特集 SFC バイオの軌跡［招待論文：総説・レビュー論文］

20 Years of the Minimal Cell’s Risefrom Theory to Practice
Yo Suzuki

Assistant Professor, Synthetic Biology and Bioenergy Group, J. Craig Venter Institute
Correspondence to: ysuzuki@jcvi.org

J. Craig Venter

Founder, Chairman, and Chief Executive Officer, J. Craig Venter Institute

Abstract:

In any discipline, simple systems are desirable for understanding the basic
workings in the groups of systems they represent. In biology, cells are established
as units for life, but even the simplest cells in nature are complex systems withuncharacterized functions beyond simply living. Making a minimal model cell in thelaboratory was therefore contemplated and then achieved. Many pioneering efforts,
including those at Keio University, shaped the process. This endeavor created thesynthetic genomics field and a worldwide community to understand the cell andexplore its applications in biotechnology and medicine.
サイエンスでは単純な実験系を使うことで、飛躍的に研究が進むことが多い。細胞は生命の単位と考えられるが、自然界に存在する最も単純な細胞でさえ、人の理解を超える未解明の機能を持った複雑な系を構築している。そこ
で生命を解析するためのモデルとして、実験室での最小細胞作成が検討され、その目標が達成された。この過程で慶應義塾大学は先駆的な役割を果たした。
この研究は合成ゲノミクスという新分野を生み出し、最小細胞を通じて生命を理解し、産業と医学に応用するための世界的な動きにつながった。

Keywords: synthetic genomics, synthetic biology, synthetic cell
合成ゲノミクス、合成生物学、人工細胞

1

Naturally minimal cells
Harold Morowitzʼs pursuit of a simple cell for understanding life has identiﬁed

mycoplasmas as model organisms (Morowitz, 1984). Mycoplasmas are small cellswith the smallest genomes of all known bacteria that are culturable in an axenic
condition. Morowitzʼs group uncovered some fundamental molecular constraints42

【第 1 章 先端生命科学】
20 Years of the Minimal Cell’s Rise from Theory to Practiceabout cellular life. His foresight is noteworthy, as the original review (Morowitz,
1984) delineated what were to come including genome sequencing, computationalmodeling, and the development of deﬁned culture media. Arguably, the work is stillongoing 38 years later.
Mycoplasmas were considered to be useful for determining the minimal numberof genes required for life (Peterson et al., 1991), but were initially studied as naturalorganisms containing whole, yet uncharacterized, genomes. Mycoplasma genitalium
has the smallest genome among all mycoplasma species examined. Its genome
sequence therefore was of great interest and was one of the ﬁrst to be determined
completely (Fraser et al., 1995). However, the sequence did not reveal functions formany genes, given the limited information available at the time. Studies of whole
mycoplasma cells led to Luis Serranoʼs pioneering systems biology work (e.g., Guellet al., 2009). Those studies generally showed the surprising complexity of theorganisms, despite their limited genetic content.

2

Theoretically minimal cells
Starting around 1995, a research focus emerged to establish minimal genomes asconcepts, departing from the constraints of natural organisms, through abstraction, as
well as clever genetic experiments and bourgeoning bioinformatics. The 20-year
progression toward minimal cells coincided with 20 years of incessant technology
development efforts at Institute for Advanced Biosciences (IAB) at Keio University,
which enhanced research at J. Craig Venter Institute (JCVI) and in the systems
biology and synthetic biology ﬁelds at large.
In 1995, Mitsuhiro Itaya published a study describing systematic mutagenesis in
Bacillus subtilis (Itaya, 1995). Mutagenesis studies to estimate the number of genes
required for a given biological process were common, but Itaya had a uniqueperspective. He analyzed 79 randomly generated mutants and found that 73 wereviable (Table 1); only six sequences tested were considered to be essential for life.
Itaya went on to make multi-mutants with up to 33 loci simultaneously disrupted.
This experiment suggested that synergistic lethal phenotypes among randomly
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selected mutations are rare. He estimated that all the essential genes needed for life
can be contained within roughly 350 kb, 8% (6/73) of the 4.2-Mb wild-type genome
size in B. subtilis.
Table 1 Frequencies of viable mutants in various species
SpeciesNon-lethal Normal
growthReference

Note

Bacillus subtilis

92%

Itaya, 1995

Escherichia coli

93%

“Vast
majority”

Baba et al.,2006

Saccharomyces
cerevisiae

83%

60%

Winzeler et al.,1999

Mycoplasma
genitalium

21%

Mycoplasma
mycoides JCVIsyn1.0

73%

48%

Hutchison etal., 2016

Non-lethal: nonessential &quasi-essential categories;
normal growth: nonessential
category;> 13% (54/432) of non-lethalʼs
are presumed to be involved insynergistic lethality

Mycoplasma
mycoides JCVIsyn3.0 (minimal
cell)

25%

8%

Hutchison etal., 2016; thisstudy

Non-lethal: nonessential &quasi-essential categories;
normal growth: nonessential
category

80% of non-lethalʼs havesynergistic growth phenotypes(Tong et al., 2004)

Glass et al.,2006

The size of this estimated essential fraction is just under the smallest
mycoplasma genome sizes, giving validity to Itayaʼs suggestion of a link to theminimal genome. The low frequency of essential genes in B. subtilis was surprising,but was later supported by similar observations with systematic gene knockout
studies in Escherichia coli (Baba et al., 2006) and Saccharomyces cerevisiae(Winzeler et al., 1999; Table 1). However, a set of essential genes identiﬁed with any
single-gene inactivation technique would not be sufficient for enabling life at least forB. subtilis or any of other typical microbes researched.
What is the difference between such an essential gene set and a gene set of a
viable minimal genome? A major difference would be that the “essential” set is44

【第 1 章 先端生命科学】
20 Years of the Minimal Cell’s Rise from Theory to Practicemissing functionally redundant genes. The evidence comes from a double knockout
study in S. cerevisiae, where 80% of non-lethal single-gene deletions were shown tohave at least one gene deletion partner to exhibit a synergistic growth defective
phenotype (Tong et al., 2004). Because non-lethal single-gene deletions are a large
fraction of the genome (Winzeler et al., 1999; Table 1), the majority of the genes in
the genome would be involved in essential life processes. The specific synergistic
lethal combinations would rarely be found randomly because the search space for
combinatorial genotypes is large. However, the prevalence of genes involved insynergistic lethal interactions would limit the extent of maximal genome reduction toa fewfold for typical microbes and less for mycoplasmas.
After the predecessor of JCVI completed sequencing the first two bacterial
genomes, the genomes of Haemophilus influenzae (Fleischmann et al., 1995) and M.
genitalium (Fraser et al., 1995), Eugene Koonin and Arcady Mushegian determinedthrough bioinformatics which M. genitalium genes were conserved in H. influenzae(Mushegian and Koonin, 1996). Considering the large evolutionary distance between
the two organisms, the genes conserved between them had to be essential andcollectively sufficient for life. They proposed a 256-gene minimal genome. This
genome contained 22 genes that were restored from M. genitalium to ﬁll the gaps inpathways, to account for possible cases where genes with unrelated sequences
evolved to perform the same function in organisms compared. A caveat would be that
if genes with functions that are unknown fall into this so-called nonorthologous genedisplacement category, they would be missed in their approach. Nevertheless, thisstudy greatly motivated the quest for minimal genomes. The mycoplasma-centric
approach likely enabled them to predict a small gene count for a minimal genome.
In 1999, Masaru Tomita developed the ﬁrst computational platform to simulate
whole-cell behaviors (Tomita et al., 1999). This system termed E-CELL was a perfectmatch with the simple biological program in M. genitalium revealed by the genome
sequencing project. Only known molecular mechanisms were included, and processes
such as DNA replication and cell division were omitted in the simulation.
Nevertheless, insights were gained into the life and death of the cell. A research
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trajectory was proposed to conduct a model-guided study to understand genes of
unknown function. The spirit of this approach was passed onto multiple computermodeling studies (e.g., Breuer et al., 2019; Karr et al., 2012).
By 1999, Hutchison et al. developed a method to determine the sequences
around transposon insertions in a high-throughput manner and applied it to the
genomes of M. genitalium and M. pneumoniae to identify genes that can be disrupted(Hutchison et al., 1999). This was followed by a study where mycoplasma cells witha transposon insertion were isolated, archived, and then used to determine the site oftransposon insertion (Glass et al., 2006). These efforts established that 382 of the 482
genes (79%) are likely to be essential in M. genitalium (Table 1). This is a large
fraction when compared to earlier results from other organisms, leaving little room
for synergistic lethal interactions. Redundant genes would have had to be much morethan proportionally reduced in the evolution of M. genitalium and closely related
mycoplasmas, when the yeast result is used as a reference. It is understandable that
redundant genes were ﬁrst to be lost when a selective pressure was applied in nature
to favor genome reduction. The ﬁrst multi-gene knockout attempt resulting in a ~10%
reduction of the genome size in M. mycoides did not reveal any synergistic
interactions, supporting the view that synergistic lethal interactions are rare in
mycoplasmas (Suzuki et al., 2015). The shortage of genetic redundancy might havefacilitated the construction of viable reduced genomes described below.

3

Birth of synthetic genomics
With hundreds of deletions to make in a genome, a top-down approach to

conduct engineering on the wild-type genome would be cumbersome. The initial
success with the bottom-up approach of synthesizing and activating the genome of
the bacteriophage ΦX174 (Smith et al., 2003) set a stage for a conceptually similar
approach for bacterial genomes. Gibson et al. developed a hierarchical assembly
method and generated a whole genome of M. genitalium from synthetic DNA
fragments (Gibson et al., 2008). The final genome was established in yeast as aplasmid, but they were unable to activate it to produce any bacterial cell.

46

【第 1 章 先端生命科学】
20 Years of the Minimal Cell’s Rise from Theory to PracticePreceding this development, Itaya had undertaken a unique approach to genome
engineering. First, parts of the genome of B. subtilis were gradually replaced withgenomic fragments from related B. subtilis var. natto within B. subtilis cells (Itaya
and Matsui, 1999). The recipient organisms acquired phenotypes related to the
production of natto, a traditional food in Japan. This is a partial conversion of anorganism, as the resulting organism had some of the old genome sequences, as wellas old functions. Expanding on this work, Itaya then introduced a whole 3.5-Mb
cyanobacterial genome into the 4.2-Mb genome of B. subtilis to generate a 7.7-Mb
hybrid genome within B. subtilis (Itaya et al., 2005). The organism with the hybrid
genome was propagated as B. subtilis, but was also tested for growth incyanobacterium medium and culturing condition. This marked the first attempt toactivate a whole genome of a species that is different from the host in the laboratory.
Lartigue et al. succeeded in transferring the natural M. mycoides genome fromM. mycoides to M. capricolum recipient cells and obtaining M. mycoides cells(Lartigue et al., 2007). They then showed that an equivalent M. mycoides genome
maintained in yeast also rendered M. mycoides cells upon transplantation (Lartigue etal., 2009). This breakthrough discovery cleared a path for the ﬁrst synthetic cell. The
essentially full-length M. mycoides genome was generated from synthetic DNA
fragments via the hierarchical assembly strategy, established in yeast, andtransplanted to the M. capricolum recipient cells to generate M. mycoides cells having
an entirely synthetic genome (Gibson et al., 2010). The watermarks introduced in the
genome conﬁrmed that the generated cells were a genuine JCVI product derived fromthe synthetic genome.
The origin of this cell lineage is unusual. It is not a M. mycoides cell. The initialcytoplasm was from the recipient M. capricolum, but was quickly replaced byproducts made from instructions in the new genome after the transplantation event.
This new genome came from yeast, but had originated in a DNA synthesizer, basedon the design in a computer. This challenges Louis Pasteurʼs theory, “Omne vivum ex
vivo (all life from life).” The escape from this constraint promised great ﬂexibility ingenome sequences that can be manipulated to create reduced genomes or massively
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reorganize genes within genomes (Hutchison et al., 2016; Venter et al., 2022).
Genome transplantation has been expanded to a useful mycoplasma-likeorganism Mesoplasma florum (Baby et al., 2018). M. capricolum has been adependable recipient with a capability to take in mega-sized DNA, without
deleterious homologous recombination activity (Labroussaa et al., 2019). At this stageof development, the main constraint seems to be that the cytoplasm and the cellularchassis of the recipient need to be compatible with the incoming genome.
Understanding the molecular mechanisms of genome activation would be importantfor further expanding the range of organisms that can be engineered with synthetic
genomics. The approach to gradually replace a native genome with a synthetic
genome using synthetic DNA pieces has been practiced in organisms such as E. coli
(Fredens et al., 2019) and S. cerevisiae (Annaluru et al., 2014).

4

Practically minimal cells
To make a minimal cell, we used repeated cycles to design, build, and test cells

having reduced genetic content. The design relied on gene essentiality data with
Hutchisonʼs transposon bombardment method (Hutchison et al., 1999) adapted fornext-generation sequencing to identify genes to discard. An improved hierarchicalassembly of synthetic DNA was then conducted to generate the next version of the
genome. The test was to identify viable organisms after genome transplantation. A
modular eight-segment construction enabled acquisition of viable organisms withsubsets of reduced segments in the wild-type background, providing materials for the
next cycles of reduction. After three design-build-test cycles, we established thesynthetic bacterium JCVI-syn3.0, “a working approximation of a minimal cell”(Hutchison et al., 2016). The genome had 473 genes in 531.5 kb, smaller than the M.
genitalium genome and 50% smaller than the M. mycoides wild-type genome. The
resulting cell has the highest fraction of the genes characterized that has beenreported for any organism.
A key ﬁnding in this work was the need to include quasi-essential genes thatwere revealed after transposon bombardment by analyzing populations at two stages48

【第 1 章 先端生命科学】
20 Years of the Minimal Cell’s Rise from Theory to Practiceof outgrowth. They appeared dispensable in the early population, but essential in thelater population. (Some of these had been classified as nonessential in earlier
experiments.) They may be needed for fast growth or only in limited occasions (e.g.,with the gene products made before gene disruption lasting multiple generations orwith the genes needed to withstand any irregular processes during the culture).
However, including quasi-essential genes in subsequent genome constructs ensured
viable genomes. It may be that having multiple slow-growth deletions compounded
to make the growth rate too slow to be detected in our transplantation assay.
A thorough analysis of functionally redundant gene combinations was notconducted during the construction of the JCVI-syn3.0 cells, but a partial search
identified roughly 26 genes that were possibly involved in synergistic lethalinteraction (Hutchison et al., 2016). Of the 432 singly nonessential genes in thestarting genome, 26 (or 52 if one redundant partner is assumed to be present for eachof the 26 genes; Table 1) makes a small fraction, consistent with the rarity ofsynergistic gene interactions in mycoplasmas.
Loss of functional redundancy during evolution probably correlated withmassive genome reduction in mycoplasmas, but more studies are needed tounderstand the molecular basis. Mycoplasmas import many nutrients such as aminoacids and nucleotides from the growth media. Removing biosynthetic pathways
would greatly promote genome reduction and loss of redundancy. Elimination of thecell wall probably helped remove complicated interactions among gene products.
Small cell size might have allowed mycoplasmas to delete many structural proteins
involving complex regulation. For several reasons, and as predicted from earlier
theoretical considerations, mycoplasmas might have been especially suited for the
genome minimization work.
The JCVI-syn3.0 cell had 149 genes (32%) that were uncharacterized(Hutchison et al., 2016). As many of them are essential for life, studying these genes
could uncover new paradigms for biology in mycoplasmas and beyond. Collaboration
between Zaida Luthey-Schulten and the JCVI group is making good progress on thisfront (Breuer et al., 2019; manuscript in preparation). Experimental functional
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characterization is required when sequences do not reveal any useful information
about their functions. For this purpose, collaboration involving Tomitaʼs group led tothe development of CRISPRi as a tool for gene inactivation (Mariscal et al., 2018).
Mycoplasmas are not ancient cells; they recently evolved in a clade of Gram-positivebacteria. However, they seem to possess multiple characteristics expected for theprimordial cell (Morowitz, 1984). Adding to this list, a recent microfluidics study
showed that JCVI-syn3.0 cells exhibit variable elongated or enlarged morphologies
attributable to defective cell division, but with DNA replication and the general
growth rate not severely affected (Pelletier et al., 2021). This characteristic may be
expected for a primordial cell without a well-developed cell division machinery. It is
intriguing to think that minimal cells kept basic functions inherited from theprimordial cell through evolution and engineered deletion.
With the JCVI cell distributed to 60 laboratories around the world (J. Glass,personal communication), the ﬁeld is expanding in multiple directions. An exciting
development is to advance minimal cell research toward the industrial realm. As
synthetic genomics makes introducing new pathways easier and faster, we will beable to generate numerous genetic modules for desired properties found in any
organism and test their fit to the minimal cell (Venter et al., 2022). Such an effort
would lead to minimal cell factories or devices that can be precisely controlled due tothe cellʼs simplicity. Michael Guarnieriʼs US Department of Energy-funded project at
National Renewable Energy Laboratory (Arnolds et al., 2021) recognizes JCVIsyn3.0 as a future platform for bioproduction. In this collaboration, we are developing
biocontainment strategies for the JCVI cell. The cell requires many nutrients in theenvironment for growth, and they are unlikely to escape. However, the situation may
change after various genetic parts are added to the cells. The minimal cell enables
imagining a powerful biocontainment strategy on a blank slate. This could involve
recoding the genome, mitigating the effects of mutations, or developing a killingmechanism that is distanced from industrially relevant cellular processes. Currently,
JCVI-syn3.0 is a cell factory to convert glucose to lactate. Our collaboration with
SyntheticGestalt, an artiﬁcial intelligence company based in Japan, aims to produce50

【第 1 章 先端生命科学】
20 Years of the Minimal Cell’s Rise from Theory to Practicevarious chemicals with computer model-guided metabolic engineering. Introductionof metabolic pathways such as those for amino acids would make minimal cells morerobust and less reliant on media ingredients, reducing the costs of culturing. With
multi-gene engineering, synergy among genetic parts can be incorporated inbioproduction or in any engineered cell behavior such as biocontainment to increase
the efficiency of the bioprocess of interest. Whole collections of minimal cells
tailored to speciﬁc purposes may assume critical roles in future biobased industries.
Acknowledgments
We thank Clyde Hutchison, Kim Wise, John Glass, Yu Atsumi, Diana Hernandez
Hernandez, and Nobuaki Kono for helpful comments on the manuscript. This review was
prepared with support from U.S. Department of Energy, Office of Science, Office of Biologicaland Environmental Research, Genomic Science Program under Secure Biosystems Design
Science Focus Area IMAGINE BioSecurity: Integrative Modeling and Genome-scale
Engineering for Biosystems Security, under contract number DE-AC36-08GO28308.

Conflicts of interest
The authors declare no competing interests.

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