Norikazu Ichihashi

Improving the reconstituted translation system is a key requirement for bottom-up synthetic biology. Here, we developed a two-step in vitro evolutionary method that can be used for improving translational proteins. In this method, two distinct conditions were sequentially applied while maintaining genotype-phenotype linkage in water-in-oil droplets. Using this method, we performed in vitro evolution of four translation factors, IleRS, PheRS, EF-G, and EF-Tu, and identified mutations that modestly enhanced translation activity in in vitro expression assays. One of the EF-G mutations (P610S) increased activity per protein approximately 2-fold for the recombinant protein purified from E. coli. This selection method is useful for improving translational proteins for bottom-up synthetic biology.

Diversification of replicators from simple self-replicating RNA molecules is a major challenge in prebiotic evolution. In our previous evolution experiment using an RNA replication system derived from the Qβ phage, a single RNA species spontaneously diversified into multiple coexisting host and parasite RNA lineages through arms race dynamics, suggesting that host–parasite interactions may promote diversification in molecular replicator ecosystems. To test this idea more directly, we evolved six independent RNA populations under high-parasite and low-parasite conditions. High-parasite populations showed accelerated mutation fixation and greater sequence divergence between populations. However, contrary to expectation, host diversification within populations was limited under high-parasite conditions, whereas transient diversification into at most three host lineages occurred under low-parasite conditions. Sequence and biochemical analyses indicate that this diversification was consistent with relaxed selection among host RNAs that achieved similar replication yields under compartmentalized replication. In contrast, coexistence with parasites helped maintain high replication capacities by continuously imposing selection for improved replication. These results suggest that antagonistic host–parasite interactions help maintain functional activity and promote divergence between populations while constraining diversification within populations. Such opposing effects may have shaped the balance between functional optimization and diversification during early molecular evolution.

All living systems use an almost identical standard genetic code (SGC), in which 20 amino acids are assigned non-randomly. According to the error minimization theory, amino acids are arranged to minimize the mutational effect on protein function, while experimental verification remains limited. Here, we constructed 10 non-standard genetic codes (non-SGCs) in vitro by reassigning three amino acids (Ala, Ser, and Leu) in vacant codons of the minimal genetic code consisting of 21 tRNAs. Most of these non-SGCs have a higher cost of amino acid replacement than the SGC, calculated based on three amino acid properties: polar requirement (PR), molecular volume (MV), and hydropathy index (HI). The protein function of three reporter genes expressed using these non-SGCs decreased similarly when random mutations were introduced into the genes, implying that the effect of mutations was similar across all the non-SGCs tested here. This result provides direct experimental evidence that mutational robustness does not significantly change in individual reporter protein activity within the range of mutational cost tested in this study (Cost _PR : 5.29–5.77, Cost _MV : 1848–2348, and Cost _HI : 3.27–5.10), which covers approximately 18.4% (PR), 37.6% (MV), and 50.8% (HI) of the possible cost range achievable among one million randomly-generated genetic codes.

The development of molecular systems capable of self-regeneration, much like living organisms, is a major goal in synthetic biology. Previous efforts to expand the repertoire of regenerating proteins have relied on empirical optimization without a theoretical foundation, fundamentally limiting the scalability of this approach. Here, we developed the first theoretical framework, based on measurable parameters of translational proteins, to rationally predict the dilution rate that enables sustainable regeneration and the steady-state translation level. Using this framework, we successfully demonstrated sustainable regeneration of EF-Tu, the most abundant translation factor, for up to 15 rounds of serial dilution. We further extended this framework to the co-regeneration of multiple translation factors and demonstrated sustainable co-regeneration of EF-Tu and IF3, another major essential translation protein, for up to 20 rounds. These results establish a rational methodology for systematically expanding the number of regenerating proteins, providing a clear path toward the realization of a fully self-regenerating system.

All living systems use an almost identical genetic code, the standard genetic code, in which 20 amino acids are assigned to 61 codons non-randomly. According to the error minimization theory, amino acids are arranged to minimize the mutational effect on protein function, while experimental verification remains limited. In this study, we constructed 10 non-standard genetic codes in vitro by reassigning three amino acids (Ala, Ser, and Leu) in vacant codons of the minimal genetic code, which consists of 21 tRNAs. Most of these non-standard genetic codes have a higher cost of amino acid replacement than the standard genetic code, calculated based on three amino acid properties: polar requirement (PR), molecular volume (MV), and hydropathy index (HI). The protein function of three reporter genes expressed using these non-standard genetic codes decreased similarly when random mutations were introduced into the genes, implying that the effect of mutations was similar across all the non-standard genetic codes tested here. This result provides direct experimental evidence that mutational robustness does not significantly change in individual reporter protein activity when the genetic code is altered within the range of mutational cost tested in this study (CostPR: 5.29 – 5.77, CostMV: 1848 – 2348, and CostHI: 3.27 – 5.10), which covers approximately 18.4% (PR), 37.6% (MV), and 50.8% (HI) of possible cost range achievable among one million randomly-generated genetic codes.

Improving the reconstituted translation system is a key requirement for bottom-up synthetic biology. Here, we developed a two-step in vitro evolutionary method that can be used for improving translational proteins. In this method, two distinct conditions were sequentially applied while maintaining genotype-phenotype linkage in water-in-oil droplets. Using this method, we performed in vitro evolution of four translation factors, IleRS, PheRS, EF-G, and EF-Tu, and identified mutations that modestly enhanced translation activity in in vitro expression assays. One of the EF-G mutations (P610S) increased activity per protein approximately 2-fold for the recombinant protein purified from E. coli. This selection method is useful for improving translational proteins for bottom-up synthetic biology.

Theoretical studies have proposed that coevolution with parasitic replicators plays a critical role in the evolution of primitive life; however, experimental verification of the potential outcomes of such coevolutionary dynamics remains limited. We previously conducted a coevolutionary experiment using an RNA–protein replication system that resulted in the spontaneous diversification of host and parasitic RNAs into five distinct lineages with robust coreplication. Here, we report contrasting evolutionary outcomes from a second long-term coevolutionary experiment. Using a droplet flow reactor (FR) system with increased dilution frequency over 5,000 h (1,600 generations), we observed reduced diversity and frequent extinctions in later experimental stages. Coreplication assays of RNA clones revealed that the primary cause of this diversity loss was the shortened reaction time resulting from frequent dilution. Further analysis of RNA clones that emerged during evolution suggested that the frequent extinctions resulted from the appearance of highly competitive parasite species and the dominance of host species that exhibited reduced replication ability. These findings demonstrate that coevolution between host and parasitic replicators can result in diversity loss and frequent extinctions depending on dilution conditions, highlighting the critical role of environmental parameters, such as dilution ratio and frequency, in enabling primitive replicators to evolve sustainably toward the emergence of life.

The error catastrophe refers to the proliferation of nonfunctional molecules in conditions where molecular replication has low accuracy, which is likely to correspond to conditions present at the Origin of Life. This error catastrophe can be avoided thanks to transient compartmentalization, provided that the compartments are sufficiently tight to prevent molecular leakage. Typically, transient compartmentalization models assume that the content of the compartments is completely pooled periodically, resulting in the complete loss of the compositional memory of the compartment. Furthermore, previous models that include the possibility of ecological interactions between molecular parasites and replicators within compartments generally do not study the effect of transient compartmentalization on their coevolution. To address both issues, we develop a framework that accounts for the coevolution of molecular replicators and parasites, along with specific compartmentalization dynamics that are transient yet partial, allowing compositional memory to accumulate from one round of compartmentalization to the next. We benchmark our model with a serial dilution experiment that displays complex oscillatory dynamics among four well-characterized RNA replicators. We also perform experiments to quantify the level of mixing in compartments when stronger stirring tends to homogenize their composition. We then model stirring-induced mixing and show how stirring alters the dynamics of compartmentalized replicators. We conclude that compositional memory arising from transient compartmentalization plays a major role in the dynamics of early molecular systems.

The error catastrophe refers to the proliferation of non-functional molecules in conditions where molecular replication has low accuracy, which is likely to correspond to conditions present at the Origin of Life. This error catastrophe can be avoided thanks to transient compartmentalization, provided that the compartments are sufficiently tight to prevent molecular leakage. Typically, transient compartmentalization models assume that the content of the compartments is completely pooled periodically, resulting in the complete loss of the compositional memory of the compartment. Furthermore, previous models that include the possibility of ecological interactions between molecular parasites and replicators within compartments generally do not study the effect of transient compartmentalization on their coevolution. To address both issues, we develop a framework that accounts for the coevolution of molecular replicators and parasites, along with specific compartmentalization dynamics that are transient yet partial, allowing compositional memory to accumulate from one round of compartmentalization to the next. We benchmark our model with a serial dilution experiment that displays complex oscillatory dynamics among four well-characterized RNA replicators. We also perform experiments to quantify the level of mixing in compartments when stronger stirring tends to homogenize their composition. We then model stirring-induced mixing and show how stirring alters the dynamics of compartmentalized replicators. We conclude that compositional memory arising from transient compartmentalization plays a major role in the dynamics of early molecular systems.