra‐ and inter‐protocell communication, exhibit collective behaviors like oscillations and deformation, and their promising applications in biomedicine. By bridging the gap between synthetic cells and functional tissue‐like systems, this review provides insights into the design, fabrication, and potential applications of prototissues in synthetic biology and regenerative medicine.

Living organisms take in matter and energy from their surroundings, transforming these inputs into forms that cells can use to sustain metabolism and power various functions. A significant advancement in the development of protocells and life‐like materials has been the creation of cell‐like microcompartments capable of evolving into higher‐order structures characterized by hierarchy and complexity. In this study, a smart emulsion system is designed to digests chemical substrates and generates organic or inorganic products, driving the self‐organization and structuration of microcompartments. Central to this system is a lipase‐derived peptide that undergoes amyloid fibrillation, exhibiting hydrolase‐like activity and stabilizing Pickering emulsions. Through catalytic hydrolysis or silicatein‐inspired mineralization, these emulsion microcompartments generate self‐organized surfactant layers from organic substrates or silica scaffolds from inorganic substrates at the oil–water interface, respectively, helping to prevent coalescence. This process further facilitates a structural evolution into high‐internal phase emulsion gels that are suitable for direct‐ink‐writing 3D printing. The findings underscore the potential for designing self‐evolving soft materials that replicate the structures and functions of living organisms.

Mechanical sensing and transduction are integral to biological systems and have inspired research into the regulation of supramolecular self‐assembly via mechanical forces. This review presents an inaugural discussion on mechanically active supramolecular systems. It focuses on two primary mechanisms for modulating these systems: the incorporation of mechanophores and the application of mechanical forces to modulate non‐covalent interactions. Challenging the traditional view of mechanical forces as solely destructive, their constructive potential when harnessed through sophisticated design is showcased. Investigation is done on how external forces like ultrasound, stirring, vortex, tension, and compression can induce fluorescence in π‐conjugated systems, initiate hydrogelation, propel non‐equilibrium self‐assembly, and affect the structure of vesicles. The review also casts light on the prospective uses of mechanically active supramolecular systems in areas such as protein activation, drug delivery, and stress sensing, illustrating the nuanced role of mechanical forces as both disruptors and enablers in the creation of functional materials.