The present invention relates to methods and compositions for chromosome integration of nucleic acids into Deinococcus bacteria. The invention more particularly relates to IS- mediated multicopy gene insertion or IS targeting methods for chromosome engineering in Deinococcus bacteria, the resulting bacteria, and the uses thereof.

Introduction Deinococcus is a gram positive bacterium that was isolated in 1956 by Anderson and collaborators. This extremophile organism is resistant to DNA damage by UV and ionizing radiations or by cross-linking agent (mitomycin C) and is tolerant to desiccation. WOO 1/023526 shows the unusual resistance of Deinococcus to radiation and further proposes their engineering and use in bioremediation. WO2009/063079 shows that Deinococcus bacteria can resist to solvents and transform biomass to generate biofuels. WO2010/130806 further discloses recombinant Deinococcus strains wherein ethanol biosynthesis genes have been inserted. These recombinant strains do exhibit improved performance in the production of ethanol.

The present invention discloses novel compositions and methods for genetically modifying Deinoccocus bacteria. More specifically, the invention provides improved Insertion Sequence-based methods for genetically modifying Deinoccocus bacteria.

Summary of the invention

The invention relates to methods and constructs for gene recombination or chromosome engineering in Deinococcus bacteria. More specifically, the invention relates to IS-based methods and constructs for gene insertion or amplification in Deinococcus bacteria, or to IS-mediated genetic modification of Deinococcus bacteria. An object of the invention therefore relates to a method for introducing a nucleic acid into the genome of a Deinococcus bacterium, comprising introducing said nucleic acid into said genome by IS-mediated insertion. In preferred embodiments, the nucleic acid is introduced into the genome of the bacterium by homologous recombination with an IS present in the genome, by intron-mediated insertion into an IS, or by IS-mediated transposition.

A further object of the invention resides in a method for producing a recombinant Deinococcus bacterium comprising one or several copies of a gene of interest inserted into its genome, the method comprising introducing said gene of interest into the genome of said bacterium by IS-mediated insertion and, optionally, amplifying the copy number by subjecting said bacterium or a descendant thereof to a gene amplification treatment.

The invention also relates to a method for inducing (or increasing) chromosomal rearrangement(s) or shuffling in a Deinococcus bacterium, the method comprising expressing (or increasing expression of) at least a transposase gene in said bacterium. The invention also relates to a Deinococcus bacterium obtained by IS-mediated insertion of a nucleic acid, or a descendant of said bacterium.

Another object of the invention is a Deinococcus bacterium comprising one or several copies of a nucleic acid inserted into an IS element.

A further object of the invention resides in a nucleic acid molecule comprising a gene of interest flanked, on one or both sides, by (i) a sequence homologous to a sequence of Deinococcus IS element or (ii) by a sequence of an inverted repeat sequence of Deinococcus IS element.

The invention also relates to a recombinant Deinococcus bacterium comprising one or several copies of a transposase gene under control of a promoter. The invention may be performed with any Deinococcus bacteria and can be used to engineer bacteria with improved genotypes or phenotypes, particularly bacteria which express recombinant genes of interest. Legend to the Figures Figl : Gene insertion by IS-mediated homologous recombination in Deinococcus. Fig2: Insertion of Ethanol pathway DNA construct into IS66 of D. geothermalis. Fig3: Gene insertion by IS-mediated artificial transposition in Deinococcus.

Detailed Description of the invention

The invention relates to IS-mediated gene insertion or chromosome engineering in Deinococcus bacteria, the resulting bacteria, and the uses thereof.

Insertion Sequences (IS) are transposable genetic elements identified in certain prokaryotic organisms. They have a typical length ranging from 300 to 3000bp (Mahillon and Chandler, 1998; Chandler and Mahillon, 2002). In bacteria, IS are frequently found as part of natural plasmids. IS typically possess one or two open reading frames (ORFs) that encode a transposase, an enzyme that is necessary for their transposition. This (these) ORF(s) is (are) surrounded by linker regions that frequently end with short-terminal inverted repeats (IRs) ranging typically from 7 to 50 bp in length. Some IS may carry multiple repeated sequences at both ends, which may represent transposase binding sites (Nagy Zita and Michael Chandler, Research in Microbiology 155 (5) p387-398). Contrary to transposons, IS do not contain ORF encoding drug resistance. Upon insertion, IS often undergo short directed repeats from 2 to 14 bp immediately outside the IRs.

Despite sequence divergence, ISs have been grouped into families based on similarities and identities in the primary sequence of their transposases (Tpases) and in their genetic organization (Robinson, Lee, & Marx, 2012). This includes the disposition of their open reading frames (ORFs), length and similarity of the terminal inverted repeats, and the characteristic number of base pairs in the target DNA which they duplicate upon insertion (Mahillon, Leonard, & Chandler, 1999). Depending on the IS, insertion may be target site selective (Craig, 1997 ; Tobes & Pareja, 2006). Through transposition, IS can interrupt the coding region of a gene, or disrupt promoter regions and alter gene expression. Given that there can be several copies of the same IS in a genome, IS can also serve as sites of DNA rearrangements such as deletions, duplications and inversions of adjacent DNA segments through homologous recombination (Robinson et al, 2012). Insertion sequences contribute to the variability of the prokaryotic genomes and phenotypes, and are thought to play an important role in the adaptability of prokaryotes to the environment (Schneider & Lenski, 2004).

IS elements have been identified in D. radiodurans (Makarova et al, 2001 ; Islam et al, 2003 ; Mennecier, Servant, Coste, Bailone, & Sommer, 2006 ; Pasternak et al, 2010). The present invention discloses the characterization of particular IS sequences in Deinococcus bacteria, as well as the uses thereof for genetic modification or shuffling of such bacteria. More specifically, the inventors analyzed the presence and occurrence of different IS families in Deinococcus sp genomes. A total of 11 IS families were found in 5 tested Deinococcus species, which are presented in Table 1. IS families IS4, IS5, IS1 and IS701 were found to be the most scattered among Deinococci, the largest family being IS4, which contains a total of 68 members in Deinococci. Surprisingly, the genome of D. geothermalis DSM11300 and D. geothermalis MX6-1E possess a larger number of IS elements compared to the other Deinococcus strains (Table 1). In these strains, seventy six IS elements belonging to 10 distinct families were detected (DSM11300 strain) and fifty five spread into 7 IS families (MX6-1E strain).

The present invention therefore shows an unexpected high level number of insertion sequences in thermophile Deinococcus strains such as D. geothermalis (table 1). This discovery brings new tools for genetic manipulation of thermophile Deinococcus strains and, in particular, allows multicopy integration of genes into Deinococcus bacteria to increase their expression. The presence of different IS families in a single Deinococcus genome even allows the introduction of different DNA constructs (one type of construct targets one family of IS). The present invention therefore provides a novel method to insert or spread or amplify a desired gene into a Deinococcus genome using Deinococcus insertion sequences.

The present invention also provides a method for chromosomal engineering of a Deinococcus bacterium by expressing or overproducing in said bacterium a transposase that targets an IS present (preferably in several copies) on the chromosome or a plasmid of said bacterium.

Definitions

Within the context of the present invention, the term "Insertion Sequence" or "IS" element designates a transposable genetic element comprising at least one transposase gene and a flanking terminal Inverted Repeat. IS are devoid of drug resistance gene. ISs have a typical length ranging from 300 to 3000bp, the terminal inverted repeats (IRs) ranging typically from 7 to 50 bp in length.

Preferred IS elements for use in the invention are Deinococcus IS elements, i.e., IS elements having the sequence of an IS present in one or, preferably, several copies in the genome of a Deinococcus bacterium. Specific examples of Deinococcus IS elements according to the invention are IS200/IS605 , IS630 ; IS701; IS607; IS982; IS3; IS1; IS6; IS5; IS4; or IS66. The sequence of these IS is provided in the sequence listing.

"IS-based" methods or "IS-mediated" methods designates any method for inserting a gene in a Deinococcus bacterium which uses all or part of an IS element. Insertion is typically targeted, i.e., site-specific or site-controlled. In particular, because IS-mediated insertion generally follows target selectivity of the IS element, the gene insertion is not random but obeys the same rule.

The term "gene" designates any nucleic acid molecule (e.g., DNA fragment) of interest, such as preferably a nucleic acid comprising an ORF encoding a product (e.g., RNA or polypeptide) of interest. The gene may be natural, recombinant or synthetic. A gene may be single or double-stranded, typically a DNA molecule. In a particular embodiment, the gene preferably encodes a protein, such as an enzyme. The gene may further comprise one or several regulatory elements, operably linked to the ORF, such as a promoter, terminator, intron, etc. The term "chromosomal engineering" designates any modification or rearrangement of a chromosome, such as a deletion, translocation, duplication, or inversion of one or several sequences within a chromosome or episome. Chromosomal engineering may result in novel chromosomes or episomes, thus creating novel biological pathways and/or genetic diversity in bacteria, leading e.g., to bacteria having improved characteristics.

IS-mediated genetic modification of a Deinococcus

As indicated the invention resides in IS-mediated genetic modification of Deinococcus bacteria, typically to produce recombinant or genetically improved bacteria. The invention is particularly advantageous since IS-mediated insertion is effective, can be site-selective, and allows insertion of multiple copies of a selected gene. IS-mediated insertion may comprise preferably the introduction of the gene into the genome of the bacterium by homologous recombination with a selected IS present (or inserted or amplified) in the genome of the bacterium; or by intron-mediated insertion into an IS present (or inserted or amplified) in the genome of the bacterium; or by IS-mediated transposition. The method may involve the use of an insertion cassette, the construction of artificial transposon, or the retro-homing mechanism of group II intron as described below, in combination or not with DNA-damaging treatments (UVs, gamma, x- irradiation). Preferably, the IS-mediated insertion of a gene in a Deinococcus bacterium includes the (targeted) introduction of said gene into an IS sequence present in the genome of said bacterium.

The invention may be used to genetically modify any Deinococcus containing an IS element, preferably any Deinococcus strain that contains or that can accept at least two copies of an IS element. Examples of Deinococcus host strains that can be engineered according to the present invention include, without limitation, D. geothermalis, D. radiodurans, D. cellulolysiticus, D. murrayi, D. guilhemensis, D. aerius ,D. aerolatus ,D. aerophilus ,D. aetherius ,D. alpinitundrae ,D. altitudinis, D. apachensis ,D. aquaticus ,D. aquatilis ,D. aquiradiocola ,D. caeni„D. claudionis ,D. daejeonensis ,D. depolymerans ,D. deserti ,D. erythromyxa ,D. ficus ,D. frigens ,D. gobiensis ,D. grandis ,D. hohokamensis ,D. hopiensis ,D. humi, ,D. indicus ,D. maricopensis ,D. marmoris ,D. misasensis ,D. mumbaiensis ,D. navajonensis ,D. papagonensis ,D. peraridilitoris ,D. pimensis ,D. piscis ,D. proteolytics ,D. radiodurans ,D. radiomollis ,D. radiophilus ,D. radiopugnans ,D. reticulitermitis ,D. roseus ,D. saxicola ,D. sonorensis, D. wulumuqiensis ,D. xibeiensis ,D. xinjiangensis ,D. yavapaiensis , and D. yunweiensis. Preferred acceptor bacteria are thermophile Deinococcus. Most preferred bacteria are Deinococcus comprising at least 2 copies of an IS element, preferably at least 3 copies thereof. The copies may be present in the chromosome, or induced in said chromosome.

Accordingly, the method of the invention typically comprises the following steps: a) provision of a Deinococcus bacterium that contains at least 1, preferably at least 2 copies of a target IS element, and; b) IS-mediated insertion of a gene into said target IS element.

Preferentially, the targeted IS is an IS present in more than one copy on the genome (chromosome and/or plasmid) of the selected Deinococcus bacterium. The targeted IS is more preferably one of the IS elements selected by the inventors which are listed in Table 1. The identification of several distinct IS elements, in addition, allows the propagation and expression of different genes of interest in a Deinococcus genome. A preferred target IS is selected from IS200/IS605 , IS630 ; IS701; IS607; IS982; IS3; ISl; IS6; IS5; IS4; or IS66.

The Deinococcus bacterium may contain said at least 2 copies naturally, or may be treated to amplify the copy number of a target IS element, prior to step b), or thereafter. Accordingly, the invention generally comprises the following steps: a) provision of a Deinococcus bacterium for which genetic modification is desired; b) selection, in said Deinococcus bacterium, of at least one target IS element present in the chromosome of said bacterium, preferably in at least two copies; c) optionally treating the bacterium to amplify the copy number of said selected target IS element; and d) inserting a gene into said bacterium by IS-mediated insertion into said selected target IS element. In a particular embodiment, the step of treating the bacterium to amplify the copy number of the selected target IS element is conducted after the insertion step. Also, the amplification step can be performed both before and after the insertion step. The treatment may comprise any treatment which allows or increases expression or activity of a transposase and/or causes a cell stress, such as a thermal shock or an irradiation of the cells, which may be selected from UV, gamma and/or X ray irradiation, either alone or in combinations, most preferably UV irradiation(s). Irradiation treatment typically comprises subjecting the microorganisms to one or several sequential irradiations (e.g., from 1 to 5), which may be of the same or different nature, preferably of the same nature. Repeated irradiation treatments are typically carried out at an interval of between 1 and 8 hours, preferably 3 to 5 hours, and more preferably of about 4 hours. A particularly preferred treatment comprises subjecting the sample to a UV, X, or gamma irradiation. Such a treatment indeed allows to amplify IS copy numbers and to stimulate chromosomal engineering. Particular UV treatments are typically of between 0.5 and 400 mJ/cm2, more preferably of between 1 and 200 mJ/cm2, typically between 1 and 100 mJ/cm2, applied for a period of time of about 5" to 5'. A preferred UV treatment is 4 mJ/cm2 for 30 seconds.

During the whole process, the cells may be placed in a suitable culture medium such as, without limitation, PGY (Bacto-peptone lOg/L, Yeast extract 5g/L, glucose 20g/L) or LB (Bacto-tryptone lOg/L, Yeast extract 2.5g/L, Sodium chloride lOg/L). It should be understood that other suitable culture media are known to the skilled person (Buchanan et al, 1974, Difco, 1995)) or may be prepared by the skilled person from such known media.

For targeted insertion by homologous recombination into an IS, the gene to be inserted (or amplified) is typically assembled as a recombination cassette, which may be cloned or not in an appropriate vector, such as pMD66. In this regard, the invention shows that linear DNA molecules can be used directly for transformation of Deinococcus bacteria. The recombination cassette typically comprises the gene flanked, on one or both sides, by a HR1 region and/or a HR2 region (of about 100-1000, more preferably about 200- 700, such as 300-600, typically about 500 pb each), said HR1 and HR2 regions being homologous, respectively, to a 5' and 3' DNA sequence of the targeted IS element. HR1 and HR2 can be any part of the sequence of the target IS. HR1 and/or HR2 allow insertion of the gene of interest into the chromosome by specific homologous recombination. The recombination cassette may comprise, in addition, e.g., a marker gene (such as a drug resistance gene). In such a case, the two genes (gene of interest and marker gene) may be placed under the control of either one promoter (operon structure) or of distinct separate promoters. Examples of marker genes include, e.g., antibiotic resistance genes such as genes conferring resistance to e.g., kanamycin, chloramphenicol, bleocin, oxytetracyclin, hygromycin, erythromycin, puromycin, orthiamphenicol. The recombination cassette (or the vector carrying the same) is introduced into the selected Deinococcus strain. Introduction may be performed using techniques such as transformation, lipofection, calcium-mediated precipitation, electroporation, etc. The presence of the recombination cassette or vector in the cell can be verified by e.g., detection of the gene or marker gene. After introduction in the host strain, integration of the recombination cassette in the IS target site(s) occurs by homologous recombination. In this regard, in a preferred embodiment, insertion is induced or stimulated by thermal shock. Indeed, upon thermal shock, the vector is lost and recombinant strains expressing the gene have therefore inserted the gene into their chromosome. The results presented in the examples show effective insertion by IS-targeted homologous recombination. They further show that transformation of Deinococcus is effective with a linear DNA construct. They further show that IS-targeted homologous recombination into Deinococcus can be performed with very large recombinant cassettes. Indeed, as shown in Example C, a recombinant cassette of more than 6 kb (e.g., comprising 4 distinct genes) can be successfully inserted into a Deinococcus bacterium by IS-targeted homologous recombination.

In this regard, the invention also relates to a method for intrioducing a DNA into a

Deinococcus bacterium, the method comprising:

. providing a linear DNA molecule, and

. introducing said molecule into a Deinococcus bacterium.

More preferably, the linear DNA molecule comprises a HR1 and/or HR2 region as defined above and the method further comprises a step of maintaining the Deinococcus under conditions allowing homologous recombination. In a particular embodiment, the linear DNA molecule comprises more than 2kb, even more than 3, 4, 5 or even 6 kb. In an alternative embodiment, IS-mediated insertion is performed by construction of an artificial transposon containing the gene, and introduction of the transposon into the selected Deinococcus bacterium, leading to IS-mediated insertion into the chromosome. The artificial transposon preferably comprises the gene, a transposase gene of a Deinococcus IS element, optionally a marker gene, and one or two IR elements of a Deinococcus IS element. The artificial transposon of the present invention can be constructed using regions or sequences of any IS element as described in Table 1 as starting material. Preferably, the transposase gene and IR sequences are derived from (e.g., have a sequence of a domain of) a same IS element. The transposase gene may be located inside the artificial transposon, that is the transposase gene may be located between the two inverted repeats. Alternatively the transposase gene may be located outside the artificial transposon. In that case, it may be on the transposon-carrying vector, or on the chromosome, or on a distinct vector. The expression of the transposase may be under the control of its own promoter, a constitutive promoter, or an inducible promoter. The marker gene, when present, can be e.g., any gene conferring resistance to an antibiotic such as kanamycin, chloramphenicol, bleocin, oxytetracyclin, or hygromycin. The constructed artificial-transposon is typically cloned into a suitable vector, such as pmD66, and introduced into the selected Deinococcus host strain. Upon introduction, IS-mediated insertion of the transposon occurs. If desirable, DNA-damaging agents such as gamma and/or X-irradiations and/or UV treatments or, more generally, any treatment allowing or increasing expression or activity of a transposase, may be applied to enhance the transposition and increase integration and amplification of the artificial transposon in the Deinococcus host cells. Indeed, it has been shown that irradiation can induce transposition in Escherichia coli (Eichenbaum & Livneh, 1998).

Another alternative embodiment for performing IS-mediated multicopy insertion of a gene into the chromosome of a Deinococcus bacterium is to use group II introns targeting IS sequences. Mobile group II introns are catalytic RNA elements present in a wide range of prokaryotic and eukaryotic organisms (Michel & Feral, 1995). Some of these introns can mobilize autonomously at a high frequency to allelic sites in a process known as homing. Mobile group II introns possess an intron-encoded protein (IEP) that has reverse transcriptase, R A splicing ("maturase"), and DNA endonuclease activities (Frazier, Filippo, Lambowitz, & Mills, 2003). Mobility initiates when the IEP helps the intron RNA fold into the catalytically active RNA structure to promote splicing, resulting in ligated exons and an intron lariat-IEP ribonucleoprotein (RNP) complex. The RNP complex recognizes specific DNA target sites and promotes integration by reverse splicing of the intron RNA directly into one strand of the target DNA. The IEP then cleaves the opposite strand and uses it as a primer for target DNA-primed reverse transcription of the inserted intron RNA. The resulting cDNA copy of the intron is integrated into genomic DNA by cellular recombination or repair mechanisms. DNA target site recognition by the RNP complex involves the base pairing of intron sequences denoted EBS1 and -2 (exon binding sites 1 and 2) and δ to sequences denoted IBS1 and -2 (intron binding sites 1 and 2) and δ' in the DNA target site. The EBS sequences can be mutagenized to retarget the intron to invade a selected IS sequence in Deinococcus. In the present invention, a plasmid such as pMD66 carrying the IEP protein (reverse transcriptase)-encoding gene and the group II intron which contains in its sequence a multiple cloning site allowing the cloning of the gene of interest was used. The expression of group II intron harboring the gene of interest is controlled by a constitutive or inducible promoter, such as the T7 constitutive promoter whereas the IEP (reverse transcriptase)-encoding gene is under the control of an inducible or constitutive promoter. Alternatively, the group II introns harboring the gene of interest is integrated into the Deinococcus chromosome and the reverse transcriptase encoding-gene carried by a replicative plasmid is under the control of either a constitutive or inducible promoter. This system that uses intron-mediated insertion or amplification does not rely on homologous recombination to achieve multicopy integration and facilitate stable chromosomal genes delivery without selection (Rawsthorne, Turner, & Mills, 2006).

A further object of the invention resides in a method for producing a recombinant Deinococcus bacterium comprising one or several copies of a gene of interest inserted into its genome, the method comprising introducing said gene of interest into the genome of said bacterium by IS-mediated insertion and, optionally, amplifying the copy number by subjecting said bacterium or a descendant thereof to a gene amplification treatment.

The invention also relates to a Deinococcus bacterium obtained by IS-mediated insertion of a nucleic acid molecule (e.g. DNA fragment), or a descendant of said bacterium. The invention further relates to a Deinococcus bacterium comprising one or several copies of a nucleic acid molecule (e.g. DNA fragment) inserted into an IS element.

A further object of the invention is a nucleic acid molecule (e.g. DNA fragment) comprising a gene of interest flanked, on one or both sides, by a sequence homologous to a sequence of Deinococcus IS element, as well as a vector comprising such a nucleic acid. Still another object of the invention is a nucleic acid molecule (e.g. DNA fragment) comprising a gene of interest flanked, on one or both sides, by a sequence of an inverted repeat sequence of Deinococcus IS element, as well as a vector comprising such a nucleic acid molecule.

Chromosomal Engineering As indicated above, another object of the invention resides in a method for inducing (or increasing) chromosomal engineering (e.g., rearrangement or shuffling) in a Deinococcus bacterium, the method comprising expressing in said bacterium a transposase gene. More particularly, the method comprises: a) causing or inducing expression of at least one transposase in a Deinococcus bacterium ; and b) selecting a Deinococcus bacterium of step a) having a reengineered chromosome.

In step a), the transposase may be expressed on a vector, or chromosome, or supplied as a protein. The transposase is preferably a transposase of an IS element present in said bacterium. Expression of the transposase may be combined with a treatment of the cells to amplify a gene copy number, such as irradiation. For example a strain expressing one or several transposases can be submitted to an increasing selection pressure (e.g., increasing ethanol concentration to strengthen its resistance to ethanol). A shuffling of the genome will occur due to the expression of the transposases and the most resistant clones will be selected. The method of the invention may be used to insert any gene of interest into a Deinococcus strain, in one or more copies, allowing an increase of its expression. The gene may encode any product of interest, such as an R A (mRNA, tR A, siR A, etc) or a polypeptide (protein, peptide, etc). Examples of such polypeptides include, without limitation, enzymes involved in metabolism, any biologically active polypeptide, etc. The polypeptide may be a polypeptide having pharmaceutical and/or agro-chemical interest. In a particular embodiment, the polypeptide is a pharmaceutical compound (e.g., suitable for use in human or veterinary medicine). Specific examples of such a compound include, without limitation, antibiotics, bacteriostatic compounds, anti-metabolite, chemotherapeutic compounds, antioxidants, anti-inflammatory, polysaccharides, anti- parasitic agents, anti-fungal agents, anti-viral compounds, cytokine-activity compounds, cell-growth factors, hormones, anti-depressives, anti-migraine, anti-asthma, contraceptives, anti-diabetics, psychotropic, anti-arythmics, enzyme-inhibitors, or adjuvants.

The polypeptide may also have utility e.g., in cosmetics or agriculture, such as pigments, insecticides, pesticides, chemical-degrading compounds, etc.

Examples of enzymes include biomass-degradation enzymes or fermentation enzymes, such as laccases, xylanases, amylases, ADH (alcohol dehydrogenase), PDC (pyruvate decarboxylase), etc. Further examples of polypeptides include enzymes of biological biosynthetic pathways, particular enzymes involved in the synthesis of antibiotics. Further aspects and advantages of the invention will be disclosed in the following experimental section, which is illustrative. Examples

A. Characterization of Deinococcus IS sites

A search and compilation of IS sequences present in Deinococcus strains was performed by the inventors. A complete list of all identified IS sequences found in different 5 Deinococcus species is presented in Table 1 below.

Table 1 - IS distribution among Deinococcus species

Deinococcus sp. IS200 IS630 IS701 IS607 IS982 IS3 IS1 IS6 IS5 IS4 IS66 Total Genome

/ of IS Size

IS605 (Mb)

D.deserti

0 2 1 0 2 5 0 0 1 4 0 15 3.86

VCD115

D.geothermalis

3 1 17 1 1 0 19 8 12 8 6 76 3.25

DSM11300

D. geothermalis

6 0 6 0 0 0 12 2 1 18 10 55 3.25

MX6-1E

D.maricopensis

0 0 0 0 0 0 0 0 1 0 0 1 3.5

DSM21211

D.proteolyticus

0 0 0 0 2 0 0 0 5 13 0 23 2.89

MRP

D.radiodurans 9 10 0 0 0 0 0 0 2 25 1 47 3.28

Total number of

IS among 18 13 24 1 5 5 31 10 22 68 17 217 -

Deinococcus sp.

10 B. Gene insertion by IS-mediated homologous recombination in Deinococcus

We built Deinococcus plasmids (pMD66-type for replicative, pUC-type for non- replicative) harboring IS-targeting cassettes (see Fig 1). A DNA fragment containing a gene encoding resistance to hygromycin (grey rectangle) under the control of its own promoter was flanked by two 500 bp regions named HR1 and HR2 (black rectangle), that are homologous to N-term and C-term sequences, respectively, of the insertion sequence IS66 of the Deinococcus geothermalis . The nucleic acid sequences of HR1 and HR2 are provided as SEQ ID NO: 11 and 12, respectively. The IS targeting vector is then transformed into D. geothermalis and strains expressing the hygromycin gene are selected using the embedded marker on PGY-agar plate containing 800 μg/ml of hygromycine. In an alternative experiment, a linear DNA fragment containing HRl-hygromycine-HR2 was used directly to transform Deinococcus cells, the selection of recombinants being carried out as described above. The clones that showed resistance to hygromycine were selected. Such clones are recombinant bacteria having inserted the target nucleic acid into an IS element. In this regard, in order to verify the insertion of the hygromycine resistance-encoding gene into the IS66, the clones were subjected to PCR amplification with primers designated "A" and "B", which anneal specifically to the region upstream the IS66 and to the 5 '-end of hygromycine gene, respectively generating a DNA PCR fragment of about 700 bp (see Fig.l).

The results presented Fig 1 confirm the insertion of the recombinant nucleic acid into the targeted IS. C. Ethanol pathway genes insertion by IS-targeted homologous recombination in

Deinococcus geothermalis

An IS-targeting cassette was constructed comprising a PDC gene and two alcohol dehydrogenase encoding genes. The cassette also comprises an antibiotic resistance marker to hygromycine. The cassette is flanked by two 500 bp regions HR1 (SEQ ID NO: 11) and HR2 (SEQ ID NO: 12) (Fig.2, black rectangle), that are homologous to N-term and C-term sequences of the insertion sequence IS66 of the Deinococcus geothermalis, respectively. The cassette comprises 6548pb. The IS-targeting cassette (linear molecule) or a vector containing the cassette, are transformed into D. geothermalis and recombinant strains having inserted the cassette into their chromosome are selected using the embedded marker on PGY-agar plate containing 800 μg/ml of hygromycine. The mapping of the cassette in the genome is confirmed by PCR using primers "C" and "D", primer C being specific of each upstream sequence of IS66 and D being specific of 5' end of PDC. The results are presented in Fig. 2. They show the construction has been found integrated into four different loci of IS66: CDS 1696, CDS 1721, CDS 2881 and CDS 2557 (Fig 2), into the chromosome or into native plasmid 1 of Deinococcus.

These results confirm the efficacy of the method with linear DNA construct. They confirm the specificity of the method since the cassette is found in the targeted IS. They also demonstrate the efficacy of the method with very large expression cassettes.

D. Gene insertion by IS-mediated artificial transposition in Deinococcus An IS-propagating cassette is prepared (Fig 3). The gene of interest is flanked by 2 Inverted repeats sequences (black, IRs) that can be recognized by a Transposase. A Deinococcus replicative vector allowing for Transposase thermosensitive-expression is transformed into the GOI IRs strain. Upon Transposase expression (grey), the gene is inserted and propagated into the chromosome.

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