JPH05502797A - Corynebacterial integron, method for transforming Corynebacterium using the integron, and obtained Corynebacterium - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Corynebacteria intigulon, the form of Corynebacterium according to the intigulon Transformation method and obtained corynebacteria The present invention relates to the genome of Corynebacteria. The invention relates to the incorporation of predetermined DNA sequences.

This incorporation has two main purposes. On the other hand, Corynebacterium strains This means that the protein is not normally expressed in the relevant strain. either because the strain does not produce homologous proteins or because it causes the strain to overproduce homologous proteins. desired. But on the other hand, it is possible to block the expression of a gene by interfering with the gene. , which is expected to cause deactivation of the corresponding enzyme activity and accumulation of the reactive substrate in the cell. Ru.

In addition to Corynebacterium, Brevibacterium (Brevibacter I) Corynebacteria, including the Initially, most or all of the methods that allowed their transformation were and - transformation using DNA from other bacterial sources is unlikely. Significant limiting barriers exist as a result of the fact that they are often ineffective.

Recently, the electroporation (elecHopo+ation) method has been used to We were able to demonstrate the possibility of transforming Cacteria. however Although the transformation method using self-replicating vectors is useful, it is difficult to integrate into the chromosome. Transformed available bacteria, i.e. with respect to the copy number of the integration element and its location. In most cases, it is important to have strains that are stable over time with respect to Favorable on scale.

The purpose of the present invention is to propose a system for integration into the genome of Corynebacteria. Is Rukoto.

The present invention relates to Corynebacterium intiglones, which are - In the above Corynebacterium, effective Genes ensuring selection - containing homologous sequences of the genomes of the above Corynebacterium; characterized in that the above sequence is adapted to the above bacteria.

“Intigron” has the property of being integrated into the genome of Corynebacterium. Understood as meaning a non-replicating vector, this intigron can be linear or An annular shape is also possible.

However, intigrons can be found in different hosts, such as the large intestine (E+chcric It is usually obtained from self-replicating plasmids that can be synthesized in Hia coli.

However, before the integration step, all traces of DNA of non-corynebacterial origin are preferably removed except for the selection gene and especially all sequences involved in replication. Yes.

The selection genes that are effective in the above Corynebacteria are sensitive to specific substances, especially antibiotics. genes related to resistance to; or a gene that confers a clearly identifiable phenotype, e.g. coloration and/or complementation; It is.

Selection for antibiotic resistance is particularly useful in the present invention. For this reason ・ Aph II+ gene conferring kanamycin resistance, designated as -KmR -Cm” Cat gene conferring resistance to chloramphenicol can be used. However, other genes, especially those related to erythromycin resistance, may Denden may also be used.

“Homologous sequences” correspond to those present in the transformed Corynebacterium or sequences exhibiting a homology level of 80% or more; may be sequences from the same species or from other species, and these sequences may be further It may be synthetic.

The sequences should or should not be made compatible, i.e. they should be made, e.g. Consider the problem of limiting barriers present in Corynebacteria using the methods described below. should be taken into consideration.

Preferably, this intigron is a plaza containing a replication region in addition to the intigron. obtained from Sumid, the inchigron has a restriction site that allows its excision, Preferably restriction sites not present in the intigron, such as Not I, Bs It is flanked by an inverted repeat sequence corresponding to tXJ or 5acI. For this reason, the above Intigron may be obtained directly by digestion with enzymes that recognize restriction sites; Moreover, the resulting ends are complementary, so if necessary, Corynebacterium may be circularized before being used for transformation.

Therefore, within the system according to the invention, intigrons are replicates placed in the replication region. A part of a plasmid vector that can be replicated using a source, i.e. a replicon. It is preferable that there be. Depending on the nature of the replicons present in this replication cassette , this complex plasmid is called coli if it consists of only one endogenous replicon. exclusively in Nebacteria or two different replicons assembled on a plasmid. If combined, each can replicate in its own host, then and is replicated in a foreign host, such as E. coli.

In this case, the assembly of the plasmid is carried out in a system different from that of Corynebacteria. This can also be done with Corynebacterium and Brevibacterium. May be useful sometimes when there are assembly difficulties.

Thanks to the presence of homologous sequences co-existing in intiglon and the genome, intiglon The DNA is inserted into the chromosome through recombination. Therefore, inside the -next transformant, The first gene cloned at the Intigron multiple cloning site was It is duplicated within the bacterial chromosome (see schematic Figure 2a).

Such duplication indicates that the doubling of a gene affects the activity encoded by this gene, especially the corresponding enzyme. It has technical utility in that it causes an increase in elementary activity.

- Next integrant (iniegraα0 structure consists of homologous sequences surrounding the selection gene) Since this corresponds to direct tandem duplication, it is possible to detect strains that overexpress selected genes. Amplifying this structure by selecting primary integrant propagators in the field. Can be done. Therefore, if the selection gene is a gene related to antibiotic resistance, the Strains that are resistant to antibiotics are selected on media with increased antibiotic content; Compatible with any gene or DNA sequence inserted within the intigron, not just the sequence. suitable for overproduction of the relevant gene.

Naturally, intigron has useful sequences in addition to homologous sequences, especially homologous, i.e. obtained from Corynebacterium or heterologous, i.e. obtained from other bacterial species , useful peptides or proteins which may be of eukaryotic or synthetic origin. It would be preferable to include sequences encoding the same.

These sequences preferably contain the requirements that ensure their expression in Corynebacteria. or they are in frame so that they can be expressed by expression elements of the host bacterium. will be inserted.

In the case of Corynebacterium, for example, certain enzymes, particularly gltA or gdhA, It may be useful to obtain overexpression.

As mentioned above, the use of intigron according to the present invention ensures gene blockage. It is possible to use this system to Blocking or as described in Figure 2b The substitution inactivates the corresponding gene, thereby inhibiting the enzyme encoded by the corresponding gene. cause overproduction of the original substrate.

In general, Inchigron is designed in the form of a built-in cassette, i.e. in some cases In addition to the selection gene and homologous sequences, which may be identical, the desired DNA sequence and/or a sequence containing several cloning sites to allow insertion of the gene. is obtained.

In all cases, for the purpose of genetic restriction, the species lacking replicative DNA from other species is Attempts are made to provide antiguron.

It is also possible to provide more complex embedded systems, especially embedded systems that further include arrays of transposed elements. It is possible. The sequence of the transposable element encodes the Corynebacterium-encoded protein. All sequences that ensure rearrangement except for those in transboses It may also be a transposable element that lacks the coding sequence for the transposable element.

Among the transposable elements, mention may be made in particular of the Mu phage in the form of a mini-Mu phage. below In the case of transposable elements such as the mini-Mu phage found in or markers that are part of a different origin were used, e.g. colored markers. .

The present invention relates to transposition enzymes obtained from various Corynebacteria, especially Brevibacterium. In addition, more specifically, it can also be used for intiglon using rsaBl as described in Figure 9. related.

Example 10 shows the characteristics of element l5aB1, and example 11 shows the selection of this type of transposed element. We present a general method that allows for the identification of

The invention relates to repressors of rearranged elements, particularly transboses and/or rearrangements. It also relates to intigrons containing arrays obtained from elements that code for.

Intigrons according to the invention correspond to all or part of the relevant sequence, in particular l5aB1. may contain an array.

This type of set contains a fragment of the mini Mu phage, a homologous DNA sequence and a selection gene. The inclusive structure allows overexpression of a specific gene when necessary, as in the case using the above structure. It is possible to obtain either a gene or a disruption of the gene.

The latter structure, although different from the former one, is nevertheless “introduced” for simplicity. It is called ``Tigron''.

The present invention particularly provides a method for introducing intigrons by electrotransformation. It also relates to Corynebacterium strains obtained by integrative transformation using Ron.

Among the corynebacterial strains that may be used, the following are further identified due to their industrial utility: Knee B, lactofermentum (lactofe+menta) m)-B, flavum -〇, glutamicuml-〇, Melassicola (melas +ecola) cloning is the final host of Inchigron, Corynebacterium In cases where intigron transfer is carried out in a host different from the intigron Assemble inside Corynebacterium to make Ron compatible with Corynebacterium. may take advantage of the possible replicative nature of the body. Therefore, the operation is Just introduce a plasmid containing the replication region in addition to intigron into the strain. This intigron thus adapted was then subjected to plasmid extraction. It is recovered by extraction, then digested with enzymes that release intigulone, and then refined. The generated fragments may or may not be self-ligated, and the ligated products may be used for integrative transformation. In this case, the limiting barrier is no longer an issue.

If it is useful to go through an intermediate Corynebacterium, especially if the DNA In the case of origin, Inchigron is caused by Corynebacterium melassecola. Adapt it to Brevibacterium lactofermentum before adapting it to Brevibacterium lactofermentum That would be useful.

Finally, the invention particularly provides for protein or metabolic production using intigron according to the invention. Use of Corynebacteria according to the invention in industrial processes relating to the production of products Regarding.

The advantages of the invention will be explained in more detail by the following example.

In the attached drawings Part 1 is a schematic diagram of the production of intigron starting from a replicating plasmid.

Part 2 is: ・By single recombination (a) ・Due to double recombination (b) FIG. 2 is a schematic diagram of the insertion of an inigron.

- Figure 3 is a schematic diagram of the structure of pcGL519.

- Figure 4 is a diagram of the kanamycin resistance rate as a function of time for the two transformed strains. It is.

Part 5 is a mini Mu phage: ・Mu dllll 6 8 1 ・　Mudll1681-Cat FIG.

Part 6 is a plasmid: ・pcGL107 and ・pCGL107: :Mud+ FIG.

Part 7 is a schematic diagram of the incorporation of a preferred intigron with or without mini-Mu. Ru.

Part 8 was cloned from an insert at the 3' end of the lac operon. Insertion of Brevibacterium lactofermentum CGL2005 (B115) Represents a restriction map of input elements.

Part 9 represents the I5aB1 sequence.

- Figure 10 represents the restriction map of l5aBl.

- Figure 11 represents the restriction map of the pcGL330 plasmid.

- Figure 12 represents the restriction map of the pCGL331 plasmid.

C, chromosomal DNA of Melassicola strain ATCC17965 was analyzed by Atzubel et al. (1987).

MboI restriction endonuclease [Boehringer (Boeh+1nBr): controlled cleavage by following the procedure described by Maniatis et al. (1982). The test was carried out using 10 μg of this DNA.

This DNA fragment was analysed, as described by Au5obel et al. (1987). separated according to their size on a base gradient.

Fragments between 6 and 15 kb in size were selected for library assembly.

pUN121 cloning plasmid (llil++on et al.

Bi+nbaim and Obtained by the method of Doly (1979). The plasmid was transformed into a BclI restriction endonuclease. linearized with enzyme (Boehringer).

The library was linearized with 1 μg of pUN121 plasmid with BclI and From 2 μg of 6-15 kb DNA fragments, the method described by Aosubel et al. (1987) The DNA was assembled by ligation with T4 DNA ligase (Boehringer) under controlled conditions. Conclusion The mixture was electrolyzed according to the procedure described by Dove et al. (1988). It was introduced into E. coli strain DH5alpha by poration. recombinant plasmid The E. coli clone with directly selected based on their ability to do so. All tetracycline resistant clones A plasmid of this type was obtained by the method of Bi+nboim and DOfy (1979). all These plasmids assembled together correspond to a DNA library.

C. E. coli strain W620 lacking citrate synthase activity.

Transformation was performed with Melassicola ATCC17965 DNA library. Tetrasa Select E. coli W620 transformed clones that can grow on minimal selective medium containing icrin. I chose.

This clone carries the pcGL508 recombinant plasmid. various subclones At the same time, C. and Melassicola DNA fragments containing the complete gltA gene were combined with two Bi. It can be shortened to a range-restricted 3.5 kb DNA fragment with the ndI11 restriction site. came.

A manufacturing diagram of Inchigron is shown in FIG.

The a　p　　　Ill gene was chosen as the selection gene; it is Resistance to kanamycin within 600μg/m1 when integrated into the chromosome give gender.

25 μg/ml is commonly used. C obtained in Example 1, quene of Mela secola 3.5 kb Hi with gltA structural gene encoding acid synthase n dllll fragment as a homologous DNA fragment of the Corynebacterial genome at the beginning. Select and insert into one of the unique sites of the multiple cloning site (Hindl11 site) did. Limiting the scope of intigrons, which are considered to be the most widely used in embedded strategies. The restriction sites include a NotI site and a BstX1 site corresponding to an 8-nucleotide sequence. It is the rank. The enzyme BstXI recognizes the sequence (CCAN5NTGG); , it cleaves as frequently as 6-nucleotide enzymes and produces only a few fragments. It can be expected that it will be born. However, the released fragment is inside a different Bs tXI site. It is recombined depending on the nature of the sequence, but from now on, the starting fragment alone is reassembled. Should.

The pcGL519 plasmid (Figure 3) is a versatile plasmid capable of producing intiglon. This is an example of C. The installation of the built-in cassette in Melasecola is No. E. coli pcGL5 consisting of two replication and integration fragments restricted at the tI site. The plasmid originally obtained from No. 19 was used for testing. The first fragment is polyclonin code for programming site, ap h III selection gene and citrate synthase Inchglobin containing the homologous Htndlll fragment of the chromosome carrying the gltA gene corresponds to The second fragment is the pBLI plasmid that is replicated in Corynebacteria. Replication region (3kb Ssp I-Hpa I fragment), replicated in E. coli orj p15A. The replication region of the generated pAcY184 plasmid and the trans-complementary M13 fur Contains a source of reproductions. The pcGL519 plasmid was inserted into the pCGL243 vector. Constructed in E. coli by inserting a 3.5 kb HindII+ fragment containing the ltA gene Ta.

As shown in Figure 1, after cloning, pCGL519 was used to transform the cells. Brevibacterium lactofermen Simultaneously with the transfer of pcGL519 into TamCGL2002, the replication region of pBLI was Since it was inactivated in E. coli, the Notl fragment containing the replication origin was replaced.

This further illustrates the advantages of the cassette construction.

Corynebacterium melassicola ATCC17 highly restrictive to E. coli Transfer of pcGL519 to 965 is shown in B. The same strain extracted from Lactofermentum This was done using a similar plasmid. The proposed system is completely restrictive for E. coli However, B, Lactofermentum is only partially affected by C, Melat. It was possible to isolate the pcGL519 plasmid from Secora strain ATCC17965. make it possible. In this way, an integration cassette carrying the changes in the recipient strain was obtained.

C, pcGL519 plasmid obtained from Melassicola ATCC17965 After cutting with Notl restriction endonuclease (Boehringer), the gltA gene Intigron containing the progeny and Aph+11 selection gene was grown in a low melting point agarose gel. isolated and purified from The intigron thus purified is then combined with Subjected to automatic recyclization. The binding mixture was then electroporated (Bonam y et al., 1990) into C. melassecolla strain ATCC17965. Kana A C6 Melassicola clone resistant to 25 μg 101 of mycin was subjected to analysis.

Five hundred transformants were obtained. 31 out of 50 analyzed were pcGL519 plus 20 of them were analyzed by Southern blot after XbaI cleavage. .

They all follow a similar integration event carried out by homologous recombination in the gltA region. Equivalent to. The nature of the conjugation product (cyclic monomer molecules or linear or cyclic polymers) Depending on the molecule), -order integrants can be obtained from single- or double-crossovers. It is interpreted as Pulse field analysis after NotI cleavage followed by glt Hybridization with a probe corresponding to the 3.5 kb Hi nd Ill fragment containing A A pad formation was performed. Incorporation of a single copy of intigron is confirmed in this analysis.

The model involves duplication of a wild copy of the gltA gene: measurements of enzyme activity showed that It increases by a factor of 1.82, which is consistent with the interpretation of the plot and indicates that the copied copy has not been inactivated. The results showed that wild-type strains and replication They are summarized in Table 1 for lasmid-transformed strains. built-in structure Stability is determined by the percentage of kanamycin-resistant cells after approximately 30 generations (Figure 4) and the enzymatic activity of stabilized citrate synthase.

Amplification of integrated structures is achieved by selection of growth on dishes containing excess kanamycin. , 800 μg/ml, then 1000 μg/ml and then kanamycin. and neomycin selection at 1000 μg/ml. direct tandem increase I got breeding. Despite the stability of the propagated construct and kanamycin resistance, The initially high level of enzyme activity obtained in the case of phosphate synthase is subsequently maintained. I couldn't. It is possible that specific inactivation of the gltA gene occurred.

The Mu derivatives selected in this example have sizes of 1'4.8 kb and 16.6 kb. Mudll1681 and Mudll1681-Cat (each with KmR and CmR) (Fig. 5). Mi=MuMudll1681-Cat is trans It is a derivative of poson Mudll1681 (Cattilho et al., 1984) . It contains the elements necessary for dislocation (excluding HU), which have already been shown, as well as the heat-sensitive replenishment. saucer gene C (regulator of transposase A and B expression), antibiotic Genes related to quality tolerance (aphll and cat, respectively) and 1acA, 1acY, and It has the 1acZ-gene.

The latter starts at the 8th codon and is tagged when M ud is inserted in the open reading frame. Enables detection of protein translational fusions.

These transposons were introduced into Corynebacteria. inside the chromosome After integration of the transposon, the method described in Example 8 amplifies the integrated copies. I was able to Along with this amplification, the target gene of the integration event (glutamate des Structural genes related to hydrogenase) also correspond within 25 times in many cases. amplified by increased enzyme activity.

” Assembly of vectors for mini-Mu transfer Integration vector (pcGL107-Fig. 6) is the kanamycin resistance marker Km (aph[lll, pUN12 luprico (Nil+son et al., 1983) and tetracycline (Te g blocked by genes conferring resistance to ampicillin (AmpR) and ampicillin (AmpR). This vector contains the dhA gene (Gdh”). ・If it is not replicated within Lactofermentum, it is due to the phase of the gdhA gene. It is integrated into the same site in a single “crossover”.

Mudll1681-Cat was added to the MC4100 strain from E. coli. into the integration vector pCGL107 by minimnduction. I entered. Among the various inserts obtained, some showed a Lac-phenotype in E. coli. (1)CGL107::Mud+, Figure 6) was selected. transposase Disabled M u d in which genes related to A and B are deleted by Hindll cleavage (pCGL107.:Mud-1 Figure 6) was obtained from this same plasmid.

Other transfer strategies were tested; It was introduced into Corynebacteria by inserting a transposon inside.

- Mudl11681 is a derivative of pUc18 lacking the lac gene introduced into it. Suicide vector (non-replicating, non-integrating) pEVI 1::Mud -pBL lupricon (Hindlll-Hpal fragment), p15A replicon and a shuttle vector (pCGL229) containing the Tn9ca gene, Mudll1681 was shut down by minimuduction in E. coli Rec book. was introduced into the Levector. Among the various inserts obtained, Lac-phenotype (pCGL229::Mud+) was selected. transbozase Disabled MudSpCGL2 in which genes related to A and B have been deleted by PstI cleavage 29::Mud- was obtained from this vector.

Escherichia coli (DH5alpha) strain and two Brepinocterium lactofer Mentum strains CGL2002 and CGL2005 (B115) with the above vector. Electrotransformed. These two strains are partial to the DNA obtained from E. coli. permissible. The experiment gave the results shown in Table 2. The following comment was added Runi -Transformation efficiency is substantially reduced in E. coli because Mud+ is present in the vector. In the case of shuttle vector pCGL229 or vector if present - This is the case of pcGL107 (which can replicate there) and the transposed gene is deleted. This is not the case. Typical for replicating plasmids with active Mu derivatives This phenomenon is due to the very high expression of Mu transisase in its natural host. giving. This is the Mud+h transbozosis M used in the above assembly. u dllll 681 and Mudll1681-Cat) effectively activate thealco I'll show you.

Shuttle vector pCG in all Corynebacterium strains tested L229::Mud+ or = (or suicide vector pEV11::Mud ) cannot be obtained. In the case of pCGL229 derivatives In this case, pBL lupricon was not present during minimuduction into E. coli Rec+. This is due to the fact that B. vector's incompetence.

- The transformants are B, Lactococcus with the integration vector pcGL107 and its derivatives. tofermentum CGL2005 (B115). pcGL107:Mu The transformation efficiencies obtained with d+ or pcGL107::Mud- were similar. This means that mini Mu Mudl11681-Cat A+E+ is an integration vector. B, which is not translocated with high efficiency when introduced into lactofermentum using It shows that

Transformation efficiency of two pCGL107 derivatives compared to pcGL107 itself. The observed decrease (20 factors) in This is probably due to a decrease in the size (10 kb in the case of pcGL107, 10 kb in the case of pcGL10 7:::26.7kb in case of Mud+ and pcGL107::Mud - study of events related to the incorporation of pcGL107:-Mud+ into the case of pcGL107::Mud+ (hereinafter referred to as pcGL320) 147 kanamycin-resistant clones were obtained from the transformation of strain CGL2005 (B115). We were able to obtain a lawn (25 μg/ml), but the transformant was No results were obtained when selecting for mufenicol (5 μg/ml) resistance.

However, 103 of these clones appeared after replication on chloramphenicol. is resistant to chloramphenicol. Therefore, the Tn9cat gene It appears that a single copy of is not expressed sufficiently to perform primary selection of colonies. conversely, this expression can be used to investigate the resistance phenotype in subsequent tests using streaks. is sufficient. All clones obtained correspond to the phenotype observed in E. coli. The phenotype is 1ac-.

Two types of inch grounds were obtained (type 1 transformants, KmRCm8 and T. The type 2 transformant, KmRCmR), has inherited the gdhA gene by a double “crossover” event. Integration of the complete plasmid into the gdhA site by child replacement and a single “crossover” event (Figure 7). Observations that favor this interpretation are as follows.

- Glutamate dehydrogenase assay in type 1 and 2 transformants ( M! ers et al., 1970) (Table 3), seven type 1 transformants. Five of these (e.g. 2 and 3, Table 3) do not have gdh activity; This is consistent with replacement of the gdhA gene by a mutated gene.

Four of the five type 2 transformants had gdh activity similar to the control. (e.g. KO2 and KO2, Table 3).

One type 1 transformant (K2) and type 2 transformant (KO2 and KO2) Southern blot molecular analysis corresponding to BamHI cleavage and plasmid probe p Detection of characteristic bands (shown in Figure 7) by cGL107 (results not shown).

They show that the molecular structure of gdh-transformants (K2) follows gene replacement. . According to them, gdh ten transformants [type 2 events (KO2 and KO2) and Some type 1 events (Kl): ] occur during plasmid integration at the gdhA site. Also check what you can get.

Type 2 transformants (Km and CmR) were treated with 5 μg/ml of chloramphenicol. Add chloramphenicol resistance gene to obtain growth of isolated colonies in the presence of The gene is not fully expressed. Therefore, we have Mu (with the cat gene) In these transformants, subclone C was created with the hope of selecting for the transposition of d. I searched for mR.

These subclones were obtained at a frequency close to 1 per 105 cells. Xgal After replication, these clones exhibit a full range of colors from white to blue (30% are clearly blue color). This result is confirmed by measuring β-galactosidase activity (Table 4). It is caused by Mud transposition, amplification of chloramphenicol resistance and insertion site. It will demonstrate the generation of β-galactosidase activity by protein fusion. actually Similar experiments conducted with , pcGL107::Mud- showed that these events were not transferred. Similar results were obtained, indicating that this was not due to an event.

Example 9 pcGL107::Demonstration of tandem amplification in Mud+ plasmid chromosome Almost all of these previously isolated Lac+ clones (except KC3T4) were expanded. It also appears to have enhanced gdh activity (Table 4). Additionally, one exception (Kc3T4) except for β-galactosidase (examined according to Mille+, 1972) and and gdh activity are approximately balanced. A similar evaluation is given to chloramphenicol acetate. Assay for transferase (according to the method of Shaw, 1975. Table 5) You can also go about it. This result does not coincide with the dislocation of M u d, and the dislocation Never carry adjacent sequences. Rather, it was range-limited by sequence homology Tandem amplification in the chromosome of the repeat unit pUN-Mud-gdh is shown. This point strains KC3, KC3T1 and KC by BamHI, NotI and XbaI This was confirmed by cutting the 3T3 genomic DNA. B a m HI inside M u d band (equal to 7kh), but also undergoes tandem amplification and prevents dislocation events. A band (llkb and 2.4kb) containing the end of Mud is also amplified. Furthermore, NotI cleavage (cut only once in Mu dll 681-Cat) and X baI cutting (cutting only once in gc (h-) effectively repeats the 24 kb band Amplify the unit.

This amplification indicates that β-galactosidase activity is initially active after 15 generations without selection pressure. If it is at a level of 70% of , it seems to be relatively stable. Cat and Gdh Activity also shows a 30% loss after 25 generations. This tandem amplification is carried out by Bacillus subb. In the squirrel (Bacillus 5ubtilis), Albertini et al. (1985) and Iaaniere et al. (1985). amplification The β-galactosidase activity of the clones was shown to be present in B. lactofermentum. Amplified parasitic translation that cannot be detected in E. coli (lac operon fused protein) Research and characterization of the insertion element l5aB1 that contributes to the open reading frame of the ampicillin resistance gene Signs The insertion element was created by extracting the E. coli DH5alpha plasmid from the amplified DNA of KC3T4. It was isolated by collection.

B, Lactofermentum strain CGL2005 (Bi12), some derivatives and Genomic DNA of other strains of Corynebacteria has previously been isolated from proteins containing inserted elements. It was explored using a probe. First, the DNA amplified in KC3T4 was The 3.5 kb P v u II fragment in Mu containing the same insertion element is an inch graph. BamHI-cut DNA from a variety of amplified strains (including KC3T4) An initial plot was used to probe the plot.

Because the insert does not contain a Bam HI site, at least one Reveal BamHI genomic fragments containing two inserts or one insert be able to.

K1- strain (pcGLl　07　:　:　: Type 1 substitution inch obtained from Mud- (corresponding to ground), five bands appear, which corresponds to the insert Indicates the existence of several copies (complete or incomplete).

B, Genome D obtained from Lactofermentum CGL2005 (B115) In BamHI digestion of NA, four bands (each with a size of 1 8 kb.

5.9kb, 5kb and 4.5kb); other strains K1-1KCI- and The fifth band (6.5 kb in size) observed in KO2 was induced by these strains. In the strain CGL2005 (B115) segregant (seHegan+) It may indicate dislocation.

Two different Brevibacterium lineages, (i) Brevibacterium lactofer; Mentum CGL2005 (B115) and (11) Brevibacterium lac The sizes of 18kb and 4.5kb are common to Tofermentum CGL2002. Two bands appear; on the contrary, strain CGL2002 shows no other bands. this is Indicates the mobility of the element. Corynebacterium melassecolla strain is a weak hybrid generates a code-forming signal, and its inserts contain other distinct but related sequences. Indicates the presence in the species.

B. Identification and cloning of lactofermentum-specific first-transfer insertion elements. It was named I5aB1, but several copies (2-5 copies) It may be present in its genome. It differs in the amplified region It can be rotated several times at the site.

The detailed restriction map is publicly known. Different but related sequences in other Corynebacterium It is present in the terrier genome.

I5aB1 consists of 1288 base pairs; the end of I5aB1 is Hcdig a+ e(al, (Biochemi+tr7 Proc, Natl.

Acad, Sci, USA, 82.1982) It was identified because it was inserted into a fragment corresponding to the terminal region (3') of the peron. good. l5aB1 is 55 nucleotides overlapping the 5 bp target sequence (CCGAT) It was inserted between 75 and 5576 (Fig. 8). The complete sequence of I5aB1 is shown in Figure 9. The restriction map is shown in FIG. Transposase structural inheritance according to sequence analysis Two open reading frames corresponding to the child and transposed repressor genes were identified. .

Example 11 Trapping vectors for insertion elements and transposons I 5aB1 insert is B, Lactofermentum CGL2005 (B11 5) was obtained through gene amplification research in the chromosome. Vine transposed in Corynebacteria To isolate all insert elements, the specific integration vectors pcGL330 and p CGL331 was assembled (Figures 11 and 12). These two vectors are pUN1 It consists of the first fragment derived from 21. pUN121 plasmid replicates in E. coli and confers ampicillin resistance: it combines the lambda phage PL promoter with Lambda phage inhibits expression of operon fusion with tetracycline resistance gene It has a sequence encoding the cl repressor. Insertion into cI gene results in the inactivation of the repressor, thereby under the control of P. to express the tetracycline resistance gene expressed in corynebacteria. I'm going to growl. Insertion events may therefore be selected for tetracycline resistance.

Vector pUN121 was linearized at the 5sp1 site and vector pUN121 was linearized with EcoRI. Fused with the second fragment obtained by cGL107 cleavage and supplemented with Flenow fragment. After transformation with E. coli D H5alpba, the vectors pcGL330 and pc GL331 were obtained, which differ with respect to their cloning direction. PC The EcoRI fragment derived from GL107 can be used for direct transformation of Corynebacteria. Selection gene for conversion (aph If! gene that confers kanamycin resistance) ) and gdhA, which functions as an integration site within the chromosome of Corynebacteria. Contains a fragment containing the homologous region of the gene (glutamate dehydrogenase structural gene). I'm reading.

B, Lactofermentum strain CGL2005 (Bi12) and Hi CGL 20 02 for kanamycin resistance using pcGL330 and pCGL331 plasmids. and electrotransformed. The transformation frequency was approximately 1o3/μg in each case However, this is compatible with plasmid integration.

(CGL2005::pcGL330 and CGL2005::pCG L331) transformants show that the regulation of P by cI is successful in the transformants. It is sensitive to tetracycline, making sure it works. tetracyc The frequency of phosphorus-resistant segregants was determined after approximately 10 generations.

Mutations that inactivate the cl gene are associated with integration at a frequency of 2 x 10'/generation. It occurs in strains with mid pcGL330 and pCGL331.

Plasmid recovery was carried out using strains CGL2005: pCGL331 and CGL20. 05:: Tetracycline-resistant Segregan isolated from pCGL330 The analysis was performed using genomic DNA extracted from the plant and cut with Pstl enzyme.

These cut DNAs were ligated together and the ligated product was used to investigate tetracycline resistance. E. coli strain DH5alpha was transformed. Analyze the recovered plasmid As a result, insertion elements were confirmed in 7 out of 9 tetracycline-resistant clones. did. In one case (arising from CGL2005::pcGL331) An insertion element identical to l5aB1 and located within cI was identified (size 1.2 kb, with unique AccI, EcoRV and XhoI sites). One more In one case (resulting from CGL2005::pcGL330), l Insertion elements different from 5aB1 (size 1. Okb, EcoRV or XhoI sites) However, it was confirmed that the ``AccI site'' was found to have an AccI site.

Transposon trapping vectors are functional; in most cases Therefore, the resulting mutation is an insertion.

The frequency of tetracycline resistance therefore reflects the frequency of rearrangements fairly accurately: Therefore, most mobile elements were identified; among them, l5aB1 was We isolated and identified another insertion element distinct from l5aB1.

The strains described have the following origins: Escherichia coli -DH5alpha: Give: 7 (Gibco) B RL・M C4100 :　Ca+adaban(f976)・OR1836:　Re1e+ Brevibacterium lactofermentum CG L 2002: Bon am4 et al. (1990)・CGL2005 (Bl 15) +Bonna++ie (1990) Corynebacterium melassecolla ・ATCC17965:0R3AN ・ATCC17965::gltA: (this application) Four of these strains are 19 Collection Nationale of the Institut Pasteur (Paris) dated July 23, 1991. ・Do Culture Do Microorganism (Collection) Nationale de Cultures de Mic+oorgani+ me+) (CNCM) CC17965::gltA:n' l-1124-E. coli 0R1836:n' l-1125-Brevibacterium lactofermentum CGL2005 (B115):n'l-1126-Brevibacterium lactofermenta Mu CGL2002:nol-1127 Strain D H5alpha is from Clontic Laboratories Catalog fclonN cb Laboratories Catxloque), no.

Available from C1021-1 (Palo Alto, CA, USA), strain M C410 0 is no, available from ATCC as 35695.

Table 1 Specific activity of citrate synthase Citric acid syntax for μmol Co A S H/win/mg protein specific activity of ! ! 2. Transformation efficiency of vectors containing Nimini Mu) transposon pCG1229 5XIQ7105106pCGL229::Mud+ 2xl O’ On, d.

pCGL229::Mud-4X LQ60 n, d.

pcGLI07 10” 3X10” 10’pcGLIO7::Mud+ 5 Kari02 [14 Kari02pCGLIG7::Mud-lff5n, d, 5 Kari02 table 1. - Assay of glutamate dehydrogenase in transformants Initial strain KmSCmS CGL2005 (B115) 2.18 transformant KmRCmS Kl　2・O K2　≦0.06 3≦0.06 4≦0.06 5 ≦ 0.06 6 2. +8 7≦0.06 Transformant Km” CmR KC72,06 assay for enzyme and glutamate dehydrogenase Initial strain KIIISOmS CGL20D5 (B115) 3.7 2.48KC34,52,[15 to C2Tl 7□4 2.73 Table 5 = Assay of CAT, β-GAL and GDH activities in amplified strains Initial strain KIIISCmS CGI, 2H5 (B115) 3.7 ≦0.07 2.18-order integran RcmS K2 ≦0.07 ≦11. (106 3 ≦0.07 ≦0.006 -Next inch ground KIRCIIIR KC34,52,722,06 KC73, 41, 82 amplified inch ground KC3T1 27.9 19.7 18.2 KC3734g, 2 33.3 4 9.6KC3T4 32.2 18.4 2.24 C3T5 19.7 13 ．． Ii 8.55 References Albertini, A. M., and Galizi, (1985).

Amplification or a chromo+ooal regio n in Bacillus sub+if order (staining in Bacillus subtilis) Amplification of color body region); J, 8acterio1. , 162:1203-12 11Ausubel　M,A,,B+ent　R,, niing+ton　R,E ,,Moose D,D.

Seidman J, G,, Sm1th J, A, and 5trobl K, (1987) in’Current protocols in Mo1e cular Biolog7' (current protocols in molecular biology), G +aane Publishing As + ociates and Wiley -1nfe+5science 'Birnboimt(, C, and Dol y　1. , (1979), A + apidalkaline extract ion procedure for +c+eening+ecombina nt plasmid DN person (for screening recombinant plasmid DNA) rapid alkaline extraction operations).

Nucleie Ac1d R2H,, 7115+3-3-1523Bona C,,Guyonva+ch A,,Reye+O,,David F,an dLeblon G, (1990), Intezpeciz select ot+an+to+mationin Co+ynebac+++ia (Corine Cross-species electrotransformation in bacteria), FEMS Mic+obilog y　Levers, 66:263-270Bonna++i+　S,,O+eg lia 1. , TrautveNe+ A, and Sicard A, M, (1 990), I+lation and cha+ac+e+i+ationo f a rest “1ction and modification def icien+ mu+antat B+evibacte+iom lacto lermemtum (restrictions and changes of Brevibacterium lactofermentum) Isolation and characterization of conjugation-defective mutants), FEMS Microbiolog 7 Lev ers, 72:143-146Ca+adaban M,],,T+anspo +1lion and fusion of thelac genz to +e [ected promoters in E, coli asingba c+e+iophage+ lambda and Mu, (bacteriophage lac gene to a selective promoter in E. coli using dilambda and Mu transposition and fusion), I, Mo1. Biol.

Cas+1lho B person, 01json P, and Ca5abada n　M, 1. (1984).

Plasmid 1nsertion mutagenesi+ and la c genefusionwitb miniMu bacteriophag e+ran+po+ons (plasmid insertion mutagenesis and mini Mu bacterium lac gene fusion with phage transcis boson), 1. Bacje+io 1. , 15g: 488-Dowe+ W, J, , Milie+ j, F, and Ragsdxla C, L, HighEflicienc7 +ransfo rmation o! E, eoli bl high voltage ele ct+opo+ajion (High efficacy of E. coli by high voltage electroporation) rate transformation), Nocleic Ac1d Rer.

+6 (1,988) 6127-6145janniere L,, N1aud et B, Pie+re E, Eh "1ich S, D (1985), 5t able gene amplification in the chloroI Ilosomeol Bacillus+ +abtilis Stable gene amplification in the chromosome of Chilis), Gtne, 40:47-55 Maniati+T, Fr1t+ch Ed, and Samb+ook J. (1982:.

Mo1acular clo, ning: A1 laboratory manu al (Molecular Cloning Experiment Manual), Co1d Spring Har barLabo+atoB Press, Cotd Spring [Ia+b or, NYM+ez 1. L,, Tempe+t D, W, and Brown C, M,, Glut+mine(amide):2-Oxoglut2-0x o Am1no Transferase 0xide-raduc+ase( NADP), an EnBme 1nvolvcd in tbeSynth! +i+　o【Glujama+e　b7　Some BacNria　(Some An enzyme involved in the synthesis of glutamic acid by bacteria, glutamine (amide) = 2-o xoglutamate aminotransferase oxido-reductase (NADP )), I.

General Microbiology, 64 (1970H87-194M itler J. (1972), E! pe+iment+ in Mo1ec ularGe++etic+ (Experiments in molecular genetics) (Cold Sp+ 1BHarbor Labo+a+ory, Col1d Spring)Ia+ bor, NY) p352-355 Nilsson B, Uhlen M, I o+eph+on S,, Gatenbeck S.

and Ph1lipson L, (1983), An improved positive+electlion plasmid vector con rt+ucted b7aligonucleo+ide mediated mutagenc+is (modified protein assembled by oligonucleotide-mediated mutagenesis) Good positive selection plasmid vector), NDC, Ac1d Red, 11.8 019-8030 Shav W, V, (1975), Chloramphen icol ac! +Y1transferase from Chloramp henicol resinantbacteria (chloramphenicol) chloramphenicol acetyltransferase from Le-resistant bacteria), Me Th.

in Enr,, 43 near 37-755%7D-=:? '1FF41/:A'7 6 Big flattery mnri D-niji;”? Shino no lk 挾■C21Tn941 27t magshishi sex jiri and × mudll 1681 mudll 1681-Cat Member udll j58L Café S [Q O) t40: 1 Type sequence length of nucleotide EF and its corresponding protein: 1288 bases versus , number of strands, double-stranded arrangement shown with only strands from left to right in the 5'-3' direction, linear chain Type of molecule Genomic DNA Biological source Brevibacterium lactofermentum strain CGL2005 (Bi 12) 1o Direct experimental source, clone characteristics isolated by plasmid recovery 1-30 left end 65-355 code distribution on the designated route Jsu15 487-124 on the designated route 8 chords 1258-1288 (reverse sequence homologous to the right end) Confirmation method Characteristics, chromosomal mobile insertion EVI of Brevibacterium lactofermentum I20cacycxcecctart-rcGtcaxcAccTTc, input AA ccAccAtc^rccrGGGswcGTyvc dingcc6O AtCTCCCAeacQ^reeyeaceCctcc^C〒C^^aact c↑CT dingC^eC^eCc8^^^racτTCY5455Mtlelectronic f龜ap almllaL@wglyal Todoroki tqwanlymahav number 1ph@tar thr9Lu&-inside q1ycy+1kiCOGGCC enter ^(+eeC^Te^e eecte α in ^C〒C in ^^G^CC^cctcc^i [^ya^cecccτ ＾Enter＾Ding 60U tyr41yaha1y*atqLLathraim@lml@IIIV-Oshima Within 1λ▼Turtle 1-Jiichi h1 Ichimushihata-2τO heritage la*nec? CjlmeAfC+ CceTCuτTYXkAtlCmTding? CCζr^CacaMeAAAecc 666hljly@atqvKamegoalaargwaLmtat@marlawl yslawphellytYfthrlyely+atqlO enter＾aateAc eM:eAeycrcreaI:A7 people^^^^c’e^^ku^CkGW dingte▼C C↑Ro^(Ccho↑C〒CroceccV26 5ysvaithrthrtthvalnetam@lye-htlyathe va▲pillptllas-λ threat @vanll7&tQ included＾GTicAl:e Ce F enter ^7AJke (M:^^A?cAccteaT^eCttGkuqαAgu^ T(＾COT＾ζCdingccec＾f?V66 lysph@thtalaaanly+preass@hawa▲tyrwas qsyageInthe+yrxewpeall@! ? ++AtGCtZffe AA? ＾Ding CT＾ccrace↑＾COGτC＾Ding↑C＾CTGCT＾ttce ecc＾uTTG610846Ismlaa groan q 5 Vl@r*gMMEtyrj @wdatj+rWa1118jtpC7atftI@tmtqy91*vya kCue1 11C? ATCc+cACATCAekTGCe? MM:tecC TCC’MCABGACflCOGC dingCATGe(f9Q5q1yph@sa t1ゑ@alaaaphlamtat@tkrmatlm++va1qlnm4 -▲15muLaumtala20^Ctyete^uguC [^idingCCAeW^ CAee161AAA’aAcCTC (hctZAT^me Torishintea^TeC C^tc^P026 tlta●thro auaph 音IL RalptMeY@lyemapLaw91 YLλsat@9▲marmtllyrat＾tc κ＾αokakut ccc c^CAA?　cyc　act　aSekiTel:　TπAACzu＾ccx　a tく虞C〃Cυ108611林qlythrtar-5aamp&#llahm l. @11alallwnorpmAlllbha-5-1*vlylarqq lmeyec completeCCACC＾ffleekAG＾C＾dingdingcAtc入＾cc＾C τtacccτG1ccccrocc＾cateiyeccccllS6 25　vai　@Ⅱ　啼工内　一 groan　-●t　1ye　the　pha-s et am ＾ 喀 5 n Lau pt● eya at 啼 |C 喀　breath up　v ml pha arrrTGOIG1ACCCl. (τ^CAAC^DingcG?G cGcececitteCtoetCrAAAw^ycDingcaceccDingcca 12O5 trpaysthrergtyramnmtwaxarcatqhatantt ryeyelymgrlulla^-prealaGTG11tonGMrAA(; eel:TO? CC↑CCTATECetcIJaTe↑actyceDingcaI2 5lwmjph@qλWryIhtqeyeyesbsaLiel++u571 1 @falaN work t{-0 soku J-j:5.47/aph111. Hlnd1l 1. Clml. 5a11. Mlvl. Ncal. Bglll Potsuno J-1 + 6.22 taphm. For hUnd. CIal. Sall. MIul. Ncol. Bgllll summary book Corynebacterial integrons exhibit efficient selection in their corynebacteria. Genes that ensure selection, homologous sequences in the genomes of the above corynebacteria, and characterized by containing the above sequence adapted to A. Applicable to the above integrons an integron if it contains the coding sequence for the protein of interest. This is especially true for the production of proteins by cultures of Corynebacteria transformed with Ru.

international search report lfila1Ml+e″+-^”hc#+”’　” Pr/F’R9110%S 6 International Search Report FR91006b6 SA　50791

Claims (26)

[Claims] 1. an integron of Corynebacterium , - to the Corynebacterium mentioned above; Genes that ensure effective selection in - Contains a homologous sequence to the genome of the Corynebacterium, and the sequence is An integron characterized by being adapted to um. 2. obtained from a plasmid containing a replication region in addition to an integron; lon is flanked by inverted repeats corresponding to restriction sites not present in the integron. The integron according to claim 1. 3. The replication region contains a valid replication source in a bacterium that is not a Corynebacterium. The integron according to claim 2. 4. 4. The integron of claim 3, wherein the source of replication is effective in E. coli. 5. Any of claims 2 to 4, wherein the replication region comprises a replication source effective in Corynebacteria. The integron described in item 1. 6. The integron of claim 1 further comprising an array of transposed elements. 7. 7. The method of claims 1-6, wherein the transposable element comprises a sequence obtained from Corynebacterium. An integron as described in any one of the above. 8. 8. An integron according to claim 7, wherein the sequence is obtained from Brevibacterium. 9. 9. The sequence according to claim 8, wherein the sequence is obtained from ISaB1 as described in FIG. integron. 10. Transposition that ensures transposition, except for proteins encoded by Corynebacterium. 10. An integron according to any one of claims 6 to 9, further comprising an array of positional elements. 11. The array of transposed elements lacks the sequence encoding for transposase , the integron according to any one of claims 6 to 10. 12. An integron according to any one of claims 1 to 11, comprising a useful sequence. . 13. A claim that the useful sequence encodes a useful peptide or protein. The integron according to claim 12. 14. 14. The integ according to claim 13, wherein the useful protein is a homologous protein. Ron. 15. 15. The protein according to claim 14, wherein the useful protein is a heterologous protein. Gron. 16. According to any one of claims 13 to 15, the useful protein is an enzyme. integron. 17. The following genes encode useful proteins: -gltA -gdhA 17. The integron of claim 16 selected from. 18. Restriction site not present in integron: -NotI -BstXI 18. An integron according to any one of claims 2 to 17 selected from. 19. Any of claims 1 to 15, wherein the sequence is adapted to a strain of Corynebacterium. The integron described in paragraph 1. 20. The sequence was transferred to a strain of Brevibacterium before being adapted in Corynebacterium. 20. The integron according to claim 19, wherein the integron is transfected. 21. 21. The integron of claim 20, wherein the sequence is amplified. 22. 22. The amplified integron of claim 21, wherein the amplification produced a stable amplified sequence. . 23. A code that can be integrated into the chromosome of Brevibacterium lactofermentum The integ according to claim 1, comprising a homologous sequence of Lineobacterium mellassecola. Ron. 24. Corynebacterium with the integron according to any one of claims 1 to 23. 1. A method for transforming a strain of P. elegans, comprising: characterized in that the integron is introduced into the above strain by electroporation. Method. 25. Corynebacterium obtainable by the method according to claim 24. 26. Stocks below: -B. Lactofermentum -B. flavum -C. glutamicum -C. Mela secola The Corynebacterium according to claim 25.