Microbial diversity and complexity pose challenges in understanding the voluminous genetic information produced from whole-genome sequences, bioinformatics and high-throughput ‘-omics’ research. These challenges can be overcome by a core blueprint of a genome drawn with a minimal gene set, which is essential for life. Systems biology and large-scale gene inactivation studies have estimated the number of essential genes to be ∼300–500 in many microbial genomes. On the basis of the essential gene set information, minimal-genome strains have been generated using sophisticated genome engineering techniques, such as genome reduction and chemical genome synthesis. Current size-reduced genomes are not perfect minimal genomes, but chemically synthesized genomes have just been constructed. Some minimal genomes provide various desirable functions for bioindustry, such as improved genome stability, increased transformation efficacy and improved production of biomaterials. The minimal genome as a chassis genome for synthetic biology can be used to construct custom-designed genomes for various practical and industrial applications.

Introduction

The genetic basis of ‘life’ is dictated by the interplay of diverse and complex nucleotides, proteins and metabolites. Although this diversity and complexity might bestow on the biotechnological industry an array of natural products, considerable effort and labour are required for obtaining such products by metabolic engineering and synthetic biology. In addition, researchers often encounter unexpected results after cell engineering, because the insertion of foreign genes might cause a disturbance in normal cellular function, and the effect of an integrated gene depends on the interaction with other genes [1]. The inconsistency arises from a complex genetic network consisting of thousands of genes. The complexity is particularly problematic in synthetic biology applications, for example, building a genetic circuit. A genetic circuit employs many regulatory elements, sensors and reporters. The genetic parts should be independent (reactive only with their cognate pairs) of other cellular components for precise operation of the circuit [2]. However, the orthogonality of the system is often disrupted because of cross-talk with endogenous signal transduction and regulatory frameworks [3]. Therefore a predictable, controllable, transformable and measurable chassis genome is needed for synthetic biology and cellular engineering [47]. A minimal genome, which contains the smallest set of genes allowing the organism to replicate in a given environment, is highlighted as the chassis genome [8].

In 1984, Morowitz [9] first proposed the idea of using mycoplasmas as models for the construction of a minimal genome in a living cell. Mycoplasmas are autonomously replicating parasites with the smallest genome [7] and have evolved by reductive evolution from Gram-positive bacteria [10]. Subsequently, Koob et al. [11] proposed a strategy to construct a minimal genome from the free-living micro-organism Escherichia coli. The E. coli genome contains approximately 4300 protein-coding genes with over 1000 predicted genes [12], whereas Mycoplasma genitalium has only ∼470 protein-coding genes in a 580-kb-long genome [13]. Although E. coli has a relatively large genome compared with mycoplasmas, it was first chosen for minimal genome construction experiments because of its easy genetic engineering, favourable growth characteristics and usage as an industrial host [12,14].

The aims of minimal genome construction include clarifying the definition of life and the construction of chassis genomes for industrial applications. The two main approaches for the construction of a minimal genome are the top-down approach (genome reduction), using free-living bacteria, and the bottom-up approach (genome synthesis), based on the genetic information of mycoplasmas. Regardless of the approach chosen, the creation and study of minimal microbial genomes can help to increase our understanding of the genetic complexity and provide a basis for the design of custom bacterial strains.

Information of the core genome

Functional analysis of the microbial genome has revealed that bacteria use only a fraction of their genes for growth and production of important biological compounds under a given set of conditions [15,16]. Identifying the essential protein-coding and non-coding DNA sequences for maintaining and replicating a free-living cellular organism is the logical first step for the construction of a minimal genome. Several approaches have been used to identify essential and non-essential genes in microorganisms under given conditions [14] (Table 1).

Table 1.
Number of estimated essential genes
Method Strain Number of essential genes/ total ORFs (%) Reference 
Systems biology M. genitalium and H. influenzae 256 [17
 COGs+NOGD 63 [19
Global transposition M. genitalium 265–350/468 (79%) [22
 M. mycoides 473/901 (52.5%)* [27
 H. influenzae RD 670/1703 (38%) [24
 E. coli K12 620/4296 (14%) [26
 P. aeruginosa 678/5500 (12%) [23
 H. pylori 255/1590 (16%) [95
 C. glutamicum658/2990 (22%) [25
Single-gene deletion or knockout S. cerevisiae 1105/5916 (19%) [96
 B. subtilis 168 271/4099 (6.8%) [28
 Salmonella enterica serovar Typhimurium LT2 490/4597 (11%) [97
 E. coli K12 303/4296 (7%) [29
Method Strain Number of essential genes/ total ORFs (%) Reference 
Systems biology M. genitalium and H. influenzae 256 [17
 COGs+NOGD 63 [19
Global transposition M. genitalium 265–350/468 (79%) [22
 M. mycoides 473/901 (52.5%)* [27
 H. influenzae RD 670/1703 (38%) [24
 E. coli K12 620/4296 (14%) [26
 P. aeruginosa 678/5500 (12%) [23
 H. pylori 255/1590 (16%) [95
 C. glutamicum658/2990 (22%) [25
Single-gene deletion or knockout S. cerevisiae 1105/5916 (19%) [96
 B. subtilis 168 271/4099 (6.8%) [28
 Salmonella enterica serovar Typhimurium LT2 490/4597 (11%) [97
 E. coli K12 303/4296 (7%) [29

COG, clusters of orthologous groups of proteins; NOGD, non-orthologous gene replacement.

*Includes 233 quasi-essential genes for robust cell growth.

Identification of essential genes in silico

Mushegian and Koonin [17] found that the 256 genes conserved between the 468 genes of Gram-positive M. genitalium and 1703 genes of Gram-negative Haemophilus influenzae are almost essential genes. Analysis of these genes suggested minimal functions necessary for growth and replication, such as protein translation, gene transcription, DNA replication, recombination and repair; chaperone-like proteins and machinery for protein export and metabolite transport; and nucleotide salvage pathways [17]. Welch et al. [18] compared genome sequences from the laboratory strain E. coli MG1655, uropathogenic E. coli CFT073 and enterohaemorrhagic DEL933 and found only 2996 genes common to all three strains. When such an analysis was carried out with ∼100 genomes, only 63 genes were conserved, most of which were related to the basic components of the central dogma of molecular biology [19]. The limitation of this sequence comparison for identifying a minimal gene set is that proteins with the same function do not necessarily share detectable sequence similarity [14]. Therefore, researchers have identified essential genes by other approaches because comparative genomics approaches might underestimate the size of the minimal gene set [20]. Recently, Yang et al. [21] defined a core proteome consisting of 356 gene products–44% of the E. coli mass-based proteome–using a systems biology-based genome-scale model of metabolism and expression. It includes 212 genes not found in previous comparative genomics approaches [21].

Identification of essential genes in vivo

For the identification of non-essential genes by large-scale gene inactivation, global transposition mutagenesis has also been used. The positions of 2209 transposon insertions in M. genitalium genomes were determined by sequencing across the junctions of transposons and genomic DNA. The analysis of these junctions suggested that 265–350 of the 470 protein-coding genes of M. genitalium are essential under laboratory conditions, including ∼100 genes of unknown function [22]. Global transposition mutagenesis in Pseudomonas aeruginosa, H. influenzae, Corynebacterium glutamicum and E. coli has identified 678, 670, 658 and 620 genes respectively as essential for growth under laboratory conditions [2326]. However, with global transposition, the essential gene set might be overestimated because of unsaturation of transposition into the genome. For saturation of transposon insertion, Hutchison et al. [27] isolated and sequenced 80000 transposons inserted into the Mycoplasma mycoides genome using next-generation sequencing. The analysis indicated 240 genes from 901 protein- and RNA-coding genes as essential.

With single-gene disruptions of the complete microbial genome, Kobayashi et al. [28] identified ∼270 genes of Bacillus subtilis as non-essential for growth in a rich medium at 37°C. Among 4288 of the E. coli genes targeted, Baba et al. [29] identified 303 genes, including 37 of unknown function, as being indispensable in Luria–Bertani (LB) medium culture. The functional groups of these 303 essential genes were divided into protein translation, ribosomal structure, cell division, lipid metabolism, transcription and cell envelope biogenesis. Only 205 of them (67%) overlapped with those in the essential gene set predicted by global transposition [26,29]. These differences might be attributed to the use of different mutagenesis strategies and growth conditions. However, because the global transposition system measures the effect of mutations on cell populations, a mutation that causes very slow growth can appear to be lethal and hence be falsely classified as essential. Hutchison et al. [27] tried to synthesize a minimal genome chemically with essential genes, but failed to construct a viable cell. They therefore added quasi-essential genes for robust cell growth for the construction of a minimal genome. Identifying essential genes does not imply easy and direct construction of a minimal genome. There is a gap between the identification of essential genes and the construction of a minimal genome because of epistasis, a genetic interaction in which the genotype of one gene results in a phenotype that masks the effect of another gene [3032]. One example of epistasis is the existence of ‘synthetic lethal’ [33] or ‘mutually essential’ genes [34]. Some deletions that are viable individually are not viable when combined with other deletions [35,36]. Moreover, the deletion of large non-essential genomic segments causes slow growth, instability and SOS system induction [37]. The unexpected cellular behaviours by genome deletion highlight the uncertainty in the construction of a minimal genome using the information of essential gene sets. However, the understanding of epistatic effects in genes could help to construct minimal genomes with more certainty [38,39].

Construction of a minimal genome

Two approaches for the construction of minimal genomes have been proposed [4042] (Figure 1). The top-down approach reduces the genome by deleting genomic segments selected randomly or identified as unnecessary. The bottom-up approach creates a minimal genome from synthetic oligonucleotides and inserts it into a cellular envelope that allows replication and metabolism after transplantation [4345].

Schematic diagram of the construction of a minimal chassis genome.

Figure 1
Schematic diagram of the construction of a minimal chassis genome.

Identification of essential gene sets under experimental conditions, genesis of a minimal genome by a top-down or bottom-up approach, evaluation and readjustment of the minimal genome.

Figure 1
Schematic diagram of the construction of a minimal chassis genome.

Identification of essential gene sets under experimental conditions, genesis of a minimal genome by a top-down or bottom-up approach, evaluation and readjustment of the minimal genome.

Top-down approach: genome reduction

The top-down approach is more feasible than the bottom-up approach because it can be performed with incomplete genetic information [1]. Since 2002, E. coli minimal genomes (5–30% smaller than the wild-type genome) have been constructed by various genomic DNA deletion techniques such as Cre/loxP site-specific recombination, DSB-mediated repair, and homologous recombination using CSMs. The genomes of B. subtilis, C. glutamicum, Streptomyces and yeasts have also been reduced for the construction of minimal-genome factories [46].

Using dual transposition with a Tn5 derivative, Goryshin et al. [47] developed a method for random deletion of genomic segments, which can be applied to simultaneous gene essentiality identification and minimal genome construction. They reduced 5.6% (262 kb) of the E. coli genome by 20 repeated deletions. Yu et al. [35] reduced the E. coli genome using the transposon-coupled Cre/loxP excision system [35]. By repeated Cre/loxP-mediated excision and P1 transduction, they produced an E. coli mutant (CDΔ3456) with four large deletions (totalling 313 kb, 287 genes) in the genome. These systems have a drawback of remnant scars in the genome after the deletion of genomic segments. Therefore the selectable marker or remaining scar (loxP or FRT site) needs to be eliminated from the deletion mutants. To construct minimal genomes without heterologous DNA sequences, E. coli genomes have also been reduced by sequential large deletions using a combination of λ Red-mediated and DSB-stimulated recombination. To construct a stable E. coli genome, genomic regions containing unnecessary functions, such as K-islands (genomic segments in K-12 only) and mobile DNA elements, including ISs, prophages, transposases, integrases and site-specific recombinases, were deleted [48]. These deletions were serially introduced into a single strain by P1 transduction, resulting in the generation of genome-reduced E. coli strains MDS12, MDS42 and MDS43, which lack 8.1%, 14.3% and 15.3% of the genome respectively [49]. Another E. coli strain, Δ16, with a highly reduced genome (29.7% reduction) was constructed [50] by deleting each target region by two serial λ Red-mediated recombinations, followed by accumulation in a single genome by serial P1 transductions. Phenotypic analysis revealed that Δ16 has slower growth and longer cells compared with the parental cell. Mizoguchi et al. [51] reconstructed a novel minimal genome strain, MGF-01, by sequentially juxtaposing each deletion target region by a P1 transposon. During the accumulation of the deleted region into a single genome, growth-defective strains were excluded.

Minimal genomes in bacteria other than E. coli have also been constructed. In B. subtilis, a model organism for the production of useful enzymes [52], the genome-reduced strain Δ6, having a 7.7% (0.53 Mb) reduction in the genome, has been constructed using a suicide plasmid−based chromosomal integration-excision system from wild-type B. subtilis 168 [53]. Ara et al. [54] deleted all prophage and prophage-like sequences from the B. subtilis genome to yield an MG1M strain that lacked 0.99 Mb of the wild-type genome. However, Δ6 cells show no phenotypically unique properties. Thus another B. subtilis minimal genome strain, MGB874 (20.7% genome reduction), was constructed by sequential deletions of 28 regions, single deletions of which did not affect cell growth [55]. Reduced genomes (lacking 190 kb of the wild-type genome) of C. glutamicum, an industrially important producer of amino acids and organic acids, were generated by a modified Cre/loxP recombination system [5659]. Tsuge et al. [60] generated C. glutamicum mutant R (11.9% genome reduction) by combining transposition and Cre/loxP excision. These genome-reduced C. glutamicum strains exhibited normal growth under standard laboratory conditions. In the fission yeast Schizosaccharomyces pombe, more than 500 kb from the genome was deleted by repeated deletion of a large genomic region using linear DNA homologous recombination [61]; this mutant was dedicated to heterologous protein production [62]. Murakami et al. [63] constructed a reduced genome of Saccharomyces cerevisiae by PCR-mediated chromosome splitting. This mutant lost ∼5% (531.5 kb) of the wild-type genome [64]. In the mutant SUKA 17 of the antibiotic- and secondary metabolite-producer Streptomyces avermitilis, 18.5% of the genome (1.67 Mb) was reduced at sub-telomeric regions by the Cre-mediated large-deletion method [65].

Although it has not yet been used for the construction of reduced genomes, the recently developed CRISPR/Cas-mediated recombination system [66,67] might be used for the construction of minimal genomes as a next-generation genome engineering toolkit.

Bottom-up approach: genome synthesis

Construction of an artificial minimal genome from synthetic oligonucleotides (the bottom-up approach) is difficult because it requires the complete information of the minimal gene set of the cell and it is hard to assemble a large chromosome without errors and introduce it into the recipient cells. Before 2008, genome reduction was the main technique for minimal genome construction. However, the development of gene synthesis and assembly technologies such as Gibson assembly, golden gate assembly and DNA assembler [68]–invented during rewriting poliovirus cDNA [69], bacteriophages [70], 1918 Spanish influenza pandemic virus [71], B. subtilis genome vector [72], M. genitalium [73,74], M. mycoides [75], mouse mitochondrial genome [76] and yeast chromosome [77]–enabled minimal genome synthesis and genome modification from scratch. Recently, the minimal genome JCVI-syn3.0, containing only the genes essential for life, was designed and constructed by chemical synthesis [27] from the ∼1-Mb genome of M. mycoides, a parasite found in cattle. First, saturated global transposition was performed to identify essential genes. The collected essential gene information was used to construct a minimal genome, but it failed to produce a viable cell. The researchers revealed the importance of a class of quasi-essential genes described previously as persistent genes [78,79]; these are otherwise non-essential genes but are needed for robust growth. Through repeated redesigning, building and testing, they produced a viable minimal genome JCVI-syn3.0 (531 kb, 473 genes, including 149 genes with unknown function). The putative functions of some of the 149 unknown genes were identified recently [80], which will certainly benefit the understanding the minimal genome. For the bottom-up approach, the chemical transplantation of the constructed genome into a recipient cell is important. There is therefore a need for a cell that is compatible with the chromosomal DNA generated to build up genomes [81,82].

In addition, the Sc2.0 project aims to generate the synthetic yeast genome [77,83]; it is, therefore, not a minimal genome construction project. For the synthesis of the complete genome of S. cerevisiae with some modification, symmetric loxP sites were integrated into the genome for genome structure and evolution studies. Additionally, although it is not a chemical genome construction, all amber stop codon TAGs in the genome were replaced with TAA to free up a codon, as reported in an E. coli codon expansion study [84,85]. The study can be applied to the minimal genome for incorporating non-canonical amino acids for the development of novel proteins with enhanced activity. Recently, E. coli with only 57 codons was synthesized and tested as 87 genomic fragments [86]. The fragments were based on genome-reduced E. coli MDS42 to minimize cost of genome synthesis. Although 87 segments had not been assembled into a full genome, the research highlights the advantages of minimal genome for industrial applications.

Applications of the minimal genome

To construct the minimal E. coli genome for industrial applications, Mizoguchi et al. [87] targeted regions that were not expected to affect the growth or primary metabolism of the bacteria by deletion. Approximately 100 regions of the E. coli genome (total size, 1.8 Mb) were deleted independently using a scarless deletion system [51], and the individual deletions were transferred to a single chromosome by P1 transduction. Throughout the genome size-reduction process, the growth properties of each intermediate strain were analysed, and only the strains displaying no growth deficiency were selected for subsequent deletions. The final genome-reduced strain, MGF-01, displayed a 1.5-fold higher final cell density than that of the wild-type strain, without any change in doubling times. Its threonine production was twice that of the wild-type parental strain when the genetic circuit for threonine production was integrated into the chromosomes. Another group reported over 1.8-fold increased amino acid production in the reduced genome [88]. Relative to the parent strain MG1655, deletion mutants of IS-free MDS42 [49] and MS56 [89] display increased genome stability and transformation efficiency two orders of magnitude higher than that of the wild-type. Although many genome-reduced strains have shown no significant difference from wild-type strains [35,62], B. subtilis minimal genome strain MGB874 produced 1.7- and 2.5-fold higher cellulase and protease respectively, compared with the wild-type parental strains [55]. A S. cerevisiae mutant displayed increased ethanol (1.8-fold) and glycerol (2-fold) production [64], and the engineered Streptomyces strain SUKA17 showed increased productivity of streptomycin and cephamycin C [1,65] (Table 2).

Table 2.
Characteristics of minimal genomes constructed
Strain Deletion size Characteristics relative to the wild-type Reference 
E. coli MS56 1.06 Mb (22.2%) Increased genome stability and transformation efficiency [89
E. coli MGF-01 1.03 Mb (22%) Increased yield (2-fold) by insertion of threonine production cassette [87
B. subtilis MGB874 873.5 kb (20.7%) Increased extracellular cellulase (1.7-fold) and protease (2.5-fold) production [55
S. avermitilis SUKA17 1.67 Mb (18.5%) Increased antibiotic production [45
S. cerevisiae MFY1160 531.5 kb (5%) Increased ethanol (1.8-fold) and glycerol (2-fold) production [64
JCVI-syn3.0 548 kb (50.8%) Synthesized chemically, minimized genome [27
Strain Deletion size Characteristics relative to the wild-type Reference 
E. coli MS56 1.06 Mb (22.2%) Increased genome stability and transformation efficiency [89
E. coli MGF-01 1.03 Mb (22%) Increased yield (2-fold) by insertion of threonine production cassette [87
B. subtilis MGB874 873.5 kb (20.7%) Increased extracellular cellulase (1.7-fold) and protease (2.5-fold) production [55
S. avermitilis SUKA17 1.67 Mb (18.5%) Increased antibiotic production [45
S. cerevisiae MFY1160 531.5 kb (5%) Increased ethanol (1.8-fold) and glycerol (2-fold) production [64
JCVI-syn3.0 548 kb (50.8%) Synthesized chemically, minimized genome [27

The minimal genome can be used for the construction of a platform genome for synthetic biology, wherein cellular metabolites and energy sources are efficiently optimized and directed towards the production of desired gene products [90]. Furthermore, metabolic waste can be minimized, and the quality and stability of its products can be maximized in chassis genomes [41,48,52]. The minimal genome can also be a platform for investigating the core functions for sustaining life and for exploring whole-genome design [27]. The minimal genome information can also provide a valuable reference for astrobiology research and efforts related to the examination of samples brought back from planetary satellites (http:://www.nap.edu/read/9638).

Conclusions and future perspectives

To construct hosts for industrial applications and create minimal genomes to understand the essential components of life, researchers have attempted to identify essential gene sets, developed large genomic segment editing techniques and generated minimal genomes by reducing the existing bacterial genomes and chemical synthesis. Some of the minimal genomes constructed have shown improved genome stability or increased production of industrial products, or both, when compared with the wild-type. In the last 20 years, basic technologies for the construction of minimal genomes have been developed, and, in future, researchers will construct ideal robust hosts using the minimal genome for scientific and industrial purposes. Minimal genome research might provide us insights into the study of genome evolution, better understanding of the genomes of more complex modern organisms, reconstruction of metabolic pathways and custom-designed microbes with fewer waste products, etc. The reduced genomes of free-living bacteria might be reduced further by deletion of non-essential genomic segments or non-essential genes identified by gene-knockout experiments to construct minimized genomes that can be used as chassis genomes. For this research, real non-essential regions–those not affecting cellular growth, because robust growth of reduced chassis genomes is important for industrial application–may be identified using a system for random deletion of large genomic regions in specific culture conditions, e.g. multiple transposition system [47,91]. Using synthetic biology techniques, we can easily design genetic circuits by computational automation [92], low-cost DNA synthesis [93] and the integration of this DNA into the minimal genome. This will speed up the construction of customized industrial genomes for bioremediation of environmental toxins, production of useful pharmaceuticals and chemicals [6,94], or the creation of renewable energy sources (Figure 2).

Applications of a minimal chassis genome.

Figure 2
Applications of a minimal chassis genome.

Minimal chassis genome constructed by the deletion of non-essential genes will provide insights for scientific and industrial purposes such as bacterial evolution and custom-designed microbes. Robust industrial genomes produced by the integration of functional modules of interest will be used for bioremediation of environmental toxins, production of useful pharmaceuticals and chemicals or the creation of renewable energy sources.

Figure 2
Applications of a minimal chassis genome.

Minimal chassis genome constructed by the deletion of non-essential genes will provide insights for scientific and industrial purposes such as bacterial evolution and custom-designed microbes. Robust industrial genomes produced by the integration of functional modules of interest will be used for bioremediation of environmental toxins, production of useful pharmaceuticals and chemicals or the creation of renewable energy sources.

Summary

  • Essential genes for sustaining life have been identified from commonly conserved sequences of various genomes and global transposition and single-gene knock out approaches.

  • For the construction of minimal genomes by the top-down approach, selected or random target regions are deleted by large-scale genome editing techniques and cumulative single-gene deletion.

  • Recent advances in DNA synthesis and assembly methods enable us to synthesize a designed genome. A minimal genome of Mycoplasma has been chemically synthesized with essential and quasi-essential genes for robust growth.

  • Minimal genomes can be used as chassis genomes for constructing custom-designed genomes for producing value-added biochemicals. The synthetic genome can be created by the integration of functional modules designed automatically in silico.

Abbreviations

     
  • Cas

    CRISPR-associated

  •  
  • CRISPR

    clustered regularly interspaced short palindromic repeats

  •  
  • CSM

    counter-selection marker

  •  
  • DSB

    double-strand break

  •  
  • IS

    insertion sequence

Funding

This work was supported by the Intelligent Synthetic Biology Center of Global Frontier Project [2011-0031957 to B.-K.C. and 2015M3A6A8065831 to B.H.S.] through the National Research Foundation (NRF) of Korea funded by the Ministry of Science, ICT and Future Planning.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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