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  • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
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  • C12N2800/00—Nucleic acids vectors
  • C12N2800/90—Vectors containing a transposable element

Definitions

• CORYNEBACTERIA INTEGRON
• the present invention relates to the integration of predetermined DNA sequences into the genome of corynebacteria.
• This integration can have two main purposes. On the one hand, it may involve producing a particular protein from a corynebacterium strain, either that this protein is not normally expressed by the strain in question, or else making it overproduce a homologous protein. But, on the other hand, it can be a question of blocking the expression of a gene by carrying out an interruption of this one, which will lead to the cancellation of the corresponding enzymatic activity and to the accumulation of the substrate of the reaction in the cell.
• corynebacteria which group, in addition to Corynebacterium, Brevibacterium, are bacteria whose handling has so far proved to be rather delicate:
• selection gene a gene ensuring selection, hereinafter called “selection gene”, effective in said corynebacterium
• integron is intended to denote a non-replicating vector having the property of integrating into the genome of a corynebactery. This integron can be in linear or circular form.
• the integron generally comes from a self-replicating piasmide which allows synthesis in a different host, Escherichia coli for example.
• the integration step it is preferable to delete all traces of DNA of non-corynebacterial origin, except the selection gene, and in particular all the sequences involved in replication.
• genes can be used, notably resistance to erythromycin.
• homologous sequence is intended to denote sequences which correspond to those present in the transformed corynebacterium or which have a homology rate greater than 80%, they may be sequences of the same species or not, these sequences may moreover be synthetic.
• sequences will have to be adapted or not, that is to say to take into account the problems of restriction barriers existing in corynebacteria, by methods some of which are described below.
• this integron comes from a piasmid which comprises, in addition to the integron, a replicative part, the integron being flanked by restriction sites allowing its excision, and preferably from reverse repeat sequences corresponding to a restriction site not present in the integron. for example Notl, BstXI or SacI.
• restriction sites for example Notl, BstXI or SacI.
• the integron therefore preferably forms part of a piasmidic vector which is capable of replicating with the aid of rephcation origins. or repiicon, placed in the repiicative part.
• the composite plasmid can replicate either only in corynebacteria, if it consists of a single endogenous replicon, or both in corynebacteria and in a foreign host, Eschericnia coii for example, when associated with the plasmid two different rephcons, each allowing rephcation in its own host.
• the construction of the piasmid may be carried out in different systems these corynebacteria, which can sometimes be advantageous given the difficulties of this construction ⁇ ans Corynebacterium and Brevibacterium.
• the integron v has to merge by recombination in the chromosome.
• Amsi. in the primary transformant the gene initially cloned at the multiple site of integration of the integron is found to be duplicated in the bacterial chromosome (see Figure 2a).
• duplication can be of technical interest insofar as the doubling of the gene can lead to an increase in the activity created by this gene, in particular the corresponding enzymatic activity.
• the structure of the primary integrant corresponds to a direct tandem duplication of the homologous sequences flanking the selection gene, it is possible to provide for an amplification of this structure by selecting the growth of the primary integrant on a medium making it possible to detect the marigolds overexpressing the selection gene.
• the selection gene is the antibiotic resistance gene
• the most resistant strains can be selected, on media with an increasing antibiotic content, which strains must correspond to an overexpression of the genes corresponding to the homologous sequences, but also any gene or DNA sequence that has been inserted into the integron.
• the integron will preferably comprise, in addition to the homologous sequence or sequences, a sequence coding for a sequence of interest, in particular for a peptide or protein of interest which may be homologous, that is to say come from a corynebacterium, or else heteroiogue, that is to say come from other bacterial species, but also be of eukaryotic or synthetic origin.
• sequences will preferably include the elements ensuring their expression in the corynebacteria or else will be inserted in phase so as to be able to be expressed by the expression elements of the host bacterium.
• the integron will be designed in the form of an integration cassette, that is to say that in addition to the selection gene and the homologous sequence, which in certain cases may be confused, provision will be made for sequence comprising several cloning sites which will allow the insertion of DNA sequences and / or genes at will.
• sequences of transposable elements can be all of the sequences in. ensure the transposition, with the exception of the proteins coded by the corynebacterium, but they can also be transposables which are devoid of the sequences coding for the transposases.
• transposable elements include phage Mu, in particular in the form of a miniMu phage.
• markers have been used which may be part of the toll or of different origin, for example colored markers.
• the invention also relates to integrons using transposable elements originating from different corynebacteria. in particular this Brevibacterium. in particular ISaBl as described in figure 9.
• Example 10 presents the characterization of the ISaBi element and Example 11 gives a general method for selecting and identifying this type of transposable element.
• the present invention also relates to integrons comprising a sequence providing transposed elements, in particular elements coding for transposases and / or transposition repressors.
• the integrons according to the inv ention can understand all or part of these sequences in question, in particular that corresponding to ISaBi.
• This type of integration structure comprising a miniMu phage fragment. a homologous DNA sequence and a gene this selection can make it possible, as with the structures described above, to obtain either an overexpression of a particular gene, or the disruption of a gene when this proves necessary.
• the present invention also relates to strains of corynebacteria obtained by integrative transformation using the integrons described above, in particular when the integron has been introduced by electrotransformation.
• the transfer of the integron can take advantage of the possible replicative properties of the construction in the corynebacterium in order to adapt the integron to the corynebacterium.
• the piasmide comprising, in addition to the integron, a replicative part, in the same strain where the integration will take place, then this integron thus adapted will be recovered by extraction of the piasmide then digestion -by the enzyme (s) which release the integron, the purified fragment then being self-ligated or not and the ligation product used for the integrative transformation; in this case the restriction barriers no longer constitute a difficulty.
• the invention relates to the use of Corynebacteria according to the invention in the context of industrial processes, in particular for the preparation of proteins or me ⁇ abolites involving the integron according to the invention.
• FIG. 1 shows diagrammatically the preparation of an integron starting from a replicative piasmid
• Figure 2 shows schematically the insertion of an integron.
• FIG. 3 diagrams the structure of pCGL519
• FIG. 4 diagrams the percentage of resistance to kanamycin as a function of time for two transformed strains.
• Figure 5 shows schematically the structure of the miniMu.
• FIG. 7 shows schematically the integration of the desired integrons whether or not containing the miniMu.
• FIG. 8 represents the restriction map of the insertion element of Brevibacterium lactofermentum CGL2005 (B 115) cloned from an insertion in the 3 'terminal end of the lac operon,
• FIG. 9 represents the ISaBi sequence
• FIG. 10 represents the restriction map of ISaBi
• the chromosomal DNA of the C. melassecola ATCC 17965 strain was prepared according to the method adapted from Ausubel et al. (1987). Digestion managed by the restriction enolonuclease Mbol (Boehringer) was carried out on 10 ⁇ g of this DNA following the protocol described in Maniatis et al. (1982). The DNA fragments were separated according to their size on a sucrose gradient as described by Ausubel et al. (1987). Fragments between 6 and 15 kb in size were used for the construction of the library.
• the cloning plasmid pUN 121 (Nilsson et al., 1983) was prepared by the method of Birnboim and Doly, (1979) from the strain of E. coli GM2199 available freely.
• the piasmid was linearized by the Bell restriction endonuclease (Boehringer).
• the bank was built by hgation with T4 DNA iigase
• the E. coli W620 strain deficient for citrate synthase activity was transformed with the DNA library of C. melassecola ATCC17965.
• a transforming clone of E. coli W620 capable of growing on minimum selection medium containing tetracycline was selected.
• This clone carries a recombinant plasmid pCGL508.
• Different subcloning made it possible to shorten the DNA fragment of C. melassecola carrying the complete gltA gene to a DNA fragment of 3.5 kb delimited by two HindIII restriction sites.
• the aphlll gene was chosen as the selection gene; it confers resistance up to 600 ⁇ g / ml of kanamycin when it is integrated into a copy in the chromosome. We usually work at 25 ⁇ g / ml.
• the 3.5 kb HindIII fragment carrying the structural gene gltA coding for the citrate synthase of C. melassecola obtained in Example 1 was initially chosen as a DNA fragment homologous to the genome of the Corynebacteria and inserted into one unique sites of the multiple cloning site (the HindIII site).
• the restriction sites bordering the integron intended to be the most used in the integration strategy are the NotI sites which correspond to an 8 nucleotide sequence, and the BstXI sites.
• the enzyme BstXI recognizes the sequence (CCAN 5 NTGG); therefore it cuts as frequently as a 6 nucleotide enzyme and we can expect to generate several fragments. However, the fragments released are reassociated according to the nature of the internal sequences of the different BstXI sites, which must ultimately lead to the only reconstitution of the starting fragment.
• the pCGL519 piasmid ( Figure 3) is an example of a versatile piasmid capable of generating an integron.
• pCGL519 consists of two replicative and integrative fragments bordered by the NotI sites.
• the first fragment corresponding to the integron which comprises a multiple cloning site, the selection gene aph111 and a HindIII fragment homologous to the chromosome carrying the gltA gene which codes for citrate synthase.
• the second fragment includes the replicative part of the piasmid pBLI (SspI-HpaI fragment of 3 kb) which replicates in corynebacteria, the replicative part of the piasmid pACY184 which replicates in E. coli, ori p15A, and the origin of replication of the phage M13 complementable in trans.
• the plasmid pCGL519 was constructed in E. coli by insertion of a HindIII fragment of 3.5 kb containing the glt.A gene in a vector pCGL243.
• the model involves the duplication of the wild copy of the gltA gene: the enzymatic activity has been measured, it is multiplied by a factor of 1.82 in agreement with the interpretation of the blots and shows that the integrated copy is not inactivated .
• the results are collated in Table 1 with respect to the wild strain and to the strain transformed with a replicating plasmid.
• the stability of the integrated structure was measured as for the percentage of resistant Kanamycin cells after about thirty generation ( Figure 4) and as for the enzymatic activity of the citrate synthase which remain stable.
• the amplification of the integrated structure was carried out by growth selection on a dish containing an excess of kanamycin, selection at 800 ⁇ g / ml then i000 ⁇ g / ml then 1000 ⁇ g / ml of kanamycin and neomycin. Direct tandem amplification was obtained. Despite. the stability of the amplified structure and of the resistance to kanamycin, the high level of enzymatic activity obtained at the start in the case of citrate synthase was not maintained subsequently. It is possible that a specific inactivation of the gltA gene has occurred.
• the Mu derivatives chosen in this example are MudII 1681 and
• MudII 1681-Cat (respectively Km R and Cm R ) which have a size of 14.8 kb and 16.6 kb respectively (Figure 5).
• the miniMu MudII 1681-Cat is a derivative of the MudII 1681 transposon (Castilho et al., 1984). They have the elements necessary for transposition stated above (except HU) as well as the gene for the heat-sensitive repressor C (regulator of the expression of transposases A and B), a gene for resistance to antibiotics (respectively aphll and cat) and the genes lacA lacY and lacZ '. The latter begins at the 8th codon and makes it possible to detect translational fusions of proteins when Mud fits into a reading frame.
• transposons We transferred these transposons to the Corynebacteria. After integration of the transposons into the chromosome, a method, described in Example 8, made it possible to amplify the integrated copy. Associated with this amplification, the target gene for the integration event (the structural gene for glutamate dehydrogenase) has in many cases also been amplified with an increase up to a factor of 25 in the corresponding enzymatic activity.
• the target gene for the integration event the structural gene for glutamate dehydrogenase
• the integrative vector (pCGL 107 - Figure 6) contains the gdhA gene (Gdh ') interrupted by the kanamycin resistance marker
• Km (aphlll), the replicon (ori) of pUN121 (Nilsson et al., 1983) and the genes conferring resistance to tetracycline (Tet R ) and to ampicillin (Amp R ).
• This vector is not replicative in Brevibacterium lactofermentum and integrates by simple "crossing-over" at the site of homology of the gdhA gene.
• MudII 16S l-Cat was introduced into the integrative vector pCGL107 by minimuduction from E. coli OR 1836 in the strain
• MudII 1681 was introduced in Corynebacteria by placing the transposon on two other types of vectors:
• Suicide vector (non-replicative, non-integrative) pEV11 is a derivative of pUC 18 lacking d3S lac genes into which MudII 168 1 has been introduced.
• MudII 1681 was introduced into the shuttle vector by minimuduction in E. coli Rec +. Among the various insertions obtained, one of them was selected giving a Lac- phenotype (pCGL229 :: Mud +). From this vector, a disarmed pCGL229 :: Mud- Mud was prepared in which the genes of transposases A and B were deleted by Pst1 digestion.
• Km R Cm S and type 2 may correspond respectively to the substitution of the gdh A gene by double crossing-over event and to the integration of the complete piasmid at the gdhA site by single event
• K2 conforms to a gene substitution. It also confirms that the gdh + transformants (type 2 events (KC2 and KC4) and some type 1 (K l) are obtained by integration of the piasmide at the gdhA site.
• the type 2 transformants do not sufficiently express the chloramphenicol resistance gene to obtain the growth of isolated colonies in the presence of chloramphenicol 5 ⁇ g / ml.
• An insert was isolated by recovering plasmids in E. coli DH5aipha from the amplified DNA of KC3T4.
• the 3.5 kb PvuII fragment internal to Mu derived from DNA amplified in KC3T4 and containing the insertion element, was used to probe the initial blot which contains the digests of DNA by BamHI various integrants and amplified strains (including KC3T4).
• Brevibacterium lactofermentum CGL2005 B1 15
• (ii) Brevibacterium lactofermentum CGL2002 two common bands appear in size 18 kb and 4.5 kb; on the other hand, the CGL2002 strain has no other bands. This reflects mobility of the element.
• the strains of Corynebacterium melassecola give weak hybridization signals with the insertion reflecting the existence of other different but related sequences in this species.
• ISaB1 lactofermentum has been identified and cloned; called ISaB1, it can be present in several copies (2 to 5 copies) in the genome. It is capable of transposing several times to different sites in an amplified region. Its fine restriction map is known. Different but related sequences exist in the genome of other corynebacteria.
• ISaBi consists of 1288 base pairs; the ends can be identified because ISaBi is inserted into a fragment which corresponds to the terminal region (3 ') of the lac operon which has been sequenced by Hediger et al. (Biochemistry Proc. Natl. Acad. Sci. USA 82, 1985). ISaBi was inserted between nucleotide 5575 and 5576 by duplicating a target sequence of 5 bp (CCGAT) ( Figure 8). The entire sequence of ISaBi is given in Figure 9 and the restriction map in Figure 10. Two open reading phases have been identified which could correspond, given the sequence analyzes, to the transposase structural gene and to the gene transposition repressor.
• the ISaBi insertion was obtained during gene amplification studies in the chromosome of B. lactofermentum (B1 15).
• B1 15 B. lactofermentum
• pCGL330 and pCGL331 Figures 11 and 12. These two vectors consist of a first fragment derived from the vector pUN 121.
• the piasmide pUN 121 is replicative in E.
• coli and carries resistance to ampicillin; it carries a sequence coding for the repressor cI of phage lambda which inhibits the expression of an operon fusion between the promoter P L of phage lambda and a resistance gene of tetracycline. Insertion into the cl gene will thereby inactivate the repressor which will then allow expression of the tetracycline resistance gene which is expressed in corynebacteria under the control of P L. The insertion events can thus be selected by resistance to tetracycline.
• the EcoRI fragment derived from pCGL 107 contains a selection gene for the direct transformation of coryneoacteria (the aphlll gene which confers resistance to kanamycin) and a fragment containing a homologous part of the gdhA gene (structural gene for ia glutamate dehydrogenase) which will serve as a point of integration into the chromosome of corynebacteria.
• the B. lactofermentum CGL2005 (BL 15) and CGL2002 strains were electrotransformed for resistance to kanamycin by the plasmids pCGL330 and pCGL331.
• the transformation frequency was approximately 103 per ⁇ g in each of the cases, which is compatible with integration of the plasmids.
• the transformants (like
• CGL2005 :: pCGL330 and CGL2005 :: pCGL331) are sensitive to tetracycline, which confirms that the regulation of P L by cl works well in transformants.
• the frequency of tetracycline resistant segregants was measured after approximately 10 generations.
• Mutations inactivating the C1 gene appear in strains with the integrated plasmids pCGL330 and pCGL331 at a frequency of 2 ⁇ 10 6 per generation.
• a piasmid recovery was carried out from genomic DNA extracted from resistant tetracycline segregants isolated from the CGL2005 :: pCGL331 and CGL2005 :: pCGL330 strains and digested with the enzyme Pstl. These digested DNAs were ligated and the ligation product was used to transform the DH5alpha strain of E. coli for resistance to tetracycline. The recovered plasmids were analyzed and in 7 cases out of 9 tetracycline-resistant clones, an insert was identified. In one case (from CGL2005 :: pCGL331), an insertion element identical to ISaBl was identified (1.2 kb in size, having the unique AccI sites,
• the transposon trap vector works; in the majority of cases, the mutation obtained is an insertion; the frequencies of resistance to tetracycline therefore fairly correctly measure the transposition frequencies; the most mobile elements have therefore been identified; of these, ISaBl has been re-isolated and another element of insertion different from ISaBl has been identified.
• ATCC17965 ORSAN
• ATCC 17965 :: gltA: (this request)
• the DH5alpha strainer is available in the catalog of
• NADPH 2 consumed / min / mg protein
• KC2T1 7.4 2.73 TABLE 5: DETERMINATION OF CAT, BGA1 and GDH ACTIVITIES

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