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.

Significance The directed evolution of proteins in vitro has generated highly functional proteins and has contributed to elucidating the sequence–function relationship of proteins. However, the available methods consider globular proteins, not membrane proteins, despite the biological and pharmaceutical importance of the latter. We report the development of a method called liposome display, which enables the in vitro evolution of membrane proteins. We applied the method to evolve the pore-forming activity of α-hemolysin from Staphylococcus aureus and obtained a mutant with 30-fold higher activity than the WT. Given its high degree of controllability, liposome display allows for the rapid and efficient evolution of a wide range of membrane proteins, thereby improving the field of membrane protein engineering.

Liposome display is a novel method for in vitro selection and directed evolution of membrane proteins. In this approach, membrane proteins of interest are displayed on liposome membranes through translation from a single DNA molecule by using an encapsulated cell-free translation system. The liposomes are probed with a fluorescence indicator that senses membrane protein activity and selected using a fluorescence-activated cell sorting (FACS) instrument. Consequently, DNA encoding a protein with a desired function can be obtained. By implementing this protocol, researchers can process a DNA library of 10(7) different mutants. A single round of the selection procedure requires 24 h for completion, and multiple iterations of this technique, which take 1-5 weeks, enable the isolation of a desired gene. As this protocol is conducted entirely in vitro, it enables the engineering of various proteins, including pore-forming proteins, transporters and receptors. As a useful example of the approach, here we detail a procedure for the in vitro evolution of α-hemolysin from Staphylococcus aureus for its pore-forming activity.

Liposomes are widely used as cell-sized compartments for encapsulation of biochemical reaction systems to construct model cell systems. However, liposomes are usually diverse in both size and structure, resulting in highly heterogeneous properties as microreactors. Here, we report the development of a strategy to investigate the internal structure of giant multilamellar vesicles (GMLVs) formed by the freeze-dried empty liposomes (FDEL) method as containers of an in vitro transcription/translation system. To evaluate the occurrence of the protein synthesis reaction in GMLVs, we designed a cascade reaction system in which a synthesized enzyme hydrolyzes the fluorescent substrate, and thus the space where the reaction takes place in liposomes becomes fluorescent. We found that only a part of the liposome was reactable and not the entire internal volume, i.e., the hydrolysis reaction took place in only a part of the fractured compartment volumes in GMLVs. Simultaneous measurement of the whole internal volume of the liposomes and the quantity of reaction product of more than 100 000 liposomes using a fluorescence-activated cell sorter (FACS) revealed that the distribution of reactable volume was proportional to the whole internal volume regardless of the liposome size, i.e., the relation between the quantity of whole and reactable volume in GMLV was found to be scale-free. This information would allow us to reduce the geometric parameters of GMLV for quantitative analysis of reaction kinetics in liposomes. The present measurement and analysis method will be an indispensable tool for exploring high-dimensional properties of a model cell system based on giant liposomes.

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.

We have isolated a cDNA encoding UDP-glucose pyrophosphorylase from a cDNA library of immature potato tuber using oligonucleotide probes synthesized on the basis of partial amino acid sequences of the enzyme. The cDNA clone contained a 1,758-base-pair insert including the complete message for UDP-glucose pyrophosphorylase with 1,431 base pairs. The amino acid sequence of the enzyme inferred from the nucleotide sequence consists of 477 amino acid residues. All the partial amino acid sequences determined protein-chemically [Nakano et al. (1989) J. Biochem. 106, 528-532] confirmed the primary structure of the enzyme. An N-terminal-blocked peptide was isolated from the proteolytic digest of the enzyme protein, and the blocking group was deduced to be an acetyl group by fast atom bombardment-mass spectrometry. On the basis of the predicted amino acid sequence (477 residues minus the N-terminal Met plus an acetyl group), the molecular weight of the enzyme monomer is calculated to be 51,783, which agrees well with the value determined by polyacrylamide gel electrophoresis. In the cDNA structure, the open-reading frame is preceded by a 125-base-pair noncoding region, which contains a sequence being homologous with the consensus sequence for plant genes, and is followed by a 174-base-pair noncoding sequence including a polyadenylation signal. Amino acid sequence comparisons revealed that the potato UDP-glucose pyrophosphorylase is homologous to the enzyme from slime mold, Dictyostelium discoideum, but not to ADP-glucose pyrophosphorylases from rice seed and Escherichia coli.

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.

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