The ability to evolve is a key characteristic that distinguishes living things from non-living chemical compounds. The construction of an evolvable cell-like system entirely from non-living molecules has been a major challenge. Here we construct an evolvable artificial cell model from an assembly of biochemical molecules. The artificial cell model contains artificial genomic RNA that replicates through the translation of its encoded RNA replicase. We perform a long-term (600-generation) replication experiment using this system, in which mutations are spontaneously introduced into the RNA by replication error, and highly replicable mutants dominate the population according to Darwinian principles. During evolution, the genomic RNA gradually reinforces its interaction with the translated replicase, thereby acquiring competitiveness against selfish (parasitic) RNAs. This study provides the first experimental evidence that replicating systems can be developed through Darwinian evolution in a cell-like compartment, even in the presence of parasitic replicators.

In all living systems, the genome is replicated by proteins that are encoded within the genome itself. This universal reaction is essential to allow the system to evolve. Here, we have constructed a simplified system involving encapsulated macromolecules termed a "self-encoding system", in which the genetic information is replicated by self-encoded replicase in liposomes. That is, the universal reaction was reconstituted within a microcompartment bound by a lipid bilayer. The system was assembled by using one template RNA sequence as the information molecule and an in vitro translation system reconstituted from purified translation factors as the machinery for decoding the information. In this system, the catalytic subunit of Qbeta replicase is synthesized from the template RNA that encodes the protein. The replicase then replicates the template RNA that was used for its production. This in-liposome self-encoding system is one of the simplest such systems available; it consists of only 144 gene products, while the information and the function for its replication are encoded on different molecules and are compartmentalized into the microenvironment for evolvability.

To date, various cellular functions have been reconstituted in vitro such as self-replication systems using DNA, RNA, and proteins. The next important challenges include the reconstitution of the interactive networks of self-replicating species and investigating how such interactions generate complex ecological behaviors observed in nature. Here, we synthesized a simple replication system composed of two self-replicating host and parasitic RNA species. We found that the parasitic RNA eradicates the host RNA under bulk conditions; however, when the system is compartmentalized, a continuous oscillation pattern in the population dynamics of the two RNAs emerges. The oscillation pattern changed as replication proceeded mainly owing to the evolution of the host RNA. These results demonstrate that a cell-like compartment plays an important role in host-parasite ecological dynamics and suggest that the origin of the host-parasite coevolution might date back to the very early stages of the evolution of life.arious functions of living organisms have been reconstituted in vitro from biological molecules to understand the basic principles underlying complex biological phenomena observed in the cell or in nature (1, 2). Several types of continuous systems of self-replication of genetic information have been constituted in vitro from RNA alone (3-6), and from the combinations of RNA and proteins (7) or of DNA, RNA, and proteins (8). One of the next important challenges is the reconstitution of interacting networks of self-replicating species, including the investigation of how such interactions generate complex ecological behaviors such as those observed in nature.Organisms in the wild self-reproduce and interact with each other to form ecosystems. Such interaction generates complex ecological behaviors such as the oscillation dynamics observed in prey-predator or host-parasite populations (9-12). The causes and results of such periodic patterning have been a focus of intensive debate for decades (13)(14)(15)(16)(17). Such interactions and the resultant coevolution of the interacting species are thought to be an important factor explaining the high degree of extant biodiversity and complexity (16,18). However, to date, the in vitro reconstitution of such ecological behavior has been limited; one example is of prey-predator oscillation dynamics constructed using a combination of small hybridizing DNA fragments and several enzymes, although the DNA fragment did not evolve in this system (19).Previously, we had constructed a simple translation-coupled RNA replication system in which an artificial genomic RNA replicated through translation of the self-encoded RNA replicase and evolved (7). This system consists of a reconstituted translation system of Escherichia coli and an RNA encoding the RNA replicase (Qβ replicase), which is composed of the catalytic β-subunit and the translational factors, EF-Tu and EF-Ts. We found that parasitic RNAs, which had lost their replicase encoding region, spontaneously appeared in this system at ...

Lysylphosphatidylglycerol (LPG) is a basic phospholipid in which L-lysine from lysyl-tRNA is transferred to phosphatidylglycerol (PG). This study examined whether the Staphylococcus aureus mprF gene encodes LPG synthetase. A crude membrane fraction prepared from wild-type S. aureus cells had LPG synthetase activity that depended on PG and lysyl-tRNA, whereas the membrane fraction from an mprF deletion mutant did not. When S. aureus MprF protein was trans-expressed in wild-type Escherichia coli cells, LPG synthesis was induced, whereas it was not observed in E. coli pgsA3 mutant cells in which the amount of PG is significantly reduced. In addition, LPG synthetase activity and a 93 kDa protein whose molecular size corresponded to that of MprF protein were co-induced in the crude membrane fraction prepared from E. coli cells expressing MprF protein. The K m values of the LPG synthetase activity for PG and for lysyl-tRNA were 56 mM and 6?9 mM, respectively, consistent with those of S. aureus membranes. These results suggest that the MprF protein is LPG synthetase.

A major challenge in constructing artificial cells is the establishment of a recursive genome replication system coupled with gene expression from the genome itself. One of the simplest schemes of recursive DNA replication is the rolling-circle replication of a circular DNA coupled with recombination. In this study, we attempted to develop a replication system based on this scheme using self-encoded phi29 DNA polymerase and externally supplied Cre recombinase. We first identified that DNA polymerization is significantly inhibited by Cre recombinase. To overcome this problem, we performed in vitro evolution and obtained an evolved circular DNA that can replicate efficiently in the presence of the recombinase. We also showed evidence that during replication of the evolved DNA, the circular DNA was reproduced through recombination by Cre recombinase. These results demonstrate that the evolved circular DNA can reproduce itself through gene expression of a self-encoded polymerase. This study provides a step forward in developing a simple recursive DNA replication system for use in an artificial cell.

Cooperation among independently replicating molecules is a key phenomenon that allowed the development of complexity during the early evolution of life. Generally, this process is vulnerable to parasitic or selfish entities, which can easily appear and destroy such cooperation. It remains unclear how this fragile cooperation process appeared and has been sustained through evolution. Theoretical studies have indicated that spatial structures, such as compartments, allow sustainable replication and the evolution of cooperative replication, although this has yet to be confirmed experimentally. In this study, we constructed a molecular cooperative replication system, in which two types of RNA, encoding replication or metabolic enzymes, cooperate for their replication in compartments, and we performed long-term replication experiments to examine the sustainability and evolution of the RNAs. We demonstrate that the cooperative relationship of the two RNAs could be sustained at a certain range of RNA concentrations, even when parasitic RNA appeared in the system. We also found that more efficient cooperative RNA replication evolved during long-term replication through seemingly selfish evolution of each RNA. Our results provide experimental evidence supporting the sustainability and robustness of molecular cooperation on an evolutionary timescale.

All living organisms have a genome replication system in which genomic DNA is replicated by a DNA polymerase translated from mRNA transcribed from the genome. The artificial reconstitution of this genome replication system is a great challenge in in vitro synthetic biology. In this study, we attempted to construct a transcription- and translation-coupled DNA replication (TTcDR) system using circular genomic DNA encoding phi29 DNA polymerase and a reconstituted transcription and translation system. In this system, phi29 DNA polymerase was translated from the genome and replicated the genome in a rolling-circle manner. When using a traditional translation system composition, almost no DNA replication was observed, because the tRNA and nucleoside triphosphates included in the translation system significantly inhibited DNA replication. To minimize these inhibitory effects, we optimized the composition of the TTcDR system and improved replication by approximately 100-fold. Using our system, genomic DNA was replicated up to 10 times in 12 hours at 30 °C. This system provides a step toward the in vitro construction of an artificial genome replication system, which is a prerequisite for the construction of an artificial cell.

Increasingly complex reactions are being constructed by bottom-up approaches with the aim of developing an artificial cell. We have been engaged in the construction of a translation-coupled replication system of genetic information from RNA and a reconstituted translation system. Here a mathematical model was established to gain a quantitative understanding of the complex reaction network. The sensitivity analysis predicted that the limiting factor for the present replication reaction was the appearance of parasitic replicators. We then confirmed experimentally that repression of such parasitic replicators by compartmentalization of the reaction in water-in-oil emulsions improved the duration of self-replication. We also found that the main source of the parasite was genomic RNA, probably by nonhomologous recombination. This result provided experimental evidence for the importance of parasite repression for the development of long-lasting genome replication systems.