特集 SFC バイオの軌跡［招待論文：総説・レビュー論文］

Past, Present, and Future of Genome
Modification in Escherichia coli
大腸菌ゲノム改変の過去・現在・未来
Hirotada Mori

Professor Emeritus, Nara Institute of Science and Technology (NAIST)

森 浩禎
奈良先端科学技術大学院大学名誉教授
Correspondence to: hmori@gtc.naist.jp

Abstract:

Escherichia coli K-12 is one of the most well-studied species of bacteria. This
species, however, is much more difficult to modify by homologous recombination(HR) than other model microorganisms. Research on HR in E. coli led to abetter understanding of the molecular mechanisms of HR, resulting in technicalimprovements and rapid progress in genome research, and allowing whole-genome
mutagenesis and large-scale genome modifications. Developments using λ Red(exo, bet, and gam) and CRISPR-Cas have made E. coli as amenable to genome
modification as other model microorganisms, such as Saccharomyces cerevisiae andBacillus subtilis. This review describes the history of recombination research in E.
coli, as well as improvements in techniques for genome modification by HR. This
review also describes the results of large-scale genome modification of E. coli using
these technologies, including DNA synthesis and assembly. In addition, this article
reviews recent advances in genome modification, considers future directions, anddescribes problems associated with the creation of cells by design.
大腸菌（Escherichia coli）K-12 株は、最もよく研究されている細菌種の 1つである。しかし、この種は他のモデル微生物に比べ、相同組換え（HR）による改変が非常に困難であった。大腸菌の HR の研究は、HR の分子機構の理解を深め、ゲノム研究の技術的向上と急速な進展をもたらし、全ゲノム変異誘発と大規模なゲノム改変を可能にしてきた。λRed（exo、bet、gam）や
CRISPR-Cas を用いた開発により、大腸菌は酵母や枯草菌といった他のモデル微生物と同様にゲノム改変を行うことができるようになった。本総説では、大腸菌の組換え研究の歴史と、HR によるゲノム改変技術の向上について述べる。また、
これらの技術を用いた大腸菌の大規模なゲノム改変の成果として、DNA 合成やアセンブリーなどについて解説する。さらに、最近のゲノム改変の進展を概観し、今後の方向性を考察するとともに、新規細胞の設計に伴う問題点についても述べる。

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Keywords: E. coli, genome modification, synthetic biology
大腸菌、ゲノム改変、合成生物学

1Introduction
The discoveries of bacterial conjugation (Lederberg and Tatum, 1946) and ofgeneralized transduction (Lennox, 1955) have enabled genetics research in
Escherichia coli K-12. Subsequent genetic investigations of E. coli K-12 and its
bacteriophages have increased knowledge of gene structure and function, and have
led to the emergence of molecular biology. Although DNA transfer by transformation
had been described previously (Griffith, 1928) and was shown to occur naturally in
Pneumococcus (Griffith, 1928), Haemophilus (Alexander and Leidy, 1950) andBacillus subtilis (Spizizen, 1958), E. coli proved to be recalcitrant. Treatment with
CalCl2 allowed the transformation (transfection) of E. coli with bacteriophage(Mandel and Higa, 1970) and plasmid DNA (Cohen et al., 1972), but nottransformation by (linear) chromosomal DNA. Based on a hypothesis that an
endogenous exonuclease in E. coli degrades linear DNA, E. coli recBCD mutants,
which lack the RecBCD exonuclease, were found to be transformable bychromosomal DNA, provided the strain carried a cryptic prophage encoding the
SbcBC(D) recombinase (Cosloy and Oishi, 1973). Expression of the λ Red
recombinase (exo, bet, and gam) was shown to increase the efficiency of homologous
recombination (HR) with linear DNA (Murphy, 1998), leading to the use of λ Red
recombinase in highly efficient systems for direct modification of chromosomal genes
via HR (Datsenko and Wanner, 2000; Yu et al., 2000).
Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas
systems participate in acquired immunity in Archea and Bacteria (Ishino et al., 2018).
Although these unusual repetitive DNA sequences were first described in 1987
(Ishino et al., 1987), molecular understanding of their function was first determined
25 years later (Jinek et al., 2012), leading to dramatic advances in the technology ofgenome modification (Wanner et al., 2014).
Genome research has become increasingly important in the 21st century.
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Technological innovations in the 1990s increased the efficiency and reduced the costsof genome modification and analysis, such as DNA sequencing and DNA synthesis.
Construction of a minimal genome enabled improvements in the ability to synthesize
antibiotics and produce other valuable materials.
To date, large-scale deletions of genes other than those that are clearly
unnecessary, such as prophage, transposon regions, and insertion sequences, havebeen unsuccessful (Kolisnychenko et al., 2002). Knowledge of the principles ofgenome construction is still incomplete even in model organisms such as E. coli.
Although a fully chemical synthetic bacterial genome has been constructed in
Mycoplasma (Gibson et al., 2010; Hutchison et al., 2016), it was not completed bydesign. The development of whole-cell metabolic models has been steadily
progressing (Edwards and Palsson, 2000; Feist et al., 2007; Monk et al., 2017; Orth etal., 2011; Reed et al., 2003), and these models have become platforms for genome
design. These models, even those in E. coli, contain large numbers of genes withunknown or incomplete functions (Orth and Palsson, 2012). Metabolic and wholecell models have been constructed for Mycoplasma (Karr et al., 2012) and areprogressing steadily for E. coli (Macklin et al., 2020), although additional research is
required to create an E. coli model cell for genome design. This review summarizes
the historical background of technological improvements, shows examples of pastand ongoing research, and considers the current status and future of this research.

2

Historical perspective of E. coli as a biological research tool

2.1

Before the molecular biology era
The discovery of conjugation in E. coli K-12 (Lederberg and Tatum, 1946),

which was thought to be sexless and to grow monogamously, and of transduction(Lennox, 1955), showed that genetic traits could be transferred between bacteria,leading to the use of E. coli K-12 as a model cell. Studies of the molecularmechanism of conjugation showed that it required a fertility factor and that DNA istransferred by the Type IV secretion system (Lawley et al., 2003), the detailed
molecular structure of which was visualized by cryo-electron microscopy (Hu et al.,264

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2019). Results showing that genetic transformation requires extracellular DNA, notprotein or RNA (Griffith, 1928), provided proof that genes are composed of DNA.
Moreover, bacterial species, such as Pneumococcus (Griffith, 1928), Haemophilus
influenzae (Alexander and Leidy, 1950), and Bacillus subtilis (Spizizen, 1958), wereshown to have the ability to take up extracellular DNA.
The discovery of conjugation and transduction in E. coli made genetics possible.
HR methods based on λ Red have been expanded for genome-scale functional
analyses of E. coli and its phages (Marinelli et al., 2012; Murphy, 2016).
2.2

Genetic and genomic engineering in the molecular biology era
Following studies showing that recBCD mutations suppress linear DNA

degradation and sbcA mutations activate the recET pathway, HR was developed forcloning PCR products (Oliner et al., 1993). Findings showing that λ Red markedly
increased the efficiency of HR in E. coli (Murphy, 1998) led to the development oftools for genetic modification and their use in E. coli and other microorganisms(Datsenko and Wanner, 2000; Fels et al., 2020; Yu et al., 2000). The method
illustrated in Fig. 1 requires only 35 bp of homology for efficient recombination and

Fig. 1 Targeted deletion by homologous recombination
Two methods have been used to block RecD exonuclease: (1) using recBC mutations and (2) λRed Gam synthesis. Cells are transformed with linear double-stranded (ds) DNA encoding anantibiotic resistance (AbR) cassette and ends homology regions (hr) of upstream (up) anddownstream (down) regions of the target.
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was adopted by the Japan genome project to construct the Keio collection of singlegene E. coli deletions (Baba et al., 2006). Greater understanding of the molecularmechanism of λ Red HR led to many improvements for its use in E. coli, phages,
and other bacteria (Ellis et al., 2001; Li et al., 2013; Marinelli et al., 2008; Wang etal., 2006; Wannier et al., 2020). Targeted replacement by HR requires suppression or
inhibition of the RecBCD exonuclease.
A strategy was subsequently developed for constructing “markerless” (scarless)gene replacements (deletions or substitutions) (Pósfai et al., 1999). This strategy
involved the introduction of I-SceI sites into the E. coli chromosome, which had beenabsent from the genome. This scarless protocol was subsequently combined with λRed HR to create scarless deletions (Fig. 2A).
Fig. 2B illustrates an alternative protocol for constructing scarless mutations.
Unwanted sequences may be removed from genomes using a counter-selection
method. A killing gene, also called a suicide gene, which can provide strong negative
selection, can be introduced into cells under permissive conditions when the geneproduct is inactive. Treating cells with an inducer of its synthesis or a compound that
inhibits cells harboring its product provides selection against cells bearing theinhibitor gene. This method has been used to target different regions of the E. coli
chromosome, including sacB (Metcalf et al., 1996), tetA (Metcalf et al., 1996), andtolC (Gregg et al., 2014). Two-step protocols in Fig. 2B have provided powerful tools
for recombineering E. coli and related bacteria with single-stranded (ss) and doublestranded (ds) DNA (Leatham-Jensen et al., 2012).
2.3

Genome-scale modification in the genome research era

2.3.1

Deletion of a large genomic region by random Tn insertion

Site-specific recombination at the loxP site resulted in two different types of Tn
insertion mutations. Tn insertions located at both ends to be deleted were selectedfrom each insertion mutation library and combined on one genome using P1transduction. In the presence of overexpressed Cre protein, the fragment locatedbetween the two types of Tn was removed by Cre-loxP site-specific recombination266

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Fig. 2A) Scarless deletion/substitution of the target
Scarless deletion using I-SceI nuclease. The drug-resistant fragment flanked by I-SceI restriction
enzyme sites is amplified with up and the next to the downside homology regions (u and hr) andintroduced into the genome by λ Red homologous recombination (Kolisnychenko et al., 2002).
The I-SceI-flanked segment is eliminated by expressing the meganuclease I-SceI, resulting in a
double-strand break (DSB), DSB-stimulated DNA repair, and RecA-dependent recombination
between the d direct repeats.
B) Two-step scarless deletion by homologous recombination
The Ab (antibiotic resistance gene) with the killing gene, such as sacB, ccdB, parE, or phage T7
0.7 under the control of the tightly regulated promoter, such as rhaBp (Haldimann et al., 1998)
is amplified with 36- to 40-nt homology region (hr) to target. The amplified fragment has thenbeen transformed into a strain expressing λ Red to insert into the genome. Double-stranded“Substitution Sequence (SS)” with flanking hrs is transformed into the Ab resistant fragment
integrated strain expressing λ Red. The transformants are selected in the presence of
L-rhamnose, preferably with L-rhamnose as the sole carbon source.
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特集 SFC バイオの軌跡(Fig. 3A) (Yu et al., 2002). These transposons were used to construct separate largescale Tn insertion libraries, which were subsequently combined in the same strain by
P1 transduction to yield a double Tn mutant. A site-specific recombinase wasintroduced to remove the chromosome segments between the Tn elements. Thismethod has been used to construct large-scale genomic deletions by, for example,
deleting 60 to 120 kb between pairs of Tn elements and choosing those mutants that
did not impair cell growth. Cre was subsequently used to eliminate segments between
loxP sites, followed by the introduction via P1 transduction of Tn elements for an
additional large deletion and the repeat of the entire process (Fig. 3A).
Another approach for deleting large chromosomal segments randomly relies on
a complex transposon with one Tn element inside another, Tn-in-Tn, carrying two
types of Tn elements with opposite terminal repeat directions (Fig. 3B) (Goryshin etal., 2003). Following random transposition of Tn-in-Tn into the chromosome,synthesis of the transposase for the internal element is induced, leading to its
transposition to a new site and elimination of DNA between the original and newsites. When this process was repeated 20 times, the average deletion length was foundto be about 10 kb, with a total of about 200 kb being successfully deleted (Goryshin
et al., 2003).
2.3.2

Large-scale deletion by HR
E. coli is thought to have acquired many genes to survive in diverse

environments. Shrinking the E. coli genome is thought to improve the efficiency ofmetabolic functions and reduce redundancy in genomic and regulatory structures(Fehér et al., 2008; Pósfai et al., 2006). Using HR, the E. coli K-12 genome lacking
K-islands, which were identified by comparative genomics as recent horizontal
acquisitions to the genome, was reduced (Fig. 2A) (Kolisnychenko et al., 2002). Thismethod, which was based on the accumulation of scarless deletions by HR and DSB,
allowed elimination of 15% of the E. coli K-12 genome. Twelve K-islands, containing
fragments of cryptic phage, transposons, disrupted pseudogenes, and genes of
unknown function, were deleted. Ultimately, 9.3% of genes in the genome, including268

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Fig. 3A) Two random insertion mutagenesis by Tn and site-specific recombination
Mutant Tns with less sequence specificity for insertion sites on chromosomes have beendeveloped, with transposons such as Tn5, Tn10, and Mariner often used. Two different Tns of
two different drug resistance genes were randomly mutated, and the location of insertion on the
genome was determined by PCR and sequencing. E. coli strains with insertions at appropriate
positions were selected and combined into a single E. coli strain using the P1 transduction
method. This method used site-specific recombination at each Tn and induced recombination byincreasing the production of site-specific recombinase and deleting the region between Tns.
B) Random insertion and deletion by Tn-in-Tn
A complex with Tnp that recognizes IE is introduced into the cell to obtain the first random
insertion mutation. Then, synthetic induction of Tnp recognizing the internal ME is performed
to obtain a transition mutation; the direction of the second transition results in a deletion
between two different Tn insertion sites.
C) Random insertion by Tn and DSB by CRISPR-Cas
Insertion of a Tn fragment into the genome, followed by CRISPR-Cas cutting of the inside of
the Tn fragment. This yielded a strain in which nuclease activity deleted the periphery.

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24 of 44 transposon regions, were deleted (Kolisnychenko et al., 2002). Furthermore,strains with large deletions grew as well on minimal medium as wild-type strains,confirming that these K-islands did not contain essential genes.
These results suggest that mobile elements, such as IS, which may drive
evolution but induce genomic instability, can be deleted, as can genes with
unnecessary function, and groups of genes that adversely affect the bacterial growthenvironment, including in humans. However, it is not easy to predict which genes
have those functions. A comparison of genomes of different E. coli strains enabled
the selection of genes that were present in K-12 but absent in other E. coli strains.
This resulted in the identification of a set of candidate genes, constituting about 20%of the genome, for deletion. This is an example of purposefully designed deletions
that contain unstable factors and gene groups that are not necessary for bacterial
growth. Moreover, the strains with large deletions, such as MDS42 and MDS43, grewalmost as well as wild-type, with the stability of their genomes and theirtransformation efficiency being improved. An improved E. coli strain MDS69 withadditional deletions is commercially available from Scarab Genomics.
Another study first compiled a list of predicted essential genes, followed by theuse of an HR method to delete regions between these genes (Hashimoto et al., 2005).
Using λ Red HR, alternate Ab resistance cassettes were inserted into intergenic
regions between two essential genes, followed by combining them by P1 transductionand eliminating the inserted cassettes with λ Red HR. About 30% of the E. coligenome was deleted by combining the largest deletions between essential genes using
P1 transduction, with the resulting phenotypes analyzed by determining their cell
shape and nucleoid organization.
2.4

Genome-scale genetic modification in systems and synthetic biology

2.4.1

CRISPR-Cas application

Technological developments since the elucidation of the CRISPR-Cas
mechanism have marked a major turning point in the biological sciences, just as thediscovery of junctions, restriction enzymes, and vectors paved the way for molecular270【第 5 章 IAB の 20 年】
Past, Present, and Future of Genome Modification in Escherichia Colibiology. CRISPR-Cas provides a process to design cleavage sites, called doublestrand breaks (DSBs), at will. Fig. 2A shows how targeting the I-SceI site to a specificlocation generates a DSB that leads to the formation of the designed deletion. Fig. 3Cshows how random Tn mutagenesis can be used to generate DSBs and nearly random
deletions by CRISPR-Cas (Ma et al., 2022).
CRISPR-Cas is most often used to create DSBs at locations governed by thesequence of guide RNA (gRNA). Accumulation of DSBs if unrepaired is lethal. In
eucaryotes, most broken DNA ends are bridged by nonhomologous end-joining(NHEJ) (Wyman and Kanaar, 2006). NHEJ, however, is usually not possible inbacteria due to lack of the key NHEJ proteins Ku and Ligase-D. In E. coli, DSBs aremost often repaired by HR and less frequently by alternative end-joining. An
alternative end-joining (A-EJ) mechanism of repairing DSBs involves end-resection
by RecBCD, end synapsis via microhomologies, and ligation of DNA ends
(Bhattacharyya et al., 2018; Chayot et al., 2010) by LigA (Fig. 3C). Combining
CRISPR-Cas with λ Red HR permits fashioning scarless deletions, insertions, or
substitutions by design, limited only by occurrences of PAM sequences (Fig. 4A)(Adli, 2018).
Development of mutant Cas proteins lacking endonuclease activity has allowed
precise base editing at very limited target sites in genomes by fusion of the enzyme
cytosine deaminase to an inactive Cas subunit (Banno et al., 2018), and various point
mutations and small insertions/deletions by fusion of the reverse transcriptase to asingle active Cas (nickase) subunit (Park et al., 2021) (Fig. 4B). This subject has beenreviewed previously (Dong et al., 2021).
The generation of catalytically inactive Cas by mutation has allowed
repurposing CRISPR as an RNA-guided platform that can specifically interfere withtranscription elongation, RNA polymerase binding, or transcription factor binding(CRISPR interference; CRISPRi) using a single guide RNA (sgRNA) chimera (Cui etal., 2018; Peters et al., 2016; Qi et al., 2013; Rousset et al., 2018; Wang et al., 2018).
CRISPRi was used to analyze a group of essential genes in B. subtilis, although
this approach did not focus directly on the genome modification (Peters et al., 2016).
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Fig. 4A) Scarless deletion/substitution using CRISPR-Cas
DSB was induced by CRISPR-Cas, and DNA fragments were transformed by bridging
homologous regions at both ends of each double-strand break, resulting in genome repair andyielding to the circular genome.
B) Editing by CRISPR-Cas fused with other enzymes
Genome editing by fusion protein with a function different from that of Cas protein. Left panel:fusion of cytosine deaminase to a Cas protein with mutation-inactivated DNase activities(Banno et al., 2018). Right panel: fusion of reverse transcriptase to a Cas, which inactivates only
the nick on the other strand, providing a template for repairing the nick site and introducing themutation by a reverse-transcribed sequence (Tong et al., 2021).

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Past, Present, and Future of Genome Modification in Escherichia ColiCRISPRi screening of E. coli was performed by synthesizing a library of 92,000
sgRNA sequences covering the entire genome, with PAM sequences as the only
constraint (Cui et al., 2018), thus identifying E. coli essential genes and genes
essential for phage l growth (Rousset et al., 2018). In a separate study, 60,000
sgRNAs were evaluated to test essentiality while also assessing the design of sgRNAs
for all genes, including those that did not encode RNA (Wang et al., 2018).
Essentiality was tested with a pooled library, with the results evaluated bydetermining the relative change in read count by next-generation sequencing (NGS).
2.4.2

A cceleration of evolution under the constraint of mutation direction byoligo DNAThe mutation of many genes simultaneously can result in the synthetic lethalityof genetic interactions, in which one mutation affects other genes, making theaccumulation of mutations difficult. Therefore, a method was devised to accelerate
the evolution of mutagenesis by adding the constraint of viability and utilizing theprinciple of HR of λ Red, while restricting sequences using synthetic DNA. This
Multiplex Automatable Genome Engineering (MAGE) method uses the b protein of l
Red and long (90 nt) synthetic ssDNAs, allowing acquisition of mutations on a
genome-scale without lethal or severe growth-defect mutations because mutant
selection is based on cell growth (Fig. 5) (Wang et al., 2009). Although MAGE was
originally performed robotically, it can also be performed manually. MAGE has
provided a powerful tool for genome-wide codon replacement (Isaacs et al., 2011),
metabolic engineering (Wang and Church, 2011), and other biological functions (Fig.
5) (Lajoie et al., 2013). λ Red HR is increased in mismatch repair mutants andshows strand bias (Costantino and Court, 2003; Li et al., 2003). MAGE technology
has been further improved by using MAGE and λ Red HR to stimulate the evolutionof host E. coli primase and helicase (Lajoie et al., 2012), which control the length of
Okazaki lagging-strand fragments (Balakrishnan and Bambara, 2013).

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Fig. 5 Acceleration of in vivo evolution by λ Red β and ssDNAs
Introduction of an ssDNA about 90 bp in length to be mutated in the cell via the induction of λRed λ protein, which promoted the introduction of mutations on the lagging strand during
DNA replication and accelerated the introduction of mutations throughout the genome.

3

Genome-scale projects

3.1

Minimal genome projects
The minimal genome project, which began in 1997 based on a concept ofminimal gene sets (Koonin, 1997; Mushegian and Koonin, 1996), has been ongoingfor many years but has not yet been completed. The main reasons for non-completion
are the existence of alternative pathways and compensatory circuits in theintracellular functional network, and the existence of orphan enzymes whose genes
have not yet been identified. The minimal genome concept was later linked to theconcept of chassis genomes in synthetic biology (Danchin, 2012).

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3.1.1

Large-scale deletion by random transposon insertion

Strains with large-scale deletions have been constructed using loxP site-specificrecombination sites embedded in two types of Tn5 and a fragment of Tn5 randomly
inserted into the E. coli genome (Yu et al., 2002). For example, two types of Tn5incorporating separate Kan and Chl antibiotic resistance genes and loxP site-specificrecombination sites were constructed and used to generate random insertion mutant
libraries. Mutants were selected from each Kan- and Chl-resistant library that flanked
the region to be deleted, and the two insertion mutations combined into a single
genome by P1 transduction. Addition of the Cre protein allowed the large-scaledeletion of the genomic region between the loxP site-specific recombination sites.
This method was used to introduce large-scale genomic deletions in six strains by
combining two types of transposons, deleting 60 to 120 kb between them, andselecting mutations that did not impair the growth of the deleted cells. The selection
marker between loxP sites were subsequently removed from the strains by Cre,followed by the introduction of another large deletion region into a single genome by
P1 transduction, resulting in the construction of a minimal genome.
By contrast, Tn5 derivatives have been used to create Tn-in-Tn system togenerate a large genomic deletion (Goryshin et al., 2003). Because the average lengthof each deleted region was about 10 kb, repeating this process 20 times successfully
introduced deletions into a ~200 kb region.
3.1.2

Large scarless deletion by HR

The reduced genome lacking K-islands (Kolisnychenko et al., 2002) has beenfurther improved (Pósfai et al., 2006). Although E. coli was originally described as anintestinal bacterium, it has acquired a diverse set of genes, enabling it to survive invarious environments. Shrinking of the genome is thought to improve the efficiencyof metabolic functions and reduce redundancy in genomic and regulatory structures(Pósfai et al., 2006). Mobile elements, such as IS, which may drive evolution but
induce genomic instability, genes with unnecessary function, and groups of genes thatadversely affect the bacterial growth environment, including in humans, can be
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deleted. However, predicting the genes that have these functions is difficult. A
comparison of the genomes of different E. coli strains identified selected genes thatwere present in K-12 but absent from other E. coli strains, resulted in the selection of
a set of candidate genes, comprising about 20% of the genome, for deletion.
The deletion method (Kolisnychenko et al., 2002) was based on theaccumulation of scarless deletions by HR and DSB, resulting in the successful
deletion of 15% of the E. coli K-12 genome. This was an example of purposefully
designed deletions of sequences that are unstable factors and gene groups that are notnecessary for the growth of E. coli.
The growth of the final strains with large deletions, MDS42 and MDS43, were
almost the same as that of the wild type, although these deletions improved genome
stability and transformation efficiency, making these strains practical reduced-genome
E. coli. MDS69, an improved E. coli strain with additional deletions, is currently
available commercially from Scarab Genomics.
Another approach of scarless deletion of a large region between essential genes
involved the performance of two HR events (Fig. 2B) (Hashimoto et al., 2005). Thismethod resulted in the deletion of the largest possible region from the essential
intergenic region and the accumulation of deletions by P1 transduction to yield aminimal genome with large deletions.
Analyses of the phenotypes of E. coli from which about 30% of the genome had
been deleted showed that growth rate was inversely associated with the size of thedeleted region (Hashimoto et al., 2005). Deletion also altered cell morphology, withchanges in cell length and width and in nucleoid organization. Attempts to combine
these large deletions showed some could not be combined (Kato, J., personalcommunication), perhaps because the combination resulted in synthetic lethality.
Therefore, it is still difficult to determine the associations between combinations ofgene deletions and specific phenotypes. This deletion project has since become a joint
project with Kyowa Hakko Co. Ltd.
Efforts have been made to develop bacteria with beneficial genomes for the
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cell growth. One strain, MGF-01, was generated by deleting 1.03 Mb from 53 regions
using P1 (Mizoguchi et al., 2008). These deletions increased glucose consumption
1.44-fold and acetate accumulation 0.09-fold, confirming the efficacy of this method(Mizoguchi et al., 2008).
The strain MS56 has a genome reduced by 23% (Park et al., 2014). It was
generated by removing IS and other factors that may cause instability in plasmids
containing foreign genes, and its stability and efficiency of expression of foreign geneproducts was analyzed (Park et al., 2014). Evaluation of the scarless HR deletion
method (Kolisnychenko et al., 2002; Pósfai et al., 2006) with human tumor necrosis
factor-related apoptosis-inducing ligand (TRAIL) and bone morphogenetic protein-2(BMP2) showed its superiority. This led to the development of genome-reduced
strains useful for biosynthesis in industrial applications.
3.2

Recoding genome

3.2.1

Recoding genome by recombineering and ssDNA accelerated evolution

Despite the significant progress of the Minimal genome project in accumulating
individual genes with specific functions, it is still almost impossible to fully design agenome with deletion of many genes. These drawbacks may be overcome by geneticinteraction analysis of systematic double deletion strains.
Evolutionary methods have also been explored. Mutants that are not viable orgrow very slowly are eliminated during the selection process. Clarification of themolecular mechanism of RED recombination has shown that ssDNA predominantly
introduces mutations into the replicating lagging strand through the activity of thebeta-protein alone (Ellis et al., 2001). These findings led to the development of theMultiplex Automated Genome Engineering (MAGE) method using in vivo evolution
with multiple types of designed ssDNA and b proteins (Fig. 5) (Wang et al., 2009).
The running of 5-35 MAGE cycles resulted in the ~105 mutant strains and increased
the production of the target product, isoplenoid lycopen, up to 5-fold within 3 days
(Wang et al., 2009).
This development has enabled genome modification by deliberately limiting the
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direction of mutation and accelerating evolution by recombination of many parts ofthe genome at once. This technology has since been further improved, allowing its
use on a larger scale. For example, a recoding genome was constructed by replacing
the TAG termination codons on all 314 E. coli genes bearing these codons with TAA
termination codons (Isaacs et al., 2011). Because these 314 genes are scatteredthroughout the genome, the genome was divided into 32 regions, and the MAGE
cycle was run for each region to obtain evolutionary mutant strains. The Conjugative
Assembly Genome Engineering (CAGE) method was also developed to integrate themutated chromosomal sites into a single E. coli strain using conjugation, resulting inan E. coli strain in which all terminal TAGs were replaced by TAAs. Although
recoding was expected to eliminate the need for TAG codons, prfA, the geneencoding releasing factor (RF1), which recognizes TAG codons, was deleted, shows
the ability to replace TAG codons with other codons. This technology was further
improved by developing primase and helicase mutant strains, which containmutations that control the lengths of Okazaki fragments synthesized by the lagging
strand (Lajoie et al., 2012).
It may also be possible to replace codons for a specific amino acid, rather thantermination codons. Forty-two highly expressed essential genes were selected, andrare codons in these genes were replaced by DNA synthesis; if this method was
unsuccessful, these codons were replaced using MAGE. Ultimately, 405 codons on
42 highly expressed essential genes were replaced, resulting in reductions in cell
growth. These results showed that genome-wide codon replacement is feasible andthat codons can be replaced using MAGE, with no or minimal effect on cell growth.
Providing artificially modified organisms with a genetic code that does not exist in
the natural would thus ensure the safety of these organisms, even if they are released
to the outside world.
3.2.2

Recoding genome by synthesis

A method has been developed to replace a target region of the genome with afragment of synthetic DNA designed for recoding by assembly in vivo, including278

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Past, Present, and Future of Genome Modification in Escherichia Coli

recoding of the entire E. coli genome (Fig. 6). For example, replacement of thecodons UAG (stop), AGG-AGA (Arg), AGC-AGU (Ser), and UUG-UUA (Leu) withother synonymous codons from the genome resulted in the construction of an E. coligenome with 57 codons. Similarly, replacing the codons TCG, TCA (Ser), and TAG(stop) resulted in the construction of an E. coli genome with 61 codons. Both methods
used designed synthetic DNA, with the genome-reduced strain MDS42 used as theparent strain.
Although these methods showed some differences in their details, both involved
assembly of the genome by HR in yeast cells and its transfer to E. coli cells. In one
method, the target region on the genome was removed, the assembled recoded
genome was inserted into the target region using attL-attP site-specific recombination,and the vector region was deleted by CRISPR (Ostrov et al., 2016). In the otherFig. 6 Assembly of synthesized DNA and homologous recombination.
Introduction of synthetic DNA fragments into cells, generally yeast cells, assembly of thefragments by in vivo homologous recombination. After assembling, the assembled fragment iscollected and transformed into E. coli cell and replacement of the target region in the λ Redinduced strain by homologous recombination.
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method, fragments that accumulated on the BAC vector were integrated into therecipient genome by transferring them to the recipient using conjugation, althoughassembly in yeast cells was the same (Wang et al., 2016). This method resulted in theconstruction of a partial recoded genome by linearizing both ends of the fragmentsthat had accumulated on the vector by CRISPR double-strand breaks and replacing
them with the target regions of the genome by HR using λ RED recombinase. This
step was repeated to construct the entire recoded genome (Fredens et al., 2019). The
resulting E. coli strain Syn61 with a recoded genome was found to grow more slowly
and have a longer cell length than the parental strain MDS42.
3.3

Genome-scale analysis towards genome design platform
In genomics, the construction of mutants is an important first step in analyzing

the biological functions of genes and their products. As of September 2020, the E.
coli genome, annotated as GenBank entry U00096.3, included 4609 genes, with 4285
of these genes encoding proteins, many of which have unknown functions. In
addition, this genome was found to include genes encoding small proteins and noncoding RNAs (Hobbs et al., 2010). Genome-scale metabolic models of E. coli havebeen developed, such as iJE660 (Edwards and Palsson, 2000), iJR904 (Reed et al.,2003), iAF1260 (Feist et al., 2007), iJO1366 (Orth et al., 2011), and iML1515 (Monket al., 2017), with others still being developed and improved. Refinements of thesemodels have shown the present of as yet unidentified alternative pathways andisozymes and gaps in metabolic networks (orphan reactions) (Orth and Palsson, 2012;
Orth and Palsson, 2010). Knowledge of E. coli is also incomplete (Orth et al., 2011;
Orth and Palsson, 2012; Orth and Palsson, 2010).
The minimal gene set concept (Koonin, 1997) has become the minimal genome
project, but it is still far from complete. By contrast, the minimal genome concept has
expanded to the concept of a minimal genome factory that optimizes the genome to
produce valuable products (Mizoguchi et al., 2007). These concepts have now
expanded to include the concept of the chassis genome in synthetic biology (Calero
and Nikel, 2019; Danchin, 2012).

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Past, Present, and Future of Genome Modification in Escherichia Coli

4

Discussion and perspective

4.1

Transition of biological concepts
About 50 years have elapsed since recombinant E. coli gene modification

technology was initially introduced, from the elucidation of its molecular mechanism
to the development of methods based on phage recombination mechanisms andimprovement of the technology. This has enabled almost any type of sequencemodification, from genome-scale large modification to base-level modification.
Genome research in the 1990s may therefore represent a turning point in biology bothtechnologically and conceptually. This situation was similar in the 1970s, when
genetic modification techniques were developed and molecular biology began tomake significant progress.
Often, new concepts are not immediately accepted. One such example is thedifference between “forward genetics” and “reverse genetics”, which was initiallydeveloped by physicists, but was not fully accepted by researchers in genetics (Yura,T., personal communication). Differences in acceptance were not likely due todifferences in ways of thinking, but increased understanding was likely due toexperimental efforts. The throughput of sequencing technology has expanded about
1,000-fold from the start of the project in 1990 to the completion of the first draft
sequence of the human genome. With the technology available at that time, thegenome of E. coli took 7 years to complete. However, the 21st century has seen thedevelopment of sequencing technologies based on novel concepts, and sequencing is
now more than a billion times more efficient than it was in 1990. The driving force
behind this development dates back to the 1990s. Researchers understood theimportance of comparative analysis of individual human genomes and the need forfurther development of sequencing technology as the next step after the completionof the human genome.
Technological innovation has not only affected the speed of analysis, but has
made possible more precise and diverse analyses, and at lower cost. For example, therange of applications of sequencing technology has rapidly expanded to include
analyses of gene expression, protein-DNA interactions, protein-protein interactions,
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nucleoids, and species distribution in populations. Moreover, genome editing in the
21st century has been revolutionized by determining the molecular mechanism
underlying CRISPR.
Although the recombination efficiency of E. coli is lower than that of othermodel microorganisms, such as B. subtilis and yeast, the tooling of λ Red
recombinase made possible the modification of E. coli by genome-scale
recombination, increasing its recombination efficiency. The availability of genome
modification techniques by recombination in Gram-positive, Gram-negative, andeukaryotic unicellular organisms has created a favorable research environment forcomparative analysis.
Progress in genome analysis led to the development of research resources inyeast, the creation of databases, the construction of gene clone and deletion strain
libraries, and their sharing as community assets (Botstein and Fink, 2011; Giaeverand Nislow, 2014). For example, participating institutions in Europe and Japan
worked together as a community to build a B. subtilis deletion strain library(Kobayashi et al., 2003; Vagner et al., 1998).
Although a similar comprehensive experimental resource community has been
proposed for E. coli, few laboratories agreed to participate. Groups at Keio
University, the Nara Institute of Science and Technology, and Purdue University
therefore agreed to develop this resource. Although the time to completion wasundetermined, the development of many innovations resulted in the completion of
the entire project in 3 years. These developed resources were subsequently sharedwith the research community as open resources. E. coli could therefore be positioned
as a model microorganism alongside B. subtilis and yeast.
The importance of putting all the pieces together is enormous. The importanceof analyzing the structure and function of target genes and proteins in detail throughindividually targeted analyses will likely remain unchanged. However, looking at theentire picture revealed aspects that could not be determined on individual studies of anarrow range of targets. Molecular biology in the 20th century has been described asa very precise “science of parts”, whereas genome research, starting in the 1990s and282

【第 5 章 IAB の 20 年】
Past, Present, and Future of Genome Modification in Escherichia Coliextending into the 21st century, can be described as “science as a system”. Alignmentof the two will likely greatly advance our understanding of the whole picture of life.
4.2

Concept of minimal genome
The minimal genome project, which started with the minimal gene set, has beena long-term effort to realize the minimal genome concept. In particular, the largescale deletion efforts showed that, although deletion of individual genes in a region
did not significantly affect the growth of an organism, simultaneous deletion of many
gene clusters significantly affects growth and may even be lethal. These findings
emphasized the importance of determining genetic interactions under conditions ofsynthetic lethality and sickness, and may have a major impact on network biology.
Indeed, findings showing that cell lethality was due to multiple gene deletions andquantitative analyses of the effects of single-gene deletions on intracellular conditions(Ishii et al., 2007) suggested that many genes that are considered non-essential may
repeatedly interact with each other to maintain intracellular stability, at least at the
metabolite level. Efforts are underway to analyze genetic interactions through thesystematic construction of double gene deletion strains. The saying in Japan, “If thewind blows, the bucket makers prosper”, is equivalent to the “Butterfly Effect” inchaos theory. Cells dynamically regulate transcription, translation, and enzyme
activities while optimizing the balance between the genetic elements of the cell andthe environmental factors affecting growth.
The minimal genome concept has also expanded to optimize the practical use ofcells for industrial purposes (Mizoguchi et al., 2007; Park et al., 2014). Moreover, thebeginning of synthetic biology has resulted in expansion of the concept of the“chassis genome” (Calero and Nikel, 2019; Danchin, 2012).
4.3

Dramatic changes in quality and quantity of data require a variety ofanalyses
The existence of comprehensive research resources and the development ofanalytic methods have led to a rapid increase in data. Effective use of these data
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requires mathematical analyses and information processing technologies such as
statistical analysis and modeling. Although analyzing the data generated from a single
comprehensive study provides many hints on physiological functions and molecularmechanisms, these hints may not be successfully verified by experimental methods.
Although data registered in databases and accessible through Web systems have beenused for informatics analysis, the contribution of these enormous amounts of data to
the progress of individual research is unclear. The situation may be dependent on thepresence of a framework for sharing information that may provide direct material forexperimental validation, such as proposals for individual molecular mechanisms
obtained from informatics analysis. Another factor may result from the same researchgroup performing comprehensive analyses and individual targeted research. The
accumulation of experience is important in deepening research in individual studies,making it difficult to complete both comprehensive and individual analyses in asingle laboratory. Establishment of a community-oriented framework is likely
necessary to share comprehensive data, along with interpretations and/or suggestions.
For example, a group at McMaster University in Canada has specialized indiscovering anti-microbial antibiotics and has developed research using
comprehensive resources. As soon as resources become available, they are used for
exhaustive screening (Pathania et al., 2009), leading to acceleration of research anddevelopment, and further expansion (Brown and Wright, 2016). This group istherefore a good example of the successful use of both comprehensive and individual
approaches in a single laboratory.
References
Adli, M. (2018) “The CRISPR tool kit for genome editing and beyond”, Nature
Communications. 9(1):1911.
Alexander, H.E. and Leidy, G. (1950) “Transformation of type specificity of Hemophilus
influenzae”, AMA Am J Dis Child . 80(5), pp. 877-878.
Baba, T., et al. (2006) “Construction of Escherichia coli K-12 in-frame, single-gene knockout
mutants: the Keio collection”, Mol Syst Biol . 2(1):2006 0008.
Balakrishnan, L. and Bambara, R.A. (2013) “Okazaki fragment metabolism”, Cold Spring Harb
Perspect Biol. 5(2).

284【第 5 章 IAB の 20 年】
Past, Present, and Future of Genome Modification in Escherichia Coli
Banno, S., et al. (2018) “Deaminase-mediated multiplex genome editing in Escherichia coli”,
Nature Microbiology. 3(4), pp. 423-429.
Bhattacharyya, S., et al. (2018) “Phage Mu Gam protein promotes NHEJ in concert with
Escherichia coli ligase”, Proc Natl Acad Sci U S A. 115(50):E11614-E11622.
Botstein, D. and Fink, G.R. (2011) “Yeast: an experimental organism for 21st Century biology”,
Genetics. 189(3), pp. 695-704.
Brown, E.D. and Wright, G.D. (2016) “Antibacterial drug discovery in the resistance era”,
Nature. 529(7586), pp. 336-343.
Calero, P. and Nikel, P.I. (2019) “Chasing bacterial chassis for metabolic engineering: aperspective review from classical to non-traditional microorganisms”, Microbial
Biotechnology. 12(1), pp. 98-124.
Chayot, R., et al. (2010) “An end-joining repair mechanism in Escherichia coli”, Proc Natl
Acad Sci U S A. 107(5), pp. 2141-2146.
Cohen, S.N., Chang, A.C. and Hsu, L. (1972) “Nonchromosomal antibiotic resistance inbacteria: genetic transformation of Escherichia coli by R-factor DNA”, Proc Natl Acad Sci
U S A . 69(8), pp. 2110-2114.
Cosloy, S.D. and Oishi, M. (1973) “Genetic transformation in Escherichia coli K12”, Proc Natl
Acad Sci U S A. 70(1), pp. 84-87.
Costantino, N. and Court, D.L. (2003) “Enhanced levels of λ Red-mediated recombinants inmismatch repair mutants”, Proc Natl Acad Sci U S A. 100(26), pp. 15748-15753.
Cui, L., et al. (2018) “A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9”,
Nature Communications. 9(1):1912.
Danchin, A. (2012) “Scaling up synthetic biology: Do not forget the chassis”, FEBS Lett.
586(15), pp. 2129-2137.
Datsenko, K.A. and Wanner, B.L. (2000) “One-step inactivation of chromosomal genes in
Escherichia coli K-12 using PCR products”, Proc Natl Acad Sci U S A. 97(12), pp. 66406645.
Dong, H., Cui, Y. and Zhang, D. (2021) “CRISPR/Cas Technologies and Their Applications inEscherichia coli”, Front Bioeng Biotechnol . 9:762676.
Edwards, J.S. and Palsson, B.O. (2000) “The Escherichia coli MG1655 in silico metabolic
genotype: its definition, characteristics, and capabilities”, Proc Natl Acad Sci U S A. 97(10),pp. 5528-5533.
Ellis, H.M., et al. (2001) “High efficiency mutagenesis, repair, and engineering of chromosomal
DNA using single-stranded oligonucleotides”, Proc Natl Acad Sci U S A. 98(12), pp. 67426746.
Fehér, T., et al. (2008) “Scarless engineering of the Escherichia coli genome”, Methods Mol
Biol. 416, pp. 251-259.
Feist, A.M., et al. (2007) “A genome-scale metabolic reconstruction for Escherichia coli K-12
MG1655 that accounts for 1260 ORFs and thermodynamic information”, Mol Syst Biol.
3(1), 121.
Fels, U., Gevaert, K. and Van Damme, P. (2020) “Bacterial Genetic Engineering by Means ofRecombineering for Reverse Genetics”, Frontiers in Microbiology. 11:548410.
Fredens, J., et al. (2019) “Total synthesis of Escherichia coli with a recoded genome”, Nature.
569(7757), pp. 514-518.
Giaever, G. and Nislow, C. (2014) “The yeast deletion collection: a decade of functional
genomics”, Genetics. 197(2), pp. 451-465.
Gibson, D.G., et al. (2010) “Creation of a bacterial cell controlled by a chemically synthesized

KEIO SFC JOURNAL Vol.22 No.2 2022285

特集 SFC バイオの軌跡
genome”, Science. 329(5987), pp. 52-56.
Goryshin, I.Y., et al. (2003) “Chromosomal deletion formation system based on Tn5 doubletransposition: use for making minimal genomes and essential gene analysis”, Genome Res.
13(4), pp. 644-653.
Gregg, C.J., et al. (2014) “Rational optimization of tolC as a powerful dual selectable marker for
genome engineering”, Nucleic Acids Res. 42(7), pp. 4779-4790.
Griffith, F. (1928) “The Significance of Pneumococcal Types”, J Hyg (Lond) . 27(2), pp. 113159.
Haldimann, A., Daniels, L.L. and Wanner, B.L. (1998) “Use of new methods for construction oftightly regulated arabinose and rhamnose promoter fusions in studies of the Escherichia
coli phosphate regulon”, J Bacteriol. 180(5), pp. 1277-1286.
Hashimoto, M., et al. (2005) “Cell size and nucleoid organization of engineered Escherichia coli
cells with a reduced genome”, Mol Microbiol. 55(1), pp. 137-149.
Hobbs, E.C., Astarita, J.L. and Storz, G. (2010) “Small RNAs and small proteins involved inresistance to cell envelope stress and acid shock in Escherichia coli: analysis of a barcoded mutant collection”, J Bacteriol . 192(1), pp. 59-67.
Hu, B., Khara, P. and Christie, P.J. (2019) “Structural bases for F plasmid conjugation and F
pilus biogenesis in Escherichia coli”, Proc Natl Acad Sci U S A. 116(28), pp.14222-14227.
Hutchison, C.A., 3rd, et al. (2016) “Design and synthesis of a minimal bacterial genome”,
Science. 351(6280):aad6253.
Isaacs, F.J., et al. (2011) “Precise manipulation of chromosomes in vivo enables genome-widecodon replacement”, Science. 333(6040), pp. 348-353.
Ishii, N., et al. (2007) “Multiple high-throughput analyses monitor the response of E. coli toperturbations”, Science. 316(5824), pp. 593-597.
Ishino, Y., Krupovic, M. and Forterre, P. (2018) “History of CRISPR-Cas from Encounter with a
Mysterious Repeated Sequence to Genome Editing Technology”, J Bacteriol . 200(7).
Ishino, Y., et al. (1987) “Nucleotide sequence of the iap gene, responsible for alkaline
phosphatase isozyme conversion in Escherichia coli, and identification of the geneproduct”, J Bacteriol. 169(12), pp. 5429-5433.
Jinek, M., et al. (2012) “A programmable dual-RNA-guided DNA endonuclease in adaptive
bacterial immunity”, Science. 337(6096), pp. 816-821.
Karr, J.R., et al. (2012) “A whole-cell computational model predicts phenotype from genotype”,
Cell. 150(2), pp. 389-401.
Kobayashi, K., et al. (2003) “Essential Bacillus subtilis genes”, Proc Natl Acad Sci U S A.
100(8), pp. 4678-4683.
Kolisnychenko, V., et al. (2002) “Engineering a reduced Escherichia coli genome”, Genome
Res. 12(4), pp. 640-647.
Koonin, E.V. (1997) “Big time for small genomes”, Genome Res. 7(5), pp. 418-421.
Lajoie, M.J., et al. (2012) “Manipulating replisome dynamics to enhance λ Red-mediatedmultiplex genome engineering”, Nucleic Acids Res. 40(22):e170.
Lajoie, M.J., et al. (2013) “Genomically recoded organisms expand biological functions”,
Science. 342(6156), pp. 357-360.
Lawley, T.D., et al. (2003) “F factor conjugation is a true type IV secretion system”, FEMS
Microbiol Lett. 224(1), pp. 1-15.
Leatham-Jensen, M.P., et al. (2012) “The streptomycin-treated mouse intestine selects
Escherichia coli envZ missense mutants that interact with dense and diverse intestinal
microbiota”, Infection and Immunity. 80(5), pp. 1716-1727.

286

【第 5 章 IAB の 20 年】
Past, Present, and Future of Genome Modification in Escherichia Coli
Lederberg, J. and Tatum, E.L. (1946) “Gene recombination in Escherichia coli”, Nature.
158(4016), 558.
Lennox, E.S. (1955) “Transduction of linked genetic characters of the host by bacteriophage
P1”, Virology. 1(2), pp. 190-206.
Li, X.T., et al. (2003) “Identification of factors influencing strand bias in oligonucleotidemediated recombination in Escherichia coli”, Nucleic Acids Res. 31(22), pp. 6674-6687.
Li, X.T., et al. (2013) “Bacterial DNA polymerases participate in oligonucleotide
recombination”, Mol Microbiol . 88(5), pp. 906-920.
Ma, S., et al. (2022) “Reduction of the Bacterial Genome by Transposon-Mediated Random
Deletion”, ACS Synthetic Biology. 11(2), pp. 668-677.
Macklin, D.N., et al. (2020) “Simultaneous cross-evaluation of heterogeneous E. coli datasets
via mechanistic simulation”, Science. 369(6502).
Mandel, M. and Higa, A. (1970) “Calcium-dependent bacteriophage DNA infection”, J Mol
Biol. 53(1), pp. 159-162.
Marinelli, L.J., Hatfull, G.F. and Piuri, M. (2012) “Recombineering: A powerful tool for
modification of bacteriophage genomes”, Bacteriophage. 2(1), pp. 5-14.
Marinelli, L.J., et al. (2008) “BRED: a simple and powerful tool for constructing mutant andrecombinant bacteriophage genomes”, PLoS One. 3(12):e3957.
Metcalf, W.W., et al. (1996) “Conditionally replicative and conjugative plasmids carrying lacZα
for cloning, mutagenesis, and allele replacement in bacteria”, Plasmid. 35(1), pp. 1-13.
Mizoguchi, H., Mori, H. and Fujio, T. (2007) “Escherichia coli minimum genome factory”,
Biotechnol Appl Biochem. 46(Pt 3), pp. 157-167.
Mizoguchi, H., et al. (2008) “Superpositioning of deletions promotes growth of Escherichia coli
with a reduced genome”, DNA Res. 15(5), pp. 277-284.
Monk, J.M., et al. (2017) “iML1515, a knowledgebase that computes Escherichia coli traits”,
Nat Biotechnol . 35(10), pp. 904-908.
Murphy, K.C. (1998) “Use of bacteriophage λ recombination functions to promote genereplacement in Escherichia coli”, J Bacteriol. 180(8), pp. 2063-2071.
Murphy, K.C. (2016) “λ Recombination and Recombineering”, EcoSal Plus. 7(1).
Mushegian, A.R. and Koonin, E.V. (1996) “A minimal gene set for cellular life derived bycomparison of complete bacterial genomes”, Proc Natl Acad Sci U S A. 93(19), pp. 1026810273.
Oliner, J.D., Kinzler, K.W. and Vogelstein, B. (1993) “In vivo cloning of PCR products in E.
coli”, Nucleic Acids Res. 21(22), pp. 5192-5197.
Orth, J.D., et al. (2011) “A comprehensive genome-scale reconstruction of Escherichia coli
metabolism--2011”, Mol Syst Biol. 7(1), p. 535.
Orth, J.D. and Palsson, B.Ø. (2010) “Systematizing the generation of missing metabolic
knowledge”, Biotechnol Bioeng. 107(3), pp. 403-412.
Orth, J.D. and Palsson, B.Ø (2012) “Gap-filling analysis of the iJO1366 Escherichia coli
metabolic network reconstruction for discovery of metabolic functions”, BMC Systems
Biology. 6(1), 30.
Ostrov, N., et al. (2016) “Design, synthesis, and testing toward a 57-codon genome”, Science.
353(6301), pp. 819-822.
Park, J., et al. (2021) “Enhanced genome editing efficiency of CRISPR PLUS: Cas9 chimeric
fusion proteins”, Scientific Reports. 11(1):16199.
Park, M.K., et al. (2014) “Enhancing recombinant protein production with an Escherichia coli
host strain lacking insertion sequences”, Appl Microbiol Biotechnol. 98(15), pp. 6701-6713.

KEIO SFC JOURNAL Vol.22 No.2 2022

287

特集 SFC バイオの軌跡
Pathania, R., et al. (2009) “Chemical genomics in Escherichia coli identifies an inhibitor ofbacterial lipoprotein targeting”, Nat Chem Biol. 5(11), pp. 849-856.
Peters, J.M., et al. (2016) “A Comprehensive, CRISPR-based Functional Analysis of Essential
Genes in Bacteria”, Cell. 165(6), pp. 1493-1506.
Pósfai, G., et al. (1999) “Markerless gene replacement in Escherichia coli stimulated by adouble-strand break in the chromosome”, Nucleic Acids Res. 27(22), pp. 4409-4415.
Pósfai, G., et al. (2006) “Emergent properties of reduced-genome Escherichia coli”, Science.
312(5776), pp. 1044-1046.
Qi, L.S., et al. (2013) “Repurposing CRISPR as an RNA-guided platform for sequence-specificcontrol of gene expression”, Cell. 152(5), pp. 1173-1183.
Reed, J.L., et al. (2003) “An expanded genome-scale model of Escherichia coli K-12 (iJR904
GSM/GPR)”, Genome Biol. 4(9):R54.
Rousset, F., et al. (2018) “Genome-wide CRISPR-dCas9 screens in E. coli identify essential
genes and phage host factors”, PLoS Genet. 14(11):e1007749.
Spizizen, J. (1958) “Transformation of Biochemically Deficient Strains of Bacillus Subtilis by
Deoxyribonucleate”, Proc Natl Acad Sci U S A. 44(10), pp. 1072-1078.
Tong, Y., et al. (2021) “A versatile genetic engineering toolkit for E. coli based on CRISPRprime editing”, Nature Communications. 12(1):5206.
Vagner, V., Dervyn, E. and Ehrlich, S.D. (1998) “A vector for systematic gene inactivation in
Bacillus subtilis”, Microbiology (Reading). 144 ( Pt 11), pp. 3097-3104.
Wang, H.H. and Church, G.M. (2011) “Multiplexed genome engineering and genotyping
methods applications for synthetic biology and metabolic engineering”, Methods Enzymol.
498, pp. 409-426.
Wang, H.H., et al. (2009) “Programming cells by multiplex genome engineering and accelerated
evolution”, Nature. 460(7257), pp. 894-898.
Wang, J., et al. (2006) “An improved recombineering approach by adding RecA to λ Red
recombination”, Mol Biotechnol. 32(1), pp. 43-53.
Wang, K., et al. (2016) “Defining synonymous codon compression schemes by genome
recoding”, Nature. 539(7627), pp. 59-64.
Wang, T., et al. (2018) “Pooled CRISPR interference screening enables genome-scale functional
genomics study in bacteria with superior performance”, Nature Communications.
9(1):2475.
Wanner, B.L., Teramoto, J. and Mori, H. (2014) “What hath DNA wrought? CRISPR-CAS genesilencing and engineering from bacteria to humans: comment on “Diversity, evolution, andtherapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems” by
Edwin L. Cooper and Nicola Overstreet”, Physics of Life Reviews. 11(1), pp. 144-145;
discussion pp. 149-151.
Wannier, T.M., et al. (2020) “Improved bacterial recombineering by parallelized protein
discovery”, Proc Natl Acad Sci U S A. 117(24), pp. 13689-13698.
Wyman, C. and Kanaar, R. (2006) “DNA double-strand break repair: all's well that ends well”,
Annu Rev Genet. 40, pp. 363-383.
Yu, B.J., et al. (2002) “Minimization of the Escherichia coli genome using a Tn5-targeted Cre/
loxP excision system”, Nat Biotechnol. 20(10), pp. 1018-1023.
Yu, D., et al. (2000) “An efficient recombination system for chromosome engineering inEscherichia coli”, Proc Natl Acad Sci U S A. 97(11), pp. 5978-5983.
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