Controlled exchange of protein and nucleic acid signals from and between synthetic minimal cells.

Synthetic minimal cells are a class of bioreactors that have some, but not all, functions of live cells. Here, we report a critical step toward the development of a bottom-up minimal cell: cellular export of functional protein and RNA products. We used cell-penetrating peptide tags to translocate payloads across a synthetic cell vesicle membrane. We demonstrated efficient transport of active enzymes and transport of nucleic acid payloads by RNA-binding proteins. We investigated influence of a concentration gradient alongside other factors on the efficiency of the translocation, and we show a method to increase product accumulation in one location. We demonstrate the use of this technology to engineer molecular communication between different populations of synthetic cells, to exchange protein and nucleic acid signals. The synthetic minimal cell production and export of proteins or nucleic acids allows experimental designs that approach the complexity and relevancy of natural biological systems. A record of this paper's transparent peer review process is included in the supplemental information.

Biofunctional coacervate-based artificial protocells with membrane-like and cytoplasm-like structures for the treatment of persistent hyperuricemia.

Coacervate droplets formed by liquid-liquid phase separation have attracted considerable attention due to their ability to enrich biomacromolecules while preserving their bioactivities. However, there are challenges to develop coacervate droplets as delivery vesicles for therapeutics resulting from the lack of physiological stability and inherent lack of membranes in coacervate droplets. Herein, polylysine-polynucleotide complex coacervate droplets with favorable physiological stability are formulated to efficiently and facilely concentrate small molecules, biomacromolecules and nanoparticles without organic solvents. To improve the biocompatibility, the PEGylated phospholipid membrane is further coated on the surface of the coacervate droplets to prepare coacervate-based artificial protocells (ArtPC) with membrane-like and cytoplasm-like structures. The ArtPC can confine the cyclic catalytic system of uricase and catalase inside to degrade uric acid and deplete the toxicity of H2O2. This biofunctional ArtPC effectively reduces blood uric acid levels and prevents renal injuries in mice with persistent hyperuricemia. The ArtPC-based therapy can bridge the disciplines of synthetic biology, pharmaceutics and therapeutics.

An inorganic mineral-based protocell with prebiotic radiation fitness.

Protocell fitness under extreme prebiotic conditions is critical in understanding the origin of life. However, little is known about protocell's survival and fitness under prebiotic radiations. Here we present a radioresistant protocell model based on assembly of two types of coacervate droplets, which are formed through interactions of inorganic polyphosphate (polyP) with divalent metal cation and cationic tripeptide, respectively. Among the coacervate droplets, only the polyP-Mn droplet is radiotolerant and provides strong protection for recruited proteins. The radiosensitive polyP-tripeptide droplet sequestered with both proteins and DNA could be encapsulated inside the polyP-Mn droplet, and form into a compartmentalized protocell. The protocell protects the inner nucleoid-like condensate through efficient reactive oxygen species' scavenging capacity of intracellular nonenzymic antioxidants including Mn-phosphate and Mn-peptide. Our results demonstrate a radioresistant protocell model with redox reaction system in response to ionizing radiation, which might enable the protocell fitness to prebiotic radiation on the primitive Earth preceding the emergence of enzyme-based fitness. This protocell might also provide applications in synthetic biology as bioreactor or drug delivery system.

Advancing Biomimetic Functions of Synthetic Cells through Compartmentalized Cell-Free Protein Synthesis.

Synthetic cells are artificial constructs that mimic the structures and functions of living cells. They are attractive for studying diverse biochemical processes and elucidating the origins of life. While creating a living synthetic cell remains a grand challenge, researchers have successfully synthesized hundreds of unique synthetic cell platforms. One promising approach to developing more sophisticated synthetic cells is to integrate cell-free protein synthesis (CFPS) mechanisms into vesicle platforms. This makes it possible to create synthetic cells with complex biomimetic functions such as genetic circuits, autonomous membrane modifications, sensing and communication, and artificial organelles. This Review explores recent advances in the use of CFPS to impart advanced biomimetic structures and functions to bottom-up synthetic cell platforms. We also discuss the potential applications of synthetic cells in biomedicine as well as the future directions of synthetic cell research.

Construction of Membraneless and Multicompartmentalized Coacervate Protocells Controlling a Cell Metabolism-like Cascade Reaction.

In recent years, there has been growing attention to designing synthetic protocells, capable of mimicking micrometric and multicompartmental structures and highly complex physicochemical and biological processes with spatiotemporal control. Controlling metabolism-like cascade reactions in coacervate protocells is still challenging since signal transduction has to be involved in sequential and parallelized actions mediated by a pH change. Herein, we report the hierarchical construction of membraneless and multicompartmentalized protocells composed of (i) a cytosol-like scaffold based on complex coacervate droplets stable under flow conditions, (ii) enzyme-active artificial organelles and a substrate nanoreservoir capable of triggering a cascade reaction between them in response to a pH increase, and (iii) a signal transduction component based on the urease enzyme capable of the conversion of an exogenous biological fuel (urea) into an endogenous signal (ammonia and pH increase). Overall, this strategy allows a synergistic communication between their components within the membraneless and multicompartment protocells and, thus, metabolism-like enzymatic cascade reactions. This signal communication is transmitted through a scaffold protocell from an "inactive state" (nonfluorescent protocell) to an "active state" (fluorescent protocell capable of consuming stored metabolites).

Motility of Synthetic Cells from Engineered Lipids.

Synthetic cells are artificial systems that resemble natural cells. Significant efforts have been made over the years to construct synthetic protocells that can mimic biological mechanisms and perform various complex processes. These include compartmentalization, metabolism, energy supply, communication, and gene reproduction. Cell motility is also of great importance, as nature uses elegant mechanisms for intracellular trafficking, immune response, and embryogenesis. In this review, we discuss the motility of synthetic cells made from lipid vesicles and relevant molecular mechanisms. Synthetic cell motion may be classified into surface-based or solution-based depending on whether it involves interactions with surfaces or movement in fluids. Collective migration behaviors have also been demonstrated. The swarm motion requires additional mechanisms for intercellular signaling and directional motility that enable communication and coordination among the synthetic vesicles. In addition, intracellular trafficking for molecular transport has been reconstituted in minimal cells with the help of DNA nanotechnology. These efforts demonstrate synthetic cells that can move, detect, respond, and interact. We envision that new developments in protocell motility will enhance our understanding of biological processes and be instrumental in bioengineering and therapeutic applications.

Biomimetic Protocells Featuring Macrophage-Like Capture and Digestion of Protein Pathogens.

Modern medical research develops interest in sophisticated artificial nano- and microdevices for future treatment of human diseases related to biological dysfunctions. This covers the design of protocells capable of mimicking the structure and functionality of eukaryotic cells. The authors use artificial organelles based on trypsin-loaded pH-sensitive polymeric vesicles to provide macrophage-like digestive functions under physiological conditions. Herein, an artificial cell is established where digestive artificial organelles (nanosize) are integrated into a protocell (microsize). With this method, mimicking crossing of different biological barriers, capture of model protein pathogens, and compartmentalized digestive function are possible. This allows the integration of different components (e.g., dextran as stabilizing block) and the diffusion of pathogens in simulated cytosolic environment under physiological conditions. An integrated characterization approach is carried out, with identifying electrospray ionization mass spectrometry as an excellent detection method for the degradation of a small peptide such as ß-amyloid. The degradation of model enzymes is measured by enzyme activity assays. This work is an important contribution to effective biomimicry with the design of cell-like functions having potential for therapeutic action.

Bottom-Up Synthetic Biology Using Cell-Free Protein Synthesis.

Technical advances in biotechnology have greatly accelerated the development of bottom-up synthetic biology. Unlike top-down approaches, bottom-up synthetic biology focuses on the construction of a minimal cell from scratch and the application of these principles to solve challenges. Cell-free protein synthesis (CFPS) systems provide minimal machinery for transcription and translation, from either a fractionated cell lysate or individual purified protein elements, thus speeding up the development of synthetic cell projects. In this review, we trace the history of the cell-free technique back to the first in vitro fermentation experiment using yeast cell lysate. Furthermore, we summarized progresses of individual cell mimicry modules, such as compartmentalization, gene expression regulation, energy regeneration and metabolism, growth and division, communication, and motility. Finally, current challenges and future perspectives on the field are outlined.

Exploring Giant Unilamellar Vesicle Production for Artificial Cells - Current Challenges and Future Directions.

Creating an artificial cell from the bottom up is a long-standing challenge and, while significant progress has been made, the full realization of this goal remains elusive. Arguably, one of the biggest hurdles that researchers are facing now is the assembly of different modules of cell function inside a single container. Giant unilamellar vesicles (GUVs) have emerged as a suitable container with many methods available for their production. Well-studied swelling-based methods offer a wide range of lipid compositions but at the expense of limited encapsulation efficiency. Emulsion-based methods, on the other hand, excel at encapsulation but are only effective with a limited set of membrane compositions and may entrap residual additives in the lipid bilayer. Since the ultimate artificial cell will need to comply with both specific membrane and encapsulation requirements, there is still no one-method-fits-all solution for GUV formation available today. This review discusses the state of the art in different GUV production methods and their compatibility with GUV requirements and operational requirements such as reproducibility and ease of use. It concludes by identifying the most pressing issues and proposes potential avenues for future research to bring us one step closer to turning artificial cells into a reality.

A Photo-degradable Crosslinker for the Development of Light-responsive Protocell Membranes.

The achievement of light-responsive behaviours is an important target for protocell engineering to allow control of fundamental protocellular processes such as communication via diffusible chemical signals, shape changes or even motility at the flick of a switch. As a step towards this ambitious goal, here we describe the synthesis of a novel poly(ethylene glycol)-based crosslinker, reactive towards nucleophiles, that effectively degrades with UV light (405 nm). We demonstrate its utility for the fabrication of the first protocell membranes capable of light-induced disassembly, for the photo-generation of patterns of protocells, and for the modulation of protocell membrane permeability. Overall, our results not only open up new avenues towards the engineering of spatially organised, communicating networks of protocells, and of micro-compartmentalised systems for information storage and release, but also have important implications for other research fields such as drug delivery and soft materials chemistry.

Compartmentalized Cell-Free Expression Systems for Building Synthetic Cells.

One of the grand challenges in bottom-up synthetic biology is the design and construction of synthetic cellular systems. One strategy toward this goal is the systematic reconstitution of biological processes using purified or non-living molecular components to recreate specific cellular functions such as metabolism, intercellular communication, signal transduction, and growth and division. Cell-free expression systems (CFES) are in vitro reconstitutions of the transcription and translation machinery found in cells and are a key technology for bottom-up synthetic biology. The open and simplified reaction environment of CFES has helped researchers discover fundamental concepts in the molecular biology of the cell. In recent decades, there has been a drive to encapsulate CFES reactions into cell-like compartments with the aim of building synthetic cells and multicellular systems. In this chapter, we discuss recent progress in compartmentalizing CFES to build simple and minimal models of biological processes that can help provide a better understanding of the process of self-assembly in molecularly complex systems.

Interfacing Coacervates with Membranes: From Artificial Organelles and Hybrid Protocells to Intracellular Delivery.

Compartmentalization is crucial for the functioning of cells. Membranes enclose and protect the cell, regulate the transport of molecules entering and exiting the cell, and organize cellular machinery in subcompartments. In addition, membraneless condensates, or coacervates, offer dynamic compartments that act as biomolecular storage centers, organizational hubs, or reaction crucibles. Emerging evidence shows that phase-separated membraneless bodies in the cell are involved in a wide range of functional interactions with cellular membranes, leading to transmembrane signaling, membrane remodeling, intracellular transport, and vesicle formation. Such functional and dynamic interplay between phase-separated droplets and membranes also offers many potential benefits to artificial cells, as shown by recent studies involving coacervates and liposomes. Depending on the relative sizes and interaction strength between coacervates and membranes, coacervates can serve as artificial membraneless organelles inside liposomes, as templates for membrane assembly and hybrid artificial cell formation, as membrane remodelers for tubulation and possibly division, and finally, as cargo containers for transport and delivery of biomolecules across membranes by endocytosis or direct membrane crossing. Here, recent experimental examples of each of these functions are reviewed and the underlying physicochemical principles and possible future applications are discussed.

A Synthetic Signaling Network Imitating the Action of Immune Cells in Response to Bacterial Metabolism.

State-of-the-art bottom-up synthetic biology allows to replicate many basic biological functions in artificial-cell-like devices. To mimic more complex behaviors, however, artificial cells would need to perform many of these functions in a synergistic and coordinated fashion, which remains elusive. Here, a sophisticated biological response is considered, namely the capture and deactivation of pathogens by neutrophil immune cells, through the process of netosis. A consortium consisting of two synthetic agents is designed-responsive DNA-based particles and antibiotic-loaded lipid vesicles-whose coordinated action mimics the sought immune-like response when triggered by bacterial metabolism. The artificial netosis-like response emerges from a series of interlinked sensing and communication pathways between the live and synthetic agents, and translates into both physical and chemical antimicrobial actions, namely bacteria immobilization and exposure to antibiotics. The results demonstrate how advanced life-like responses can be prescribed with a relatively small number of synthetic molecular components, and outlines a new strategy for artificial-cell-based antimicrobial solutions.

A Self-Regenerating Artificial Cell, that is One Step Closer to Living Cells: Challenges and Perspectives.

Controllable, self-regenerating artificial cells (SRACs) can be a vital advancement in the field of synthetic biology, which seeks to create living cells by recombining various biological molecules in the lab. This represents, more importantly, the first step on a long journey toward creating reproductive cells from rather fragmentary biochemical mimics. However, it is still a difficult task to replicate the complex processes involved in cell regeneration, such as genetic material replication and cell membrane division, in artificially created spaces. This review highlights recent advances in the field of controllable, SRACs and the strategies to achieve the goal of creating such cells. Self-regenerating cells start by replicating DNA and transferring it to a location where proteins can be synthesized. Functional but essential proteins must be synthesized for sustained energy generation and survival needs and function in the same liposomal space. Finally, self-division and repeated cycling lead to autonomous, self-regenerating cells. The pursuit of controllable, SRACs will enable authors to make bold advances in understanding life at the cellular level, ultimately providing an opportunity to use this knowledge to understand the nature of life.

Minimal Out-of-Equilibrium Metabolism for Synthetic Cells: A Membrane Perspective.

Life-like systems need to maintain a basal metabolism, which includes importing a variety of building blocks required for macromolecule synthesis, exporting dead-end products, and recycling cofactors and metabolic intermediates, while maintaining steady internal physical and chemical conditions (physicochemical homeostasis). A compartment, such as a unilamellar vesicle, functionalized with membrane-embedded transport proteins and metabolic enzymes encapsulated in the lumen meets these requirements. Here, we identify four modules designed for a minimal metabolism in a synthetic cell with a lipid bilayer boundary: energy provision and conversion, physicochemical homeostasis, metabolite transport, and membrane expansion. We review design strategies that can be used to fulfill these functions with a focus on the lipid and membrane protein composition of a cell. We compare our bottom-up design with the equivalent essential modules of JCVI-syn3a, a top-down genome-minimized living cell with a size comparable to that of large unilamellar vesicles. Finally, we discuss the bottlenecks related to the insertion of a complex mixture of membrane proteins into lipid bilayers and provide a semiquantitative estimate of the relative surface area and lipid-to-protein mass ratios (i.e., the minimal number of membrane proteins) that are required for the construction of a synthetic cell.

DNA-Based Artificial Receptors as Transmembrane Signal Transduction Systems for Protocellular Communication.

The ability to reproduce signal transduction and cellular communication in artificial cell systems is significant in synthetic protobiology. Here, we describe an artificial transmembrane signal transduction through low pH-mediated formation of the i-motif and dimerization of DNA-based artificial membrane receptors, which is coupled to the occurrence of fluorescence resonance energy transfer and the activation of G-quadruplex/hemin-mediated fluorescence amplification inside giant unilamellar vesicles. Moreover, an intercellular signal communication model is established when the extravesicular H+ input is replaced by coacervate microdroplets, which activate the dimerization of the artificial receptors, and subsequent fluorescence production or polymerization in giant unilamellar vesicles. This study represents a crucial step towards designing artificial signalling systems with environmental response, and provides an opportunity to establish signalling networks in protocell colonies.

Signal Transduction in Artificial Cells.

In recent years, significant progress has been made in the emerging field of constructing biomimetic soft compartments with life-like behaviors. Given that biological activities occur under a flux of energy and matter exchange, the implementation of rudimentary signaling pathways in artificial cells (protocells) is a prerequisite for the development of adaptive sense-response phenotypes in cytomimetic models. Herein, recent approaches to the integration of signal transduction modules in model protocells prepared by bottom-up construction are discussed. The approaches are classified into two categories involving invasive biochemical signals or non-invasive physical stimuli. In the former mechanism, transducers with intrinsic recognition capability respond with high specificity, while in the latter, artificial cells respond through intra-protocellular energy transduction. Although major challenges remain in the pursuit of a sophisticated artificial signaling network for the orchestration of higher-order cytomimetic models, significant advances have been made in establishing rudimentary protocell communication networks, providing novel organizational models for the development of life-like microsystems and new avenues in protoliving technologies.

The Long Road to a Synthetic Self-Replicating Central Dogma.

The construction of a biochemical system capable of self-replication is a key objective in bottom-up synthetic biology. Throughout the past two decades, a rapid progression in the design of in vitro cell-free systems has provided valuable insight into the requirements for the development of a minimal system capable of self-replication. The main limitations of current systems can be attributed to their macromolecular composition and how the individual macromolecules use the small molecules necessary to drive RNA and protein synthesis. In this Perspective, we discuss the recent steps that have been taken to generate a minimal cell-free system capable of regenerating its own macromolecular components and maintaining the homeostatic balance between macromolecular biogenesis and consumption of primary building blocks. By following the flow of biological information through the central dogma, we compare the current versions of these systems to date and propose potential alterations aimed at designing a model system for self-replicative synthetic cells.

Membranized Coacervate Microdroplets: from Versatile Protocell Models to Cytomimetic Materials.

Although complex coacervate microdroplets derived from associative phase separation of counter-charged electrolytes have emerged as a broad platform for the bottom-up construction of membraneless, molecularly crowded protocells, the absence of an enclosing membrane limits the construction of more sophisticated artificial cells and their use as functional cytomimetic materials. To address this problem, we and others have recently developed chemical-based strategies for the membranization of preformed coacervate microdroplets. In this Account, we review our recent work on diverse coacervate systems using a range of membrane building blocks and assembly processes. First, we briefly introduce the unusual nature of the coacervate/water interface, emphasizing the ultralow interfacial tension and broad interfacial width as physiochemical properties that require special attention in the judicious design of membranized coacervate microdroplets. Second, we classify membrane assembly into two different approaches: (i) interfacial self-assembly by using diverse surface-active building blocks such as molecular amphiphiles (fatty acids, phospholipids, block copolymers, protein-polymer conjugates) or nano- and microscale objects (liposomes, nanoparticle surfactants, cell fragments, living cells) with appropriate wettability; and (ii) coacervate droplet-to-vesicle reconfiguration by employing auxiliary surface reconstruction agents or triggering endogenous transitions (self-membranization) under nonstoichiometric (charge mismatched) conditions. We then discuss the key cytomimetic behaviors of membranized coacervate-based model protocells. Customizable permeability is achieved by synergistic effects operating between the molecularly crowded coacervate interior and surrounding membrane. In contrast, metabolic-like endogenous reactivity, diffusive chemical signaling, and collective chemical operations occur specifically in protocell networks comprising diverse populations of membranized coacervate microdroplets. In each case, these cytomimetic behaviors can give rise to functional microscale materials capable of promising cell-like applications. For example, immobilizing spatially segregated enzyme-loaded phospholipid-coated coacervate protocells in concentrically tubular hydrogels delivers prototissue-like bulk materials that generate nitric oxide in vitro, enabling platelet deactivation and inhibition of blood clot formation. Alternatively, therapeutic protocells with in vivo vasoactivity, high hemocompatibility, and increased blood circulation times are constructed by spontaneous assembly of hemoglobin-containing cell-membrane fragments on the surface of enzyme-loaded coacervate microdroplets. Higher-order properties such as artificial endocytosis are achieved by using nanoparticle-caged coacervate protocell hosts that selectively and actively capture guest nano- and microscale objects by responses to exogenous stimuli or via endogenous enzyme-mediated reactions. Finally, we discuss the current limitations in the design and programming of membranized coacervate microdroplets, which may help to guide future directions in this emerging research area. Taken together, we hope that this Account will inspire new advances in membranized coacervate microdroplets and promote their application in the development of integrated protocell models and functional cytomimetic materials.

The limits of metabolic heredity in protocells.

The universal core of metabolism could have emerged from thermodynamically favoured prebiotic pathways at the origin of life. Starting with H2 and CO2, the synthesis of amino acids and mixed fatty acids, which self-assemble into protocells, is favoured under warm anoxic conditions. Here, we address whether it is possible for protocells to evolve greater metabolic complexity, through positive feedbacks involving nucleotide catalysis. Using mathematical simulations to model metabolic heredity in protocells, based on branch points in protometabolic flux, we show that nucleotide catalysis can indeed promote protocell growth. This outcome only occurs when nucleotides directly catalyse CO2 fixation. Strong nucleotide catalysis of other pathways (e.g. fatty acids and amino acids) generally unbalances metabolism and slows down protocell growth, and when there is competition between catalytic functions cell growth collapses. Autocatalysis of nucleotide synthesis can promote growth but only if nucleotides also catalyse CO2 fixation; autocatalysis alone leads to the accumulation of nucleotides at the expense of CO2 fixation and protocell growth rate. Our findings offer a new framework for the emergence of greater metabolic complexity, in which nucleotides catalyse broad-spectrum processes such as CO2 fixation, hydrogenation and phosphorylation important to the emergence of genetic heredity at the origin of life.