Cell-free expression of RuBisCO for ATP production in the synthetic cells.

Recent advances in bottom-up synthetic biology have made it possible to reconstitute cellular systems from non-living components, yielding artificial cells with potential applications in industry, medicine and basic research. Although a variety of cellular functions and components have been reconstituted in previous studies, sustained biological energy production remains a challenge. ATP synthesis via ribulose-1,5-diphosphate carboxylase/oxygenase (RuBisCO), a central enzyme in biological CO2 fixation, holds potential as an energy production system, but its feasibility in a cell-free expression system has not yet been tested. In this study, we test RuBisCO expression and its activity-mediated ATP synthesis in a reconstituted Escherichia coli-based cell-free translation system. We then construct a system in which ATP is synthesized by RuBisCO activity in giant vesicles and used as energy for translation reactions. These results represent an advance toward independent energy production in artificial cells. Graphical Abstract.

Emergence of linkage between cooperative RNA replicators encoding replication and metabolic enzymes through experimental evolution.

The integration of individually replicating genes into a primitive chromosome is a key evolutionary transition in the development of life, allowing the simultaneous inheritance of genes. However, how this transition occurred is unclear because the extended size of primitive chromosomes replicate slower than unlinked genes. Theoretical studies have suggested that a primitive chromosome can evolve in the presence of cell-like compartments, as the physical linkage prevents the stochastic loss of essential genes upon division, but experimental support for this is lacking. Here, we demonstrate the evolution of a chromosome-like RNA from two cooperative RNA replicators encoding replication and metabolic enzymes. Through their long-term replication in cell-like compartments, linked RNAs emerged with the two cooperative RNAs connected end-to-end. The linked RNAs had different mutation patterns than the two unlinked RNAs, suggesting that they were maintained as partially distinct lineages in the population. Our results provide experimental evidence supporting the plausibility of the evolution of a primitive chromosome from unlinked gene fragments, an important step in the emergence of complex biological systems.

Minimal RNA self-reproduction discovered from a random pool of oligomers.

The emergence of RNA self-reproduction from prebiotic components would have been crucial in developing a genetic system during the origins of life. However, all known self-reproducing RNA molecules are complex ribozymes, and how they could have arisen from abiotic materials remains unclear. Therefore, it has been proposed that the first self-reproducing RNA may have been short oligomers that assemble their components as templates. Here, we sought such minimal RNA self-reproduction in prebiotically accessible short random RNA pools that undergo spontaneous ligation and recombination. By examining enriched RNA families with common motifs, we identified a 20-nucleotide (nt) RNA variant that self-reproduces via template-directed ligation of two 10 nt oligonucleotides. The RNA oligomer contains a 2'-5' phosphodiester bond, which typically forms during prebiotically plausible RNA synthesis. This non-canonical linkage helps prevent the formation of inactive complexes between self-complementary oligomers while decreasing the ligation efficiency. The system appears to possess an autocatalytic property consistent with exponential self-reproduction despite the limitation of forming a ternary complex of the template and two substrates, similar to the behavior of a much larger ligase ribozyme. Such a minimal, ribozyme-independent RNA self-reproduction may represent the first step in the emergence of an RNA-based genetic system from primordial components. Simultaneously, our examination of random RNA pools highlights the likelihood that complex species interactions were necessary to initiate RNA reproduction.

Investigation of Compatibility between DNA Replication, Transcription, and Translation for in Vitro Central Dogma.

Recent advances in in vitro synthetic biology have made it possible to reconstitute various cellular functions in a test tube. However, the integration of these functions remains a major challenge. This study aimed to identify a suitable condition to achieve all three reactions that constitute the central dogma: transcription, translation, and DNA replication. Specifically, we investigated the effect of the concentrations of 11 nonprotein factors required for in vitro transcription, translation, and DNA replication on each of these reactions. Our results indicate that certain factors have opposing effects on the three reactions. For example, while dNTP is necessary for DNA replication, it inhibited translation, and both rNTP and tRNA, which are essential for transcription and translation, inhibited DNA replication with several DNA polymerases. We also found that these opposing effects were partially alleviated by optimizing the magnesium concentration. Using this knowledge, we successfully demonstrated transcription/translation-coupled DNA replication with higher levels of transcription and translation while maintaining a certain level of DNA replication. These findings not only provide useful insights for the development of a complex artificial system with the central dogma but also raise the question of how natural cells overcome the incompatibility between the three reactions.

Progresses in Cell-Free In Vitro Evolution.

Biopolymers, such as proteins and RNA, are integral components of living organisms and have evolved through a process of repeated mutation and selection. The technique of "cell-free in vitro evolution" is a powerful experimental approach for developing biopolymers with desired functions and structural properties. Since Spiegelman's pioneering work over 50 years ago, biopolymers with a wide range of functions have been developed using in vitro evolution in cell-free systems. The use of cell-free systems offers several advantages, including the ability to synthesize a wider range of proteins without the limitations imposed by cytotoxicity, and the capacity for higher throughput and larger library sizes than cell-based evolutionary experiments. In this chapter, we provide a comprehensive overview of the progress made in the field of cell-free in vitro evolution by categorizing evolution into directed and undirected. The biopolymers produced by these methods are valuable assets in medicine and industry, and as a means of exploring the potential of biopolymers.

In Vitro Transcription/Translation-Coupled DNA Replication through Partial Regeneration of 20 Aminoacyl-tRNA Synthetases.

The in vitro reconstruction of life-like self-reproducing systems is a major challenge in in vitro synthetic biology. Self-reproduction requires regeneration of all molecules involved in DNA replication, transcription, and translation. This study demonstrated the continuous DNA replication and partial regeneration of major translation factors, 20 aminoacyl-tRNA synthetases (aaRS), in a reconstituted transcription/translation system (PURE system) for the first time. First, we replicated each DNA that encodes one of the 20 aaRSs through aaRS expression from the DNA by serial transfer experiments. Thereafter, we successively increased the number of aaRS genes and achieved simultaneous, continuous replication of DNA that encodes all 20 aaRSs, which comprised approximately half the number of protein factors in the PURE system, except for ribosomes, by employing dialyzed reaction and sequence optimization. This study provides a step-by-step methodology for continuous DNA replication with an increasing number of self-regenerative genes toward self-reproducing artificial systems.

Genetic analysis of Bacillus subtilis stable L-forms obtained via long-term cultivation.

Various bacteria can change to a spherical cell-wall-deficient state, called L-from, in the presence of antibiotics that inhibit cell wall synthesis. L-forms are classified into two types: unstable and stable L-forms. Unstable L-forms revert to a normal walled state in the absence of antibiotics, while stable L-forms remain in their wall-deficient state. The conversion from unstable to stable L-forms has been often observed during long-term cultivation. However, the genetic cause for this conversion is not yet fully understood. Here, we obtained stable Bacillus subtilis L-form strains from unstable L-form strains via three independent long-term culturing experiments. The whole genome sequencing of the long-cultured strains identified many mutations, and some mutations were commonly found in all three long-cultured strains. The knockout strain of one of the commonly mutated genes, tagF, in the ancestral strain lost the ability to revert to walled state (rod shape), supporting that eliminating the function of tagF gene is one of the possible methods to convert unstable L forms to a stable state.

Plausible pathway for a host-parasite molecular replication network to increase its complexity through Darwinian evolution.

How the complexity of primitive self-replication molecules develops through Darwinian evolution remains a mystery with regards to the origin of life. Theoretical studies have proposed that coevolution with parasitic replicators increases network complexity by inducing inter-dependent replication. Particularly, Takeuchi and Hogeweg proposed a complexification process of replicator networks by successive appearance of a parasitic replicator followed by the addition of a new host replicator that is resistant to the parasitic replicator. However, the feasibility of such complexification with biologically relevant molecules is still unknown owing to the lack of an experimental model. Here, we investigated the plausible complexification pathway of host-parasite replicators using both an experimental host-parasite RNA replication system and a theoretical model based on the experimental system. We first analyzed the parameter space that allows for sustainable replication in various replication networks ranging from a single molecule to three-member networks using computer simulation. The analysis shows that the most plausible complexification pathway from a single host replicator is the addition of a parasitic replicator, followed by the addition of a new host replicator that is resistant to the parasite, consistent with the previous study by Takeuchi and Hogeweg. We also provide evidence that the pathway actually occurred in our previous evolutionary experiment. These results provide experimental evidence that a population of a single replicator spontaneously evolves into multi-replicator networks through coevolution with parasitic replicators.

Experimental evidence for the correlation between RNA structural fluctuations and the frequency of beneficial mutations.

RNA has been used as a model molecule to understand the adaptive evolution process owing to the simple relationship between the structure (i.e., phenotype) and sequence (i.e., genotype). RNA usually forms multiple substructures with similar thermodynamic stabilities, called structural fluctuations. Ancel and Fontana theoretically proposed that structural fluctuation is directly related to the ease of change in structures by mutations and thus works as a source of adaptive evolution; however, experimental verification is limited. Here, we analyzed 76 RNA genotypes that appeared in our previous in vitro evolution to examine whether (i) RNA fluctuation decreases as adaptive evolution proceeds and (ii) RNAs that have larger fluctuations tend to have higher frequencies of beneficial mutations. We first computationally estimated the structural fluctuations of all RNAs and observed that they tended to decrease as their fitness increased. We next measured the frequency of beneficial mutations for 10 RNA genotypes and observed that the total number of beneficial mutations was correlated with the size of the structural fluctuations. These results consistently support the idea that the structural fluctuation of RNA, at least those evaluated in our study, works as a source of adaptive evolution.

Transfer RNA Synthesis-Coupled Translation and DNA Replication in a Reconstituted Transcription/Translation System.

Transfer RNAs (tRNAs) are key molecules involved in translation. In vitro synthesis of tRNAs and their coupled translation are important challenges in the construction of a self-regenerative molecular system. Here, we first purified EF-Tu and ribosome components in a reconstituted translation system of Escherichia coli to remove residual tRNAs. Next, we expressed 15 types of tRNAs in the repurified translation system and performed translation of the reporter luciferase gene depending on the expression. Furthermore, we demonstrated DNA replication through expression of a tRNA encoded by DNA, mimicking information processing within the cell. Our findings highlight the feasibility of an in vitro self-reproductive system, in which tRNAs can be synthesized from replicating DNA.

How evolution builds up complexity?: In vitro evolution approaches to witness complexification in artificial molecular replication systems.

How can evolution assemble lifeless molecules into a complex living organism? The emergent process of biological complexity in the origin of life is a big mystery in biology. In vitro evolution of artificial molecular replication systems offers unique experimental opportunities to probe possible pathways of a simple molecular system approaching a complex life-like system. This review focuses on experimental efforts to examine evolvability of molecules in vitro from the pioneering Spiegelman's experiment to our latest research on an artificial RNA self-replication system. Genetic translation and compartmentalization are shown to enable sustainable replication and evolution. Latest studies are revealing that coevolution of self-replicating "host replicators" and freeloading "parasitic replicators" is crucial to extend evolvability of a molecular replication system for continuous evolution and emergence of diversity. Intense competition between hosts and parasites would have existed even before the origin of life and contributed to generating complex molecular ecosystems. This review article is an extended version of the Japanese article "An in vitro evolutionary journey of an artificial RNA replication system towards biological complexity" published in SEIBUTSU-BUTSURI Vol.61, p.240-244 (2021)."

Evolutionary transition from a single RNA replicator to a multiple replicator network.

In prebiotic evolution, self-replicating molecules are believed to have evolved into complex living systems by expanding their information and functions open-endedly. Theoretically, such evolutionary complexification could occur through successive appearance of novel replicators that interact with one another to form replication networks. Here we perform long-term evolution experiments of RNA that replicates using a self-encoded RNA replicase. The RNA diversifies into multiple coexisting host and parasite lineages, whose frequencies in the population initially fluctuate and gradually stabilize. The final population, comprising five RNA lineages, forms a replicator network with diverse interactions, including cooperation to help the replication of all other members. These results support the capability of molecular replicators to spontaneously develop complexity through Darwinian evolution, a critical step for the emergence of life.

Continuous Cell-Free Replication and Evolution of Artificial Genomic DNA in a Compartmentalized Gene Expression System.

In all living organisms, genomic DNA continuously replicates by the proteins encoded in itself and undergoes evolution through many generations of replication. This continuous replication coupled with gene expression and the resultant evolution are fundamental functions of living things, but they have not previously been reconstituted in cell-free systems. In this study, we combined an artificial DNA replication scheme with a reconstituted gene expression system and microcompartmentalization to realize these functions. Circular DNA replicated through rolling-circle replication followed by homologous recombination catalyzed by the proteins, phi29 DNA polymerase, and Cre recombinase expressed from the DNA. We encapsulated the system in microscale water-in-oil droplets and performed serial dilution cycles. Isolated circular DNAs at Round 30 accumulated several common mutations, and the isolated DNA clones exhibited higher replication abilities than the original DNA due to its improved ability as a replication template, increased polymerase activity, and a reduced inhibitory effect of polymerization by the recombinase. The artificial genomic DNA, which continuously replicates using self-encoded proteins and autonomously improves its sequence, provides a useful starting point for the development of more complex artificial cells.

Primitive Compartmentalization for the Sustainable Replication of Genetic Molecules.

Sustainable replication and evolution of genetic molecules such as RNA are likely requisites for the emergence of life; however, these processes are easily affected by the appearance of parasitic molecules that replicate by relying on the function of other molecules, while not contributing to their replication. A possible mechanism to repress parasite amplification is compartmentalization that segregates parasitic molecules and limits their access to functional genetic molecules. Although extent cells encapsulate genomes within lipid-based membranes, more primitive materials or simple geological processes could have provided compartmentalization on early Earth. In this review, we summarize the current understanding of the types and roles of primitive compartmentalization regarding sustainable replication of genetic molecules, especially from the perspective of the prevention of parasite replication. In addition, we also describe the ability of several environments to selectively accumulate longer genetic molecules, which could also have helped select functional genetic molecules rather than fast-replicating short parasitic molecules.

Exchange of Proteins in Liposomes through Streptolysin O Pores.

Liposomes, which are vesicles surrounded by lipid membranes, can be used as biochemical reactors by encapsulating various reactions. Accordingly, they are useful for studying cellular functions under controlled conditions that mimic the environment within a cell. However, one of the shortcomings of liposomes as biochemical reactors is the difficulty of introducing or removing proteins due to the impermeability of the membrane. In this study, we established a method for exchanging proteins in liposomes by forming reversible pores in the membrane. We used the toxic protein streptolysin O (SLO); this forms pores in membranes made of phospholipids containing cholesterol that can be closed by the addition of calcium ions. After optimizing the experimental procedure and lipid composition, we observed the exchange of fluorescent proteins (transferrin Alexa Fluor 488 and 647) in 9.9 % of liposomes. We also introduced T7 RNA polymerase, a 98-kDa enzyme, and observed RNA synthesis in ∼8 % of liposomes. Our findings establish a new method for controlling the internal protein composition of liposomes, thereby increasing their utility as bioreactors.

Relaxed Substrate Specificity in Qß Replicase through Long-Term In Vitro Evolution.

A change from RNA- to DNA-based genetic systems is hypothesized as a major transition in the evolution of early life forms. One of the possible requirements for this transition is a change in the substrate specificity of the replication enzyme. It is largely unknown how such changes would have occurred during early evolutionary history. In this study, we present evidence that an RNA replication enzyme that has evolved in the absence of deoxyribonucleotide triphosphates (dNTPs) relaxes its substrate specificity and incorporates labeled dNTPs. This result implies that ancient replication enzymes, which probably evolved in the absence of dNTPs, could have incorporated dNTPs to synthesize DNA soon after dNTPs became available. The transition from RNA to DNA, therefore, might have been easier than previously thought.

Translation-coupled RNA replication and parasitic replicators in membrane-free compartments.

We report RNA self-replication through the translation of its encoded protein within membrane-free compartments generated by liquid-liquid phase separation. The aqueous droplets support RNA self-replication by concentrating a genomic RNA and translation proteins, facilitating the uptake of small substrates, and preventing the replication of parasitic RNAs through compartmentalization.

Emergence and diversification of a host-parasite RNA ecosystem through Darwinian evolution.

In prebiotic evolution, molecular self-replicators are considered to develop into diverse, complex living organisms. The appearance of parasitic replicators is believed inevitable in this process. However, the role of parasitic replicators in prebiotic evolution remains elusive. Here, we demonstrated experimental coevolution of RNA self-replicators (host RNAs) and emerging parasitic replicators (parasitic RNAs) using an RNA-protein replication system we developed. During a long-term replication experiment, a clonal population of the host RNA turned into an evolving host-parasite ecosystem through the continuous emergence of new types of host and parasitic RNAs produced by replication errors. The host and parasitic RNAs diversified into at least two and three different lineages, respectively, and they exhibited evolutionary arms-race dynamics. The parasitic RNA accumulated unique mutations, thus adding a new genetic variation to the whole replicator ensemble. These results provide the first experimental evidence that the coevolutionary interplay between host-parasite molecules plays a key role in generating diversity and complexity in prebiotic molecular evolution.

Minimization of Elements for Isothermal DNA Replication by an Evolutionary Approach.

DNA replication is one of the central functions of the cell. The complexity of modern DNA replication systems raises a question: is it possible to achieve a simpler continuous isothermal DNA replication using fewer proteins? Here, we searched such replication using an evolutionary approach. Through a long-term serial dilution experiment with phi29 DNA polymerase, we found that large repetitive DNAs spontaneously appear and continuously replicate. The repetitive sequence is critical for replication. Arbitrary sequences can replicate if they contain many repeats. We also demonstrated continuous DNA replication using expressed polymerase from the DNA for 10 rounds. This study revealed that continuous isothermal DNA replication can be achieved in a scheme simpler than that employed by modern organisms, providing an alternative strategy for simpler artificial cell synthesis and a clue to possible primitive forms of DNA replication.

Structural transition of replicable RNAs during in vitro evolution with Qß replicase.

Single-stranded RNAs (ssRNAs) are utilized as genomes in some viruses and also in experimental models of ancient life-forms, owing to their simplicity. One of the largest problems for ssRNA replication is the formation of double-stranded RNA (dsRNA), a dead-end product for ssRNA replication. A possible strategy to avoid dsRNA formation is to create strong intramolecular secondary structures of ssRNA. To design ssRNAs that efficiently replicate by Qß replicase with minimum dsRNA formation, we previously proposed the "fewer unpaired GC rule." According to this rule, ssRNAs that have fewer unpaired G and C bases in the secondary structure should efficiently replicate with less dsRNA formation. However, the validity of this rule still needs to be examined, especially for longer ssRNAs. Here, we analyze nine long ssRNAs that successively appeared during an in vitro evolution of replicable ssRNA by Qß replicase and examine whether this rule can explain the structural transitions of the RNAs. We found that these ssRNAs improved their template abilities step-by-step with decreasing dsRNA formation as mutations accumulated. We then examine the secondary structures of all the RNAs by a chemical modification method. The analysis of the structures revealed that the probabilities of unpaired G and C bases tended to decrease gradually in the course of evolution. The decreases were caused by the local structural changes around the mutation sites in most of the cases. These results support the validity of the "fewer unpaired GC rule" to efficiently design replicable ssRNAs by Qß replicase, useful for more complex ssRNA replication systems.