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Synthetic cellularity based on non-lipid
micro-compartments and protocell models
Mei Li, Xin Huang, T-Y Dora Tang and Stephen Mann
This review discusses recent advances in the design and
construction of protocell models based on the self-assembly or
microphase separation of non-lipid building blocks. We focus
on strategies involving partially hydrophobic inorganic
nanoparticles (colloidosomes), protein–polymer globular nanoconjugates (proteinosomes), amphiphilic block copolymers
(polymersomes), and stoichiometric mixtures of oppositely
charged biomolecules and polyelectrolytes (coacervates).
Developments in the engineering of membrane functionality to
produce synthetic protocells with gated responses and control
over multi-step reactions are described. New routes to
protocells comprising molecularly crowded, cytoskeletal-like
hydrogel interiors, as well as to the construction of hybrid
protocell models are also highlighted. Together, these
strategies enable a wide range of biomolecular and synthetic
components to be encapsulated, regulated and processed
within the micro-compartmentalized volume, and suggest that
the development of non-lipid micro-ensembles offers an
approach that is complementary to protocell models based on
phospholipid or fatty acid vesicles.
Addresses
Centre for Protolife Research, Centre for Organized Matter Chemistry,
School of Chemistry, University of Bristol, Bristol BS8 1TS, UK
Corresponding author: Mann, Stephen ([email protected])
Current Opinion in Chemical Biology 2014, 22:1–11
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.2014.05.018
1367-5931/# 2014 Elsevier Ltd. All rights reserved
the most plausible scenario for the origin of life, as
enzyme catalysis [6], enzymatic-mediated RNA synthesis
[7] and replication [8], polymerase chain reaction (PCR)induced DNA amplification [9], and gene expression of
single components [10] or cascading networks [11,12]
have been successfully demonstrated in lipid-based or
surfactant-based micro-compartments. However, such
processes are often limited by the high impermeability
of the lipid membrane, which prevents continuous
activity within the vesicles due to mass transfer and
osmotic pressure restrictions. Moreover, the absence of
internal structuration within the vesicle micro-compartments restricts the ability of these synthetic models to
mimic cell-like behaviours associated with molecular
crowding or cytoskeletal assembly and disassembly.
And in terms of general utility, the widespread instability
of fatty acid vesicles to pH, ionic strength, multi-valent
cations and temperature implies that these chemical
micro-ensembles have limited technological application
particularly in adverse environments. As a consequence,
alternative types of synthetic cell-like entities are being
developed based on the self-assembly of inorganic (colloidosomes), polymer (polymersomes) or protein (proteinosomes) membranes, or phase separation of liquid
micro-droplets (membrane-free coacervates). Integrated
micro-systems based on these new types of protocells
should benefit from greater chemical stability and mechanical robustness, enhanced biocompatibility, and
increased scope for designing membrane functionality.
Moreover, a membrane-free protocell model based on
coacervate micro-droplets could contribute to alternative
scenarios for the origin of cellular life on the early Earth.
Here, we discuss these new approaches to synthetic
cellularity, focusing particularly on recent work published
within the last two or three years.
Protocell models based on inorganic
nanoparticle self-assembly
Introduction
Compartmentalization of primitive biochemical reactions
within microscale water-filled environments is a promising route towards synthetic cellular systems, and an
essential step in elucidating the origin of life [1,2]. Conventional studies on synthetic protocell models have
focused on minimal systems that confine and support
biological reactions in localized volumes of aqueous
space, which are delineated by organic boundaries usually
in the form of membrane-bounded lipid or fatty acid
vesicles [3–5]. To date, such an approach appears to offer
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Colloidosomes (also known as Pickering emulsions) are
microscale capsules that are produced from the spontaneous self-assembly of inorganic or organic colloidal
particles in water/oil biphasic systems. Assembly is driven
by a decrease in total free energy associated with placement of the particles specifically at the liquid/liquid
interface [13], and produces stabilized water or oil droplets that are dispersed in an oil or water continuous
phase, respectively. Since the seminal paper by Weitz
et al. in 2002 [14], a wide range of colloidosomes have
been prepared using inorganic particles, such as silica
[15,16], clays [17], CaCO3 [18], graphene oxide [19],
Current Opinion in Chemical Biology 2014, 22:1–11
2 Synthetic biology
TiO2 [20], Fe3O4 [21], cerium oxide [22], gold particles
[23], metal organic frameworks [24] and polyoxometalateorganic hybrid particles [25]. In most cases, the colloidosomes are several tens of micrometres in diameter, and
consist of a monolayer of closely packed particles in the
form of a continuous, enclosed membrane. Significantly,
the interstices between the particles form an array of
pores that exhibit high levels of selective permeability
based on solute size-exclusion. As a consequence, colloidosomes offer great technological potential in macromolecular storage and for controlling matter transport
between internal and external microscale reaction
environments.
Recently, a series of experiments have been initiated in
which semipermeable inorganic colloidosomes were
utilized as micro-compartments for housing biological
or biomimetic functions [15]. In principle, this
approach should provide new routes to the engineering
of artificial cellular systems and alternative membranebounded protocells based on inorganic nanoparticle selfassembly, which could have applications in synthetic
biology, bionanotechnology and origins of life research.
Water-filled colloidosomes, tens of micrometres in
diameter, were prepared in oil by using partially hydrophobic silica nanoparticles, only 20–30 nm in size
(Figure 1a). Many types of biomolecules could be
entrapped without denaturation within the inorganic
compartments; for example a large ensemble of
enzymes, amino acids, nucleic acids, ribosomes, plasmids, among others, were successfully encapsulated and
used as a cell-free gene expression system for spatially
confined in vitro protein synthesis [15] (Figure 1b).
Three examples of sustained enzyme catalysis using
entrapped proteins and oil-soluble membrane-permeable substrates were also demonstrated.
The permeability of the colloidosome shell has been
tailored by engineering the nature of the membrane
components. For example, control over the pore size
and release rate of encapsulated dye molecules can be
achieved by using organic colloids in the form of temperature-dependent polymer microgel particles that exhibit
swelling/deswelling behaviour upon changing temperature [26]. Alternatively, sintering the temperature responsive polymer particles [27], coupling a pH-responsive
crosslinker to the membrane components [28], or varying
the size and shape of the nanoparticles [29] can be used to
tune the permeability of the colloidosomes. However,
these methods are only successful in suppressing the
transport of molecules larger than a crucial value; in
contrast, the diffusion of small molecules through the
colloidosome monolayer is difficult to regulate over a
broad range of membrane particle diameters [30]. This
is a major challenge for the use of colloidosomes as waterdispersible, semipermeable inorganic protocells, as the
rapid discharge of small molecule substrates from the
Current Opinion in Chemical Biology 2014, 22:1–11
micro-compartment would severely restrict their potential as primitive artificial cells.
As a step towards overcoming this problem, water-filled
protocell constructs with self-activated, electrostatically
mediated permeability have been recently prepared and
investigated in continuous aqueous media [31]. Before
transfer from dodecane into water, silica nanoparticlebased colloidosomes were crosslinked with tetramethoxysilane (TMOS) and then functionalized by grafting a
pH-responsive copolymer onto the inorganic membrane
(Figure 1c). Once in water, these modifications produced
inorganic protocells with an outer polymer-based coronal
layer that was capable of electrostatically mediating the
diffusion of small molecules through the interstitial
nanospaces of the silica nanoparticle shell. Changes in
the zeta potential of the polymer (+20 and 30 mV at pH
2 and pH 10, respectively; pKa 5.76) were used to prepare
colloidosomes with net positive or negative surface
charges at low or high pH, respectively, and provide
the basis of an electrostatically gated inorganic protocell.
For example, at low pH, small molecule cationic solutes
(acriflavine hydrochloride, rhodamine 6G) were rejected
from entry or egress by electrostatic repulsions arising
from positive charges on the colloidosome membrane,
whereas at high pH small molecule anionic solutes
(calcein) were unable to diffuse across the membrane.
Significantly, this gating mechanism was exploited for
the self-regulation of an enzyme reaction within the
inorganic protocells [31,32]. The small molecule anionic substrate 4-nitrophenyl phosphate (4NPP) was
added to the continuous aqueous phase of a dispersion
of polymer-grafted, crosslinked colloidosomes containing encapsulated alkaline phosphatase, and the reaction
switched on or off by pH-mediated electrostatically
induced gating of 4NPP into the micro-compartment
interior [31]. A related strategy has been reported using
a thermally gated process to control the diffusion of small
molecules across a semipermeable colloidosome membrane [33]. For this, a thermo-sensitive triblock copolymer was encapsulated into the interior of organic
nanoparticle-based colloidosomes and used to inhibit
the diffusion of dye molecules through the membrane
pores by temperature-dependent adsorption or desorption of the polymer to the inner surface of the semipermeable shell.
In addition to tuning membrane permeability, other
recent studies have explored the possibility of preparing
colloidosome-based protocells with molecularly crowded
interiors that mimic the structural reversibility and
chemical milieu of the cytoskeletal matrix. This was
achieved by encapsulation of alkaline phosphatase and
a derivatized amino acid, fluorenylmethylcarbonyl-tyrosine-(O)-phosphate (FMOC-TyrP) into silica nanoparticle-based colloidosomes, followed by self-assembly of a
supramolecular hydrogel within the micro-compartments
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Synthetic cellularity and protocell models Li et al.
3
Figure 1
(a)
(d)
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Current Opinion in Chemical Biology
Protocell models based on inorganic nanoparticle self-assembly. (a) Optical micrograph of silica nanoparticle-stabilized water micro-droplets
(colloidosomes) dispersed in oil; scale bar = 200 mm. (b) Fluorescence microscopy image of a single colloidosome undergoing cell-free gene
expression. Plasmid pEXP5-NT/eGFP and components of a cell-free gene expression system were encapsulated within the inorganic protocells and
left for 24 hours at 37 8C, and the increase in green fluorescence due to in vitro gene-directed synthesis of the protein eGFP was monitored with time
[15]; scale bar = 100 mm. (c) Construction of water-dispersible aqueous inorganic micro-compartments with self-activated gated membrane
permeability. The scheme illustrates the design and construction of TMOS-crosslinked, polymer-grafted colloidosomes. The branched copolymer
corona is linked to the semipermeable outer surface of a closely packed shell of silica nanoparticles. pH-induced changes in the net charge associated
with cationic dimethylamino (blue) and anionic carboxylate (red) groups of the copolymer layer generate a gated response to the transfer of charged
small molecules across the inorganic membrane. The structure of the branched copolymer is shown (right). (d) Fluorescence microscopy image
recorded after 1 day from a single colloidosome containing encapsulated alkaline phosphatase and FMOC-TyrP. The intense blue fluorescence
originates from binding of Hoechst 33258 dye to amino acid nanofilaments that are self-assembled into a hydrogel matrix inside the microcompartment [34]; scale bar = 20 mm. (e) Enzyme kinetic plots for alkaline phosphatase-mediated dephosphorylation of substrate 4NPP showing
changes in initial rates with substrate concentration for reactions undertaken in colloidosomes containing a hydrogel core (30 8C, blue circles) or a
disassembled FMOC-Tyr hydrogel interior (45 8C, red triangles). Corresponding values for the maximum velocity (Vmax) and Michaelis constant (Km) are
shown [34]. (f) Optical microscopy images showing time course of growth and division of inorganic protocells. A single liquid bud occurs at a discrete
rupture point after approximately 10 minutes, and continues to develop into a 2G micro-compartment to produce a pair of conjoined inorganic
protocells [35]; scale bar = 100 mm.
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Current Opinion in Chemical Biology 2014, 22:1–11
4 Synthetic biology
due to enzyme-mediated FMOC-TyrP dephosphorylation (Figure 1d). [34]. Significantly, the kinetics of
hydrogel growth were strongly influenced by confinement
within the colloidosome micro-environment. Moreover,
increasing the temperature above the gel–sol transition
point was used to reversibly disassemble the amino acid
hydrogel nanofilaments, such that protocells with modulated internal viscosities were produced. As a consequence, auxiliary enzyme reactions inside the
protocells could be effectively switched on or off in the
absence or presence of the cytoskeletal-like internalized
matrix (Figure 1e).
Taken together, the above reports demonstrate the
potential of nanoparticle-based inorganic colloidosomes
as novel microscale bio-reactors, and suggest that studies
on bio-inorganic protocells could provide an alternative
paradigm of primitive cell formation that is not based on
lipid compartmentalization [15]. Although the design
and construction of inorganic-based micro-compartments
with biomimetic cell-like properties is clearly feasible, it
remains difficult to envisage how these non-lipid protocells could undergo processes of growth and division
analogous to the behaviour of organic vesicles. In this
regard, a recent study described a remarkable process of
primitive growth and division involving buffer-filled inorganic colloidosomes dispersed in oil [35]. Growth of the
colloidosomes was induced by organosilane-mediated
methanol formation inside the micro-compartments,
and resulted in a localized rupture of the inorganic
membrane followed by outgrowth and separation of a
second-generation protocell that was stabilized by surface
adsorption of silica nanoparticles present in the oil phase
(Figure 1f). Addition of the organosilane simultaneously
generated a slow increase in internal pressure by methanol production, as well as an increase in the membrane
rigidity via silica crosslinking of the colloidosome membrane. Under optimum conditions, the balance between
volume expansion and increased surface elasticity associated with methanol production and silica crosslinking of
the membrane, respectively, gave rise to stable growth
conditions and the inception of a single rupture point to
produce conjoined protocells that readily separated.
Multiple divisions were also achieved by changing the
amount of organosilane added to the colloidosome dispersion [35].
Proteinosome-based protocells
Very recently, a new type of membrane-bounded protocell model based on the spontaneous interfacial assembly
of amphiphilic protein–polymer nano-conjugates has
been reported [36]. The protein–polymer nano-conjugates, which consisted of three or so polymer molecules
per protein, assembled at water droplet/oil interfaces into
micrometer-scale water-filled capsules termed proteinosomes. The proteinosomes were delineated by a closely
packed monolayer of conjugated protein–polymer
Current Opinion in Chemical Biology 2014, 22:1–11
building blocks that could be crosslinked such that the
micro-compartments were transferred into water without
fragmentation (Figure 2a). As a consequence, a wide
range of guest molecules from small molecule fluorescence dyes to colloidal particles could be encapsulated at
high efficiency inside the micro-compartments. Significantly, it was possible to entrap a hundred or so components of a cell-free gene expression system within the
proteinosome internal volume (ca. 1 pL), and perform in
situ protein synthesis (Figure 2b). Moreover, the protein–
polymer membrane was sufficiently elastic and robust to
withstand partial dehydration and rehydration, and
remained structurally intact when held at a temperature
of 70 8C for 90 min, suggesting that the proteinosomes
could provide a useful protocell model system for studying the microscale confinement of thermophilic enzymes,
or for developing artificial cells capable of undertaking
temperature cycling procedures, such as PCR-mediated
amplification of entrapped genetic polymers.
A key advantage of the proteinosome system lies with the
ability to rationally design the structure and function of
the proteinosome membrane by controlling the chemistry
of the protein–polymer nanoscale building blocks. This
makes the system an attractive candidate for the development of a range of functional protocells. For example,
recent studies indicated that the proteinosome membrane was semipermeable, stimulus-responsive, enzymatically active, and mechanically elastic [36]. It also
exhibited size-selective permeability, only allowing molecules less than 40 kDa in molecular mass to enter or
leave the proteinosome interior. Moreover, conjugation of
the temperature sensitive polymer, poly(N-isopropylacrylamide) (PNIPAAm) to the protein molecules generated a
thermally gated system that operated via the swelling or
deswelling (at T > 33 8C) of the polymer chains present
on the outer surface of the proteinosome membrane. As a
consequence, enzyme-mediated peroxidation within the
proteinosomes of a small molecule initially present in the
external solution was switched on or off by thermally
gating the diffusion of the substrate through the polymer
shell barrier layer [36] (Figure 2c,d).
As a development of these ideas, a recent study has
exploited the fact that the proteinosome system provides
a means of transforming water-soluble proteins and
enzymes into membrane-bounded ensembles. Thus, provided that insertion and crosslinking into the membrane
does not seriously perturb biomolecule function, a wide
range of multi-functional, biocompatible protocellular
constructs can be envisaged. For example, glucose
amylase (GA), glucose oxidase (GO) and horseradish
peroxidase (HRP) have been used to prepare protein–
PNIPAAm amphiphilic nano-conjugates to construct
proteinosomes comprising an enzyme triad capable of
undertaking a multi-step membrane-mediated cascade
reaction using starch as the starting substrate [37]. The
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Synthetic cellularity and protocell models Li et al.
5
Figure 2
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Current Opinion in Chemical Biology
Proteinosome-based protocells. (a) AFM image of a dried proteinosome after transfer to water showing intact, collapsed micro-compartment enclosed
by a continuous and flexible protein-polymer crosslinked membrane; scale bar = 5 mm [36]. (b) Gene-directed synthesis of eGFP in proteinosomes.
The fluorescence microscopy image shows a population of proteinosomes with encapsulated plasmid pEXP5-NT/eGFP and cell-free gene expression
components recorded after 2 hours of incubation at 37 8C. Green fluorescence associated with in vitro expression of eGFP is observed in the
proteinosome micro-compartments; scale bar = 100 mm [36]. (c) Scheme showing polymer chain gating of enzyme reactions inside proteinosomes.
Access to an entrapped enzyme depends on the hydrophobicity and porosity of the protein–polymer membrane, which can be influenced by the
swelling/deswelling behaviour of the PNIPAAm chains. As a consequence, access to the internal compartment is curtailed for certain small molecule
substrates above 33 8C due to polymer chain collapse on the outer surface of the membrane. (d) Temperature-dependent catalytic reaction rates of
free myoglobin (black lines) and proteinosome-encapsulated myoglobin (red lines) in the presence of 2-methoxyphenol (5 mM) and H2O2 (20 mM),
showing membrane-gated enzyme catalysis in proteinosomes dispersed in bulk water [36]. (e) Three-step cascade reaction in multi-enzyme
proteinosomes. Summary chart of the catalytic activity of enzyme–polymer proteinosomes prepared with different membrane and internal spatial
organizations. Values are initial reaction rates of product formation (V0) at 25 8C. Grey circles represent the proteinosome membrane, and locations of
GA, GO and HRP are indicated. Starch is added to the external aqueous phase to initiate the cascade. The V0 values for the cascade reaction were
essentially unchanged when native GO or HRP was encapsulated in the interior of the proteinosomes. By contrast, V0 was reduced by 90% when
native GA was encapsulated within the aqueous interior and the GO-PNIPAAm and HRP-PNIPAAm conjugates crosslinked into the membrane [37]. (f)
Mechanical properties of hydrogel-containing proteinosomes. Optical microscopy images showing time-dependent self-healing behaviour of a single
hydrogel proteinosome subjected to deformation using a soft plastic fiber [38]; scale bar = 10 mm.
tandem reaction was regulated by the positional assembly
and spatial separation of the three enzymes within the
membrane of the protocell (Figure 2e), as well as by
temperature-dependent changes in the conformation of
the covalently attached polymer chains located on the
external surface.
In recent work, proteinosome-based protocells with new
types of higher-order functionality based on controlled
membrane disassembly and permeability, internalized
assembly of a cytoskeletal-like matrix, and self-production of a membrane-enclosing outer wall have been
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described [38]. Genetic polymers were encapsulated in
the proteinosomes and their release triggered by protease-mediated hydrolysis or chemical cleavage of the
membrane building blocks. In addition, proteinosomeencapsulated, enzyme-mediated amino acid dephosphorylation was used to generate a supramolecular amino acid
hydrogel within the interior of the protein–polymer
micro-compartments. As a consequence, the mechanical
properties of the protocells were considerably enhanced.
For example, the spherical morphology of the hydrogelcontaining proteinosomes was quickly re-established
after protocells were mechanically deformed or deflated
Current Opinion in Chemical Biology 2014, 22:1–11
6 Synthetic biology
due to dehydration (Figure 2f). Interestingly, prolonged
growth of the encapsulated hydrogel resulted in penetration of the amino acid nanofilaments through the porous
shell of the proteinosome to produce a continuous, protease-resistant outer wall that totally enclosed the
protein–polymer membrane [38], analogous in some
respects to the cell wall/cell membrane layered structure
of bacterial cells.
Polymersome-based protocells
The above sections highlight the fact that synthetic
polymers play important structural and functional roles
in the design of artificial cell-like micro-systems based on
hybrid building blocks. In particular, covalent grafting of
polymer chains to inorganic nanoparticles (colloidosomes)
or globular proteins (proteinosomes) is an effective means
to generate microscale water-filled compartments
enclosed by semipermeable monolayer membranes of
functional nanoscale components. These membranes
are therefore very different from the molecule-based
bilayer or multi-lamellar structures that are characteristic
of fatty acid and phospholipid vesicles. By contrast, the
self-assembly of amphiphilic block copolymers into
vesicle-like micro-architectures (polymersomes [39]) provides a more closely related synthetic analogy with liposome-based protocell models [40,41]. Polymersomes are
generally more stable than liposomes, can be prepared
with a wide range of membrane chemistries, and engineered towards semipermeability by introducing porosity
into the block copolymer bilayers [42–46]. For example,
channel-forming proteins can be reconstituted without
loss of function within the polymer membrane to produce
polymersomes with size-selective or substrate-specific
pores [43]. Alternatively, stimuli-responsive block copolymers can be used as pore-generating components to
control membrane-permeability, induce protocell disassembly, or regulate the activity of encapsulated enzymes
in response to changes in the external environment [44].
In an attempt to mimic the morphological diversity of
living cells, approaches have been developed to induce
controllable shape transformations in polymersomes. In
particular, kinetic manipulation of the phase behaviour of
the glassy hydrophobic segment of an amphiphilic copolymer [47,48], and crosslinked induced changes in the
composition and structure of the polymersome membrane [49], have been successfully developed. For
example, well-defined bowl-shaped polymersomes (polymer stomatocytes) were constructed using a poly(ethylene glycol)-polystyrene block copolymer [47]. The
hydrophobic polystyrene domain displays a phase transition from a solvent-swollen rubbery state to a rigid
glassy state, and kinetic manipulation of this phase behaviour was exploited to capture transient morphologies
in the shape of the polymersomes. Moreover, by selectively entrapping catalytically active platinum nanoparticles within the nanocavities the polymer stomatocytes
Current Opinion in Chemical Biology 2014, 22:1–11
were capable of moving autonomously in the presence of
aqueous hydrogen peroxide solutions [47].
It is well known that multi-compartmentalization is a key
characteristic of eukaryotic cells, and it is therefore an
interesting challenge to design models of primitive compartmentalization with integrated synthetic organelles.
Although this strategy has been developed extensively
in liposomes-in-liposome systems, it has only recently
been advanced in non-lipid models involving polymersomes [50–53]. Polymersomes-in-polymersome microarchitectures have been prepared by an emulsion/
centrifugation method, and used to develop a threeenzyme cascade reaction across multiple compartments
using either compatible or incompatible enzymes [54].
In other studies, a multi-compartment system (capsosome) was prepared by incorporating liposomes inside
the shell of a polymeric capsule using a layer-by-layer
technique [55,56]. Co-encapsulation of glutathione
reductase and disulfide-linked polymer-oligopeptide conjugates was then used to trigger the enzyme-mediated
reduction of glutathione specifically within the multicompartmentalized protocells.
Membrane-free protocell models
As discussed above and elsewhere in this edition, the
ubiquity of the cell membrane in living organisms has
stimulated the development of a range of models of
protocellular micro-compartmentalization based on the
self-assembly of amphiphilic building blocks such as
phospholipids, fatty acids, functionalized inorganic nanoparticles, block copolymers and protein–polymer nanoconstructs. Although it seems obvious that the modelling
of primitive cells would be based primarily on membrane
assembly, it is less evident that these systems have much
relevance to questions concerning the origin of life.
Indeed, except for fatty acid self-assembly, most of the
other models are implausible in this regard, although they
have considerable technological merit as platforms for
synthetic cellularity. Unfortunately, origin of life theories
of fatty acid vesicle self-assembly are also deficient in
several respects, notably in terms of low encapsulation
efficiencies, sensitivity to changes in pH and ionic
strength, and the absence of internal structuration. In
principle, these limitations can be overcome by invoking
an intriguing alternative model of prebiotic organization
based on the spontaneous formation of membrane-free,
chemically enriched liquid droplets via aqueous phase
separation or complex coacervation.
Aqueous phase separation in multicomponent solutions
containing two types of polymers or macromolecules is
driven by a change in entropy [57], which results in two
distinct thermodynamic phases comprising different polymer compositions [58]. Polymer phase separation has been
demonstrated in aqueous systems comprising neutral
polyethylene glycol/dextran solutions [59]. Formation of
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Synthetic cellularity and protocell models Li et al.
the two liquid phases, which was dependent on the
temperature, concentration and ionic strength, gave rise
to separated liquids with different polymer compositions
that could be tuned by changing the molecular weight or
stoichiometric ratio of the components [60]. Significantly,
liquid–liquid demixing can give rise to membrane-free
fluid-like organelles in living cells that are molecularly
distinct from the surrounding cytoplasm [61]. Interest
in these systems continues to grow [62,63]. Complex
coacervation also involves aqueous two-phase separation
but is typically driven by electrostatic and entropic interactions between charged polymers or macromolecules, and
results in the formation of component-enriched microdroplets suspended in a chemically deficient aqueous
continuous phase [64,65]. The spontaneous formation
of micro-droplets in these equilibrium systems provides
a facile mechanism for membrane-free compartmentalization, and as noted by Oparin in the 1930s, could be highly
relevant to the evolution of the cell on early Earth [66].
Hundreds of complex coacervate systems have been
characterised and prepared from a range of components
such as biological macromolecules, multivalent proteins
and synthetic and natural polymers [67]. However, there
are few reports of coacervate droplets produced from low
molecular weight components. In this regard, a recent
study described the formation of coacervate micro-droplets prepared by electrostatic complexation of cationic
oligolysine and anionic nucleotides such as ATP [68].
The droplets were tens or hundreds of micrometres in
size, highly enriched in peptides and nucleotides, stable
across a large pH and temperature range, and capable of
sequestering a wide range of low and high molecular
weight solutes including enzymes, substrates, nanoparticles and photoactive molecules (Figure 3a,b). The ease of
preparation, molecular simplicity of the components,
fundamental biological relevance of peptides and nucleotides, as well as the ability of the droplets to undergo
growth and disassembly, suggested that a model of prebiotic organization based on coacervation should be
seriously considered. Moreover, partitioning of molecules
into the coacervate phase was based on differences in
dielectric constant between the external aqueous solution
and the molecularly crowded interior of the droplets
[68]. As a consequence, compared with bulk aqueous
solution, the sequestration of biomolecular reactants into
polylysine/ATP coacervate micro-droplets resulted in a
significantly increased reaction rate for a glucose phosphorylation/dehydrogenation two-cascade enzyme reaction (Figure 3c). Similarly, elevated concentrations of a
minimal multi-enzyme complex actinorhodin polyketide
synthase were obtained by partitioning within the droplets, and used to increase the synthesis yield of two
complex natural products [69].
Considerable enhancements in reaction rates have also
been demonstrated in the molecularly crowded
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7
environments of aqueous two-phase systems. For
example, compared to a polymer free solution, RNA
catalysis by the hammerhead ribozyme was increased
approximately 70-fold when partitioned preferentially
into the dextran-rich phase of a polyethylene glycol/
dextran phase-separated system [60]. Similarly, urease
activity was significantly increased by preferential
sequestration into the dextran phase, and could be
further regulated by tuning the relative volumes of
the polyethylene glycol/dextran phases [70]. In other
studies, enhanced levels of gene expression were
reported in a cell lysate that was subjected to phase
separation in polyethylene glycol-rich droplets by in situ
dehydration of the aqueous phase within a microfluidic
device [71].
Hybrid protocell models
Although coacervate micro-droplets can be stabilized by
excess surface charge [72], under many conditions they
exhibit a propensity to coalesce into larger droplets or
undergo macroscopic phase separation. As a consequence,
their use as a protocell model can be compromised by
their liquid-like behaviour and low surface tension. To
circumvent this problem, and to generate a new type of
hybrid protocell, Mann and colleagues have recently
developed several methods to produce membrane-bound
coacervate micro-droplets via spontaneous assembly or
partitioning of auxiliary components on the surface of the
liquid micro-compartments [73,74,75]. For example,
partitioning of high concentrations of the organic molecule, 8-anilionapthalene sulphonate (ANS) into microdroplets prepared from mixtures of oligolysine and ATP
resulted in the formation of a distinct ANS-rich membrane on the surface of the droplet [73]. In other work, a
continuous inorganic membrane was assembled onto the
surface of polyelectrolyte/ATP coacervate micro-droplets
via electrostatic interactions between the polycationic
polymer and inorganic polyanions (phosphotungstate)
that were added to the continuous phase of the dispersion
(Figure 3d) [75]. Interestingly, formation of the inorganic
membrane was associated with sub-division of the droplet
interior to produce a novel three-tiered vesicle architecture comprising a semipermeable negatively charged
phosphotungstate/polyelectrolyte membrane, a submembrane coacervate shell enriched in polyelectrolyte
and ATP, and an internal water-filled lumen (Figure 3e).
As a consequence, proteins encapsulated in the coacervate vesicles were inaccessible to proteases, and could be
exploited for the spatial localization and coupling of twoenzyme cascade reactions.
Very recently, a fatty acid multi-lamellar membrane has
been spontaneously assembled on the surface of coacervate micro-droplets containing RNA and oligopeptides
[74]. Membrane assembly occurred under conditions
not conducive to vesicle formation in free solution
to produce well-defined fatty acid-coated droplets
Current Opinion in Chemical Biology 2014, 22:1–11
8 Synthetic biology
Figure 3
(b)
(c)
0.10
[β-NADPH]/mM
(a)
(d)
(e)
C
0.08
0.06
0.04
0.02
0.00
0.0
W
2.0
1.0
3.0
Time/min
M
(ii)
(h)
200
(f)
(g)
100
(i)
1.6
Intensity (a.u.)
300
1 x:P
0.6
0.11
0.12
0.13
0.14
0.15
q (Å–1)
Current Opinion in Chemical Biology
Membrane-free and hybrid protocell models. (a) Optical bright field image of coacervate micro-droplets prepared in water by mixing cationic
poly(diallyldimethyl ammonium chloride (PDDA) with anionic ATP. The micro-droplets also contain a sequestered low molecular weight dipeptide, Nfluorenylmethyloxycarbonyl dialanine; scale bar = 50 mm. (b) Confocal fluorescence microscopy image showing localized blue fluorescence
associated with sequestration of 8-anilionapthalene sulphonate (ANS) into oligolysine/ATP coacervate micro-droplets [73]; scale bar = 1 mm. (c)
Catalytic reactions in peptide/nucleotide coacervate droplets. Time profile of increase in b-NADPH concentration associated with a two-enzyme
cascade reaction in polylysine/ATP coacervates containing the ATP-dependent enzyme hexokinase and glucose-6-phosphate dehydrogenase (filled
dark red circles). The reaction rate is approximately doubled compared with the corresponding reaction in bulk aqueous solution (open blue circles)
[68]. (d) Optical microscopy image showing spherical inorganic membrane-bounded coacervate vesicles prepared by addition of aqueous sodium
phosphotungstate (PTA) to a vigorously stirred dispersion of PDDA/ATP coacervate droplets [75]; scale bar = 50 mm. Inset shows single coacervate
vesicle viewed between cross-polarizers showing birefringence due to ordering in the PTA/PDDA membrane. (e) Optical microscopy image showing
initial stage of development of a three-tiered micro-architecture in PTA/PDDA/ATP coacervate vesicles prepared as in (d); locations of the PTA/PDDA
membrane (M), PDDA/ATP coacervate sub-membrane layer (C), and water-filled lumen (W) are shown [75]. Image was taken 30 s after addition of PTA
to a coacervate dispersion; scale bar = 20 mm. (f) Graphic showing hybrid protocell model based on the self-assembly of a fatty acid multi-layered
membrane on the surface of coacervate micro-droplets containing ATP, RNA and cationic oligopeptides or polyelectrolytes. (g) Fluorescence
microscopy image of an oleate-coated PDDA/ATP coacervate droplet stained with lipid-soluble BODIPY FL C16 dye, and showing green fluorescent
ring associated with membrane formation [74]; scale bar = 1 mm. (h) Small angle X-ray scattering profiles of oleate/PDDA/ATP micro-droplets
prepared at different oleate/PDDA/ATP molar ratios (PDDA:ATP molar ratio = 1:0.25 = P in all cases). Peaks at 5.46 (peak (i)) and 4.77 nm (peak (ii)) are
observed; the latter is highly dependent on increasing oleate concentration (increasing x: P) and was associated with assembly of a multi-lamellar fatty
acid membrane on the surface of the coacervate droplets [74].
(Figure 3f,g). The membrane-coated droplets were stabilized with regard to coalescence and exhibited selective
uptake or exclusion of small and large molecules. Moreover, changes in the ionic strength were employed to
disassemble the coacervate interior without loss of membrane integrity. Together, the results indicated that
simple physical and chemical processes could give rise
to hybrid protocells based on membrane-compartmentalization, chemical enrichment and internalized
Current Opinion in Chemical Biology 2014, 22:1–11
structuration (molecular crowding), and that key aspects
of vesicle-based and coacervate-based models of prebiotic
compartmentalization could be successfully integrated.
Finally, we note that aqueous two-phase polymer systems
have been encapsulated in phospholipid vesicles to generate internalized sub-compartments [76]. As a consequence, lipid-based protocells exhibiting protein
localization and phase transfer [77] were produced, and
www.sciencedirect.com
Synthetic cellularity and protocell models Li et al.
in micelles and self-reproducing vesicles. J Am Chem Soc
1994, 116:7541-7547.
vesicle budding and division into compositionally different daughter vesicles demonstrated [78,79].
8.
Oberholzer T, Wick R, Luisi PL, Biebricher CK: RNA replication in
self-reproducing vesicles: an approach to a minimal cell.
Biophys Enzymatic Res Commun 1995, 207:250-257.
9.
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liposomes. Chem Biol 1995, 2:677-682.
Conclusions
This review has highlighted recent advances in the design
and construction of protocell models based on the selfassembly or microphase separation of non-lipid building
blocks. We have focused particularly on strategies involving partially hydrophobic inorganic nanoparticles (colloidosomes), protein–polymer globular nano-conjugates
(proteinosomes), amphiphilic block copolymers (polymersomes), and mixtures of oppositely charged biomolecules and polyelectrolytes (coacervates). Significant
advances have been made in the design and construction
of these micro-compartments, notably in the engineering
of membrane functionality for gated responses and control over multi-step reactions, as well as in the structuration of the interior space for molecular crowding and
cytoskeletal-like reversible hydrogel assembly. As a
result, a wide range of biomolecular and synthetic components can be encapsulated, regulated and processed
within the micro-compartmentalized space, suggesting
that these non-lipid micro-ensembles offer attractive
alternatives to phospholipid or fatty acid protocell
models. Taken together, the studies reviewed herein
exemplify a modern approach to synthetic cellularity that
advances the physical and chemical basis of cell structure
and function, proposes steps towards understanding the
emergence of primitive cells on the early Earth, and
facilitates the development of smart autonomously functioning chemical micro-compartments. These attributes
should provide novel opportunities in bioinspired microstorage and delivery, micro-reactor technologies, cytomimetic engineering, and the development of integrated
constructs for diverse procedures in synthetic biology.
References and recommended reading
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