Research

Ichihashi Group  PI: Norikazu Ichihashi, associate professor

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, humanity has 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 living 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. 1. Construction of an evolvable artificial cell
  2. 2. Design and construction of reproductive artificial cell
  3. 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 a 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 be a living cell.

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 (thereofre, 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 is considered to acceralate host evolution. To examine the possiblity that the evolutionary acceralation by parasites plays an important role in the continusou 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 prasitic RNA concentration shows a pray-predator-like oscilation dynamics and the oscilation pattern changes gradually due to the evolution of both host and parasite (Figure 2, Bansho et al 2016). Owing to the presence of parasite, the host genome RNA restart evolution and the evolution continues seemingly endlessly.

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 too replicate at a lower error rate. To date, we have constructed a transcription-translation-coupled DNA replication system using phi29 DNA polymerase (Figure 3, Sakatani et al 2015). This is the simplest DNA replication system because it requires only one gene for replication, while the replication is not recursive yet. We are now trying to make this sytem recursive.

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.

We analyzed the evolutionary process in more detail and found that the process repeats two phases: the deversification phase, in which mutations produce genetic difersity, and the selection phase, in which the average fitness increases by consuming the diversity (Figure 6, Ichihashi et al 2015).

References

    Articles

  1. 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)
  2. 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)
  3. 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)
  4. 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)
  5. 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)
  6. 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)
  7. 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)
  8. 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)
  9. Reviews

  10. Ichihashi, N., Yomo, T.
    Constructive approaches for understanding the origin of self-replication and evolution
    Life, 6(3), 26 (2016)
  11. 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)