Cooperation between catabolism and anabolism is crucial for maintaining homeostasis in living cells. The most fundamental systems for catabolism and anabolism are the glycolysis of sugars and the transcription-translation (TX-TL) of DNA, respectively. Despite their importance in living cells, the in vitro reconstitution of their cooperation through purified factors has not been achieved, which hinders the elucidation of the design principle in living cells. Here, we reconstituted glycolysis using sugars and integrated it with the PURE system, a commercial in vitro TX-TL kit composed of purified factors. By optimizing key parameters, such as glucokinase and initial phosphate concentrations, we determined suitable conditions for their cooperation. The optimized system showed protein synthesis at up to 33% of that of the original PURE system. We observed that ATP consumption in upstream glycolysis inhibits TX-TL and that this inhibition can be alleviated by the co-addition of glycolytic intermediates, such as glyceraldehyde 3-phosphate, with glucose. Moreover, the system developed here simultaneously synthesizes a subset of its own enzymes, that is, glycolytic enzymes, in a single test tube, which is a necessary step toward self-replication. As glycolysis and TX-TL provide building blocks for constructing cells, the integrated system can be a fundamental material for reconstituting living cells from purified factors.

Inside cells, various biological systems work cooperatively for homeostasis and self-replication. These systems do not work independently as they compete for shared elements like ATP and NADH. However, it has been believed that such competition is not a problem in codependent biological systems such as the energy-supplying glycolysis and the energy-consuming translation system. In this study, we biochemically reconstituted the coupling system of glycolysis and translation using purified elements and found that the competition for ATP between glycolysis and protein synthesis interferes with their coupling. Both experiments and simulations revealed that this interference is derived from a metabolic tug-of-war between glycolysis and translation based on their reaction rates, which changes the threshold of the initial substrate concentration for the success coupling. By the metabolic tug-of-war, translation energized by strong glycolysis is facilitated by an exogenous ATPase, which normally inhibits translation. These findings provide chemical insights into the mechanism of competition among biological systems in living cells and provide a framework for the construction of synthetic metabolism in vitro.

In order to recapitulate complex eukaryotic compartmentalization, synthetic biology aims to recreate cellular membrane-lined compartments from the bottom-up. Many important cellular organelles and cell-produced extracellular vesicles are in the size range of several hundreds of nanometers. Although attaining a fundamental characterization and mimicry of their cellular functions is a compelling goal, the lack of methods for controlled vesicle formation in this size range has hindered full understanding. Here, we show the optimization of a simple and efficient protocol for the production of large unilamellar vesicles (LUVs) with a median diameter in the range of 450-550 nm with high purity. Importantly, we rely on commercial reagents and common laboratory equipment. We thoroughly characterize the influence of different experimental parameters on the concentration and size of the resulting vesicles and assess changes in their lipid composition and surface charge. We provide guidance for researchers to optimize LUV production further to suit specific applications.

Methanol has gained substantial attention as a substrate for biomanufacturing due to plentiful stocks and nonreliance on agriculture, and it can be sourced renewably. However, due to inevitable complexities in cell metabolism, microbial methanol conversion requires further improvement before industrial applicability. Here, we present a novel, parallel strategy using artificial cells to provide a simplified and well-defined environment for methanol utilization as artificial methylotrophic cells. We compartmentalized a methanol-utilizing enzyme cascade, including NAD-dependent methanol dehydrogenase (Mdh) and pyruvate-dependent aldolase (KHB aldolase), in cell-sized lipid vesicles using the inverted emulsion method. The reduction of cofactor NAD+ to NADH was used to quantify the conversion of methanol within individual artificial methylotrophic cells via flow cytometry. Compartmentalization of the reaction cascade in liposomes led to a 4-fold higher NADH production compared with bulk enzyme experiments, and the incorporation of KHB aldolase facilitated another 2-fold increase above the Mdh-only reaction. This methanol-utilizing platform can serve as an alternative route to speed up methanol biological conversion, eventually shifting sugar-based bioproduction toward a sustainable methanol bioeconomy.

Cells are small, closed spaces filled with various types of macromolecules. Although it is shown that the characteristics of biochemical reactions in vitro are quite different from those in living cells, the role of the co-existence of various macromolecules in cell-size space remains still elusive. Here, using a constructive approach, it is demonstrated that the co-existence of various macromolecules themselves has the ability to tune protein localization for spatiotemporal regulation and a biochemical reaction system in a cell-size space. Both experimental and theoretical analyses reveal that enhancement of interfacial effects by a large surface-area-to-volume ratio facilitates membrane localization of molecules in the cell-size space, and the interfacial effects are alleviated by competitive binding to lipid membranes among multiple proteins even if their membrane affinities are weak. These results indicate that competition for membrane binding among various macromolecules in the cell-size space plays a role in regulating the spatiotemporal molecular organization and biochemical reaction networks. These findings shed light on the importance of surrounding molecules for biochemical reactions using purified elements in small spaces.

The ongoing COVID-19 pandemic has been brought on by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike glycoprotein (S), which decorates the viral envelope forming a corona, is responsible for the binding to the angiotensin-converting enzyme 2 (ACE2) receptor and initiating the infection. In comparison to previous variants, Omicron S presents additional binding sites as well as a more positive surface charge. These changes hint at additional molecular targets for interactions between virus and cell, such as the cell membrane or proteoglycans on the cell surface. Herein, bottom-up assembled synthetic SARS-CoV-2 miniviruses (MiniVs), with a lipid composition similar to that of infectious particles, are implemented to study and compare the binding properties of Omicron and Alpha variants. Toward this end, a systematic functional screening is performed to study the binding ability of Omicron and Alpha S proteins to ACE2-functionalized and nonfunctionalized planar supported lipid bilayers. Moreover, giant unilamellar vesicles are used as a cell membrane model to perform competitive interaction assays of the two variants. Finally, two cell lines with and without presentation of the ACE2 receptor are used to confirm the binding properties of the Omicron and Alpha MiniVs to the cellular membrane. Altogether, the results reveal a significantly higher affinity of Omicron S toward both the lipid membrane and ACE2 receptor. The research presented here highlights the advantages of creating and using bottom-up assembled SARS-CoV-2 viruses to understand the impact of changes in the affinity of S for ACE2 in infection studies.

The development and bottom-up assembly of synthetic cells with a functional cytoskeleton sets a major milestone to understand cell mechanics and to develop man-made machines on the nano- and microscale. However, natural cytoskeletal components can be difficult to purify, deliberately engineer and reconstitute within synthetic cells which therefore limits the realization of multifaceted functions of modern cytoskeletons in synthetic cells. Here, we review recent progress in the development of synthetic cytoskeletons made from deoxyribonucleic acid (DNA) as a complementary strategy. In particular, we explore the capabilities and limitations of DNA cytoskeletons to mimic functions of natural cystoskeletons like reversible assembly, cargo transport, force generation, mechanical support and guided polymerization. With recent examples, we showcase the power of rationally designed DNA cytoskeletons for bottom-up assembled synthetic cells as fully engineerable entities. Nevertheless, the realization of dynamic instability, self-replication and genetic encoding as well as contractile force generating motors remains a fruitful challenge for the complete integration of multifunctional DNA-based cytoskeletons into synthetic cells.

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.

Artificial cells are membrane vesicles mimicking cellular functions. To date, giant unilamellar vesicles made from a single lipid membrane with a diameter of 10 μm or more have been used to create artificial cells. However, the creation of artificial cells that mimic the membrane structure and size of bacteria has been limited due to technical restrictions of conventional liposome preparation methods. Here, we created bacteria-sized large unilamellar vesicles (LUVs) with proteins localized asymmetrically to the lipid bilayer. Liposomes containing benzylguanine-modified phospholipids were prepared by combining the conventional water-in-oil emulsion method and the extruder method, and green fluorescent protein fused with SNAP-tag was localized to the inner leaflet of the lipid bilayer. Biotinylated lipid molecules were then inserted externally, and the outer leaflet was modified with streptavidin. The resulting liposomes had a size distribution in the range of 500-2000 nm with a peak at 841 nm (the coefficient of variation was 10.3%), which was similar to that of spherical bacterial cells. Fluorescence microscopy, quantitative evaluation using flow cytometry, and western blotting proved the intended localization of different proteins on the lipid membrane. Cryogenic electron microscopy and quantitative evaluation by α-hemolysin insertion revealed that most of the created liposomes were unilamellar. Our simple method for the preparation of bacteria-sized LUVs with asymmetrically localized proteins will contribute to the creation of artificial bacterial cells for investigating functions and the significance of their surface structure and size.

Extracellular vesicles (EVs) are lipid membrane-enclosed compartments released by cells for intercellular communication in homeostasis and disease. Studies have shown great therapeutic potential of EVs, including but not limited to regenerative and immunomodulatory therapies. Additionally, EVs are promising next-generation drug delivery systems due to their biocompatibility, low immunogenicity, and inherent target specificity. However, clinical application of EVs is so far limited due to challenges in scaling up production, high heterogeneity, batch-to-batch variation, and limited control over composition. Although attaining a fundamental characterization of EVs\' functions is a compelling goal, these limitations have hindered a full understanding. Therefore, there is rising interest in exploiting the beneficial properties of EVs while gaining better control over their production and composition. Herein, we describe a method for the bottom-up assembly of bioinspired, fully synthetic vesicles that mimic the most important biophysical and biochemical properties of natural EVs.