Available online at www.sciencedirect.
com Current Opinion in
ScienceDirect
Designer endosymbionts: Converting free-living
bacteria into organelles
Rebecca S. Meaney, Samir Hamadachea,
Maximillian P. M. Soltysiaka and Bogumil J. Karas
Abstract
An emerging frontier in synthetic biology involves the engi-
neering of interspecies relationships, one of which could result
in the development of novel organelles. The endosymbiotic
theory is the most widely accepted model for the evolutionary
origin of mitochondria and chloroplasts, asserting that these
organelles descended from free-living bacteria. Imitating this
process experimentally, which we refer to herein as directed
endosymbiosis, could enable the development of an entirely
new class of organisms with synthetic organelles. In this
review, we discuss principles and strategies for directed
endosymbiosis and highlight current developments. We also
describe several bacterial species as candidates for converting
into organelles that would have interesting applications.
Addresses
Department of Biochemistry, Schulich School of Medicine and
Dentistry, The University of Western Ontario, London, N6A 5C1,
Ontario, Canada
Corresponding author: Karas, Bogumil J (bkaras@uwo.ca)
a
Co-second authors.
Current Opinion in Systems Biology 2020, 24:41–50
This review comes from a themed issue on Synthetic Biology (2020)
Edited by Matteo Barberis and Tom Ellis
For complete overview of the section, please refer the article collection -
Synthetic Biology (2020)
Available online 28 September 2020
https://doi.org/10.1016/j.coisb.2020.09.008
2452-3100/© 2020 The Authors. Published by Elsevier Ltd. This is an
open access article under the CC BY license (http://creativecommons.
org/licenses/by/4.0/).
Keywords
Synthetic biology, Directed endosymbiosis, Novel organelle, Nitrogen-
fixing organelle, Mycoplasma, Cyanobacteria, Rhizobia.
Introduction
Synthetic biology is the rapidly evolving practice of
designing and creating novel biological systems. This
highly interdisciplinary field is made possible by tech-
nological advances in the reading, writing, and editing of
genomes [1]. A largely unexplored realm involves the
engineering of novel endosymbiotic relationships
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Systems Biology
between two or more species. The endosymbiotic theory
is the widely accepted model for explaining the origins of
mitochondria and chloroplasts [2]. According to this
theory, these organelles descend from free-living bacteria
that took up residence within other cells and formed
symbiotic relationships with their hosts over evolutionary
time [3,4]. By imitating this evolutionary mechanism, a
technique that can be called directed endosymbiosis, it
could be possible to generate novel or synthetic organ-
elles. Not only does this provide an opportunity to
improve existing types of organelles, but there is also the
potential to equip cells with entirely new classes of or-
ganelles [5]. While this review focuses on the develop-
ment of bacterial species for endosymbiosis within yeast
hosts, directed endosymbiosis will not be limited to
these organisms. The rapid growth rate, genetic tracta-
bility, and availability of yeast strain collections will ease
the process of establishing effective methods for
directed endosymbiosis; however, we foresee a broad
range of hosts, including multicellular organisms such as
plants and animals, being adapted in the future.
Although modification of existing organelles such as
chloroplasts and mitochondria could lead to the devel-
opment of synthetic organelles, methods for cloning,
engineering, and delivery are still being developed [6e
8]. Instead, the strategy we discuss here involves
introducing bacteria (candidate endosymbionts) into
eukaryotic cells (hosts) to produce stable symbiotic
chimeras. Toward such a goal, Agapakis et al. [9] aimed
to create artificial chloroplasts. They demonstrated that
the cyanobacterium Synechococcus elongatus could be taken
up and propagated by zebrafish embryos, hamster ovary
cells, or mouse macrophages with no apparent effect on
their viability [9]. More recently, Mehta et al. (2018)
[10] transferred and maintained auxotrophic Escherichia
coli within respiration-deficient Saccharomyces cerevisiae in
an effort to mimic the evolution of mitochondria. In this
study, thiamin-deficient E. coli exported ATP through a
transgenic translocase for the S. cerevisiae Dcox2 host in
exchange for thiamin. Chimeras were then able to grow
on nonfermentable media, showing a partially rescued
respiration-competent phenotype [10]. To further
simulate organelle evolution, the group created multi-
auxotrophic strains and removed up to 46 kb from the
E. coli genome, imitating genome reduction that
Current Opinion in Systems Biology 2020, 24:41–50
42 Synthetic biology (2020)
occurred in the lineage leading to mitochondria. They
confirmed that the bacteria-containing hosts were stable
for at least 40 generations with survival assays and visual
imaging [10,11]. Furthermore, after establishing a
method for the transfer of whole bacterial genomes to
yeast, Karas et al. proposed [12] and later observed [13]
that synthetic mycoplasma cells are transiently suppor-
ted within S. cerevisiae. In this review, we highlight the
current techniques and methodologies used in
pioneering studies for the development of novel endo-
symbionts. Discussion focuses on symbiont engineering,
methods to deliver endosymbionts, as well as candidate
bacteria and their potential advantages as novel
organelles.
Designing endosymbioses
There are two conditions that aid in the initiation of
symbiosis: syntrophy and tolerance. Syntrophy requires
that neither partner can survive without the other, each
providing a necessary compound that the other cannot
synthesize or obtain on its own in a specific environment
[2]. By engineering syntrophy into the endosymbiotic
relationship, selection pressures favor chimeras over
their free-living precursors and ensure a stable rela-
tionship over evolutionary timescales [14,15]. Tolerance
refers to the symbionts having either negligible or
manageable adverse effects on the survival and fitness of
their symbiotic counterparts. In the case of endosym-
biosis, tolerance means that the host does not digest the
endosymbiont, whose presence in the cytoplasm is
nontoxic to the host [16].
So far, a straightforward empirical strategy has been
implemented to promote syntrophic relationships be-
tween symbionts. As demonstrated by Mehta et al.
(2019) [10,11], the viability of auxotrophic bacterial
endosymbiont variants can be evaluated based on the
permissive growth of chimeras in selective media.
However, to rationally engineer syntrophy, a holistic
understanding of the unique metabolic systems from
each symbiont is required [16,17]. Existing metabolic
systems biology and computational-based pipelines,
including genome-scale metabolic reconstruction tools
and constraint-based reconstruction analyses, have been
traditionally designed for modeling individual microbial
strains [18e20]. These models have proven to be
important in many studies for enabling accurate pre-
dictions of phenotypes, distributions and variability in
metabolic flux, and changes in relative expression levels
[18,21]. However, new algorithms could be written to
integrate data from both symbionts into a single hybrid
model [5,19]. Simulations of syntrophic systems could
allow for more reliable experimental designs within a
defined set of conditions, as well as aid in tuning existing
or novel organelles for optimal function by identifying
metabolic targets for upregulation or downregulation
[20,21]. Another benefit of using a systems approach is
Current Opinion in Systems Biology 2020, 24:41–50
the ability to observe and compare quantitative changes
in the metabolism and genetic content that may occur in
either symbiont over evolutionary time [22]. Aside from
identifying compounds for syntrophic exchange, it is
critical to ensure the successful transport of these
compounds from one organism to another [4]. Such
transporters could be installed in the endosymbiont as
needed [10].
For endosymbionts to withstand the host’s intracellular
environment, an extra level of engineering to defend
from autophagy- or lysosomal-mediated degradation is
required [9,10]. When Karas et al. (2013, 2019) [12, 13]
used synthetic mycoplasma cells in fusion/engulfment
experiments with yeast, the bacterial genomes were
transferred to the nucleus rather than whole bacterial
cells surviving within the yeast cytoplasm [12]. Genome
survival was enabled by inserting a yeast centromere,
selection marker, and autonomous replicating sequences
into the mycoplasma genomes. However, without addi-
tional engineering to prevent bacterial cell degradation,
the bacterial cells were only transiently observed
immediately after the cell engulfment events [13]. To
circumvent cellular digestion, both Agapakis et al. and
Mehta et al. installed genes derived from pathogenic
bacteria that encode proteins that have evolved specif-
ically to manipulate the host’s machinery. Agapakis et al.
(2011) [9] used a cytolysin from Listeria monocytogenes,
called Listeriolysin O. This pore-forming toxin disrupts
endosomal membranes, allowing escape from phag-
osomes after bacterial invasion of eukaryotic cells [23].
Alternatively, Mehta et al. (2018) [10] demonstrated
that soluble N-ethylmaleimideesensitive factor
attachment receptor (SNARE)elike proteins from
Chlamydia trachomatis (Ctr_incA and CT_813) and Chla-
mydia caviae (Cca_incA) could function synergistically to
effectively preserve E. coli symbionts over several rounds
of plating. These bacterial SNARE-like proteins contain
a coiled-coil motif that is structurally analogous to
eukaryotic SNARE proteins [24]. This cross-species
proteineprotein interaction inhibits lysosomal fusion
and proteosomal digestion [10,25]. Provided the endo-
symbiont has escaped the host defense mechanisms,
ensuring that its presence is not detrimental to the
host’s survival is a crucial step to sustaining the symbi-
otic relationship. Relative rates of cell division, the
export of cytotoxins from the endosymbiont, or deple-
tion of host metabolites are all potential concerns in
directed endosymbiosis [15].
Based on the designs developed in these foundational
studies, we propose that candidate endosymbionts can
be engineered with components for symbiosis and for
genome transfer and maintenance (Figure 1a). If Design
1 does not produce a successful symbiosis, the additional
modifications in Design 2 can serve as a control for
bacterial cell engulfment, without the need for micro-
scopy. To distinguish between two different outcomes in
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 Converting free-living bacteria into organelles Meaney et al. 43

Design 2 (Figure 1b), yeast cells need to be plated on
the appropriate media, in accordance with the yeast
selection marker, and screened with appropriate mo-
lecular biology techniques. If the symbiosis is success-
ful, continued propagation, along with consecutive
rounds of whole-genome sequencing of the recovered
chimeras, may reveal acquired mutations and horizontal
gene transfer events. Over time the symbiotic relation-
ship between both organisms may evolve to optimize
their syntrophy, tolerance, and overall fitness.
Installing endosymbionts
Methods for installing endosymbionts in host cells can
be categorized into biological, mechanical, or chemical
techniques. Biological methods involve genetically
based systems which either allow bacteria to invade cells
of other species or, conversely, enable cells to internalize
bacteria from their surroundings. In 2011, Agapakis et al.
[9] demonstrated three approaches: two biological and
one mechanical. The first biological strategy equipped
S. elongatus with recombinant invasin to invade
mammalian endothelial cells. Originating from the
pathogen Yersinia pseudotuberculosis, invasin is an outer
membrane protein that exploits mammalian b1-
integrins to invade host cells [27]. The other biolog-
ical approach harnessed the phagocytic activity of
macrophages to engulf S. elongatus cells expressing
invasin. In both cases, the S. elongatus cells also
expressed Listeriolysin O to escape lysosomal degrada-
tion. The researchers also applied microinjection to
deliver live S. elongatus cells directly into zebrafish em-
bryos [9]. Other mechanical approaches that have the
potential to enable greater precision and efficiency of
the engulfment process include micro-
electromechanical systems and microfluidics [28].
The use of polyethylene glycol (PEG) is an established
chemical method for transforming DNA and fusing
mammalian cells to create hybridomas [29]. In 1989,
Sulo et al. [30] demonstrated that PEG could be used to
introduce isolated mitochondria into respiration-
deficient yeast protoplasts. A method was established
much later by Karas et al. (2013, 2014) [12, 31] for the
transfer of whole bacterial genomes to yeast sphero-
plasts as a means to prevent genome shearing. It was
hypothesized that PEG-mediated direct genome trans-
fer from bacteria to yeast could happen via one of three
ways: (1) the release of bacterial genomes into solution
and uptake of the whole genome during yeast trans-
formation, (2) fusion between yeast and bacteria cell
membranes, or (3) whole cell engulfment [12]. The
third outcome was supported by follow-up research

Figure 1

Genome engineering strategies for directed endosymbiosis using bacteria and yeast as partners. (a) Design 1 includes the necessary elements that need
to be added to the bacterial genome to facilitate syntrophy and tolerance. This can include genes such as a membrane protein for transferring the
syntrophic product into the yeast cytosol and SNARE-like proteins to manipulate the host’s lysosomal machinery to avoid degradation (square) [10].
Design 2 includes the features of design 1 along with a yeast centromere sequence and yeast selection marker (circle). Note that yeast autonomous
replication sequences (star) must be inserted approximately every 100 kb if the G + C content of the bacterial genome is greater than 40% [26]. (b)
Potential outcomes using the two designs described in (a). When bacteria with genome design 1 are engulfed by yeast, the cell survive processing by
lysosomal vesicles, allowing the bacteria and yeast to exchange cytosolic metabolites and promote a symbiotic relationship. Using design 2 two outcomes
are possible. In 2i, engineering of the candidate endosymbiont fails to protect the cell from host degradation, but the bacterial genome is translocated to
the yeast nucleus for propagation. This demonstrates that the endosymbiont was successfully engulfed by yeast, but additional engineering will be
required for cell survival. Meanwhile 2ii, indicates a functional symbiotic relationship as is with design 1. SNARE, soluble N-ethylmaleimide–sensitive
factor attachment receptor

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44 Synthetic biology (2020)
[13]. Mehta et al. (2018) [10] used a similar method to
deliver E. coli endosymbionts into S. cerevisiae host cells
during their pursuit to model mitochondrial evolution.
This work provides evidence to suggest that PEG-
mediated cell engulfment will be an effective method
for inducing directed endosymbiosis, especially if auto-
mated like other protoplast transformation systems
[32]. While the molecular mechanisms of PEG-
mediated cell engulfment are not yet fully understood,
controls can be implemented to distinguish endosym-
biotic events from other results (Figure 1b).
Candidate endosymbionts
Mollicutes
Bacteria in the class Mollicutes are the simplest self-
replicating organisms and are established as model sys-
tems for investigating the minimal genetic re-
quirements for life [33,34]. These bacteria are well
known for their parasitic behaviors, absent cell wall, low
genomic G þ C content (25e40%), and very small ge-
nomes (0.6e2.2 Mb) due to reductive evolution [34].
Given their natural proclivity for intracellular life,
Mollicutes are an attractive foundation for developing
specialized organelles in a low genetic background; or in
other words, an endosymbiotic chassis. They may also be
useful for studying organelle genome evolution because
of their tendency to leave mutations unrepaired [35].
Mollicutes have a significantly reduced metabolic ca-
pacity and are required to outsource nearly all of their
essential nutrients from host organisms [36]. In some
Mollicutes, the UGA codon translates to tryptophan
rather than stop [34]. In directed endosymbiosis, it
could be advantageous to use a bacterium with a
nonstandard genetic code to ensure that only the
endosymbiont is responsible for producing the syntro-
phic metabolite for a host that uses the standard genetic
code, regardless of horizontal gene transfer.
Much of the pioneering research in synthetic biology
using Mollicutes was completed at the J. Craig Venter
Institute (JCVI) and focused around developing tools
for genetic manipulation of fast-growing Mycoplasma
mycoides cells. After demonstrating that the M. mycoides
subsp. capri LC GM12 genome could be transplanted
into another bacterium [37], the authors chemically
synthesized and assembled the entire 1.08 Mb (24%
G þ C) to create the first cell with a fully synthetic
genome, JCVI-syn1.0 [38]. Further work eliminated 428
genes, resulting in the self-replicating organism with the
smallest set of genes, called JCVI-syn3.0 [33]. Recently
this strain, now called JCVI-syn3A, was upgraded to
improve growth rate, and a comprehensive metabolic
reconstruction was also completed [39].
One disadvantage of using synthetic cells based upon
the M. mycoides genome is that they are classified as a
biosafety level 3 organism in some countries, making
Current Opinion in Systems Biology 2020, 24:41–50
them difficult to work with in academic and industrial
research facilities. In these cases, Mesoplasma florum
could be used instead because it is a fast-growing
mollicute that naturally lacks pathogenic activity and
has several established genetic tools [40e42]. Alterna-
tively, the mollicute Acholeplasma laidlawii could also be
developed as a candidate endosymbiont as it too has a
biosafety level 1 classification and a small 1.5-Mb
genome. Unlike M. mycoides and M. florum, A. laidlawii
uses the standard genetic code. Some synthetic biology
tools have been developed for A. laidlawii [43]. See
Supplementary File S1 for an extensive list of genetic
tools developed for the candidate endosymbionts.
Cyanobacteria
Cyanobacteria are an essential group of phototrophic
bacteria. Their ancestors are theorized to be responsible
for the initial oxygenation of Earth’s atmosphere and the
origin of eukaryotic chloroplasts [17,44]. As photosyn-
thetic organisms, cyanobacteria are desirable models for
biomanufacturing applications in terms of economic and
environmental benefits. Consequently, these bacteria
are being explored for the production of valuable com-
pounds, including native secondary metabolites (e.g.
pigments, vitamins) and heterologous metabolites (e.g.
alcohols, fatty acids, hydrocarbons) [17,21,45]. In addi-
tion, cyanobacteria can be grown in large-scale, open
bioreactors without occupying arable land or consuming
potable water, promoting environmentally sustainable
manufacturing technologies [46,47]. For the interest of
developing novel photosynthetic organelles, we will
focus the discussion around S. elongatus.
Until recently, the leading model strain of Synechococcus
was S. elongatus sp. PCC 7942; however, S. elongatus sp.
UTEX 2973 has been favored since its discovery owing
to its rapid 1.9-h doubling time. Both strains harbor
2.7 Mb (55% G þ C) genomes along with an endogenous
plasmid and are 99.8% genetically identical; however,
UTEX 2973 has a growth rate that is roughly 2.5-fold
greater [48]. Mutations to only three genes are
thought to have caused this dramatic change in growth
rate [48], as well as an increased tolerance to high light
intensity and temperature [49]. These are properties
that could substantially enhance the potential for
commercial-scale bioproduction in cyanobacteria [50].
Several synthetic biology toolboxes [44,51,52] have
been developed for these strains, including standardized
methods for modular cloning, assembly and delivery
[53], and genome editing techniques. These tools will
aid with the engineering of S. elongatus endosymbionts.
There are some notable engineering challenges inherent
to using light-dependent metabolic systems, such as
those from cyanobacteria, in synthetic organelles. Pre-
vious studies have demonstrated that characterized ge-
netic parts from more established hosts, such as E. coli
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and S. cerevisiae, do not always have robust functions
when installed in cyanobacteria and consequently
require debugging for reliable use [54]. In addition,
S. elongatus has been shown to have poor photosynthetic
efficiency, utilizing approximately 10% of solar energy
[47]. Synthetic biology approaches to widen photo-
system absorption spectra or optimize the electron
transport chain could significantly improve energy uti-
lization and, in turn, production capacity [17,47]. The
high ploidy of Synechococcus strains is another restricting
factor that can result in laborious procedures for the
identification of fully segregated mutants. For example,
PCC 7942 can carry w5e7 genome copies per cell [48].
Prochlorococcus marinus, the most abundant photosyn-
thetic organism on Earth, could be used as an alternative
because it has a small (1.7 Mb) single-copy genome that
can be cloned in yeast [55]. However, with a doubling
time of 24 h and very few established genetic tools,
further research will be required before P. marinus could
be converted into endosymbionts [56].
Rhizobia
Rhizobia are a diverse group of diazotrophic proteobac-
teria known best for their interactions with legumes.
Symbiotic rhizobia species can infect and colonize root
nodules from the soil, where they can effectively fix and
supply biologically active ammonia for the plant cell in
exchange for C4-dicarboxylates [57,58]. While native
rhizobiaeplant interactions contribute more than 65%
of biologically fixed nitrogen in crops, the continued
increase in the global population has resulted in the
mass use of nitrogen fertilizers to maximize crop yield
[58,59]. The manufacture and application of these
products, often in excess, has resulted in significant
production of greenhouse gases, aquatic eutrophication,
and billions of dollars in costs annually [57,60]. Several
strategies to address the nitrogen problem have been
researched, revolving primarily around the installation of
transgenes into nonlegume hosts; however, an intriguing
alternative would involve converting rhizobia into syn-
thetic nitrogen-fixing organelles [61]. Similar to bacte-
roids [19,58], which are differentiated endosymbiotic
rhizobia, nitrogen-fixing organelles could reside within
plant cells and provide a subcellular compartment to
house the native nitrogen-fixation genes and regulation
system (nif and fix, respectively) while modulating
oxygen concentration and generating adequate cellular
power [60,62]. Rhizobia endosymbionts could mitigate
many of the current challenges associated with adapting
highly complex and sensitive biological nitrogen-fixation
systems, as seen in recent studies implementing Kleb-
siella and Azotobacter nitrogenase systems in eukaryotic
hosts [63,64]. Furthermore, some species of rhizobia
have the capability to share their natural tolerance for
heavy metals, increased salinity, and drought with their
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Converting free-living bacteria into organelles Meaney et al. 45
host, posing another engineering opportunity to create
specialized organelles for bioremediation [65].
The model species, Sinorhizobium meliloti, is naturally
found in three distinct environments; bulk soil, the
rhizosphere, and in root nodules of alfalfa (Medicago
sativa) [66]. S. meliloti 1021 is the representative strain
and harbors a multipartite genome composed of a pri-
mary chromosome (3.6 Mb), a megaplasmid for symbi-
otic nitrogen fixation called pSymA (1.4 Mb) and an
essential chromid called pSymB (1.7 Mb) [66,67]. With
a doubling time as short as 2 h, S. meliloti has a growth
rate comparable to yeast, making it suitable for rapid and
high-throughput screening protocols for research and
biotechnological applications [68]. Several
recombination-based cloning and editing strategies [69]
have been established in S. meliloti along with many
genetic parts (promoters, terminators, ribosome binding
site (RBSs), selectable markers, origins of replication,
etc.) that have been characterized [19,57,70,71].
Another feature of S. meliloti is its abundance of diverse
inducible solute-binding transporters, a trait for adapt-
ing to various ecological settings, that may simplify en-
gineering syntrophic metabolite transfer between the
host and proto-endosymbiont [72].
The natural propensity to form symbiotic relationships
with alfalfa is a strong indication that S. meliloti could be
genetically tractable as a synthetic organelle. However,
it is likely that initiating and controlling nitrogen fixa-
tion may be challenging because rhizobiaelegume
symbioses are mediated through a highly regulated
cascade of cell signaling interactions that result in dif-
ferentiation of both symbionts [19,58,62,73]. A newly
identified strain, Bradyrhizobium sp. DOA9, may serve as
a promising secondary candidate as it can fix atmo-
spheric nitrogen in both symbiotic and free-living states
[74]. The presence of two structurally distinct activator
proteins of the nifHDK structural genes, encoded on
the chromosome (nifAc) and plasmid (nifAp), in DOA9
were shown to exhibit redundant transcriptional initia-
tion during symbiosis but not independent growth.
Upon further investigation, it was confirmed that nifAp
is dispensable for nitrogen fixation in free-living condi-
tions, whereas nifAc is not, suggesting that this intrinsic
regulation system may be easier to induce for the crea-
tion of nitrogen-fixing organelles [75].
Relevance of synthetic organelles
After a detailed discussion surrounding specific strate-
gies and species for developing synthetic organelles, a
broader perspective on their importance in the scientific
and general community should also be touched upon.
Directed endosymbiosis can be applied in several
different areas of study, including eukaryotic evolution,
Current Opinion in Systems Biology 2020, 24:41–50
46 Synthetic biology (2020)

biotechnology, cell compartmentalization, and medicine
(Figure 2). The mechanisms, microenvironment, and
duration in which the mitochondria and chloroplast
arose have been highly debated over time [14]; however,
this experimental strategy may now offer a platform to
put such hypotheses to the test [76]. Aside from
modeling the conception of current organelles, evolu-
tionary biology techniques could also be used for
directed evolution with either random or targeted
mutagenesis [77].
Natural organelles have evolved unique sets
teristics to survive and reproduce under
environmental conditions [14]. With a
knowledge of an organism’s biochemical and
composition, it may be possible to achieve further
optimized genetic networks to improve cellular pro-
cesses that occur in organelles, including oxidative
phosphorylation or carbon fixation, during respiration
and photosynthesis, respectively [47]. These improve-
ments could result in traits such as the increased growth
rate and biomass, allowing microbe and plant species to
be exploited to their full bioindustrial potential. In
addition, directed endosymbiosis can be used to solve
other biotechnologically relevant issues including
cellular containment or fine-tuning environmental con-
of charac- ditions because organelles are, by definition, isolated
particular from the rest of the cell [78].
thorough
metabolic

Figure 2

Potential applications of synthetic organelles in various areas of research. Left: superior organelles could be created based on naturally occurring
mitochondria and chloroplasts (improved or custom phenotypes). Top: synthetic organelles could be used as models for researching diseases implicating
mitochondria. In principle, engineered organelles could then be used therapeutically to replace or repopulate defective mitochondria in patients. Right:
organelles compartmentalize biochemical networks. Synthetic organelles can provide unique environmental conditions necessary to enable or optimize
highly specific or sensitive biological processes, such as nitrogen fixation. Bottom: directed endosymbiosis could be used as an experimental tool for
studying evolutionary biology and presents the opportunity to elucidate more information regarding the origin of the eukaryotic lineage and the role of
symbiogenesis in speciation.

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Lastly, synthetic organelles could have significance in
medicine for the study and treatment of organelle-
related diseases. For example, in mammals, mitochon-
dria have pivotal cellular roles in energetics, cell cycle
regulation, and apoptosis, and when such mechanisms
are disrupted or deregulated it can lead to conditions
including, cancer, diabetes, muscular degenerative and
neurodegenerative diseases [79]. Using a mammalian
host model for directed endosymbiosis, it may be
possible to develop therapeutic strategies, similar to
artificial organelles developed by Einfalt et al. [80], that
could allow for the repair or replacement of impaired
mitochondria with synthetic counterparts.
Conclusion
Directed endosymbiosis is a developing field that will
contribute new opportunities for the study of symbio-
genesis and the development of improved or original
organelles. The systems and synthetic biology methods
examined in this review provide a set of principles for
the design, construction, and analysis of endosymbiotic
systems following cell engulfment events. Such strate-
gies for implementing syntrophy and tolerance will
likely be expanded and improved upon with additional
experimental research. To aid in the evaluation of future
systems and to confirm that endosymbionts have been
sufficiently engineered to protect the cell from intra-
cellular degradation, the second design strategy
presented in Figure 1 d with elements for symbiosis
and genome transfer d could be implemented for
testing purposes. Furthermore, there are various prac-
tical techniques for endosymbiont delivery, but further
investigation of the molecular mechanisms and genetic
determinants behind each method will be instrumental
in enhancing the efficacy of studying synthetic symbi-
oses. As discussed, PEG-mediated cell engulfment
seems to be the most viable method currently available
for delivering endosymbionts.
We presented rationale for the development of three
candidate endosymbiont groups d Mollicutes,
Cyanobacteria, and Rhizobia d along with a
description of the current genetic tools available for
engineering these organisms. Foundational work to
establish directed endosymbiosis will be based on
bacterial and eukaryotic model systems, such as those
discussed here. However, increasingly complex endo-
symbiotic systems could be created, with prokaryotic
or eukaryotic endosymbionts and multicellular hosts
such as plants and animals. In basic research, directed
endosymbiosis can be used as an experimental tool
for studying areas of active debate in evolutionary
theory, such as the mechanics of endosymbiosis or its
implications for the modern synthesis [16,76]. From a
biotechnological standpoint, synthetic organelles can
provide a broad range of applications, from nitrogen-
fixing organelles for self-fertilizing plants to creating
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Converting free-living bacteria into organelles Meaney et al. 47
photosynthetic animals for extraterrestrial ecosystems
[81]. Thus, directed endosymbiosis has the potential
to gain significant experimental tractability in many
fields of study in the near future.
Author contribution
R.S.M. and S.H. wrote the first draft manuscript, and all
authors edited the final version. R.S.M. and S.H. created
draft figures, and all authors edited the final versions.
M.P.M.S wrote the supplementary file. Graphical ab-
stract and Figure 1 were created with BioRender.com.
Conflict of interest statement
Nothing declared.
Acknowledgements
This work was supported by the Government of Canada’s New Frontiers in
Research Fund (NFRF), [NFRFE-2018-01124]. In addition, research in
B.J.K. is also supported by Natural Sciences and Engineering Research
Council of Canada (NSERC), [RGPIN-2018-06172].
Appendix A. Supplementary data
Supplementary data to this article can be found online
at https://doi.org/10.1016/j.coisb.2020.09.008.
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