CN1061624A - Integron of coryneform bacterium, method for transforming coryneform bacterium with said integron and coryneform bacterium obtained therefrom - Google Patents

Abstract

The invention relates to an integrant of coryneform bacteria, characterized in that it comprises: a sequence homologous to the gene ensuring efficient selection in said coryneform bacterium and to the genome of said coryneform bacterium, wherein said sequence is suitable for said bacterium.

When said integrant contains sequences encoding useful proteins, it is particularly suitable for the production of proteins by culturing coryneform bacteria transformed therewith.

Description

The present invention relates to the integration of predetermined DNA sequences in the genome of coryneform bacteria.

This integration may have two main purposes: one is to allow the coryneform strain to produce a particular protein that it should not be expressing, or to overproduce a homologous protein. Another purpose might be to block expression of a gene by interfering with its effectiveness, resulting in loss of corresponding enzyme activity and accumulation of reaction substrates in the cell.

Corynebacteria in general, with the exception of Corynebacterium and Brevibacterium, are hitherto considered troublesome in terms of injury;

- first because there is no proper way to convert it,

- In addition, transformation with DNA from other bacterial sources is mostly ineffective due to the presence of significant restrictive barriers.

The electroporation method has recently been demonstrated to be useful for the transformation of corynebacteria. However, if this transformation technique for self-replicating vectors makes sense, it would be preferable to use industrially those bacteria that can be transformed by integration within , those strains that are very stable at this time.

The object of the present invention is to provide an integration system integrated in coryneform bacteria.

The present invention relates to a coryneform bacterium integron (integrons), characterized in that it comprises:

- a gene to ensure selection, which is effective in said coryneform bacteria, and hereinafter referred to as "selection gene",

- Homologous sequences of the genome of said coryneform bacterium, which are particularly adapted to said bacterium.

The term "integrator" refers to a non-replicative vector capable of integrating in the coryneform bacterium genome, and this integron can be linear or circular.

Generally speaking, however, the integron can be derived from a self-replicating plasmid that allows synthesis in different hosts such as E. coli. Prior to the integration step, it is desirable to remove all traces of DNA of non-coryneform origin other than the selection gene, especially those sequences involved in the replication process.

The selection genes effective in said coryneform bacteria are:

- resistance genes to specific substances, especially antibiotics,

- Genes that can provide a clearly identifiable phenotype, color and/or compensation.

More preferred among these are antibiotic resistance selectable markers, in which case it is possible to utilize:

- the gene AphIII conferring kanamycin resistance, denoted as Km ^R ,

- The gene Cat conferring resistance to chloramphenicol, denoted as Cm ^R . However, other genes may also be used, in particular the erythromycin resistance gene.

"Homologous sequence" means a sequence corresponding to, or at least 80% homologous to, a sequence present in the transformed coryneform bacterium, which may involve sequences of the same species or of a different species, and these sequences may be synthetic .

These sequences will or may not be suitable for certain methods in which the problem of restrictive barriers present in coryneform bacteria has to be taken into account.

The integron is preferably provided as a corresponding plasmid containing, in addition to the integron, a replicating part, wherein the integron may be flanked by restriction sites allowing cleavage, and preferably contains restrictions that do not correspond to those in the integron. Sex sites such as NatI, BstXl or SacI inverted repeats. In this way, when digested with an enzyme directed at a known restriction site, the integrant can be directly obtained; because the ends of the integrant are complementary, it can be ligated into a circle according to need before being used to transform coryneform bacteria.

In the system of the present invention, the integron is preferably carried as a part of a plasmid, and the plasmid vector is capable of replicating through the replication origin or replicon in the replicating part. According to the nature of the replicons present in the replication cassette, if the plasmid is composed of endogenous replicons unique to coryneform bacteria, the composite plasmid can only replicate in coryneform bacteria, but when two different, respective When combined with a plasmid, a replicon capable of replicating within its native host can replicate both in coryneform bacteria and in foreign hosts such as E. coli.

In this case, plasmids can be constructed in systems other than coryneform bacteria, which is beneficial for plasmids that are difficult to construct in corynebacterium and brevibacterium.

Due to the presence of homologous sequences in both the integron and the genome, the integron must be inserted into the chromosome by means of a recombination process. Thus, in the first transformant, the gene initially cloned into the multicloning site of the integron is replicated in the bacterial chromosome (see Fig. 2a).

Such a mode of duplication has beneficial technological value in assays where the gene is multiplied, resulting in a significant increase in the activity encoded by the gene, especially the corresponding enzymatic activity.

Given that the structure of the first integrating part corresponds to a direct tandem duplication of homologous sequences surrounding the selected gene, it is possible to preamplify this structure based on selection for the development of the first integrating part on a medium that allows detection of overexpressed selected genes . Thus, when the selection gene is an antibiotic resistance gene, the most resistant strains can be selected by increasing the antibiotic content in the medium, i.e. those strains that should be able to overexpress the corresponding gene, which is equivalent to to homologous sequences and correspond to all genes or DNA sequences that will be inserted into the integron.

Of course, the integron preferably contains a sequence encoding a useful sequence, especially a sequence encoding a useful peptide or protein, in addition to one or more homologous sequences. Said sequence may be homologous or derived from Coryneform bacteria or, alternatively, of different origin, ie from other strains of bacteria, but also of eukaryotic or synthetic origin.

These sequences preferably contain elements ensuring expression in coryneform bacteria, or are preferably inserted at a stage suitable for expression of expression elements of the host bacterium.

In the case of using Corynebacterium hosts, it is of interest to obtain overexpression of certain enzymes, in particular of gltA or gdhA.

As previously indicated, it is possible to use this system to ensure gene disruption using the integrons of the present invention. For disruption or substitution, as shown in Figure 26, the corresponding gene is inactive, resulting in an overproduction of the substrate of the enzyme encoded by the corresponding gene.

In general, integrons exist in the form of integration cassettes, that is to say, in addition to selection genes and homologous sequences, in some cases, they can also be mixed with sequences that can provide multiple cloning selection sites, allowing the insertion of any DNA sequence and/or gene.

In all cases, for genetic qualification, one also attempted to predict integrants without replicating DNA from other lines.

It is also possible to predict more complex integration systems, especially integration systems that additionally include sequences of transposable elements. Sequences of transposable elements may be assembled from sequences other than protein coding sequences of coryneform bacteria that ensure translocation and may involve translocation of non-translocase coding sequences.

Among the transposable elements, mention should be made of phage Mu, especially in the form of phage miniMu. Where transposable elements such as the phage miniMu form are used, markers, such as chromogenic markers, which may be part of the phage or part of a different origin, may be utilized as described below.

The present invention also relates to the use of integrons of transposable elements (especially ISaB1 as shown in Figure 9) obtained from different coryneform bacteria, especially bacteria of the genus Corynebacterium.

The characterization of the ISaB1 element is given in Example 10, and the general method allowing the selection and identification of this type of transposable element is given in Example 11.

The invention also relates to integrons comprising sequences derived from translocated elements, especially encoding translocase and/or translocation repressors.

The integrons of the invention may contain all or part of the sequence, in particular the sequence corresponding to ISaB1.

This type of integrated construct comprising a phage miniMu fragment, a homologous DNA sequence and a selection gene, such as the one described above, allows overexpression of specific genes or, if necessary, gene disruption.

These latter constructs differ from the earlier constructs referred to simply as "integrons".

The present invention also relates to the coryneform bacterial strain obtained by introducing the integrant through integrative transformation with the aid of the aforementioned integrant, especially through electrotransformation.

Among the available strains of coryneform bacteria, those particularly suitable for industrial production are:

Brevibacterium lactofermentum (B lactofermentum)

Brevibacterium flavum (B flavum)

Corynebacterium glutamicum (C glrtamicum)

Corynebacterium molasses (C.melassecola)

In the case of clonal selection using hosts other than coryneform bacteria as definitive hosts, possible replication properties can be engineered in coryneform bacteria to adapt integrons to coryneform bacteria. To do this, a plasmid containing a replicating part in addition to the integron can be introduced into the strain, or integrated, and the plasmid extracted and digested with one or more enzymes that release the integron to recover the adapted integrant. Alternatively, the purified fragments may or may not be ligated and the ligation products used for integrative transformation; in this case, the restriction barrier does not pose too many difficulties.

In some cases, transfer from a coryneform bacterial intermediate is beneficial, particularly where the DNA is derived from E. coli, it may be advantageous to adapt the integron to Brevibacterium lactofermentum before adaptation to C. molasses.

Finally, the present invention also relates to the application of the coryneform bacteria of the present invention in industrial production, especially the application of the integron of the present invention in the preparation of proteins or metabolites.

The following examples will further demonstrate the advantages of the present invention.

Figure 1 is a flow chart for the preparation of integrants starting from a replicating plasmid,

Figure 2 shows the insertion of integrons:

· After a single recombination (a)

· After double recombination (b),

Figure 3 shows the structure of pCGL519,

Figure 4 shows the percent kanamycin resistance of two transformed strains as a function of time,

Figure 5 shows the structure of miniMu,

· MudⅢ 1681,

· MudⅢ 1681-Cat,

Figure 6 shows plasmids pCGL107 and pCGL107::Mud+,

Figure 7 shows the integration of integrons with and without miniMu,

Figure 8 shows a restriction map of the insert of the Brevibacterium lactofermentum CGL2005 (B115) clone starting from the 3' terminal insert region of the lac operator,

Figure 9 shows the ISaB1 sequence,

Figure 10 is a restriction map of ISaB1,

Figure 11 is a restriction map of plasmid pCGL330,

Figure 12 is a restriction map of plasmid pCGL331.

Embodiment 1: Construction of the chromosomal DNA library of Corynebacterium molasses ATCC17965 and the cloning of gltA gene

Chromosomal DNA of Corynebacterium molasses ATCC17965 strain was prepared according to the method proposed by Ausubel et al. (1987). 10 µg of the above DNA was digested with restriction endonuclease MboI (Boehringer). DNA fragments were separated by size on a sucrose gradient as described by Ausubel et al. (1987). Fragments with a size of about 6-15kb are reserved for the construction of DNA libraries.

The plasmid for cloning pUN121 (Nilsson et al., 1983) was prepared from ad libitum E. coli GM2199 strain according to the method of Birnboim and Doly (1979). The plasmid was linearized by cutting with restriction endonuclease BclI (Boehringer).

Under the conditions described by Ausubel et al. (1987), 1 μg of the plasmid pUN121 linearized by BclI and 2 μg of the above-mentioned 6-15kb DNA fragment were ligated with D4 DNA ligase to construct a DNA library. The ligation mixture was introduced into E. coli strain DH5α by electroporation following the procedure described by Dower et al. (1988). Colonies with recombinant plasmids capable of growth were directly selected on LB medium containing 10 μg/ml tetracycline. Plasmids of all tetracycline-resistant clones were prepared according to the method of Birmboim and Dloy (1979). A collection of these plasmids is equivalent to a DNA library.

Escherichia coli W620 strain lacking citrate synthase activity was transformed with the DNA library of Corynebacterium molasses ATCC17965. E. coli W620 transformant clones capable of growth were selected on minimal selection medium containing tetracycline. This clone carries the recombinant plasmid pCGL508. Differences in clonal selection limited the C. molasses DNA fragment carrying the complete gltA gene to a 3.5 kb DNA fragment defined by two HindIII sites.

Example 2: integron pCGL519

The process of preparing integrons is shown in Figure 1.

The gene aphIII was selected as the selection gene; when a copy is integrated in the chromosome, it confers resistance up to 600 μg/ml kanamycin. The general situation is 25μg/ml. The 3.5 kb HindIII fragment having the structural gene gltA encoding the citrate synthase of Corynebacterium molasses, which was obtained in Example 1, was initially selected as a homologous DNA fragment of the genome of the genus Corynebacterium, and inserted into multiple cloning sites within the unique site (Hind III site). The restriction sites at the edge of the integron that are expected to be most useful for the integration strategy are the NotI site, which corresponds to a sequence of 8 nucleotides, and the BstXI site. The restriction enzyme BstXI recognizes the sequence (CCAN ₅ NTGG); therefore it also often cuts 6-nucleotide sequences and may generate multiple fragments. However, the released fragments can recombine according to the properties of fragments within different BstXI sites, resulting in a single recombination of the separated fragments.

Plasmid pCGL519 (Fig. 3) is an example of a plasmid susceptible to integron production. Integration assays for Corynebacterium molasses were initially performed in the E. coli plasmid integration cassette. pCGL519 is composed of two marginal NotI sites, replicable and integrating fragments. The first fragment corresponds to an integron containing the multiple cloning site, the selection gene aphIII and the homologous HindIII fragment of the chromosome carrying the gltA gene encoding citrate synthase. The second fragment contains the replication part of the plasmid pBLⅠ that can replicate in coryneform bacteria (3kb SspI-HpaI fragment), the replication part of the metaplasmid pACY184 that can replicate in Escherichia coli, orip15A and the origin of replication of the reverse complementary phage M13 . The 3.5kb HindIII fragment containing the gltA gene was inserted into the vector pCGL243 in Escherichia coli to construct plasmid pCGL159.

Brevibacterium lactofermentum was transformed with pCGL519 after clone selection as shown in Figure 1 . When pCGL519 was transferred into Brevibacterium lactofermentum CGL2002, the NatI fragment containing the origin of replication was replaced because the replicating part of pBLI was inactive in E. coli. This demonstrates a compensating advantage of the cassette structure.

Example 3: Integration

Starting from a plasmid extract of Brevibacterium lactofermentum, pCGL519 was transferred into Corynebacterium molasses ATCC17965 which is very restricted relative to E. coli. The proposed system allows the isolation of plasmid pCGL519 from the Corynebacterium molasses ATCC17965 strain, which is completely restricted with respect to E. coli and only partially restricted with respect to Brevibacterium lactofermentum. In this way, an integration cassette modified for the recipient strain is produced.

After digestion of plasmid pCGL519 from Corynebacterium molasses ATCC17965 with restriction endonuclease NotI (Boehringer), the integrant containing the gltA gene and the selection gene AphIII was isolated and purified on a low-melting agarose gel. The integrons thus purified were ligated to themselves to form a circle. This ligation mixture was then introduced into the C. molasses ATCC 17965 strain by electroporation (Bonamy et al., 1990). Clones of C. molasses exhibiting resistance to 25 μg/ml kanamycin were analyzed.

500 transformants were obtained. Of the 50 transformants analyzed, 31 did not possess plasmid pCGL519, of which 20 were analyzed by Southern blot after digestion with XbaI. All of them were consistent with integration by homologous recombination within the gltA region. Depending on the nature of the ligation product (cyclic monomeric molecules or linear or circular polymeric molecules), the first integrant can be interpreted based on the results of a simple or double "crossover". Analysis was performed in pulsed field after NotI digestion and hybridization with a probe corresponding to the 3.5 kb HindIII fragment containing the gltA gene. After this analysis, it was further confirmed that only one copy of the integron was integrated.

Participation in replication of the wild-type gltA gene copy is the measured enzyme activity, multiplied by a factor of 1.82, which agrees with the blot analysis and indicates that samples of the integrated copy are inactive. The results of the comparison of the wild-type strain and the strain transformed with the replicating plasmid are summarized in Table 1. The structural stability of the integration was examined, and it was found that the percentage of kanamycin-resistant cells and the enzymatic activity of citrate synthase remained stable after 30 passages.

Integrated constructs were amplified by selection on incubators containing excess kanamycin at 800 µg/ml, then 1000 µg/ml, and further containing 1000 µg/ml kanamycin and neomycin. Direct tandem amplification products were obtained. Although the amplified construct was stable with kanamycin resistance, the high level of enzymatic activity of the originally obtained citrate synthase was not retained thereafter. This may be the specific inactivation of the gltA gene.

Example 4: Construction based on miniMu

The Mu derivatives used in this example are MidII1681 and MudII1681-Cat (represented by ^KmR and ^CmR, respectively), and their sizes are 14.8kb and 16.6kb, respectively (Fig. 5). miniMu MudII 1681-Cat is a derivative of the transposon MudII 1681 (Castilho et al., 1984). They have the factors necessary for the aforementioned translocation (except HU), as well as the gene of thermosensitive repressor C (the gene that regulates the expression of translocase A and B), antibiotic resistance gene (represented by aphⅡ and cat, respectively) and the lacA, lacY and lacZ' genes. The latter starts at the 8th codon and detects transfusion fusion of the protein when the Mud is inserted in frame.

These transposons were transferred into coryneform bacteria. After integration of the transposon into the chromosome, the integrated copy can be amplified as described in Example 8. In connection with this amplification, the integrated target gene (the structural gene for glutamate dehydrogenase) can also be similarly amplified in many cases, resulting in an increase of up to 25-fold in the corresponding enzymatic activity.

Embodiment 5: the construction that is used for the carrier of miniMu transfer

The integrated vector (pCGL107, Figure 6) contains the gdhA gene (Gdh') interrupted by the kanamycin resistance ^KmR marker (aphIII), the replicon (ori) of pUN121 (Nilsson et al., 1983) and the conferring tetracycline resistance. sex (Tet ^R ) and ampicillin resistance (Amp ^R ) genes. This vector cannot replicate in Brevibacterium lactofermentum but integrates by a simple "crossover" at the homologous site of the gdhA gene (Leblon et al., 1990).

MudⅡ1681-Cat was introduced into the integration vector pCGL107 from Escherichia coli OR1836 in the MC4100 strain by minimuduction. Among the different insertions obtained, one retained the E. coli lac- phenotype (pCGL107::Mud+, Figure 6). Mud from pCGL107::Mud- was removed from the same plasmid (Fig. 6) and in which the genes for translocase A and B were deleted by HindIII digestion.

Other transfer strategies have also been tested; introducing MudII 1681 into coryneform bacteria with the transposon on two different types of vectors:

- The suicide vector (non-replicating, non-integrating) pEV11::Mud, which involves a derivative of the gene pUC18 without the introduction of MudII 1681.

- Shuttle vector (pCGL229) with the replicon of pBL1 (HindIII-HpaI fragment), the p15A replicon and the Cat gene of Tn9.

In Rec+ E. coli, MudII 1681 was introduced into the shuttle vector by minitransduction. Of the various insertions obtained, one still retained the Lac- phenotype (pCGL229::Mud+). From this vector, Mud from which pCGL229::Mud- has been removed and in which the genes for translocase A and B have been deleted by PstI digestion can be prepared.

Example 6: Constructed electroporation efficiency and its transfer in Brevibacterium lactofermentum

Escherichia coli strain (DH5α) and two Brevibacterium lactofermentum strains CGL2002 and CGL2005 (B115) were transformed with the aforementioned vectors. Both strains are only partially permissive relative to E. coli DNA. The results of these experiments are listed in Table 2, and from this the following observations can be drawn:

- In E. coli, the net efficiency of transformation is low when Mud+ is present on the vector, both in the case of the shuttle vector pCGL229 and in the vector pCGL107 (which is likely to replicate in E. coli), but when the transposon is deleted Then there is another situation. This typical phenomenon of replicating plasmids carrying active Mu derivatives may be attributed to the very strong expression of the Mu translocase in their natural hosts. This indicated that the Mud+ (MudII1681 and MudII1681-Cat) utilized in the above construction had good activity.

- In none of the coryneform bacterial strains tested, it was not possible to obtain transformants with the shuttle vector pCGL229::Mud+ or - (nor with the suicide vector pEV11::Mud). In the case of using a derivative of pCGL229, it is possible that its replicon pBL1 is inactive when microtransduced into E. coli Rec+, since this vector is not amplified in Brevibacterium lactofermentum.

- In Brevibacterium lactofermentum CGL2005 (B115), transformants can be produced using the integration vector pCGL107 and its derivatives. Similar transformations were obtained with pCGL107::Mud+ and pCGL107::Mud-. This indicated that the efficiency of MiniMu MudII1681-CatA+B+ was not high when introduced into Brevibacterium lactofermentum by means of an integrating vector.

The observed reduced transformation efficiency of the two pCGL107 derivatives compared to pCGL107 itself was indeed due to the increased length of the transformant plasmids (10 kb in the case of pCGL107 and 10 kb in the case of pCGL107::Mud+). 26.7 kb compared to 22.1 kb in the case of pCGL107::Mud-).

Example 7: Study on the integration of pCGL107::Mud+ in Brevibacterium lactofermentum CGL2005 (B115)

147 kanamycin (25 μg/ml) resistant clones were obtained from pCGL107::Mud+ (hereinafter referred to as pCGL320) transformed strain CGL2005 (B115), but none of the transformants were selected against chloramphenicol (5 μg/ml) resistance of. However, 103 of these clones were resistant to chloramphenicol after selection on chloramphenicol. Thereby it was shown that the Cat gene of Tn9 was insufficient to express a single copy allowing initial selection of clones; instead, this expression was found to be sufficient by subsequent scratch testing to detect the resistance phenotype. The resulting clones exhibited a lac-phenotype corresponding to that seen in E. coli.

The two types of integrants obtained (Km ^R Cm ^S for type 1 transformants and Km ^R Cm ^R for type 2) may correspond to the replacement of the gdhA gene by a double "crossover" and that obtained by a single "crossover" Integration of the complete plasmid at the gdhA site (Figure 7). The relevant data for this interpretation are:

- Dosage of glutamate dehydrogenase in type 1 and type 2 transformants (see technique of Meers et al., 1970) (Table 3): 5 out of 7 transformants of type 1 (Examples K2 and K3, Table 3) There was no gdh activity at all, which can be seen as a result of replacement of the gene for gdhA by the interrupted gene. Four out of five transformants of type 2 were confirmed to have similar gdh activity (examples KC2 and KC4, Table 3).

- Southern blot molecular analysis of BamHI digests corresponding to type 1 (K2) and type 2 (KC2 and KC4) transformants, and characteristic bands revealed by pCGL147 plasmid probe (results not shown). These results indicated that the molecular structure of the transformant gdh(K2) further confirmed the gene substitution. It was also confirmed that digestion of the plasmid at the gdhA site resulted in gdh+ transformants (type 2 (KC2 and KC4) and several type 1 transformants).

Example 8: Selection of possible translocations

In the presence of chloramphenicol (5 μg/ml), the isolated colonies could still grow, indicating that the type 2 transformants (Km ^R and Cm ^R ) could not sufficiently express the chloramphenicol resistance gene. To this end, we studied transformants of the ^CmR subclones and wished to select for the translocation of Mud (which carries the cat gene). These subclones were obtained at a frequency of 1 in ¹⁰⁵ cells. After replication on Xgal, these clones exhibited a color gradient from white to blue (net 30% blue). These results were further confirmed by measuring β-galactosyl activity (Table 4). This suggests that translocation of Mud amplifies chloramphenicol resistance and exhibits β-galactosidase activity due to fusion of the protein insertion site. In fact, the same experiments with pCGL107::Mud- gave the same results, indicating that these phenomena were not the result of translocation.

Example 9: Evidence of tandem amplification of plasmid pCGL107::Mud+ on chromosome

It was confirmed that almost all clones (except KC3T4) isolated previously had amplified gdh activity (Table 4). In addition, β-galactosidase (measured according to the method described in Miller, 1972) is almost proportional to the activity of gdh, except for a few cases (KC3T4). The same result was obtained in the detection of chloramphenicol acetyltransferase (according to the method described in Show, 1975, Table 5). This result contradicts Mud's translocation, because Mud never takes the adjacent sequence with it during translocation. Specifically, this indicates a tandem expansion of the repeat unit pUN-Mud-gdh on the chromosome, making it bordered by sequence homology. This was confirmed by BamHI, NotI and XbaI digestion of genomic DNA of strains KC3, KC3T1 and KC3T3. Not only the BamHI band inside Mud (equal to 7 kb), but also the bands containing the border region of Mud (11 kb and 2.4 kb) were also amplified, confirming the opposite effect of tandem amplification and translocation. In addition, digestion with NotI (cutting only once in MudII1681-cat) and XbaI (cutting only once in gdh') significantly amplified the 2.4 kb band of the repeat unit.

Under no selection pressure, β-galactosidase activity was measured to retain 70% of the activity level after 15 passages, indicating that the amplification is quite stable. Cat and Gdh activities were also lost by 30% after 25 passages. This tandem amplification is reminiscent of the results of experiments with Bacillus subtilis by Albertini et al. (1985) and Janniere et al. (1985). The β-galactosidase activity of the amplified clones can be attributed to the amplified parasitic transduction present in Brevibacterium lactofermentum but not detectable in E. outside the reading frame region of the resistance gene).

Example 10: Investigation of the properties of the inserted part ISaB1

After recovery of the E. coli DH5α plasmid, the insertion element was isolated starting from the amplified DNA of KC3T4. Genomic DNA of Lactobacillus fermentum CGL2005 (B115), certain derivatives and other strains of coryneform bacteria was probed with probes containing previously isolated insertion elements. The 3.5 kb PvuII fragment within Mu derived from DNA amplified in _KC3T4 and containing the insertion element was used to probe the _initial blot containing the integrant and the BamHI digest DNA of each amplified strain (KC3T4). Since the insert does not contain a BamHI site, this experiment can reveal BamHI genomic fragments containing at least one insert or insert. In strain K1- (corresponding to the type 1 substitution integrant from pCGL107::Mid-), 5 bands appeared, indicating the presence of multiple copies (integer or non-integer) of the insertion.

Genomic DNA from Brevibacterium lactofermentum CGL2005 (B115), whose BamHI digestion product showed four bands shared with K1- (corresponding to the size of 18kb, 5.9kb, 5kb and 4.5kb); present in other strains K1-, KC1- and the fifth band (6.5 kb in size) in KC3 showed a translocation within the segregant of strain CGL2005 (B115) in the source of these strains.

Between two different Brevibacterium lineages (i) Brevibacterium lactofermentum CGL2005 (B115) and (ii) Brevibacterium lactofermentum CGL2002, there are two common bands of 18 kb and 4.5 kb in size; in contrast, strain CGL2002 has There is no additional belt. This demonstrates the mobility of these elements. The C. molasses strains gave a very weak hybridization signal to the insert, translated into other sequences different from those present in these species.

The first migration-specific insertion element of Brevibacterium lactofermentum has been identified and cloned and designated ISaB1, which may be present in multiple copies (2 to 5 copies in the genome). Multiple translocations are readily available at different sites in the amplified region. Different but manifested sequences are present in the genomes of other coryneform bacteria.

ISaB1 is composed of 1288 base pairs; because ISaB1 is inserted into the 3' terminal region of the lac operon corresponding to the sequence determined by Hediger et al. (Biochemistry, Proc. Natl. Acad. Sci. USA, 82, 1985) within the fragment, so its surrounding parts can be identified. ISaB1 inserted between nucleotides 5575 and 5576 and duplicated a 5 bp target sequence (CCGAT) (Fig. 8). The entire sequence of ISaB1 is given in Figure 9, and its restriction map is shown in Figure 10. Two open phases of the reading frame were identified, which, based on the sequence analysis presented, suggested that they might correspond to the structural gene of the translocase and the translocation repressor gene.

Example 11: Capture vectors for insertion elements and transposons

During gene amplification studies in the chromosome of Brevibacterium lactofermentum CGL2005 (B115), an ISaB1 insertion was obtained. In order to isolate all inserted elements that were transferred in coryneform bacteria, special integration vectors were constructed: pCGL330 and pCGL331 (Figures 11 and 12). These two vectors are constructed from the first fragment derived from vector pUN121. Plasmid pUN121 can be replicated in Escherichia coli and carries ampicillin resistance; it has a sequence encoding the CI repressor of lambda phage, which can prevent the expression of fusion between the PL promoter of lambda phage and the tetracycline resistance gene. Insertion inactivation within the Cl gene, and thus the repressor, enables expression of the tetracycline resistance gene in coryneform bacteria under the control of PL. Insertions can also be selected for tetracycline resistance. Cut pUN121 linearly at the SspⅠ site, and digest it with EcoRI filled with Klenow, and fuse with the second fragment obtained from the pCGL107 vector to transform Escherichia coli DH5α to obtain vectors pCGL330 and pCGL331. The difference between the two lies in their The direction of the clone. The EcoRI fragment derived from pCGL107 contains a selection gene suitable for direct transformation of coryneform bacteria (the aphIII gene conferring kanamycin resistance) and a fragment containing the source part of the gdgdhA gene element (the structural gene of glutamate dehydrogenase), It acts as an integration point in the chromosome of coryneform bacteria.

For kanamycin resistance, strains of Brevibacterium lactofermentum CGL2005 (B115) and CGL2002 can be electrotransformed with plasmids pCGL330 and pCGL331. In each case, the transformation frequency was approximately 10 exp3 per microgram, which was compatible with the integration of the plasmid. These transformants (such as CGL2005::pCGL330 and CGL2005::CGL331) are very sensitive to tetracycline, which proves that the regulation of Cl on _PL plays a good role in the transformants. The frequency of tetracycline resistant segregants was tested after 10 passages.

Mutations that inactivate the Cl gene occur at a frequency of 2 x 10exp-6 per generation in strains with integrated plasmids pCGL330 and pCGL331.

Tetracycline-resistant segregants were isolated from strains CGL2005::pCGL331 and CGL2005::pCGL330 and digested with PstI, from which genomic DNA was extracted to recover plasmids.

To obtain tetracycline resistance, these digested DNAs were ligated, and the ligation product was used to transform E. coli strain DH5α. The recovered plasmids were analyzed and the insertion element was identified for 7 of 9 tetracycline resistant clones. In one case (from CGL2005::pCGL331 ), an insertion element (1.2 kb in size with unique AccI, EcoRV and xhoI sites) was identified from the identified IsaB1 based on localization within Cl. In another case (from CGL2005::pCGL330) an insertion element of a different ISaB1 (1.0 kb in size with AccI but no EcoRV and xho sites) was identified.

Capture vectors embody the function of transposons; in most cases, the resulting mutation is an insertion; the frequency of translocations can be detected fairly accurately from the frequency of tetracycline resistance; elements with the greatest migration have been identified; these include ISaB1 was reisolated and additional insertion elements of different ISaB1s were identified.

The sources of the strains listed are:

Escherichia coli

·DH5alpha ：GibcoBRL

Mc4100: Casadabam (1976)

·OR1836：Reyes

Brevibacterium lactofermentum

· CGL2002: Bonamy et al. (1990)

· CGL2005 (B115): Bonnassie et al. (1990)

Corynebacterium molasses

·ATCC17965 ：ORSAN

· ATCC17965::gltA: (this application)

Four of these strains have been deposited at la Collection Na-tonale de Cultures de Microorganismes (CNCM) de l'Institut Pastewr (Paris) on July 23, 1991:

- Corynebacterium molasses ATCC17965::gltA: No.Ⅰ-1124

- Escherichia coli OR1836: No. Ⅰ-1125

- Brevibacterium lactofermentum CGL2005 (B115): No.Ⅰ-1126

- Brevibacterium lactofermentum CGL2002: No.Ⅰ-1127

Strain DH5alpha can be found in the catalog of Clontech Laboratories under the accession number NO.C1021-1 (Palo Alto, CA, USA), and strain MC4100 is ATCC under the accession number No.35695.

Table 1 Specific activity of citrate synthase Strains specific activity relative activity ATCC17965 1,041 1 ATCC17965::gltA 1,893 1,82 ATCC17965 (pCGL519) 5,340 5,13

Citrate synthase specific activity (micromole CoASH/min/mg protein)

Table 2: Transformation efficiencies of vectors with MiniMu

bacterial strain

CGL2002 CGL2005 (B115)

pCGL229 5×10 ⁷ 10 ⁵ 10 ⁶

pCGL229::Mud+ 2×10 ⁴ 0 n·d·

pCGL229::Mud-4×10 ⁶ 0 n·d·

pCGL107 10 ⁵ 3×10 ³ 10 ⁴

pCGL107::Mud+ 5×10 ² 0 4×10 ²

pCGL107::Mud-10 ⁵ n·d·5×10 ²

Table 3: Activity levels of glutamate dehydrogenase during primary conversion

Bacterial strain Gdh specific activity (micromolar

NADPH ₂ consumption

/min/mg protein)

Starting strain km ² cm ²

CGL2005 (B115) 2, 18

Transformant km ² cm ²

k1 2,0

k2≤0,06

k3≤0,06

k4≤0,06

k5≤0,06

k6 2, 18

k7≤0,06

Transformant km ² cm ²

KC2 2, 24

KC3 2, 12

KC4 2, 18

KC5 3,45

KC7 2,06

Table 4: In the amplified integrants, β-galactosidase and

Activity level of glutamate dehydrogenase

Bacterial strains β-galactosidase activity Glutamate dehydrogenase specific activity

Starting strain

CGL205 (B115) 3, 7 2, 48

KC2 6, 2 2, 24

KC3 4,5 2,06

amplified integrant

KC3T1 27,9 18,2

KC3T2 20,7 16,0

KC3T3 48, 2 49, 6

KC3T4 32, 2 2, 24

KC3T5 19, 7 8, 55

KC3T6 19,0 11,9

KC3T7 34,5 28,0

KC2T1 7, 4 2, 73

Table 5: Levels of CAT, βGA1 and GDH activities in the amplified strains

Bacterial strain βgal activity eat specific activity Gdh specific activity

Starting strain

CGL2005 (B115) 3, 7 ≤ 0, 07 2, 18

primary integration

K2 ≤0,07 ≤0,006

K3 ≤0,07 ≤0,006

primary integration

KC3 4,5 2,72 2,06

KC7 3, 4 1, 82

Amplified integration

KC3T1 27, 9 19, 7 18, 2

KC3T3 48, 2 33, 3 49, 6

KC3T4 32, 2 18, 4 2, 24

KC3T5 19, 7 13, 6 8, 55

references

Albertini A.M. and Galizzi A. (1985). Amplification of a chromosomal region in Bacillus subtilis; J.Bacteriol., 162:1203-1211)

Ausubel M.A., Brent R., Kingston R.E., Moore D.D., Seidman J.G., Smith J.A., and Struhl K. (1987) in "Current protocols in Molecular Biology", Greene Publishing Associates and Ci Wiley-Inters

Birnboim H.C.and Doly J., (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA.Nucleic Acid Res.7:1513-1523

Bonamy C., Guyonvarch A., Reyes O., David F. and Leblon G. (1990) Interspecies electro-transformation in Corynebacteria. FEMS Microbiology Letters 66: 263-270

Bonnassie S.Oreglia J.Trautwetter A.and Sicard A.M. (1990) Isolation and characterization of a restriction and modification deficient mutant of Brevibacterium lactofermentum, FEMS Microbiol.Letters, vol3-142:1

Casadaban M.J.Transposition and fusion of the lac genes to selected promoters in E.coli using bacteriophages lambda and Mu.J.Mol.Biol.104(1976) 541-555

Castilho B.A., Olfson P.and Casabadan M.J. (1984) Plasmid insertion mutagenesis and lac gene fusion with miniMu bacteriophage transposons, J.Bacteriol.158:488-495

Dower W.J., Miller J.F. and Ragsdale C.W., High Efficiency transformation of E. coli by high voltage electroporation. Nucleic Acid Res. 16 (1988) 6127-6145

Janniere L., Niaudet B., Pierre E., Ehrlich S.D. (1985) Stable gene amplification in the chromosome of Bacillus subtilis, Gene 40:47-55

Maniatis T., Fritsch Ed. and Sambrook J. (1982). Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

Meers  J.L.Tempest  D.W.and  Brown  C.M.Glutamine（amide）：2-Oxoglutarate  Amino  Transferase  Oxido-reductase（NADP），an  Enzyme  Involved  in  the  Synthesis  of  Glutamate  by  Some  Bacteria  J.General  Microbiology  64（1970）187-194

Miller J. (1972) Experiments in Molecular Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) p352-355

Nilsson B., Uhlén M., Josephson S., Gatenbeck S. et Philipson L., 1983. An improved positive selection plasma vector constructed by oligonucléotide mediated mutagenesis.Nuc.Acid.Res.9-8,801

Shaw W.V. (1975) Chloramphenicol acetyl transferase from chloramphenicol resistant bacteria. Metho. in Enz. 43: 737-755

Claims (26)

1, the integron of bar shaped bacteria is characterised in that it comprises

--guarantee effective gene of selecting in said bar shaped bacteria,

--the genomic homologous sequence of said bar shaped bacteria, wherein said sequence is adapted to said bacterium.

2, according to the integron of claim 1, the plasmid that is characterised in that its source also comprises and duplicates part except that containing integron, and this integron is arranged in the both sides of the opposite tumor-necrosis factor glycoproteins that is equivalent to integron unrestriction site.

3,, be characterised in that duplicating part is included in effective replication origin in the non-bar shaped bacteria according to the integron of claim 2.

4,, be characterised in that replication origin is effective in intestinal bacteria according to the integron of claim 3.

5,, be characterised in that duplicating part is included in the bar shaped bacteria effectively replication origin according to each integron among the claim 2-4.

6,, be characterised in that it also comprises the sequence of transposable element according to the integron of claim 1.

7, according to each integron among the claim 1-6, be characterised in that transposable element comprises the sequence from bar shaped bacteria.

8,, be characterised in that this sequence is from brevibacterium sp according to the integron of claim 7.

9, integron according to Claim 8 is characterised in that this sequence is from as shown in Figure 9 ISaB1.

10, according to each integron among the claim 6-9, be characterised in that it except that comprising protein coding sequence, also comprise the transposable element sequence of assurance by the coryneform bacteria transposition.

11, according to each integron among the claim 6-10, be characterised in that these transposable element sequences lack the sequence of the translocase of encoding.

12, according to each integron among the claim 1-11, be characterised in that it comprises a useful sequence.

13,, be characterised in that peptide or protein that said useful sequence encoding is useful according to the integron of claim 12.

14,, be characterised in that useful proteins matter is homologous protein according to the integron of claim 13.

15,, be characterised in that useful proteins matter is heterologous protein according to the integron of claim 14.

16, according to each integron among the claim 13-15, be characterised in that useful proteins matter is enzyme.

17,, be characterised in that coding useful proteins sequence is selected from following gene according to the integron of claim 16:

-gltA，

-gdhA。

18, according to each integron among the claim 2-17, be characterised in that non-existent restriction site is selected from the integron:

-NotⅠ，

-BstⅩⅠ。

19, according to each integron among the claim 1-15, be characterised in that said sequence is adapted to the bar shaped bacteria bacterial strain.

20,, be characterised in that said sequence was transferred in the brevibacterium sp bacterial strain before being adapted to corynebacterium according to the integron of claim 19.

21,, be characterised in that said preface is amplified according to the integron of claim 20.

22,, being characterised in that amplification produces the sequence of stable integration according to the integron of the amplification of claim 21.

23,, be characterised in that it contains the coryneform homologous sequence of integrating of the molasses of dwelling in Brevibacterium lactofermentus according to the integron of claim 1.

24, utilize the method for each integron conversion bar shaped bacteria bacterial strain among the claim 1-23, be characterised in that through electroporation integron is introduced in the said bacterial strain.

25, the bar shaped bacteria that can make by the method for claim 24.

26,, be characterised in that it relates to following bacterial strain according to the bar shaped bacteria of claim 25:

-Brevibacterium lactofermentus,

-brevibacterium flavum,

-corynebacterium glutamicum,

-molasses the coryneform bacteria of dwelling.

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