What is considered a potential use for genetically engineered members of the genus Deinococcus?

Overview

Deinococcus radiodurans has been recorded as the world's toughest known bacterium in The Guinness Book of World Records. D. radiodurans can survive a multitude if extreme conditions, including severe droughts, lack of nutrients, as well as withstand one thousand times more radiation than humans can. As far as we can understand, D. radiodurans can survive these extreme conditions due to its ability to repair damaged DNA, as quick as a few hours, with absolute fidelity. The hardiness of D. radiodurans also makes it a very useful in bioremediation processes to detoxify toluene and mercury, both commonly found in radioactive waste sites.[2] 

History

The first strain of D. radiodurans, D. radiodurans R1 was isolated in Oregon in 1956 from canned meat that had spoiled from x-ray radiation. The culture of the bacteria produced a red, non-sporulating, gram-positive cocci. The culture showed extreme resistance to a variety of normally harmful conditions such as ionizing radiation, UV light, and hydrogen peroxide, all conditions known to damage DNA. D. radiodurans also showed a high resistance to desiccation, being able to endure conditions with practically no moisture. D. radiodurans was originally classified as a member of the micrococcus genus, but after analyzing the rRNA, it was placed in its own genus, Deinococcus.[5] 

DNA Repair

D. radiodurans' ability to efficiently repair its own DNA with a high rate of fidelity enables the bacteria to survive such unfavorable conditions. One of the advantages of D. radiodurans is that is has four copies of its genome while it is in a stationary phase, and 8-10 copies while DNA multiplication is occurring.[6] D. radiodurans performs repairs to its' genome in two main steps. The chromosome fragments are reconnected by single strand annealing, and then multiple proteins, including RecA, repair double strand breakage via homologous recombination.[4] In order to express the resistance phenotypes of D. radiodurans, the bacterium requires a functional RecA protein. The RecA protein traditionally binds to RecB and RecC proteins, that are a part of the RecBCD enzymes found in Escherechia coli for recombinational processes, but D. radiodurans' genome doesn't include genes for proteins RecB and RecC. In D. radiodurans, RecA is normally expressed at relatively low levels, until it suffers heavy DNA damage.[3]  

Applications

Thanks to D. radiodurans' extremophile properties, it is a great candidate for treating toxic chemical waste. D. radioduran is genetically engineered to decompose heavy metals and toxic solvents in radioactive environments. One example is the mercuric reductase gene from E. coli is inserted in to the genome of D. radioduran to detoxify mercury produced from the manufacturing of nuclear weapons.[1] A strain of D. radiodurans has also been developed to detoxify both mercury and toluene.[7]

References

1. Brim H, McFarlan SC, Fredrickson JK, Minton KW, Zhai M, Wackett LP, Daly MJ (2000). "Engineering Deinococcus radiodurans for metal remediation in radioactive mixed waste environments" (PDF). Nature Biotechnology. 18 (1): 85–90. doi:10.1038/71986. PMID 10625398. S2CID 28531.
2. DeWeerdt, S. E. (2002, July 05). The world?s toughest bacterium. Retrieved March 03, 2021, from //www.genomenewsnetwork.org/articles/07_02/deinococcus.shtml
3. Kim, J. I., & Cox, M. M. (2002). The RecA proteins of Deinococcus radiodurans and Escherichia coli promote DNA strand exchange via inverse pathways. Proceedings of the National Academy of Sciences of the United States of America, 99(12), 7917–7921. //doi.org/10.1073/pnas.122218499
   

4. Levin-Zaidman, S., Englander, J., Shimoni, E., Sharma, A., Minton, K., & Minsky, A. (2003, January). Ringlike structure of THE Deinococcus RADIODURANS Genome: A key TO RADIORESISTANCE? Retrieved March 04, 2021, from //ui.adsabs.harvard.edu/abs/2003Sci...299..254L/abstract

5. Makarova, K. S., Aravind, L., Wolf, Y. I., Tatusov, R. L., Minton, K. W., Koonin, E. V., & Daly, M. J. (2001). Genome of the extremely radiation-resistant bacterium Deinococcus radiodurans viewed from the perspective of comparative genomics. Microbiology and molecular biology reviews : MMBR, 65(1), 44–79. //doi.org/10.1128/MMBR.65.1.44-79.2001
6. Moseley, B. E., & Mattingly, A. (1971). Repair of irradiation transforming deoxyribonucleic acid in wild type and a radiation-sensitive mutant of Micrococcus radiodurans. Journal of bacteriology, 105(3), 976–983. //doi.org/10.1128/JB.105.3.976-983.1971
7. Appukuttan, D., Rao, A. S., & Apte, S. K. (2006). Engineering of Deinococcus radiodurans R1 for bioprecipitation of uranium from dilute nuclear waste. Applied and environmental microbiology, 72(12), 7873–7878. //doi.org/10.1128/AEM.01362-06

Deinococcus spp are among the most radiation-resistant micro-organisms that have been discovered. They show remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation and oxidizing agents. Traditionally, Escherichia coli and Saccharomyces cerevisiae have been the two platforms of choice for engineering micro-organisms for biotechnological applications, because they are well understood and easy to work with. However, in recent years, researchers have begun using Deinococcus spp in biotechnologies and bioremediation due to their specific ability to grow and express novel engineered functions. More recently, the sequencing of several Deinococcus spp and comparative genomic analysis have provided new insight into the potential of this genus. Features such as the accumulation of genes encoding cell cleaning systems that eliminate organic and inorganic cell toxic components are widespread among Deinococcus spp. Other features such as the ability to degrade and metabolize sugars and polymeric sugars make Deinococcus spp. an attractive alternative for use in industrial biotechnology.

Keywords: biochemical engineering, biotechnology, Deinococcus, extremophile, fermentation biotechnology

Deinococcus, originally identified as Micrococcus, are coccoid or rod-shaped nonsporulating bacteria known for their resistance to multiple stresses and their capacity to repair DNA damage with unparalleled efficiency compared to other known bacterial species. Deinococcus radiodurans strain R1 was the first discovered deinobacteria and was isolated in 1956 (Anderson et al. 1956) from X ray-irradiated canned meat. More than 60 different species have been isolated from very diverse environments, such as air dust (Weon et al. 2007; Yang et al. 2009, 2010) and air sample (Yoo et al. 2010), activated sludge (Im et al. 2008), desert soils (De Groot et al. 2005; Rainey et al. 2005), arsenic polluted water (Suresh et al. 2004), cold environments in Antarctica (Hirsch et al. 2004), hot springs or biofilms at the surface of paper machines (Ferreira et al. 1997; Kolari et al. 2002), radioactive sites (Siebert and Hirsch 1988) faeces (Brooks and Murray 1981), gut of a wood-feeding termite (Chen et al. 2012) and Phoenix spacecraft surface (Stepanov et al. 2014). These species grow at temperatures ranging from 4 to 55°C and have been regrouped in a distinct (Rainey et al. 2005) eubacterial phylogenetic lineage related to the Thermus genus. The closest Deinococcus relatives are Trueperaceae, Thermales (Marinithermus, Thermus, Oceanithermus and Vulcanithermus); no members of this genus have been implicated as pathogens.

Robust cells that are able to assimilate all of the most common sugars and synthesize the biological macromolecules necessary for growth from cheap carbon sources without auxotrophy are of considerable interest for biotechnological applications. Indeed, several Deinococcus species meet this requirement because they are capable of growing on minimal media and metabolizing multiple sugars. Furthermore, the well-known robustness of this bacterium and its physiological specificities makes it an attractive chassis for future biotechnological applications. However, many bacteria with attractive properties are recalcitrant to genetic manipulation because of the lack of plasmids and shuttle vectors, the scarcity of selection markers, and low transformability or narrow host range. Approximately, 900 publications referenced in Medline describe the ability of D. radiodurans R1 to resist radiation and to overcome oxidative stress. Most of the genetic tools available for Deinococcus originate or were developed for this strain, and it can be easily engineered. The second most well-studied species is Deinococcus geothermalis, with approx. 50 publications referenced in Medline, but there is only one report describing the transformation of this strain with an autonomous plasmid originally constructed for D. radiodurans (Brim et al. 2003). In comparison, 300 000 publications mention Escherichia coli, and 100 000 publications discuss Saccharomyces cerevisiae. The focus on the resistance of Deinococcus to radiation has somewhat masked the potential interest in the industrial applications of this genus. In this review, we summarize the physiological and genomic properties of Deinococcus, the tools available to engineer the different species and the most recent applications.

The sequencing of the Deinococcus genome has provided useful information regarding the biology, physiology, biological diversity and biotechnological potential of these bacteria. However, many of these areas have not yet been studied in detail.

Deinococcus radiodurans R1 contains 3195 genes and has a high GC content (67%; similar to Thermus thermophilus) and a 3·25 Mb genome (two chromosomes of 2·6 and 0·4 Mb, a megaplasmid of 0·17 Mb, and a small plasmid of 45 kb) (White et al. 1999). Deinococcus geothermalis (Makarova et al. 2007) and Deinococcus grandis have approximately the same genome size, while the genome of Deinococcus gobiensis (Yuan et al. 2012) is 4·4 Mb and that of Deinococcus hopiensis is 6·69 Mb. Deinococcus proteolyticus (Copeland et al. 2012) has the smallest published genome (2·84 Mb). This suggests a strong interspecies genome diversity (Fig.​1) and genomic plasticity compared to Escherichia (genome size 4·5–5·8 Mb). This observation was confirmed experimentally via the duplication of a selection marker flanked by a direct repeat of host DNA sequences in D. radiodurans (Smith et al. 1988). After amplification by antibiotic selection, approx. 50 copies per cell had accumulated (10% of the genome). This experiment demonstrated the plasticity of D. radiodurans and its ability to replicate and express extremely large foreign DNA pieces. Makarova estimated that 10–15% of the D. radiodurans genome originated from horizontal transfer, and most of these genes are located on the megaplasmid (Makarova et al. 2001).

A genomic comparison of Deinococcus proteolitycus (2·84 Mb), Deinococcus geothermalis (3·2472 Mb) and Deinococcus gobiensis genomes (4·41 Mb). Putative orthologous genes were determined by reciprocal best-hits BLAST with an e-value cut-off of 10e−3. Most of the core genome is conserved and located on the chromosome, but the plasmids are expectedly more divergent.

Deinococcus is polyploid and contains four copies of the genome in stationary phase and 10 copies during the exponential phase (Hansen 1978). This can contribute to the well-known genome stability of Deinococcus. However, Thermus, one of its closest phylogenic relatives, is also polyploid and is not resistant to UV or radiation, suggesting that genome stability is most likely multifactorial (Ohtani et al. 2010). The polyploidy affects genetic engineering strategies because homogenous chromosomal modification must be achieved.

Computational analysis revealed that the D. radiodurans genome possesses mobile genetic elements, including a large number of insertion sequences (ISs) elements (Makarova et al. 2001). The first IS in D. radiodurans may be related to the IS4 family of E. coli and was discovered within the uvrA gene of the mtcB mutant 2621 (Narumi et al. 1997). The IS8301 element (now named ISDra2) was later found in the pprI gene (Hua et al. 2003). Pasternak et al. 2010 showed that the frequency of ISDra2 transposition is increased 50 to 60-fold following UV or γ-radiation because both processes generate large quantities of single-stranded DNA. IS transposition appears to be a major event in spontaneous and induced mutagenesis in D. radiodurans (Mennecier et al. 2006).

Deinococcus radiodurans exhibits unparalleled resistance to oxidative stress (Slade and Radman 2011; Misra et al. 2013). Oxidative stress can be caused by reactive oxygen species produced by respiration, exposure to irradiation or chemical agents. Deinococcus radiodurans can survive high doses of ionizing radiation (up to 2000 double-stranded breaks per bacteria) without considerable irreversible DNA or protein damage, and can reassemble its chromosome with a high fidelity. The resistance of this bacterium to these stresses is due to multiple converging mechanisms.

Two different pathways for the assimilation of glucose, the pentose phosphate and glycolysis, are present and active in Deinococcus. Liedert et al. (2012) proposed that D. geothermalis grows in aerobic conditions by preferentially channelling glucose to the pentose phosphate pathway generally involved in the regeneration of NADPH rather than NADH, in agreement with our observations on exponentially growing D. geothermalis (Leonetti, J.-P., unpublished results). The pentose phosphate pathway and the generated NADPH are directly involved in repair mechanisms and resistance to oxidative stress (Juhnke et al. 1996; Rui et al. 2010). The under-utilization of glycolysis can be a mean to limit NADH formation and to avoid the generation of reactive oxygen species during its recycling by the respiratory chain. This trait is a corolar of the resistance of Deinococcus to irradiation and is of significant importance for biotechnology as NADPH is used by several anabolic pathways, such as amino acids synthesis (e.g. arginine, proline, isoleucine, methionine and lysine), vitamin synthesis (e.g. pantothenate, phylloquinone, and tocopherol), polyol synthesis (e.g. xylitol), isoprenoid synthesis and fatty acid synthesis. In addition, NADPH is the source of reducing equivalents for cytochrome P450 hydroxylation of aromatic compounds, steroids, alcohols and drugs. The most commonly used micro-organisms in bioproduction, such as E. coli and S. cerevisiae, produce primarily NADH and Deinococcus could be used as a preferential host to build NADPH-consuming pathways. However, reliable metabolic models of Deinococcus are still missing in the literature, which makes metabolic engineering more time consuming and labour intensive like for most of the new alternative chassis. Furthermore Deinococcus has never been fermented yet at a scale compatible with an industrial application.

Although Deinococcus is a nonsporulating bacteria, it is extremely resistant to desiccation. Mattimore and Battista (Mattimore and Battista 1996) have shown that D. radiodurans R1 can survive 6 weeks of desiccation, while the viability of E. coli was reduced by six logs after only 7 days. It is believed that resistance to ionizing radiation and desiccation are closely linked evolutionary processes because desiccation resistance appears to require extensive DNA repair (Mattimore and Battista 1996). Deinococcus possesses a pathway for the synthesis of trehalose from malto-oligosaccharides (Timmins et al. 2005) and can produce trehalose (unpublished results), a common osmolyte. The protective role of this osmolyte in osmotic stress, heat shock and dehydration has been well-characterized in other species (Purvis et al. 2005; Yu et al. 2012). Surviving desiccation is a major issue for the production of starter cultures for industrial applications (Hofman and Thonart 2010).

The ability of cells to tolerate stress is often limiting in industrial environments and is multifactorial. Wang & et al. (Wang et al. 2012, 2013) found that the transcription regulator irrE from D. radiodurans, when transformed into E. coli, enhanced tolerance to ethanol, butanol and acetate (10–100-fold), and conferred significant cross-tolerance to two other common inhibitors found in lignocellulosic hydrolysates, 5-hydroxymethyl-2-furaldehyde and vanillin. The irrE gene encodes a general switch transcription factor that is responsible for the extreme radioresistance of D. radiodurans (Earl et al. 2002).

Deinococcus often forms tetrads (Fig.​2a,​c). Most bacteria can be easily classified as Gram-positive or Gram-negative due to the presence or absence, respectively, of a thick cell wall (Fig.​2d) of peptidoglycan that sequesters the stain after treatment. The cell envelope of Deinococcus differs depending on the species and exhibits an unusual structure and composition. Deinococcus indicus, D. grandis and D. deserti strain YIM 007T stain Gram-negative, whereas a majority of the other Deinococcus species stain Gram-positive. The Gram-positive coloration of many Deinococcus is reminiscent of the thick peptidoglycan layers of Gram-negative bacteria that are difficult to decolorize.

Deinococcus geothermalis often forms tetrades (a, c) and presents an unusual thick cell envelope (c, d). Scanning electron microscopy (SEM) images of D. geothermalis planktonic cells (a) and cells in contact with Whatman paper (b) Transmission Electron Microscopy (TEM) images of D. geothermalis (c, d).

As reviewed by others (Makarova et al. 2001), the following six layers of the cell envelope have been identified by electron microscopy: (i) the most internal layer is the plasma membrane common to all cells and is composed of unusual lipids including alkylamine chains. (ii) it is followed by a perforated peptidoglycan cell wall. (iii) the third layer is unique and may be composed of a matrix of tiny uncharacterized compartments. (iv) the fourth layer is another plasma membrane and is the outer membrane. (v) the fifth layer is a distinct electrolucent zone. (vi) the sixth layer consists of regularly packed hexagonal protein subunits (the S-layer, or hexagonally packed intermediate layer), typical of other bacterial S-layers. Several strains of Deinococcus are surrounded by a dense carbohydrate coat.

Whether this thick unusual structure is directly involved in resistance to xenobiotics is unknown, but D. geothermalis strain T27 has been reported to survive in the presence of high concentrations of ethyl acetate, toluene and diethylphtalate provided as a nonaqueous layer to a cell suspension (Kongpol et al. 2008).

Deinococcus geothermalis forms biofilms (Fig.​2b). This species is frequently found on paper machines (Väisänen et al. 1998; Kolari et al. 2001), and it is very difficult to remove from steel surfaces. Deinococcus geothermalis E50051 was reported to persist after 1 h of washing with 0·2% NaOH or 0·5% sodium dodecyl sulphate, in contrast to other biofilm formers, such as Burkholderia cepacia F28L1, Staphylococcus epidermidis O-47 and D. radiodurans strain DSM 20539. The attachment of D. geothermalis involves thread-like structures that interact with the machinery surface (Saarimaa et al. 2006). Pili-mediated adhesion is common to multiple Gram-negative and Gram-positive pathogenic bacteria. Saarimaa et al. characterized the adhesion threads of D. geothermalis as glycosylated type IV pili that are closely related to the type II protein secretion system (Sandkvist 2001; Peabody et al. 2003). Type IV pili genes homologous to putative pil genes in D. radiodurans have also been implicated in the natural competence of T. thermophilus HB27, a close relative of Deinococcus, and in the secretion of diverse extracellular enzymes (Friedrich et al. 2002).

Bacterial biofilms can impair or enhance industrial processes. Very often biofilms are considered a defect because they can clog tubing and impair fermentation processes. However, biofilm formation can be advantageous as it can be used to increase the biomass density in a reactor because it is not dependent on cell recycling, there is no need for re-inoculation in repeated-batch fermentation, and it can prevent ‘wash out’ when using continuous processes at a high dilution rate. Furthermore, biofilms are often more resistant to extreme conditions. Biofilm formation is not inherent to the Deinococcus family or even to D. geothermalis.

An extensive genetic toolbox exists for some but not all members of the Deinococcus clade. This toolbox provides a solid technological basis for the rapid construction of genetic variants, underlying future biotechnological applications.

Genetic engineering of bacteria requires the transfer of circular or linear DNA into the intended host. The first evidence that Deinococcus was able to take up exogenous DNA was obtained in 1968 by Moseley and Setlow (Moseley and Setlow 1968). The authors co-incubated DNA conferring resistance to an antibiotic with D. radiodurans cells sensitive to the antibiotic and obtained antibiotic-resistant clones. In addition to D. radiodurans, it was later shown that D. geothermalis strain DSM11300 (Brim et al. 2003) can be transformed by a CaCl2-dependent technique at a frequency of 5 × 102 transformant per μg of DNA, and D. grandis strain ATCC43672 (Satoh et al. 2009) can be transformed by electroporation with heterologous DNA at a frequency of 1·4 × 104 transformants per μg of DNA. In our hands, chemical transformation is the most efficient procedure for transforming D. radiodurans R1 and D. geothermalis DSM11300 (unpublished results). The mechanism underlying DNA uptake remains poorly understood. The following four basic mechanisms can be used in DNA translocation: (i) natural transformation, (ii) transformation using chemical competent cells, (iii) electroporation and (iv) conjugation.

Although more than 60 Deinococcus species are presently known, most of the genetic engineering tools available are devoted to D. radiodurans (Table​1). Two large (>40 kb) shuttle vectors, pS28 and pS19 (Smith et al. 1989), built from E. coli vector pS27 and the full-length D. radiodurans stain SARK cryptic plasmids pUE10 (37 kb) and pUE11 (45 kb) have been shown to replicate in E. coli and D. radiodurans (Smith et al. 1989). Three smaller derivatives of the historical vector pUE10 have been constructed, including the 16-kb plasmid pI3 (Masters and Minton 1992), the 27-kb plasmid pMD66 (Daly et al. 1994), and pRAD1, a 6·3-kb plasmid. These vectors have been extensively used to engineer D. radiodurans (Meima and Lidstrom 2000).

An overview of the most frequently used plasmids for the transformation of Deinococcus

The plasmid pMD66 is also able to replicate into D. geothermalis (Daly et al. 1994). It was used to engineer this bacterium for use in the bioremediation of high-temperature radioactive waste environments. Deinococcus geothermalis transformation with this plasmid is 1000-fold less efficient than the D. radiodurans one (8 × 105 and 5 × 102 transformants per μg of DNA, respectively).

Versatile shuttle vectors for D. grandis have also been constructed (Satoh et al. 2009). The plasmid pZT23 can transform D. grandis at an efficiency of approx. 6·6 × 103 transformants per μg of DNA, and pZT27 and pZT29 can transform this species at efficiencies of approx. 1·1 × 104–1·4 × 104 transformants per μg of DNA respectively.

The available vectors are, however, quite large and rather species-specific. The development of new, more versatile vectors with a broader host range would be useful tools for the genetic manipulation of other Deinococcus spp.

Selection markers can confer resistance to antibiotics, antimicrobials, or heavy metals or can be metabolic markers. They are used for the selection of plasmid- or insert-containing clones from a large bacterial population. In practice, antibiotic and metabolic markers are frequently used by molecular biologists. Antibiotic resistance cassettes are often derived from broad-spectrum resistance elements and are generally active in many bacterial species. Kanamycin cassettes derived from Tn9, chloramphenicol cassettes derived from Tn9O3, and tetracycline, hygromycin (Harris et al. 2004) and spectinomycin (Bouthier de la Tour et al. 2009) resistance genes can be used to select transformants or recombinant D. radiodurans or D. geothermalis (Smith et al. 1988; Pasternak et al. 2010). The following antibiotic selection markers can also be used on D. geothermalis in our laboratory: hygromycin, oxytetracycline (but not tetracycline), bleocine, kanamycin, cloramphenicol and erythromycin (Gerber, E., unpublished results). More recently, an a-amylase-containing vector was transformed into a D. geothermalis strain lacking its endogenous α-amylase, and the transformant were selected on agar plates containing starch (unpublished results).

In contrast to selection markers, counter-selection markers serve to eliminate unwanted genetic elements such as DNA segments encoding selection markers. Counter-selection can be achieved by exploiting unique metabolic properties present in one bacterium but not another. For example, Bacillus subtilis can express the gene sacB, a levansucrase that catalyses the synthesis of high molecular weight fructose polymers in the presence of sucrose; however, the expression of the same gene in E. coli (Gay et al. 1985) is toxic and favours the elimination of sacB from the strain. Taking advantage of this property, sacB has been used in many different Gram-negative bacteria as a counter-selection marker, thus bypassing the need for an antibiotic resistance marker. This type of selection was used successfully in D. radiodurans (Pasternak et al. 2010) but produces partial phenotypes that are difficult to select in D. geothermalis (unpublished results).

Genetic engineering often necessitates the generation of chromosomal modifications. Chromosomal mutants can either be generated randomly or targeted at specific site and can involve insertions, deletions or the modification of the native DNA sequence. Chromosomal mutations make it possible to stably express genes without maintaining a plasmid in the cell. However, insertions or deletions at specific loci require detailed knowledge of the targeted genome and of the contribution of each locus to strain fitness.

The first Deinococcus chromosomal mutant was constructed by homologous recombination (Smith et al. 1988) in D. radiodurans. Homologous recombination occurs when nonreplicative plasmids encoding an antibiotic resistance marker flanked by homology regions are integrated into the bacterial chromosome. The first report demonstrating that duplication insertion can be used to create insertional mutations in D. radiodurans was published in 1999 (Markillie et al. 1999). The same technique was used to engineer D. radiodurans for organopolluant degradation by cloning the todC1C2BA operon from Pseudomonas putida F1 (Lange et al. 1998) and for metal remediation by inserting the merA gene from E. coli strain BL308 into the D. radiodurans chromosome (Brim et al. 2000). Chromosomal replacement was first used in 2000 (Meima and Lidstrom 2000) by replacing the α-amylase gene with a gene encoding 1,4-α-d-glucan glucanohydrolase. The same study showed that the use of linearized DNA greatly enhanced the occurrence of replacement recombinants.

Significant progress has been made in engineering D. radiodurans. A current goal is to engineer Deinococcus strains as resistant to higher temperatures. Deinococcus geothermalis has been identified as the best candidate strain for this purpose. Deinococcus geothermalis can express genes of interest cloned into pMD66 and its derivative at a high temperature (50°C) (Brim et al. 2003). Suicide plasmids and linear DNA fragments can be used to obtain double-crossover recombinants in D. geothermalis with an excellent efficiency (unpublished results). In our experience, despite that Deinococcus forms tetrads and harbours multiple copies of its genome, 50% of the modifications made on the chromosome of D. geothermalis are readily homogeneous after a single plating under antibiotic selection pressure. Twenty-five to thirty per cent more homogeneous clones can be obtained by serial (2–3) plating at higher antibiotic concentrations. Finally, 15–20% of the constructions cannot be homogenized, possibly because the gene is essential and cannot be completely deleted, or because the gene is toxic when expressed by all the copies. This limits the use of several technologies such as marker-free multiplexed genome editing (CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats ).

Several studies have demonstrated the robustness of this poly-extremophilic genus for use in the metabolism or bioremediation of toxic compounds such as organic molecules or heavy metals in radioactive environments.

A Deinococcus strain expressing the toluene dioxygenase from Ps. putida was able to bioremediate toluene, chlorobenzene, 3-4 dichlorobutene and indole under irradiation (60 Gy/h for 24 h) and was resistant to 1 g l−1 toluene (Lange et al. 1998). Brim et al. (2000) have also generated several D. radiodurans strains expressing the Hg (II) resistance gene (merA), a mercuric reductase gene from E. coli strain BL308. This strain resisted the bactericidal effect of ionic Hg (II) at concentrations up to 50 mmol l−1, and it was able to reduce toxic Hg (II) to the much less toxic and volatile elemental Hg (0). The authors also noted the remarkable genome plasticity of D. radiodurans, bacteria able to maintain, replicate and express extremely large segments of foreign DNA integrated in multicopy in the chromosome.

In addition to these interesting perspectives, bacteria belonging to the Deinococcus-Thermus group possess other specific characteristics. Since the first report of the genus Deinococcus, more than 50 species within this genus have been discovered from various environments. Several species can grow on and/or digest various biomasses because they encode biomass digestion enzymes and are able to assimilate a variety of sugars present in lignocellulosic biomass. Numerous reports have described the ability of D. geothermalis to form biofilms and the problems that this ability poses during the papermaking process (Väisänen et al. 1998; Kolari et al. 2003). One of the most distinctive features of D. geothermalis in comparison with D. radiodurans is its great abundance of genes encoding sugar metabolism enzymes. d-xylose, in the form of xylan polymers, is a complex polysaccharide found in lignocellulosic biomass. Deinococcus geothermalis contains genes encoding xylanases, enzymes involved in xylane and xylose metabolism most of which form a cluster in a megaplasmid. In contrast to the proteolytic lifestyle of D.  radiodurans, D. geothermalis is a proficient hydrolytic organism (Väisänen et al. 1998; Kolari et al. 2003), and most of these functionalities were acquired by horizontal gene transfer (Omelchenko et al. 2005). Several of these enzymes have been patented (Claverie et al. 2012).

The production of bio-energy and biomolecules in a sustainable fashion is becoming increasingly important. In recent years, the mechanism by which new industrial strains are generated has changed dramatically in response to massive sequencing projects that have identified new genes, the development of new bioinformatic tools, and increasing knowledge of bacterial metabolism. These advances have made it possible to construct synthetic pathways for the production of a variety of chemical compounds from sugars. Escherichia coli and S. cerevisiae remain the most commonly used chassis in industry. However, there now is an increasing demand for micro-organisms that are able to use cheaper carbon sources, such as organic wastes or lignocellulosic biomass. Because it is able to digest cellulose and xylan and assimilate all of the sugars present in lignocellulosic biomass, D. geothermalis meets this requirement and has been patented for this purpose (Leonetti and Matic 2009). Interestingly, D. geothermalis T27 (Kongpol et al. 2008) is also able to tolerate high concentrations of solvent (decane, ethyl-acetate, etc.), an unusual property that is of major interest in industrial processes. Substantial progress has been made regarding our knowledge of the Deinococcus genome, physiology and fermentation capabilities. This knowledge coupled with the ability to genetically modify different Deinococcus species makes it possible to build industrial strains with the ability use organic wastes and lignocellulosic biomass more efficiently and with improved tolerance to various abiotic and biotic stresses. However, one key aspect to engineer rapidly cost efficient strains is the understanding of the physiology of the chassis and the availability of reliable metabolic models. With only 900 publications on Deinococcus ranks far beyond E. coli and S. cerevisiae in term of scientific knowledge, which makes the strain optimization process more time consuming and labour intensive.

All the authors are employees of Deinove, a company exploiting Deinococcus in synthetic biology.

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