Research
Our group are trying to understand the origin and evolution of life through constituting an artificial cell.
During the last few decades, molecular biology has revealed all genes and chemical components in the simplest organism such as bacteria. However, researchers have not suceeded in constructing such simplest bacteria, which means our knowledge about the cell is not sufficient. Why cannot we construct a cell? What is lacking in our knowledge? One of the direct methods to answer these questions is to construct a cell from scratch. Our final target is to construct an artificial cell which is indistinguishable from living cells.
For this purpose, we are now tackling these three projects.
- 1. Construction of an evolvable artificial cell
- 2. Design and construction of reproductive artificial cell
- 3. Applications using the constructed artificial cells
1. Construction of an evolvable artificial cell
To construct a living cell as a final goal, we first focused on the ability to evolve because the ability is one of the unique characteristics of life. In the prebiotic chemical evolution on the ancient earth, it is considered that an assembly of organic molecules acquired the ability to evolve and thereby evolved to be present-day sofisticated and complex living cells. If this idea is true, when we reconstitute a chemical reaction system that has the ability to evolve, the system should evolve to become a state regarded as living.
To examine this hypothesis, we contructed an artificial reaction system, which consists of a reconstitued transalation system of E.coli and a single-stranded RNA (artificial RNA genome) which encodes RNA replicase. The whole system is encapsulated in a micro-scale water-in-oil emulsion or liposomes. The genome RNA continuously self-replicated and evolved thorugh spontaneous mutations introduced by replication error if we periodically supplied droplets containing nutrients (the translation system) in the emulsion system (Figure 1, Ichihashi et al 2013).
This artificial cell has a cell-like micro-strucutre (therefore, we call this an artificial cell). However, we did not intend to make cell-like structures initially but just intended to make a system that has the ability to evolve. As a consequence of constructing the ability to evolve, we found that the ability requires a cell-like structure (Ichihashi et al 2013). In addition, this system is able to adapt to various environments even though it has only one gene (Mizuuchi et al 2015, 2016).
Was this evolvable artificial cell able to evolve to be a living cell? Interestingly, we found that the evolution of the artificial cell is different from that of living things. The evolution of the artificial cell stopped after about 600 generations and did not get closer to living cells. This result indicates that the ability to evolve is not sufficient for the emergence of living cells. How can we make the artificial system evolve continuously? To answer this question, we are now working on the following three projects.
1-1. Coevolution with parasites
All living things in nature co-evolve with parasitic organisms such as viruses, and such parasites are considered to accelerate the host evolution. To examine the possiblity that the evolutionary acceleration by parasites plays an important role in the continuous evolution of living things, we are performing co-evolution of the artificial cell with parasitic RNAs, which spontaneously appeared in the artificial cell. We found that the host (genome RNA) and parasitic RNA concentrations represent a pray-predator-like oscillation dynamics and the oscillation pattern changed gradually due to the evolution of both host and parasite (Figure 2-1, Bansho et al 2016). Owing to the presence of parasite, the host genome RNA restarted evolution and the evolution continued seemingly endlessly.
After a long-term evolution more than 500 generations, we found that the host and parasitic RNAs changed their population dynamics from regular oscillation to unregular coreplication at a higher concentration (Figure 2-2, Furubayashi et al 2020).
More interestingly, new types of parasitic RNAs(parasitic RNAs beta and gamma), longer than the original parasitic RNA alpha, appeared at the later rounds, indicating that the parasitic RNA was diversifed. Sequence analysis of the host RNA also revealed that the host RNA was also diversified to at least two lineages (Figure 2-3, Furubayashi et al 2020).
To understand the reason of the diversification, we performed a series of competition experiments, in which we compared replication ability when we mixed a pair of repersentative host and parasite RNAs (Figure 2-4, Furubayashi et al 2020). The result shows that a new parasite adapted to the host RNA that dominated at that time and the nextly-appeared host RNA acquired the resistance to the parasite. Then the next type parasite adapted to the host RNA again. In this manner, the host and parasitic RNAs contitued to adapt to each other. We think that this evolutionary arms race produced the diversity and allowed the continuous evolution. As far as we know, this is the first example of a co-evolutionary process of a molecular replication system.
1-2. Evolution of complexity
One of the challenges to let the artificial cell evolve to become a living cell is the development of complexity through evolution. Generally, the evolution of complexity is difficult process because usually simpler replicater replicate faster and thus has higher fitness. Therefore, the artificial cell has not suceeded in obtaining new genes and new function. To understand how an artificial cell acquires a new genes and develop complexity in the replication system, we are now trying to introduce a new genome RNA which encodes another gene. This is the experimental verification of the hypercycle model proposed by Manfred Eigen about 40 years ago (Eigen 1978)
1-3. Evolution from single-stranded RNA genome to double-stranded RNA genome
We have found that strong RNA structure is critically important for a singel-stranded RNA genome to be replicated (Usui et al 2015). This indicates that a single-stranded genome cannnot encode any possible genes but only sequences that form strong RNA structures. This means that possilbe genome RNA sequences (i.e., possilbe evolutionary routes) is restricted as long as the artificial cell uses a single-stranded RNA as the genome. This may be the reason why all the present organisms use doulbe-stranded DNA as genomes. Presently, we are trying to reproduce the evolution process from single-stranded to doulbe-stranded genomes.
2. Design and construction of reproductive artificial cell
Another characteristics of living things is a recursive self-replication. All living organisms or cells are able to reproduce themselves. We are trying to develop an artificial in vitro system that has this recursive self-reproduction ability.
We are tackling these projects.
2-1. Development of a DNA genome self-replication system
Although we ahve succeeded in the development of an RNA genome replication system as a simplest self-replication system, DNA genome is preferable to encode many genes and replicate at a lower error rate. To date, we have constructed a transcription-translation-coupled DNA replication system using phi29 DNA polymerase (Figure 3-1, Sakatani et al 2015 and 2018). This is the simplest DNA replication system because it requires only two genes for replication, phi29 DNA polymerase and Cre recombinase.
We also found a simpler recursive replication almost by accident. This scheme requires only one gene, phi29 DNA polymerase gene, and repetitive long linear DNA (Figure 3-2, Okauchi et al 2020). Usually, DNA polymerase cannot replicate a lnear DNA recursively because the a newly synthesized DNA must be shorter than the template DNA according to "end replication problem." However, if the template DNA consists of repetitive sequence, the produt DNA can maintain the original size thorugh the hybridization of newly-synthesized strands at a shifted position. Because this is the simplest DNA replication scheme, we think that this may be a primitive DNA replication scheme.
2-2. In vitro reconstitution of ribosome
The present artificial cell contains the reconstituted translation system, which is supplied externally. To achive the total reproduction of the artificial cell, the translation system should be produced in the internal reaction. The largest challgenge is ribosome production. Presently, we are trying to establish a method to reconstitute 30S subunit in vitro and improve 16S rRNA though the directed evolution (Figure 4). Other than ribosome, most of the aminoacyl-tRNA synthetases has been reproduced internally (Awai et al 2015).
3. Applications using the constructed artificial cells
3-1. Directed evolution of ribosomal RNAs
The artificial cells we have developed are useful as a reactor for the directed evolution of RNA or proteins. By using the artificial cells, we do not need bacteria to express genes anymore and are able to performe directed evolution completely in vitro. This is especially useful for the genes mutations of which are harmful for the cell. We are now trying to perform the in vitro evolution of 16s rRNA (Figure 4).
3-2. Understanding the evolutionary process
One of the advantages of an evolvable artificial cell is that all the components are defined. Any natural cells has many functionally unknown genes and therefore the relationship between mutations and their effects are obscure in many cases. In contrast, the internal reaction of the artificial cell is completely identified and quantitatively understood, which allows clear understanding of the evolutionary processes.
Futhermofe, the evolution of the artificial cells occurs in a test tube, which means we can observe every step in the evolution. This allows us to analyze the evolutionary process at unprecidented resolution. Actually, we have analyzed the evolutionary process by using a next-generation sequencer and observed an unexpected evolutionary process: many genotypes with the same level of fitness appeared and competitively increase their populations (Figure 5, Ichihashi et al 2015). This process is different from the conventional model of evolution, in which a rare genotype with a higher fitness sometimes appeared and rapidly dominate the population.
References
- Furubayashi, T., Ueda, K., Bansho, Y., Motooka, D., Nakamura, S., Mizuuchi, R., Ichihashi, N.*
Emergence and diversification of a host-parasite RNA ecosystem through Darwinian evolution
eLIFE 9:e56038 (2020) PDF
- Okauchi, H., Sakatani,Y., Ohtsuka, K., Ichihashi, N.*
Minimization of elements for isothermal DNA replication by an evolutionary approach
ACS Synthetic Biology 9, 1771?1780 (2020) (2020) - Sakatani, Y., Yomo, T., Ichihashi, N.
Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination.
Sci Rep 8, 13089 (2018) PDF
- Mizuuchi, R., Ichihashi, N.*, Yomo, T. (*corresponding)
Adaptation and diversification of an RNA replication system under initiation- or termination-impaired translational conditions
Chembiochem, 17, 1229-1232 (2016) - Bansho, Y., Furubayashi, T., Ichihashi, N.*, Yomo, T.* (*co-corresponding)
Host-parasite oscillation dynamics and evolution in a compartmentalized RNA replication system
Proc Nat Sci USA, 113, 4045-4050 (2016) - Usui, K.*, Ichihashi, N.*, Yomo, T. (*equally contributed)
A design principle for a single-stranded RNA genome that replicates with less double-strand formation
Nucleic Acids Research 43, 8033-8043. (2015) - Awai, T., Ichihashi, N., Yomo, T.
Activities of 20 aminoacyl-tRNA synthetases expressed in a reconstituted translation system in Escherichia coli
Biochemistry and Biophysics Reports 3, 140-143. (2015) - Sakatani, Y., Ichihashi, N., Kazuta, Y., Yomo, T.
A transcription and translation-coupled DNA replication system using rolling-circle replication
Scientific Reports 5, 10404. (2015) - Mizuuchi, R., Ichihashi, N., Usui, K., Kazuta, Y., Yomo, T.
Adaptive evolution of an artificial RNA genome to a reduced ribosome environment
ACS Synthetic Biology 4, 292-298. (2015) - Ichihashi, N., Usui, K., Kazuta, Y., Sunami, T., Matuura, T., Yomo, T.
Darwinian evolution in a translation-coupled RNA replication system within a cell-like compartment.
Nature Communications, 4, 1-7, (2013) - Ichihashi, N., Usui, Aita, T., Motooka, D., Nakamura, S., Yomo, T.
Periodic Pattern of Genetic and Fitness Diversity during Evolution of an Artificial Cell-Like System
Molecular Biology and Evolution 32, 3205-3214. (2015) - Ichihashi, N., Yomo, T.
Constructive approaches for understanding the origin of self-replication and evolution
Life, 6(3), 26 (2016) - Ichihashi, N., Matsuura, T., Kita, H., Sunami, T., Suzuki, H., Yomo, T
Constructing partial models of cells.
Cold Spring Harbor Perspective in Biology 2, a004945 (2010)