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ScienceDirect Positive roles of compartmentalization in internal reactions Norikazu Ichihashi1,2 and Tetsuya Yomo1,2,3 Recently, many researchers have attempted to construct artificial cell models using a bottom-up approach in which various biochemical reactions that involve a defined set of molecules are reconstructed in cell-like compartments, such as liposomes and water-in-oil droplets. In many of these studies, the cell-like compartments have acted only as containers for the encapsulated biochemical reactions, whereas other studies have indicated that compartmentalization improves the rates and yields of these reactions. Here, we introduce two ways in which compartmentalization can improve internal reactions: the isolation effect and the condensation effect. These positive effects of compartmentalization might have played an important role in the genesis of the first primitive cell on early Earth. Addresses 1 Department of Bioinformatics Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan 2 Exploratory Research for Advanced Technology, Japan Science and Technology Agency, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan 3 Graduate School of Frontier Biosciences, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan Corresponding author: Yomo, Tetsuya ([email protected]) Current Opinion in Chemical Biology 2014, 22:12–17 This review comes from a themed issue on Synthetic biology Edited by Pier Luigi Luisi, Pasquale Stano and Cristiano Chiarabelli
http://dx.doi.org/10.1016/j.cbpa.2014.06.011 1367-5931/# 2014 Elsevier Ltd. All rights reserved.
Introduction All living things are composed of structural units called cells, which are vesicles typically ranging from 1 to 100 mm in diameter surrounded by a lipid membrane with membrane-bound proteins. A number of biological molecules, such as polynucleic acids, proteins, and low-molecularweight compounds, are encapsulated within the cell and perform various cellular functions, such as genome replication, translation, metabolism, signal transduction, cell growth, division, and other processes. These functions are strictly regulated by a complex genetic regulatory network, which is composed of more than 3000 interactions in Escherichia coli [1]. In other words, the cell is a micro-scale chemical reactor of enormous complexity, and artificially Current Opinion in Chemical Biology 2014, 22:12–17
preparing such a complex system is presently beyond the reach of human technology. The de novo construction of systems that perform natural cellular functions is the first step in developing technology to synthesize artificial cells. This technique is alternately called the semi-synthetic approach, the constructed approach, or in vitro synthetic biology [2–5]. The advantage of this approach compared with methods involving the modification or minimization of an extant cell is the ability to control the experimental conditions due to the use of a known set of materials, with researchers having strict control over the experimental conditions and the concentrations of all components. Using this approach, we can understand the conditions necessary to achieve a targeted cellular function without ambiguity because there are no unknown factors [6,7]. This type of knowledge could give rise to new technology that would allow us to tailor biological functions to our needs [8,9]. To date, various types of biological reactions have been constructed in cell-like compartments using a bottom-up approach. For example, polymerase chain reaction (PCR) [10], RNA synthesis [11], the expression of various proteins [12–17], RNA replication coupled with translation [18,19], and the regulation of gene expression via a riboswitch [20] or a positive feedback circuit [21] have been achieved using a discrete set of materials in phospholipid vesicles (liposomes). Another type of cell-like compartment is the water-in-oil droplet (or water-in-oil emulsion), in which the expression of green fluorescent protein (GFP) [22], the translation and selection of a methyltransferase [23], PCR [24–26], and RNA replication coupled with translation [27] have been achieved. In most of these examples, the cell-like compartments (e.g., the liposomes or water-in-oil droplets) have been used as mere containers to encapsulate the reaction solutions; therefore, the compartmentalization had no effect (or a negative effect) on the internal reactions. However, several recent studies have reported that compartmentalization can have positive effects on internal biological reactions [14,28–30], implying that the cell-like compartment plays a role beyond that of a mere container. In this review, we introduce two examples of the positive effects of compartmentalization on biological reactions: the isolation effect and the condensation effect. The isolation effect
Compartmentalization can improve the progress of an internal biochemical reaction through an isolation effect, which involves isolating a small number of inhibitory www.sciencedirect.com
Positive roles of compartmentalization Ichihashi and Yomo 13
(a)
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Our group has attempted to construct an artificial genome replication system that mimics the genome replication of RNA viruses. We have reported the construction of a translation-coupled RNA replication system composed of an artificial RNA genome encoding an RNA replication enzyme and the reconstituted translation system of E. coli [18]. In this system, the RNA replication enzyme derived from bacteriophage Qb was translated from an artificial RNA genome and was used to replicate the original genome. However, this self-replication process could not proceed for longer than a few hours despite an adequate nutrient supply due to the appearance of a ‘parasitic’ RNA that was generated from the genomic RNA as a result of recombination during the reaction. Because this parasitic RNA had lost the coding region of the replicase but retained the recognition site for the replicase, it could be replicated by the extant replicase (Figure 1b). In this sense, this small RNA was a parasite of the RNA replication system. Once this parasitic RNA appeared, it rapidly replicated until it occupied all the replicase molecules, inhibiting further genomic RNA replication after just a few hours.
Figure 1
RNA replication
factors from the major components of the reaction solution. If a reaction solution that contains a small amount of an inhibitory factor is encapsulated into a sufficient number of compartments, the inhibitory factors will be present in only a small number of the compartments, leaving most of the compartments free of the inhibitor. Therefore, on average, compartmentalization enhances the reaction rate (Figure 1a). This effect has been demonstrated by performing PCR in water-in-oil droplets (i.e., emulsion PCR), in which an extremely small amount of a target DNA fragment can be amplified without being inhibited by the non-specific amplification of smaller fragments [24,25]. The isolation effect has also been reported to significantly enhance RNA replication by an RNA-dependent RNA-polymerase (Qb replicase) [31,32]. Because RNA replication is more relevant to artificial cell technologies, we focus on this theme below.
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When we encapsulated the RNA replication solution in water-in-oil droplets of various sizes, we found that parasitic replication was repressed and that genome replication was prolonged [32]. The effect of compartmentalization depended on the compartment size: the smaller the droplet, the more the parasitic RNA was repressed and the longer the genome replication was able to proceed (Figure 1c). Interestingly, the optimum droplet size was similar to the size of E. coli cells (i.e., a few micrometers), which might be related to our use of the coliphage Qb replicase. Using this droplet size, we were also able to show that the genomic RNA selfreplicated recursively and evolved [27]. These results illustrate the role that cell-like compartments can play in recursive genome replication by excluding a parasitic replicator. www.sciencedirect.com
(a) Schematic diagram illustrating how isolation affects RNA replication. Genomic RNA replication is competitively inhibited by parasitic RNA, which appears spontaneously from genomic RNA. In a bulk reaction, the parasite inhibits the replication of all genomes, while in compartments, the parasite is confined and only inhibits a minor fraction of the genome replication (adapted with permission from the American Chemical Society [31]). (b) Schematic diagram illustrating the translation-coupled RNA replication system. The RNA replicase, translated from the genomic RNA (plus-strand), synthesizes the minus-strand using the original plusstrand as a template. The replicase subsequently replicates the plusstrand using the minus-strand as a template. The parasitic RNA, which has lost the replicase-encoding region, inhibits genome replication by successfully competing for the replicase (reproduced with permission from Elsevier [32]). (c) The effect of compartment size on RNA replication. Translation-coupled RNA replication can be performed in compartments of various sizes. In smaller compartments, the replication of the parasitic RNA is decreased and genome replication is increased (reproduced with permission from Elsevier [32]). Current Opinion in Chemical Biology 2014, 22:12–17
14 Synthetic biology
The role of cell-like compartments in isolating parasitic replicators is relevant to the origin of self-replication systems on early Earth. The emergence of a recursive replication system is considered the key step toward the origin of life. One of the major obstacles to this process is the appearance of a parasitic replicator, which can emerge in a self-replication system with a certain level of complexity and abolish the self-replication reaction [33].
Theoretically, one method to overcome the problem of parasitic replicators involves the use of spatial structures, such as compartments, that restrict the diffusion of parasitic molecules [34,35]. However, this possibility has not been experimentally verified. Our experimental results demonstrate that the isolation effect of compartments supports the role of spatial structures in the production of a recursive self-replication system.
Figure 2
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(a) A model of the accumulation of macromolecules under dilute conditions during liposome formation (reproduced with permission from John Wiley and Sons [36]). (b) Fluorescent image of liposomes stained with Nile red (right panel). Only some of the liposomes exhibit eGFP expression (left panel) (adapted with permission from John Wiley and Sons [36]). Current Opinion in Chemical Biology 2014, 22:12–17
www.sciencedirect.com
Positive roles of compartmentalization Ichihashi and Yomo 15
The condensation effect
The second example of the positive effect of compartmentalization on internal biochemical reactions is the condensation effect, in which the internalized materials are concentrated, accelerating the reaction. To date, two condensation mechanisms have been reported by two different groups. The first mechanism involves the condensation of components by cooperative encapsulation into liposomes. Stano et al. prepared a diluted cell-free transcription– translation system containing DNA that encoded GFP
but could not function under the usual bulk conditions because it was excessively dilute. However, when the diluted solution was encapsulated into liposomes, some of the liposomes exhibited GFP translation (Figure 2) [36]. The number of liposomes that exhibited GFP expression was much greater than would have been predicted assuming random encapsulation of all the components. These results indicated that the components of the transcription–translation system became condensed when they were encapsulated into some of the liposomes through cooperative incorporation, enabling GFP expression, which had previously been impossible outside of the
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(a) Schematic illustration of glucuronidase activity enhancement by compartmentalization (reproduced with permission from the American Chemical Society [29]). (b) Fluorescence image of water-in-oil droplets. (1, 4) Bright field, (2, 5) fluorescence, and (3, 6) merged images with (1, 2, 3) 0.3 nM or (4, 5, 6) 0.01 nM DNA. The scale bar represents 20 mm. Active glucuronidase exhibits green fluorescence in this experiment. Only a small fraction of the droplets exhibit glucuronidase activity at a DNA concentration of 0.01 nM (adapted with permission from the American Chemical Society [29]). www.sciencedirect.com
Current Opinion in Chemical Biology 2014, 22:12–17
16 Synthetic biology
liposomes. Luisi et al. further reported that the amount of protein incorporated into a liposome did not obey a Poisson distribution, implying that incorporation was an independent process. Rather, protein incorporation showed a power-law behavior, which implied the accumulation of solutes upon encapsulation [37]. To date, the mechanism of cooperative incorporation remains unknown. However, the condensation effect during liposome encapsulation may have been important to the origin of life. This process might have played a role in concentrating diluted biological molecules from bulk solution to enable biological functions on early Earth. The second mechanism underlying the condensation effect was revealed during the expression of a multimeric enzyme. Matsuura et al. performed transcription–translation of a glucuronidase gene in water-in-oil droplets of various sizes and found that compartmentalization of the reaction into smaller droplets facilitated the expression of glucuronidase activity when the DNA encoding the glucuronidase gene was adjusted to less than one molecule per compartment (Figure 3) [29]. At that concentration, there were two types of compartments: those that contained DNA and those that lacked DNA. In these compartments, the effective DNA concentration increased as the compartment size became smaller. The effective DNA concentration was proportional to the expression of the glucuronidase monomer, and the monomer concentration was proportional to the formation of the active tetrameric glucuronidase. Therefore, smaller compartments improved the glucuronidase activity. This effect on glucuronidase expression has also been observed in a microchamber [38]. Given that this type of condensation effect must occur when any protein that functions in a multimeric form is expressed, it should be considered when multimeric cellular proteins are expressed in an artificial cell.
Conclusions Recent advances in bottom-up approaches to generate an artificial cell have revealed that cell-like compartments can facilitate internal biochemical reactions through both isolation and condensation effects. These effects might have played important roles in the early development of primitive life because compartmentalization isolates inhibitory factors and concentrates biological polymers. These effects of compartmentalization cannot be revealed solely by analyzing extant cells because cells are already compartmentalized. Thus, the bottom-up approach is advantageous for the construction of an artificial cell because conditions that are not realized in native living organisms can be tested (e.g., reactions with or without compartments). Further development of artificial cells will provide important knowledge regarding the basic principles of life. The greatest difference between the described artificial cell models (liposomes and water-in-oil droplets) and Current Opinion in Chemical Biology 2014, 22:12–17
native cells is that most artificial cells are stable and neither grow nor divide. The stability of a compartment is important for reliable compartmentalization; however, the compartment must be unstable to some extent so that it is able to grow and divide. Preparing artificial cells possessing these conflicting properties is most likely the next important challenge. Some studies have reported the partial growth of cell-like structures. For example, Zhu et al. reported the growth and division of a lipid vesicle composed of fatty acids [39], and Kurihara et al. observed the growth of a lipid vesicle composed of artificial lipids in response to the amplification of its internal DNA [40]. Future research should focus on the construction of a lipid synthesis pathway [41] or cell division machinery [42] inside liposomes. In the process of generating artificial cell models that are closer to natural cells, we may come to understand the underlying principles necessary to prepare liposomes that mimic the dynamic properties of cellular membranes. Other fundamental roles of compartments have been proposed in theory and remain to be experimentally verified. One example is the stochastic corrector model, which is a mechanism proposed by Szathmary and Demeter in which a replication system prevents the collapse caused by the accumulation of parasitic replicators (Eigen’s paradox [43]) [44]. In this model, the combination of stochastic fluctuation in the number of encapsulated replicators and grouplevel selection leads to the selective advantage of ‘host’ replicators compared to parasitic (selfish) replicators. The principle of the model is similar to that of the isolation effect described in this review, although the isolation effect, in which parasitic replicators exist in very small numbers, represents an extreme case. In other theoretical studies, Kamimura and Kaneko proposed that a mutually catalyzing chemical reaction produces a compartment-like structure without any boundary molecules [45], and Mavelli and Stano proposed that a vesicle that reproduces itself in an autopoietic manner exhibits homeostasis in size [46]. These studies suggest that simple physical processes can produce biological phenomena without any complex biological machinery, which might be relevant to the emergence of life in the prebiotic world. The experimental verification of these theoretical proposals, including the stochastic corrector model, will be the next important challenge in in vitro synthetic biology.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Martinez-Antonio A, Janga SC, Thieffry D: Functional organisation of Escherichia coli transcriptional regulatory network. J Mol Biol 2008, 381:238-247.
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Forster AC, Church GM: Synthetic biology projects in vitro. Genome Res 2007, 17:1-6.
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Forlin M, Lentini R, Mansy SS: Cellular imitations. Curr Opin Chem Biol 2012, 16:586-592.
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Stano P, Luisi PL: Semi-synthetic minimal cells: origin and recent developments. Curr Opin Biotechnol 2013, 24:633-638.
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Szostak JW, Bartel DP, Luisi PL: Synthesizing life. Nature 2001, 409:387-390.
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Ichihashi N, Matsuura T, Kita H et al.: Constructing partial models of cells. Cold Spring Harb Perspect Biol 2010, 2:a004945.
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Doktycz MJ, Simpson ML: Nano-enabled synthetic biology. Mol Syst Biol 2007, 3:125.
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Zhang Y, Ruder WC, LeDuc PR: Artificial cells: building bioinspired systems using small-scale biology. Trends Biotechnol 2008, 26:14-20.
10. Oberholzer T, Albrizio M, Luisi PL: Polymerase chain reaction in liposomes. Chem Biol 1995, 2:677-682. 11. Oberholzer T, Wick R, Luisi PL, Biebricher CK: Enzymatic RNA replication in self-reproducing vesicles: an approach to a minimal cell. Biochem Biophys Res Commun 1995, 207:250-257. 12. Oberholzer T, Nierhaus KH, Luisi PL: Protein expression in liposomes. Biochem Biophys Res Commun 1999, 261:238-241. 13. Yu W, Sato K, Wakabayashi M et al.: Synthesis of functional protein in liposome. J Biosci Bioeng 2001, 92:590-593. 14. Nomura SM, Tsumoto K, Hamada T et al.: Gene expression within cell-sized lipid vesicles. Chembiochem 2003, 4:1172-1175. 15. Noireaux V, Libchaber A: A vesicle bioreactor as a step toward an artificial cell assembly. Proc Natl Acad Sci USA 2004, 101:17669-17674. 16. Kuruma Y, Stano P, Ueda T, Luisi PL: A synthetic biology approach to the construction of membrane proteins in semi-synthetic minimal cells. Biochim Biophys Acta 2009, 1788:567-574. 17. Soga H, Fujii S, Yomo T et al.: In vitro membrane protein synthesis inside cell-sized vesicles reveals the dependence of membrane protein integration on vesicle volume. ACS Synth Biol 2014, 3:372-379. 18. Kita H, Matsuura T, Sunami T et al.: Replication of genetic information with self-encoded replicase in liposomes. Chembiochem 2008, 9:2403-2410. 19. Ichihashi N, Matsuura T, Kita H et al.: Importance of translation– replication balance for efficient replication by the selfencoded replicase. Chembiochem 2008, 9:3023-3028. 20. Martini L, Mansy SS: Cell-like systems with riboswitch controlled gene expression. Chem Commun (Camb) 2011, 47:10734-10736. 21. Kobori S, Ichihashi N, Kazuta Y, Yomo T: A controllable gene expression system in liposomes that includes a positive feedback loop. Mol Biosyst 2013, 9:1282-1285. 22. Fiordemondo D, Stano P: Lecithin-based water-in-oil compartments as dividing bioreactors. Chembiochem 2007, 8:1965-1973. 23. Tawfik DS, Griffiths AD: Man-made cell-like compartments for molecular evolution. Nat Biotechnol 1998, 16:652-656. 24. Ghadessy FJ, Ong JL, Holliger P: Directed evolution of polymerase function by compartmentalized self-replication. Proc Natl Acad Sci USA 2001, 98:4552-4557. 25. Nakano M, Komatsu J, Matsuura S et al.: Single-molecule PCR using water-in-oil emulsion. J Biotechnol 2003, 102:117-124. 26. Kelly BT, Baret JC, Taly V, Griffiths AD: Miniaturizing chemistry and biology in microdroplets. Chem Commun 2007:1773-1788. 27. Ichihashi N, Usui K, Kazuta Y et al.: Darwinian evolution in a translation-coupled RNA replication system within a cell-like compartment. Nat Commun 2013, 4:2494. This study demonstrated the Darwinian evolution of the genomic RNA of an artificial RNA-replication system in a water-in-oil emulsion. www.sciencedirect.com
28. Pereira de Souza T, Stano P, Luisi PL: The minimal size of liposome-based model cells brings about a remarkably enhanced entrapment and protein synthesis. Chembiochem 2009, 10:1056-1063. 29. Matsuura T, Hosoda K, Kazuta Y et al.: Effects of compartment size on the kinetics of intracompartmental multimeric protein synthesis. ACS Synth Biol 2012, 1:431-437. This study demonstrated the condensation effect in a compartment using a multimeric enzyme. The expression of glucuronidase activity was enhanced in smaller droplets. 30. Kato A, Yanagisawa M, Sato YT et al.: Cell-Sized confinement in microspheres accelerates the reaction of gene expression. Sci Rep 2012, 2:283. 31. Urabe H, Ichihashi N, Matsuura T et al.: Compartmentalization in a water-in-oil emulsion repressed the spontaneous amplification of RNA by Q beta replicase. Biochemistry 2010, 49:1809-1813. 32. Bansho Y, Ichihashi N, Kazuta Y et al.: Importance of parasite RNA species repression for prolonged translation-coupled RNA self-replication. Chem Biol 2012, 19:478-487. This study demonstrated that compartmentalization repressed parasitic amplification and allowed prolonged genome replication. 33. Eigen M, Biebricher CK, Gebinoga M, Gardiner WC: The hypercycle. Coupling of RNA and protein biosynthesis in the infection cycle of an RNA bacteriophage. Biochemistry 1991, 30:11005-11018. 34. Bresch C, Niesert U, Harnasch D: Hypercycles, parasites and packages. J Theor Biol 1980, 85:399-405. 35. Takeuchi N, Hogeweg P, Koonin EV: On the origin of DNA genomes: evolution of the division of labor between template and catalyst in model replicator systems. PLoS Comput Biol 2011, 7:e1002024. 36. Stano P, D’Aguanno E, Bolz J et al.: A remarkable self organization process as the origin of primitive functional cells. Angew Chem Int Ed Engl 2013, 52:13397-13400. This study demonstrated that dilute macromolecules accumulated and functioned to express enhanced GFP (eGFP) when they were encapsulated into liposomes. This process might have been involved in overcoming the difficulty of concentrating macromolecules on the early Earth. 37. Luisi PL, Allegretti M, Pereira de Souza T et al.: Spontaneous protein crowding in liposomes: a new vista for the origin of cellular metabolism. Chembiochem 2010, 11:1989-1992. Direct observation of protein encapsulation into liposomes using cryogenic transmission electron microscopy (cryo-TEM) revealed that the amount of protein per liposome did not obey a Poisson distribution. 38. Okano T, Matsuura T, Suzuki H, Yomo T: Cell-free protein synthesis in a microchamber revealed the presence of an optimum compartment volume for high-order reactions. ACS Synth Biol 2014, 3:347-352. 39. Zhu TF, Szostak JW: Coupled growth and division of model protocell membranes. J Am Chem Soc 2009, 131:5705-5713. 40. Kurihara K, Tamura M, Shohda K et al.: Self-reproduction of supramolecular giant vesicles combined with the amplification of encapsulated DNA. Nat Chem 2011, 3:775-781. 41. Yu X, Liu T, Zhu F, Khosla C: In vitro reconstitution and steadystate analysis of the fatty acid synthase from Escherichia coli. Proc Natl Acad Sci USA 2011, 108:18643-18648. 42. Osawa M, Anderson DE, Erickson HP: Reconstitution of contractile FtsZ rings in liposomes. Science 2008, 320:792-794. 43. Eigen M: Selforganization of matter and the evolution of biological macromolecules. Naturwissenschaften 1971, 58:465-523. 44. Szathmary E, Demeter L: Group selection of early replicators and the origin of life. J Theor Biol 1987, 128:463-486. 45. Kamimura A, Kaneko K: Reproduction of a protocell by replication of a minority molecule in a catalytic reaction network. Phys Rev Lett 2010, 105. 46. Mavelli F, Stano P: Kinetic models for autopoietic chemical systems: the role of fluctuations in a homeostatic regime. Phys Biol 2010, 7:16010. Current Opinion in Chemical Biology 2014, 22:12–17
ScienceDirect Positive roles of compartmentalization in internal reactions Norikazu Ichihashi1,2 and Tetsuya Yomo1,2,3 Recently, many researchers have attempted to construct artificial cell models using a bottom-up approach in which various biochemical reactions that involve a defined set of molecules are reconstructed in cell-like compartments, such as liposomes and water-in-oil droplets. In many of these studies, the cell-like compartments have acted only as containers for the encapsulated biochemical reactions, whereas other studies have indicated that compartmentalization improves the rates and yields of these reactions. Here, we introduce two ways in which compartmentalization can improve internal reactions: the isolation effect and the condensation effect. These positive effects of compartmentalization might have played an important role in the genesis of the first primitive cell on early Earth. Addresses 1 Department of Bioinformatics Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan 2 Exploratory Research for Advanced Technology, Japan Science and Technology Agency, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan 3 Graduate School of Frontier Biosciences, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan Corresponding author: Yomo, Tetsuya ([email protected]) Current Opinion in Chemical Biology 2014, 22:12–17 This review comes from a themed issue on Synthetic biology Edited by Pier Luigi Luisi, Pasquale Stano and Cristiano Chiarabelli
http://dx.doi.org/10.1016/j.cbpa.2014.06.011 1367-5931/# 2014 Elsevier Ltd. All rights reserved.
Introduction All living things are composed of structural units called cells, which are vesicles typically ranging from 1 to 100 mm in diameter surrounded by a lipid membrane with membrane-bound proteins. A number of biological molecules, such as polynucleic acids, proteins, and low-molecularweight compounds, are encapsulated within the cell and perform various cellular functions, such as genome replication, translation, metabolism, signal transduction, cell growth, division, and other processes. These functions are strictly regulated by a complex genetic regulatory network, which is composed of more than 3000 interactions in Escherichia coli [1]. In other words, the cell is a micro-scale chemical reactor of enormous complexity, and artificially Current Opinion in Chemical Biology 2014, 22:12–17
preparing such a complex system is presently beyond the reach of human technology. The de novo construction of systems that perform natural cellular functions is the first step in developing technology to synthesize artificial cells. This technique is alternately called the semi-synthetic approach, the constructed approach, or in vitro synthetic biology [2–5]. The advantage of this approach compared with methods involving the modification or minimization of an extant cell is the ability to control the experimental conditions due to the use of a known set of materials, with researchers having strict control over the experimental conditions and the concentrations of all components. Using this approach, we can understand the conditions necessary to achieve a targeted cellular function without ambiguity because there are no unknown factors [6,7]. This type of knowledge could give rise to new technology that would allow us to tailor biological functions to our needs [8,9]. To date, various types of biological reactions have been constructed in cell-like compartments using a bottom-up approach. For example, polymerase chain reaction (PCR) [10], RNA synthesis [11], the expression of various proteins [12–17], RNA replication coupled with translation [18,19], and the regulation of gene expression via a riboswitch [20] or a positive feedback circuit [21] have been achieved using a discrete set of materials in phospholipid vesicles (liposomes). Another type of cell-like compartment is the water-in-oil droplet (or water-in-oil emulsion), in which the expression of green fluorescent protein (GFP) [22], the translation and selection of a methyltransferase [23], PCR [24–26], and RNA replication coupled with translation [27] have been achieved. In most of these examples, the cell-like compartments (e.g., the liposomes or water-in-oil droplets) have been used as mere containers to encapsulate the reaction solutions; therefore, the compartmentalization had no effect (or a negative effect) on the internal reactions. However, several recent studies have reported that compartmentalization can have positive effects on internal biological reactions [14,28–30], implying that the cell-like compartment plays a role beyond that of a mere container. In this review, we introduce two examples of the positive effects of compartmentalization on biological reactions: the isolation effect and the condensation effect. The isolation effect
Compartmentalization can improve the progress of an internal biochemical reaction through an isolation effect, which involves isolating a small number of inhibitory www.sciencedirect.com
Positive roles of compartmentalization Ichihashi and Yomo 13
(a)
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Our group has attempted to construct an artificial genome replication system that mimics the genome replication of RNA viruses. We have reported the construction of a translation-coupled RNA replication system composed of an artificial RNA genome encoding an RNA replication enzyme and the reconstituted translation system of E. coli [18]. In this system, the RNA replication enzyme derived from bacteriophage Qb was translated from an artificial RNA genome and was used to replicate the original genome. However, this self-replication process could not proceed for longer than a few hours despite an adequate nutrient supply due to the appearance of a ‘parasitic’ RNA that was generated from the genomic RNA as a result of recombination during the reaction. Because this parasitic RNA had lost the coding region of the replicase but retained the recognition site for the replicase, it could be replicated by the extant replicase (Figure 1b). In this sense, this small RNA was a parasite of the RNA replication system. Once this parasitic RNA appeared, it rapidly replicated until it occupied all the replicase molecules, inhibiting further genomic RNA replication after just a few hours.
Figure 1
RNA replication
factors from the major components of the reaction solution. If a reaction solution that contains a small amount of an inhibitory factor is encapsulated into a sufficient number of compartments, the inhibitory factors will be present in only a small number of the compartments, leaving most of the compartments free of the inhibitor. Therefore, on average, compartmentalization enhances the reaction rate (Figure 1a). This effect has been demonstrated by performing PCR in water-in-oil droplets (i.e., emulsion PCR), in which an extremely small amount of a target DNA fragment can be amplified without being inhibited by the non-specific amplification of smaller fragments [24,25]. The isolation effect has also been reported to significantly enhance RNA replication by an RNA-dependent RNA-polymerase (Qb replicase) [31,32]. Because RNA replication is more relevant to artificial cell technologies, we focus on this theme below.
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When we encapsulated the RNA replication solution in water-in-oil droplets of various sizes, we found that parasitic replication was repressed and that genome replication was prolonged [32]. The effect of compartmentalization depended on the compartment size: the smaller the droplet, the more the parasitic RNA was repressed and the longer the genome replication was able to proceed (Figure 1c). Interestingly, the optimum droplet size was similar to the size of E. coli cells (i.e., a few micrometers), which might be related to our use of the coliphage Qb replicase. Using this droplet size, we were also able to show that the genomic RNA selfreplicated recursively and evolved [27]. These results illustrate the role that cell-like compartments can play in recursive genome replication by excluding a parasitic replicator. www.sciencedirect.com
(a) Schematic diagram illustrating how isolation affects RNA replication. Genomic RNA replication is competitively inhibited by parasitic RNA, which appears spontaneously from genomic RNA. In a bulk reaction, the parasite inhibits the replication of all genomes, while in compartments, the parasite is confined and only inhibits a minor fraction of the genome replication (adapted with permission from the American Chemical Society [31]). (b) Schematic diagram illustrating the translation-coupled RNA replication system. The RNA replicase, translated from the genomic RNA (plus-strand), synthesizes the minus-strand using the original plusstrand as a template. The replicase subsequently replicates the plusstrand using the minus-strand as a template. The parasitic RNA, which has lost the replicase-encoding region, inhibits genome replication by successfully competing for the replicase (reproduced with permission from Elsevier [32]). (c) The effect of compartment size on RNA replication. Translation-coupled RNA replication can be performed in compartments of various sizes. In smaller compartments, the replication of the parasitic RNA is decreased and genome replication is increased (reproduced with permission from Elsevier [32]). Current Opinion in Chemical Biology 2014, 22:12–17
14 Synthetic biology
The role of cell-like compartments in isolating parasitic replicators is relevant to the origin of self-replication systems on early Earth. The emergence of a recursive replication system is considered the key step toward the origin of life. One of the major obstacles to this process is the appearance of a parasitic replicator, which can emerge in a self-replication system with a certain level of complexity and abolish the self-replication reaction [33].
Theoretically, one method to overcome the problem of parasitic replicators involves the use of spatial structures, such as compartments, that restrict the diffusion of parasitic molecules [34,35]. However, this possibility has not been experimentally verified. Our experimental results demonstrate that the isolation effect of compartments supports the role of spatial structures in the production of a recursive self-replication system.
Figure 2
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diluted mixture (non-reactive)
spontaneous overcrowding (reactive)
+lipids cell-like liposome
eGFP
inert liposome (b)
10 µm Current Opinion in Chemical Biology
(a) A model of the accumulation of macromolecules under dilute conditions during liposome formation (reproduced with permission from John Wiley and Sons [36]). (b) Fluorescent image of liposomes stained with Nile red (right panel). Only some of the liposomes exhibit eGFP expression (left panel) (adapted with permission from John Wiley and Sons [36]). Current Opinion in Chemical Biology 2014, 22:12–17
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Positive roles of compartmentalization Ichihashi and Yomo 15
The condensation effect
The second example of the positive effect of compartmentalization on internal biochemical reactions is the condensation effect, in which the internalized materials are concentrated, accelerating the reaction. To date, two condensation mechanisms have been reported by two different groups. The first mechanism involves the condensation of components by cooperative encapsulation into liposomes. Stano et al. prepared a diluted cell-free transcription– translation system containing DNA that encoded GFP
but could not function under the usual bulk conditions because it was excessively dilute. However, when the diluted solution was encapsulated into liposomes, some of the liposomes exhibited GFP translation (Figure 2) [36]. The number of liposomes that exhibited GFP expression was much greater than would have been predicted assuming random encapsulation of all the components. These results indicated that the components of the transcription–translation system became condensed when they were encapsulated into some of the liposomes through cooperative incorporation, enabling GFP expression, which had previously been impossible outside of the
Figure 3
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Current Opinion in Chemical Biology
(a) Schematic illustration of glucuronidase activity enhancement by compartmentalization (reproduced with permission from the American Chemical Society [29]). (b) Fluorescence image of water-in-oil droplets. (1, 4) Bright field, (2, 5) fluorescence, and (3, 6) merged images with (1, 2, 3) 0.3 nM or (4, 5, 6) 0.01 nM DNA. The scale bar represents 20 mm. Active glucuronidase exhibits green fluorescence in this experiment. Only a small fraction of the droplets exhibit glucuronidase activity at a DNA concentration of 0.01 nM (adapted with permission from the American Chemical Society [29]). www.sciencedirect.com
Current Opinion in Chemical Biology 2014, 22:12–17
16 Synthetic biology
liposomes. Luisi et al. further reported that the amount of protein incorporated into a liposome did not obey a Poisson distribution, implying that incorporation was an independent process. Rather, protein incorporation showed a power-law behavior, which implied the accumulation of solutes upon encapsulation [37]. To date, the mechanism of cooperative incorporation remains unknown. However, the condensation effect during liposome encapsulation may have been important to the origin of life. This process might have played a role in concentrating diluted biological molecules from bulk solution to enable biological functions on early Earth. The second mechanism underlying the condensation effect was revealed during the expression of a multimeric enzyme. Matsuura et al. performed transcription–translation of a glucuronidase gene in water-in-oil droplets of various sizes and found that compartmentalization of the reaction into smaller droplets facilitated the expression of glucuronidase activity when the DNA encoding the glucuronidase gene was adjusted to less than one molecule per compartment (Figure 3) [29]. At that concentration, there were two types of compartments: those that contained DNA and those that lacked DNA. In these compartments, the effective DNA concentration increased as the compartment size became smaller. The effective DNA concentration was proportional to the expression of the glucuronidase monomer, and the monomer concentration was proportional to the formation of the active tetrameric glucuronidase. Therefore, smaller compartments improved the glucuronidase activity. This effect on glucuronidase expression has also been observed in a microchamber [38]. Given that this type of condensation effect must occur when any protein that functions in a multimeric form is expressed, it should be considered when multimeric cellular proteins are expressed in an artificial cell.
Conclusions Recent advances in bottom-up approaches to generate an artificial cell have revealed that cell-like compartments can facilitate internal biochemical reactions through both isolation and condensation effects. These effects might have played important roles in the early development of primitive life because compartmentalization isolates inhibitory factors and concentrates biological polymers. These effects of compartmentalization cannot be revealed solely by analyzing extant cells because cells are already compartmentalized. Thus, the bottom-up approach is advantageous for the construction of an artificial cell because conditions that are not realized in native living organisms can be tested (e.g., reactions with or without compartments). Further development of artificial cells will provide important knowledge regarding the basic principles of life. The greatest difference between the described artificial cell models (liposomes and water-in-oil droplets) and Current Opinion in Chemical Biology 2014, 22:12–17
native cells is that most artificial cells are stable and neither grow nor divide. The stability of a compartment is important for reliable compartmentalization; however, the compartment must be unstable to some extent so that it is able to grow and divide. Preparing artificial cells possessing these conflicting properties is most likely the next important challenge. Some studies have reported the partial growth of cell-like structures. For example, Zhu et al. reported the growth and division of a lipid vesicle composed of fatty acids [39], and Kurihara et al. observed the growth of a lipid vesicle composed of artificial lipids in response to the amplification of its internal DNA [40]. Future research should focus on the construction of a lipid synthesis pathway [41] or cell division machinery [42] inside liposomes. In the process of generating artificial cell models that are closer to natural cells, we may come to understand the underlying principles necessary to prepare liposomes that mimic the dynamic properties of cellular membranes. Other fundamental roles of compartments have been proposed in theory and remain to be experimentally verified. One example is the stochastic corrector model, which is a mechanism proposed by Szathmary and Demeter in which a replication system prevents the collapse caused by the accumulation of parasitic replicators (Eigen’s paradox [43]) [44]. In this model, the combination of stochastic fluctuation in the number of encapsulated replicators and grouplevel selection leads to the selective advantage of ‘host’ replicators compared to parasitic (selfish) replicators. The principle of the model is similar to that of the isolation effect described in this review, although the isolation effect, in which parasitic replicators exist in very small numbers, represents an extreme case. In other theoretical studies, Kamimura and Kaneko proposed that a mutually catalyzing chemical reaction produces a compartment-like structure without any boundary molecules [45], and Mavelli and Stano proposed that a vesicle that reproduces itself in an autopoietic manner exhibits homeostasis in size [46]. These studies suggest that simple physical processes can produce biological phenomena without any complex biological machinery, which might be relevant to the emergence of life in the prebiotic world. The experimental verification of these theoretical proposals, including the stochastic corrector model, will be the next important challenge in in vitro synthetic biology.
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