Cell-Free Protein Expression by a Reconstituted Transcription-Translation System Energized by Sugar Catabolism.

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.

Metabolic Tug-of-War between Glycolysis and Translation Revealed by Biochemical Reconstitution.

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.

Multimolecular Competition Effect as a Modulator of Protein Localization and Biochemical Networks in Cell-Size Space.

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.

Controlling the Periodicity of a Reaction-Diffusion Wave in Artificial Cells by a Two-Way Energy Supplier.

Reaction-diffusion (RD) waves, which are dynamic self-organization structures generated by nanosize molecules, are a fundamental mechanism from patterning in nano- and micromaterials to spatiotemporal regulations in living cells, such as cell division and motility. Although the periods of RD waves are the critical element for these functions, the development of a system to control their period is challenging because RD waves result from nonlinear physical dynamics under far-from-equilibrium conditions. Here, we developed an artificial cell system with tunable period of an RD-driven wave (Min protein wave), which determines a cell division site plane in living bacterial cells. The developed system is based on our finding that Min waves are generated by energy consumption of either ATP or dATP, and the period of the wave is different between these two energy suppliers. We showed that the Min-wave period was modulated linearly by the mixing ratio of ATP and dATP and that it was also possible to estimate the mixing ratio of ATP and dATP from the period. Our findings illuminated a previously unidentified principle to control the dissipative dynamics of biomolecules and, simultaneously, built an important framework to construct molecular robots with spatiotemporal units.

Cytoplasmic delivery of siRNA using human-derived membrane penetration-enhancing peptide.

BACKGROUND: Although protein-based methods using cell-penetrating peptides such as TAT have been expected to provide an alternative approach to siRNA delivery, the low efficiency of endosomal escape of siRNA/protein complexes taken up into cells by endocytosis remains a problem. Here, to overcome this problem, we adopted the membrane penetration-enhancing peptide S19 from human syncytin 1 previously identified in our laboratory. RESULTS: We prepared fusion proteins in which the S19 and TAT peptides were fused to the viral RNA-binding domains (RBDs) as carrier proteins, added the RBD-S19-TAT/siRNA complex to human cultured cells, and investigated the cytoplasmic delivery of the complex and the knockdown efficiency of target genes. We found that the intracellular uptake of the RBD-S19-TAT/siRNA complex was increased compared to that of the RBD-TAT/siRNA complex, and the expression level of the target mRNA was decreased. Because siRNA must dissociate from RBD and bind to Argonaute 2 (Ago2) to form the RNA-induced silencing complex (RISC) after the protein/siRNA complex is delivered into the cytoplasm, a dilemma arises: stronger binding between RBD and siRNA increases intracellular uptake but makes RISC formation more difficult. Thus, we next prepared fusion proteins in which the S19 and TAT peptides were fused with Ago2 instead of RBD and found that the efficiencies of siRNA delivery and knockdown obtained using TAT-S19-Ago2 were higher than those using TAT-Ago2. In addition, we found that the smallest RISC delivery induced faster knockdown than traditional siRNA lipofection, probably due to the decreased time required for RISC formation in the cytoplasm. CONCLUSION: These results indicated that S19 and TAT-fused siRNA-binding proteins, especially Ago2, should be useful for the rapid and efficient delivery of siRNA without the addition of any endosome-disrupting agent.

Mode selection mechanism in traveling and standing waves revealed by Min wave reconstituted in artificial cells.

Reaction-diffusion coupling (RDc) generates spatiotemporal patterns, including two dynamic wave modes: traveling and standing waves. Although mode selection plays a substantial role in the spatiotemporal organization of living cell molecules, the mechanism for selecting each wave mode remains elusive. Here, we investigated a wave mode selection mechanism using Min waves reconstituted in artificial cells, emerged by the RDc of MinD and MinE. Our experiments and theoretical analysis revealed that the balance of membrane binding and dissociation from the membrane of MinD determines the mode selection of the Min wave. We successfully demonstrated that the transition of the wave modes can be regulated by controlling this balance and found hysteresis characteristics in the wave mode transition. These findings highlight a previously unidentified role of the balance between activators and inhibitors as a determinant of the mode selection of waves by RDc and depict an unexplored mechanism in intracellular spatiotemporal pattern formations.

Guanine nucleotide exchange factor DOCK11-binding peptide fused with a single chain antibody inhibits hepatitis B virus infection and replication.

Hepatitis B virus (HBV) infection is a major global health problem with no established cure. Dedicator of cytokinesis 11 (DOCK11), known as a guanine nucleotide exchange factor (GEF) for Cdc42, is reported to be essential for the maintenance of HBV. However, potential therapeutic strategies targeting DOCK11 have not yet been explored. We have previously developed an in vitro virus method as a more efficient tool for the analysis of proteomics and evolutionary protein engineering. In this study, using the in vitro virus method, we screened and identified a novel antiasialoglycoprotein receptor (ASGR) antibody, ASGR3-10M, and a DOCK11-binding peptide, DCS8-42A, for potential use in HBV infection. We further constructed a fusion protein (10M-D42AN) consisting of ASGR3-10M, DCS8-42A, a fusogenic peptide, and a nuclear localization signal to deliver the peptide inside hepatocytes. We show using immunofluorescence staining that 10M-D42AN was endocytosed into early endosomes and released into the cytoplasm and nucleus. Since DCS8-42A shares homology with activated cdc42-associated kinase 1 (Ack1), which promotes EGFR endocytosis required for HBV infection, we also found that 10M-D42AN inhibited endocytosis of EGFR and Ack1. Furthermore, we show 10M-D42AN suppressed the function of DOCK11 in the host DNA repair system required for covalently closed circular DNA synthesis and suppressed HBV proliferation in mice. In conclusion, this study realizes a novel hepatocyte-specific drug delivery system using an anti-ASGR antibody, a fusogenic peptide, and DOCK11-binding peptide to provide a novel treatment for HBV.

Characterization of the membrane penetration-enhancing peptide S19 derived from human syncytin-1 for the intracellular delivery of TAT-fused proteins.

Although cell-penetrating peptides such as the HIV-derived TAT peptide have been used as tools for the intracellular delivery of therapeutic peptides and proteins, a problem persists: the endosomal escape efficiency is low. Previously, we found that the fusogenic peptide S19, derived from the human protein syncytin-1, enhance the endosomal escape efficiency of proteins that incorporated by endocytosis via TAT. In this study, we first performed Ala-scanning mutagenesis of S19, and found that all Ile, Val, Leu and Phe with high ß-sheet forming propensities in S19 are important for the intracellular uptake of S19-TAT-fused proteins. In a secondary structure analysis of the mutated S19-TAT peptides in the presence of liposomes mimicking late endosomes (LEs), the CD spectra of V3A and I4A mutants with low uptake activity showed the appearance of an α-helix structure, whereas the mutant G5A retained both the uptake activity and the ß-structure. In addition, we investigated the appropriate linking position and order of the S19 and TAT peptides to a cargo protein including an apoptosis-induced peptide and found that both the previous C-terminal S19-TAT tag and the N-terminal TAT-S19 tag promote the cytoplasmic delivery of the fusion protein. These results and previous results suggest that the interaction of TAT with the LE membrane causes a structural change in S19 from a random coil to a ß-strand and that the subsequent parallel ß-sheet formation between two S19 peptides may promote adjacent TAT dimerization, resulting in endosomal escape from the LE membrane.

Activation of a diluted E. coli cell-free transcription-translation system within liposomes by hypertonic concentration.

We present a protocol for activating protein synthesis in liposomes encapsulating a diluted E. coli cell extract-based TX-TL (transcription-translation) system by hypertonic concentration. Protein expression is turned on in the liposome-encapsulated TX-TL system by simple treatment with a concentrated external solution. The expression of sfGFP is demonstrated here, but it can be applied to other proteins. This protocol can be applied to the development of artificial cells utilizing the switch-on mechanism to activate protein expression, responding to the outer environment. For complete details on the use and execution of this protocol, please refer to Akui et al. (2021).

Transcription-translation of the Escherichia coli genome within artificial cells.

Here we created artificial cells in which information of the genome of living cells is expressed by the elements encoded in the genome. We confirmed that the system works normally within artificial cells, which paves the way for reconstructing living cells from biomolecules.

System concentration shift as a regulator of transcription-translation system within liposomes.

Biochemical systems in living cells have their optimum concentration ratio among each constituent element to maintain their functionality. However, in the case of the biochemical system with complex interactions and feedbacks among elements, their activity as a system greatly changes by the concentration shift of the entire system irrespective of the concentration ratio among elements. In this study, by using a transcription-translation (TX-TL) system as the subject, we illustrate the principle of the nonlinear relationship between the system concentration and the activity of the system. Our experiment and simulation showed that shifts of the system concentration of TX-TL by dilution and concentration works as a switch of activity and demonstrated its ability to induce a biochemical system to confer the permeability of small molecules to liposomes. These results contribute to the creation of artificial cells with the switch and provide an insight into the emergence of protocells.

Switching ON of Transcription-Translation System Using GUV Fusion by Co-supplementation of Calcium with Long-Chain Polyethylene Glycol.

Giant unilamellar vesicles (GUVs) have been used as a material for bottom-up synthetic biology. However, due to the semi-permeability of the membrane, the need for methods to fuse GUVs has increased. To this aim, methods that are simple and show low leakage during fusion are important. In this study, we report a method of GUV fusion by a divalent cation (Ca2+ ) enhanced with a long chain polyethylene glycol (PEG20k). The methods showed significant GUV fusion without leakage of internal components of GUVs and maintained cell-free transcription-translation ability inside the GUVs without external supplementation of macromolecules. We demonstrate that the Ca-PEG method can be applied for switching ON of transcription-translation in GUVs in a fusion-dependent manner. The method developed here can be applied to extend bottom-up synthetic biology and molecular robotics that use GUVs as a chassis.

A Relationship between NTP and Cell Extract Concentration for Cell-Free Protein Expression.

The cell-free protein synthesis (CFPS) that synthesizes mRNA and protein from a template DNA has been featured as an important tool to emulate living systems in vitro. However, an obstacle to emulate living cells by CFPS is the loss of activity in the case of usage of high concentration cell extracts. In this study, we found that a high concentration of NTP which inhibits in the case of lower concentration cell extract restored the loss of CFPS activity using high concentration cell extracts. The NTP restoration was independent of the energy regeneration system used, and NTP derivatives also restored the levels of CFPS using a high concentration cell extract. Experiments using dialysis mode of CFPS showed that continuous exchange of small molecule reduced levels of NTP requirement and improved reaction speed of CFPS using the high concentration of cell extract. These findings contribute to the development of a method to understand the condition of living cells by in vitro emulation, and are expected to lead to the achievement of the reconstitution of living cells from biomolecule mixtures.

Conformational equilibrium of MinE regulates the allowable concentration ranges of a protein wave for cell division.

The Min system for determining the cell division position at the center in bacteria has a unique character that uses a protein wave (Min wave) that emerges from its components (MinD and MinE). The Min wave emerges under the coupling of chemical reactions and molecular diffusions of MinDE and appears when the concentrations of MinD and MinE are similar. However, the nanoscale mechanism to determine their concentration ranges has remained elusive. In this study, by using artificial cells as a mimic of cells, we showed that the dominant MinE conformations determined the allowable concentration ranges for the emergence of the Min wave. Furthermore, the deletion of the membrane-binding region of MinE indicated that the region was essential for limiting the concentration ranges to be narrower. These findings illustrate a parameter tuning mechanism underlying complex molecular systems at the nanoscale for spatiotemporal regulation in living cells and show a possibility that the regulation of the equilibrium among molecular conformations can work as a switch for cell division.

Microfluidic screening system based on boron-doped diamond electrodes and dielectrophoretic sorting for directed evolution of NAD(P)-dependent oxidoreductases.

We report the development of a micro total analysis system (µTAS) based on electrochemical measurements and dielectrophoretic sorting for screening of NAD(P)-dependent oxidoreductases. In this system, the activity of enzymes immobilized on microbeads, together with their encoding DNA, can be measured with a boron-doped diamond (BDD) electrode in each compartment (∼30 nL) of the microfluidic system. The 30 nL droplets containing microbead-displayed genes of enzymes with higher activity can then be recovered by dielectrophoretic sorting. Previously, we developed the NAD(P)H-measuring device containing the BDD electrode for high-throughput measurement of the activity of NAD(P)-dependent oxidoreductases. In this study, we fabricated an encapsulating device and a droplet-sorting device for nanoliter-size droplets, for the first time, and then combined these three devices to construct a µTAS for directed evolution of NAD(P)-dependent oxidoreductases. We confirmed that this system works by proof-of-principle experiments and successfully applied this system for screening of randomized libraries of NAD-dependent dehydrogenases.

Self-organization Assay for Min Proteins of Escherichia coli in Micro-droplets Covered with Lipids.

The Min system determines the cell division plane of bacteria. As a cue of spatiotemporal regulation, the Min system uses wave propagation of MinD protein (Min wave). Therefore, the reconstitution of the Min wave in cell-sized closed space will lead to the creation of artificial cells capable of cell division. The Min waves emerge via coupling between the reactions among MinD, MinE, and ATP and the differences in diffusion rate on the cell membrane and in the cytoplasm. Because Min waves appear only under the balanced condition of the reaction-diffusion coupling, special attentions are needed towards several technical points for the reconstitution of Min waves in artificial cells. This protocol describes a technical method for stably generating Min waves in artificial cells.

Cell-sized confinement controls generation and stability of a protein wave for spatiotemporal regulation in cells.

The Min system, a system that determines the bacterial cell division plane, uses changes in the localization of proteins (a Min wave) that emerges by reaction-diffusion coupling. Although previous studies have shown that space sizes and boundaries modulate the shape and speed of Min waves, their effects on wave emergence were still elusive. Here, by using a microsized fully confined space to mimic live cells, we revealed that confinement changes the conditions for the emergence of Min waves. In the microsized space, an increased surface-to-volume ratio changed the localization efficiency of proteins on membranes, and therefore, suppression of the localization change was necessary for the stable generation of Min waves. Furthermore, we showed that the cell-sized space strictly limits parameters for wave emergence because confinement inhibits both the instability and excitability of the system. These results show that confinement of reaction-diffusion systems has the potential to control spatiotemporal patterns in live cells.

Regulation of spatiotemporal patterning in artificial cells by a defined protein expression system.

Spatiotemporal patterning is a fundamental mechanism for developmental differentiation and homeostasis in living cells. Because spatiotemporal patterns are based on higher-order collective motions of elements synthesized from genes, their behavior dynamically changes according to the element amounts. Thus, to understand life and use this process for material application, creation of artificial cells with time development of spatiotemporal patterning by changes of element levels is necessary. However, realizing coupling between spatiotemporal patterning and synthesis of elements in artificial cells has been particularly challenging. In this study, we established a system that can synthesize a patterning mechanism of the bacterial cell division plane (the so-called Min system) in artificial cells by modifying a defined protein expression system and demonstrated that artificial cells can show time development of spatiotemporal patterning similar to living cells. This system also allows generation and disappearance of spatiotemporal patterning, is controllable by a small molecule in artificial cells, and has the ability for application in cargo transporters. The system developed here provides a new material and a technique for understanding life, development of drug delivery tools, and creation of molecular robots.

A dual system using compartmentalized partnered replication for selection of arsenic-responsive transcriptional regulator.

Engineering and design of genetic circuit in living cell is critical in accessing the beneficial application of synthetic biology. Directed evolution can avoid the complicated rational design of such circuit by screening or selecting functional circuit from non-functional one. Here, we proposed a positive-negative selection system for selecting a transcription factor that activates gene expression in response to arsenic in solution. First, we developed a whole cell biosensor for sensing arsenite in liquid using a regulator (ArsR) and a reporter (GFP), and evaluated its performance. Second, we developed a positive selection system for active ArsR using compartmentalized partnered replication that uses thermostable DNA polymerase as the reporter of activity. Third, we developed a negative selection system using sucrose-induced suicide gene SacB as the reporter for exclusion of inactive ArsR variants.

Engineering of DNA polymerase I from Thermus thermophilus using compartmentalized self-replication.

Although compartmentalized self-replication (CSR) and compartmentalized partnered replication (CPR) are powerful tools for directed evolution of proteins and gene circuits, limitations remain in the emulsion PCR process with the wild-type Taq DNA polymerase used so far, including long run times, low amounts of product, and false negative results due to inhibitors. In this study, we developed a high-efficiency mutant of DNA polymerase I from Thermus thermophilus HB27 (Tth pol) suited for CSR and CPR. We modified the wild-type Tth pol by (i) deletion of the N-terminal 5' to 3' exonuclease domain, (ii) fusion with the DNA-binding protein Sso7d, (iii) introduction of four known effective point mutations from other DNA polymerase mutants, and (iv) codon optimization to reduce the GC content. Consequently, we obtained a mutant that provides higher product yields than the conventional Taq pol without decreased fidelity. Next, we performed four rounds of CSR selection with a randomly mutated library of this modified Tth pol and obtained mutants that provide higher product yields in fewer cycles of emulsion PCR than the parent Tth pol as well as the conventional Taq pol.