JPH07327680A - Novel plasmid and method for obtaining mutant strain using the same - Google Patents

Abstract

(57) [Summary] [Structure] (a) Incapable of autonomous replication in coryneform bacteria,
(B) Inverted repeats present 5'-upstream and 3'-downstream of the indicator gene DNA, 5'-upstream and 3'-downstream of the open reading frame in the insertion sequence derived from the coryneform bacterium. A DNA region having at least one inverted repeat of (c)
A plasmid having a DNA region containing an open reading frame in an insertion sequence derived from a coryneform bacterium, and a method for obtaining a coryneform bacterial mutant strain using the same. [Effects] Using the mutant strain obtained in the present invention, for example, various amino acid-requiring strains are isolated, chromosomal DNAs of these mutant strains are extracted, and the expression of the marker gene is used as an index to participate in the amino acid biosynthesis system. Genes can be conveniently isolated. In addition, if a strain with reduced proteolytic activity is selected and various enzymes are expressed intracellularly using this strain as a host, the enzyme will be retained more stably than in a wild-type host, allowing efficient substance production. Will be possible.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel plasmid, and more particularly to DN containing an insertion sequence of coryneform bacteria.
The present invention relates to a recombinant plasmid having an A region and a method for obtaining a coryneform bacterial mutant strain using the recombinant plasmid. Transposable elements such as insert sequences and transposons are genetic elements found in prokaryotic cells and eukaryotic cells. The transposable element has the ability to transpose / insert itself into various sites on the chromosome, and is a highly industrially useful genetic element that can be used for improving breeding of bacteria.

[0002]

Insertion sequences, which are one of transposition sequences in microorganisms, have several characteristics. About 1-2 kb
And has inverted repeats (inverted repeats) at both ends. In addition, when inserted into the microbial chromosome, duplication of target sequences occurs.

Insertion sequences of microorganisms are those derived from Escherichia coli [Mol. Gen. Genet. vol. 122, 26
7-277 (1973)], derived from Shigella [J. B
actiol. vol. 172, 4090 to 409
9 (1990)], those derived from acetic acid bacteria [Mol. Mic
robiol. vol. 9, 211-218 (199
3)], those derived from mycoplasma [Mol. Micr
ob. vol. 7, 577 to 584 (1993)], etc., but there has been no previous finding regarding an insertion sequence derived from a coryneform bacterium.

The present inventors have previously found that an insertion sequence can be newly isolated from a certain coryneform bacterium, determined the base sequence, and proposed it (Japanese Patent Application No. 5-248459).
See description). Although various mutant strains using transposable elements and gene function analysis methods have been well studied in Escherichia coli, studies in coryneform bacteria have been delayed, and establishment of such methods is required.

[0005]

DISCLOSURE OF THE INVENTION The present invention has been made for the purpose of developing a method for easily producing a mutant strain by utilizing a transposable element in industrially important coryneform bacteria.

[0006]

As a result of intensive studies to solve the above problems, the present inventors have constructed a plasmid using the insertion sequence derived from coryneform bacterium previously proposed by the present inventors. Then, they found that a mutant strain can be easily obtained by transforming a coryneform bacterium with this plasmid, and completed the present invention.

Thus, according to the present invention, (1) (a) autonomous replication in coryneform bacteria is not possible, and (b)
5′-upstream and 3′-downstream of the indicator gene DNA,
A DNA region having at least one inverted repeat of the inverted repeats existing at the 5'-upstream side and the 3'-downstream side of the open reading frame in the insertion sequence derived from the coryneform bacterium, and (c) the coryneform bacterium A plasmid characterized by having a DNA region containing an open reading frame in the insertion sequence derived from (2) a coryneform bacterium was transformed with this plasmid, and the expression of the indicator gene of the transformant was used as an indicator, Provided is a method for obtaining a mutant strain, which comprises isolating an indicator gene expression strain.

The present invention will be described in more detail below. The "insertion sequence" used in the present invention means a DNA base sequence existing on the coryneform bacterial chromosome and capable of transposing on the chromosome or on the plasmid. An example of the basic procedure for preparing the insert sequence used in the present invention (hereinafter, sometimes abbreviated as “A fragment”) from the source microorganism, coryneform bacterium, is as follows:

The A fragment is present on the chromosome of the above coryneform bacterium, for example, Corynebacterium glutamicum ATCC31831 strain, and this strain has a sacB gene derived from Bacillus subtilis and can be replicated in the coryneform bacterium. Transformed with, for example, the plasmid pCRY30-sacB, smeared on a plate containing sucrose,
It can be isolated and obtained by extracting a plasmid from the growing sucrose-resistant strain.

First, Bacillus subtilis, such as Marburg (Ma
rburg) strain [Biochem. Biophys. R
es. Commun. , Vol. 119, 795-80
0 (1984)], chromosomal DNA is extracted from the culture. Using this chromosomal DNA as a template, a region containing known sacB and sacR (expression control gene of sacB gene) genes was subjected to PCR method [Polymerase Ch
ain Reaction method; Michael A .; I
nnis, PCR Protocols (PCR Protocol
cols), Academic Press Inc.
(1990)]. The amplified DNA fragment is introduced into a shuttle vector of Escherichia coli-Coryneform bacterium, and E. coli (Escherichia coli) JM109 (Takara Shuzo) is transformed with this vector to obtain a transformant.

From the obtained transformant, plasmid DNA is obtained.
And a coryneform bacterium such as Corynebacterium glutamicum ATCC31831 using this plasmid.
And a plasmid is extracted from a sucrose-resistant strain that grows on a plate containing sucrose. Of these plasmids, those having a larger size than the original plasmid are selected, and the DNA fragments inserted in the sacB and sacR gene regions of this plasmid are isolated to separately obtain the A fragment. be able to.

Here, the function of the A fragment of the present invention is, for example, originally present on the coryneform bacterial chromosome,
DN not present on the plasmid used for transformation
It can be confirmed by observing a phenomenon in which the A fragment is translocated from the chromosome to the plasmid under certain conditions. On the contrary, the function of the A fragment can be further confirmed by transposing the insertion sequence transposed on the plasmid onto the chromosome.

One of the A fragments thus obtained is the above-mentioned coryneform bacterium, such as Corynebacterium
A fragment existing on the chromosomal DNA of glutamicum ATCC31831 strain and having a size of about 1.5 kb can be mentioned. The number of recognition sites and the size of the cleaved fragment when the inserted sequence of about 1.5 kb was cleaved with various restriction enzymes are shown in Table 1 below.

[0014]

[Table 1] Table 1 Restriction enzyme Recognition site number Cleavage fragment size (kb) BamHI 2 0.9, 0.5, 0.1 HindIII 2 1.0, 0.4, 0.1 NcoI 10. 8, 0.7 Nsp (7524) I 1 1.1, 0.4

In the present specification, "the number of recognition sites" by a restriction enzyme means that a DNA fragment or a plasmid is completely digested with a restriction enzyme and the digested product is subjected to 1% agarose gel electrophoresis according to a method known per se and It was subjected to 4% polyacrylamide gel electrophoresis and the value determined from the number of separable fragments was adopted.

The "size of the cleaved fragment" and the size of the plasmid are the same as those of the E. coli lambda phage (λ phase) when agarose gel electrophoresis is used.
Based on the standard line drawn by the migration distance on the same agarose gel of a DNA fragment of known molecular weight obtained by cleaving NA with the restriction enzyme HindIII, and when using polyacrylamide gel electrophoresis, DN of X174 phage (φ × 174 phase)
The size of the cleaved DNA fragment or each DNA fragment of the plasmid is calculated based on the standard line drawn by the migration distance on the same polyacrylamide gel of the DNA fragment of known molecular weight obtained by cleaving A with the restriction enzyme HaeIII. The size of the plasmid is determined by adding the sizes of the cleaved fragments. In addition, in determining the size of each DNA fragment,
For the size of a fragment of 1 kb or more, the result obtained by 1% agarose gel electrophoresis was adopted and
For the size of fragments from kb to less than 1 kb, the results obtained by 4% polyacrylamide gel electrophoresis were adopted.

On the other hand, the base sequence of the insertion sequence derived from the chromosomal DNA of Corynebacterium glutamicum ATCC31831 is the plasmid pUC118 or pUC.
119 (manufactured by Takara Shuzo Co., Ltd.) using the dideoxy nucleotide enzymatic method [dideoxy chain terminal
method, Sanger, F .; et. al. , Pro
c. Natl. Acad. Sci. USA, Vol. 7
4, 5463 (1977)]. The above-mentioned DNA of about 1.5 kb determined in this way
The insert sequence having the base sequence of the fragment has the sequence shown in SEQ ID NO: 1 in the sequence listing below.

The insert sequence used in the present invention including the above-mentioned nucleotide sequence is not limited to one isolated from natural coryneform bacterium chromosomal DNA, but is also a commonly used DNA synthesizer, for example, Applied Biosystems (Appli).
ed Biosystems), model 394DN
It may be one synthesized using an A / RNA synthesizer.

The insert sequence used in the present invention obtained from the chromosomal DNA of Corynebacterium glutamicum ATCC31831 as described above has a part of the base sequence as long as it does not substantially impair the function of the insert sequence. The base may be replaced with another base or deleted, or a new base may be inserted,
Furthermore, a part of the base sequence may be transposed, and any of these derivatives is included in the insertion sequence used in the present invention.

Here, a plasmid having the sacB gene derived from Bacillus subtilis used for the isolation of the A fragment of the present invention and capable of replicating in a coryneform bacterium, for example, plasmid pCRY30-s.
A method for constructing acB, a coryneform bacterium that can be transformed with the plasmid, a method for culturing the transformant, and the like will be described.

First, as a shuttle vector for Escherichia coli-coryneform bacteria into which the sacB and sacR gene regions can be introduced, for example, plasmid pCRY30 described in JP-A-3-210184; JP-A-2-276575.
Plasmids pCRY21 and pCRY2K described in Japanese Patent Publication No.
E, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX; plasmids pCRY2 and pCRY3 described in JP-A-1-191686; pAM330 described in JP-A-58-67679; pHM1519 described in JP-A-58-77895. PAJ655, p described in JP-A-58-192900
AJ611 and pAJ1844; JP-A-57-134
PCG1 described in JP-A-500; JP-A-58-3519
PCG2 described in JP-A-7; JP-A-57-183799.
It is possible to use pCG4 and pCG11 described in the publication.

As a method for preparing the above-mentioned plasmid vector pCRY30, Brevibacterium stationii is used.
s) The plasmid pBY503 (see Japanese Patent Application Laid-Open No. 1-95785) DNA was extracted from IFO12144 (FERM BP-2515) and the size of the gene containing the gene responsible for the replication / proliferation function of the plasmid with the restriction enzyme XhoI was about 4.0.
A kb DNA fragment (replication functional region) is excised, and a DNA fragment (stabilization functional region) containing a gene having a stabilizing function of a plasmid of about 2.1 kb and having a size of about 2.1 kb is excised with restriction enzymes EcoRI and KpnI. EcoRI-K of plasmid pHSG298 (manufactured by Takara Shuzo Co., Ltd.)
By incorporating at the pnI and SalI sites,
The plasmid vector pCRY30 can be prepared.

Next, the introduction of the sacB and sacR gene regions into the shuttle vector of the Escherichia coli-coryneform bacterium is carried out, for example, by cleaving a restriction enzyme site existing at only one site in the plasmid vector with the restriction enzyme, and The fragment and the cleaved shuttle vector may be treated with S1 nuclease to make them blunt ends, or they may be ligated by DNA ligase treatment in the presence of an appropriate adapter DNA.

For example, the introduction of a DNA fragment containing the sacB and sacR gene regions into the above-mentioned plasmid pCRY30 is carried out by a restriction enzyme such as B which does not cleave the gene regions.
The amHI site was prepared by adding this restriction enzyme site to the end of the primer used when amplifying the gene region by the PCR method, and the amplified DNA fragment of the gene region was treated with the restriction enzyme BamHI, and the plasmid pC
DN digested with RY30 cleaved with the restriction enzyme BamHI
It can be performed by ligating with A ligase.

Using this plasmid, E. coli (Escherichia coli) JM109 (manufactured by Takara Shuzo) as described above.
Of the Bacillus subtilis-derived sacB gene used for the isolation of the A fragment of the present invention, thereby replicating in a coryneform bacterium by transforming Escherichia coli, obtaining a transformant, and extracting a plasmid DNA from the transformant. Propagable plasmids can be constructed.

The plasmid p constructed in this way
Approximately 2.0 kb of sacB and s in CRY30
The recombinant plasmid into which the acR gene region was introduced was transformed with p
It was named CRY30-sacB. Plasmid pCRY
Details of the method for producing 30-sacB will be described in Example 1 below.

As a host coryneform bacterium which can be transformed with a plasmid having the sacB gene derived from Bacillus subtilis and capable of replicative growth in coryneform bacteria, corynebacterium
Glutamicum ATCC31831, Brevibacterium flavum MJ-233 (FERM BP-1497)
And its origin, Brevibacterium ammoniagenes
genes) ATCC 6871, ATCC 1374
5, ATCC 13746; Brevibacterium divaritum
icatum) ATCC14020; Brevibacterium lactofermentum (Brevibacterium)
um lactofermentum) ATCC138
69 and the like can be used as the host coryneform bacterium.
These coryneform bacteria can also be used as a source microorganism of the insertion sequence used in the present invention. For the isolation of the A fragment of the present invention, it is most preferable to use Corynebacterium glutamicum ATCC31831 among the above coryneform bacteria as a source microorganism and a host.

Transformation of the above-mentioned host coryneform bacterium with the above recombinant plasmid is carried out by a method known per se, for example, C
alvin, N .; M. and Hanawalt, P. et al.
C. , Journal of Bacteria
y, Vol. 170, 2796 (1988); Ito,
K. , Nishida, T .; and Izaki.
K. , Agricultural and Biolo
musical Chemistry, 52, 293 (19)
88) and the like, for example, a pulse wave is applied to a host microorganism [Satoh, Y. et al. , J
individual ofIndustrial Micro
biology, Vol. 5, 159 (1990)].

As described above, the transformant thus obtained is smeared on a plate containing 2 to 15% of sucrose and cultured, and each sucrose-resistant strain that grows is subjected to liquid culture. A plasmid is extracted by a commonly used method known per se, for example, the alkali-SDS method, etc., and the plasmid is analyzed with an appropriate restriction enzyme. A plasmid having a size larger than the original plasmid is selected, and sacB of the plasmid and By isolating the DNA fragment inserted in the sacR gene region, the A fragment used in the present invention can be separated and obtained.

The above transformant can be cultured in a normal nutrient medium containing a carbon source, a nitrogen source, an inorganic salt and the like, and sucrose is used as the carbon source. As the nitrogen source, for example, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea, etc. may be used alone or in combination. As the inorganic salt, for example, potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and the like are used. In addition, nutrients such as various vitamins such as peptone, meat extract, yeast extract, corn steep liquor, casamino acid, biotin and the like can be added to the medium.

Culturing can usually be carried out at a temperature of about 25 ° C. to about 35 ° C. under aerobic conditions such as aeration and stirring (in the case of liquid culture). The pH during the culture can be around 7 to 8, and the pH can be adjusted during the culture by adding an acid or an alkali. The carbon source concentration at the start of culture, etc.
It is 5 to 12% by volume. The culture thus obtained is smeared on a plate containing 2 to 15% of sucrose as described above and cultured at 33 ° C. for 1 to 3 days to obtain a sucrose-resistant strain into which the A fragment has been introduced. From this sucrose-resistant strain, the A fragment can be isolated and obtained as described above, and its nucleotide sequence can be determined.

Based on the nucleotide sequence thus determined (for example, the nucleotide sequence shown in SEQ ID NO: 1), an open reading frame that controls the transposition function in the sequence (hereinafter this may be abbreviated as "ORF") A DNA region (c) containing a portion (for example, the nucleotide sequence shown in SEQ ID NO: 4) and an inverted repeat (for example, SEQ ID NO: 2) existing 5′-side upstream and 3′-downstream of the ORF.
And a DNA region (b) in which at least one inverted repeat of the nucleotide sequences shown in 3 and 3 is linked to the 5'-upstream side and the 3'-downstream side of the indicator gene DNA,
By introducing into a suitable cloning vector that does not carry the gene responsible for replication and growth in coryneform bacteria,
The plasmid of the present invention can be constructed.

By transforming a coryneform bacterium with this plasmid and isolating an indicator gene expression strain, a mutant strain in which the indicator gene is inserted / transposed into the host coryneform bacterial chromosome can be obtained. The inverted repeat and ORF in the insertion sequence derived from the coryneform bacterium used for constructing the plasmid of the present invention are SEQ ID NO: 2 respectively.
Or not only the sequences shown in 3 and 4 but also the inverted repeat or OR of the insertion sequence as described above.
As long as the function of F is not substantially impaired, some bases in the base sequence may be replaced with other bases or deleted, or new bases may be inserted. Further, a part of the nucleotide sequence may be rearranged, and any of these derivatives can be used for constructing the plasmid of the present invention.

The indicator gene is not particularly limited as long as it can be an indicator (marker) for the introduction of the indicator gene into the host due to the expression of the gene. For example, various drug resistance genes and β-galactosidase can be used. Genes (Pharmacia) can be mentioned. Further, specific examples of the drug resistance gene include a kanamycin resistance gene (manufactured by Pharmacia) and a chloramphenicol resistance gene (manufactured by Pharmacia).

The expression of the above-mentioned indicator gene can be confirmed as follows. When the indicator gene is a drug resistance gene, it can be easily carried out by culturing in a selective medium containing the drug and examining the drug sensitivity of the transformant.
It is also possible to culture the transformant in a commonly used medium and examine the expression product of the indicator gene in the medium by utilizing the properties of the expression product of the indicator gene. For example, when the indicator gene is β-galactosidase, it is cultured in a selective medium containing 5-bromo-4-chloro-3-indolyl-β-D-galactosidase (X-gal), which is a substrate for β-galactosidase. , The color of colonies formed by the transformant, that is, X-depending on the expression of β-galactosidase.
It can be distinguished by the fact that gal is decomposed and the colony turns blue.

A cloning vector into which each of the above-mentioned DNA regions [(b) and (c)] can be introduced has a gene that controls the replication / proliferation function in Escherichia coli and replicates / proliferates in coryneform bacteria. There is no particular limitation as long as it is a plasmid that does not have a gene that controls the function. For example, plasmid pHSG398 (Takara Shuzo),
pMW218 (manufactured by Nippon Gene Co., Ltd.) or the like can be used.

Next, an example of the above-mentioned plasmid of the present invention will be described. First, the isolated sequence of the isolated sequence (SEQ ID NO: 1) isolated from a coryneform bacterium, which has 24 bp of either one of the inverted repeats present 5'-upstream and 3'-downstream The inserted sequence is amplified by the PCR method with the primer having the sequence. The amplified insert sequence has a complete inverted repeat at the end. After inserting this into the SmaI site of plasmid pHSG398 (Takara Shuzo), the ORF on the inserted sequence was deleted by the restriction enzymes NheI and NaeI, and replaced with a kanamycin resistance gene cassette (Pharmacia) used as an indicator gene instead. .

On the other hand, the portion containing the ORF on the inserted sequence and inside the inverted repeat was amplified by PCR, and lac was added to the SmaI site of the plasmid pHSG398.
A plasmid inserted in the same direction as the c promoter is prepared. When the XbaI-SacI fragment derived from the previous plasmid was inserted into the XbaI site of this plasmid,
OR on the inserted sequence downstream of the lac promoter shown in
A plasmid pMV23 in which F and the kanamycin resistance gene having inverted repeats at both ends are inserted in the same direction can be prepared.

When transformation was carried out using this plasmid pMV23, due to the function of the protein encoded by the ORF, an indicator gene having an inverted repeat at the 5'-upstream side and 3'-downstream thereof (kanamycin resistance gene ) Can transpose to various positions on the host coryneform chromosome. Furthermore, since this indicator gene has no ORF after being inserted into the chromosome, transposition insertion cannot be repeated any more and is stably retained on the chromosome.

Examples of the host coryneform bacterium that can be transformed using the plasmid of the present invention include those mentioned above. Among these coryneform bacteria, use of a recA gene-deficient strain involved in chromosome / plasmid replication / repair / recombination improves the mutant strain acquisition rate and is particularly preferable as a host. The recA gene-deficient strain can be prepared as follows using a partial fragment of the recA gene derived from coryneform bacterium.

The recA gene is a gene for coryneform bacteria such as Brevibacterium flavum.
terium flavum) MJ-233 (FERM
It exists on the chromosome of the BP-1497) strain and can be isolated and obtained from this chromosome by the method described below. First, Brevibacterium flavum MJ-23
Chromosomal DNA is extracted from cultures of 3 strains. Using this chromosome as a template, Escherichia coli and Bacillus subtilis re
The gene fragment is amplified by the PCR method using the common region sequence of the cA gene as a primer.

The primer is 18 to 18 out of the recA gene common region sequences derived from Escherichia coli and Bacillus subtilis.
A 27-mer, preferably 20-23-mer base sequence can be selected and synthesized. The synthesis is carried out by a commonly used DNA synthesizer such as Applied Biosystems (A
manufactured by Applied Biosystems, model 3
It can be performed using a 94 DNA / RNA synthesizer.

The amount of the primer used in PCR is 0.05 to 0.5 μM, preferably 0.25 μM, per reaction. The PCR conditions are 9 for the denaturation step.
30 ° C. to 60 seconds, preferably 60 seconds at 4 ° C., annealing step at 55 ° C. for 30 to 120 seconds, preferably 1 hour at 55 ° C.
20 seconds, extension process at 72 ℃ 60 ~ 180
Second, preferably 72 ° C. for 120 seconds. These steps can be performed for 25 to 40 cycles, preferably 30 cycles to amplify a DNA fragment having a size of about 0.3 kb. The amplified DNA fragment can be confirmed by the agarose gel electrophoresis method described above.

Brevibacterium flavum MJ above
The nucleotide sequence of the recA gene partial fragment having a size of about 0.3 kb obtained by amplifying the 233 chromosomal DNA by the PCR method has the base sequence of the plasmid pGEM-.
It can be determined by the above dideoxynucleotide enzymatic method using a T vector (PROMEGA). The amino acid sequence thus deduced from the homology with the amino acid sequences of the genes of the microorganisms of other species with respect to the nucleotide sequence of the DNA fragment of about 0.3 kb has the sequence shown in SEQ ID NO: 5 in the sequence listing below. Which is composed of 318 base pairs encoding 106 amino acids.
It is a partial fragment of the ecA gene.

Next, the DNA fragment was treated with the plasmid pHSG3.
98 (manufactured by Takara Shuzo) is subcloned into the BamHI site, and this plasmid is used to transform Brevibacterium flavum MJ-233. This is smeared on A agar medium containing 3 μg / ml of chloramphenicol to obtain growing transformants. The strain is one in which the plasmid has been recombinantly inserted into the chromosome using the homology of the recA fragment on the plasmid,
In the transformant, the recA gene on the chromosome is destroyed.

Transformation of the above-mentioned host coryneform bacterium with the above-mentioned plasmid of the present invention can be carried out by a method known per se. By culturing the transformant thus obtained in the above-mentioned medium and isolating the indicator gene-expressing strain by the above-mentioned method, a population of coryneform bacterial mutants can be obtained. From the population of these mutants, for example, isolates of various amino acid-requiring strains,
The chromosomal DNA of these mutants can be extracted, and the genes involved in the amino acid biosynthesis system can be easily isolated by using the expression of the indicator gene as an indicator. In addition, if a strain with reduced proteolytic activity is selected and various enzymes are expressed intracellularly using this strain as a host, the enzyme will be retained more stably than in a wild-type host, and substance production will be performed efficiently. Will be possible.

[0047]

The present invention has been described above, but the present invention will be described in more detail with reference to the following examples. Example 1 Isolation of Insertion Sequence from Coryneform Bacterium and Determination of Nucleotide Sequence (1) Construction of Plasmid Replicating in Coryneform Bacteria Having SacB and sacR Gene Regions

(A) Extraction of total DNA of Bacillus subtilis Semi-synthetic medium A medium [composition: urea 2 g, (NH ₄ ) ₂ S
O ₄ 7g, K ₂ HPO ₄ 0.5g, KH ₂ PO _{4
0.5g, MgSO 4 0.5g, FeSO 4} · 7H 2
O 6 mg, MnSO ₄ .4-6H ₂ O 6 mg, yeast extract 2.5 g, casamino acid 5 g, biotin 20
0 μg, thiamine hydrochloride 200 μg, glucose 20
g, distilled water 1 liter] The above-mentioned Bacillus subtilis Marburg strain was cultivated in 1 liter until the early logarithmic growth phase, and the bacterial cells were collected. 10 mg / ml of the obtained bacterial cells
10 mM NaCl-20 mM Tris buffer (pH 8.0) -1 mM EDTA · 2Na containing lysozyme
Suspended in 15 ml of solution. Next, proteinase K was added so that the final concentration was 100 μg / ml, and the mixture was incubated at 37 ° C. for 1 hour. Further, sodium dodecyl sulfate was added so that the final concentration was 0.5%, and the mixture was kept at 50 ° C. for 6 hours for lysis. After adding an equal amount of phenol / chloroform solution to this lysate and gently shaking at room temperature for 10 minutes, the whole volume was centrifuged (5,000 × g, 20 minutes, 10 to 12 ° C.), and the supernatant was collected. Fractions were collected, sodium acetate was added to 0.3 M, and then twice the amount of ethanol was slowly added. The DNA existing between the aqueous layer and the ethanol layer was scattered with a glass rod, washed with 70% ethanol, and then air-dried. 10m to the obtained DNA
M Tris buffer (pH 7.5) -1 mM EDTA-2
Add 5 ml of Na solution and let stand overnight at 4 ° C to obtain template DNA.
Was used for PCR.

(B) Selection of primer The nucleotide sequences of the sacB and sacR gene regions of Bacillus subtilis have already been determined [Mol. Gen. Genet.
Vol. 200, 220-228 (1985)]. Based on this sequence, the following pair of primers capable of amplifying a DNA fragment containing the entire sacB and sacR gene regions were selected, and applied to Applied Biosystems (Applie).
d Biosystems), model 394 DNA
/ RNA synthesizer. A recognition site for the restriction enzyme BamHI is added to the ends of these primers.

(A-1) 5'-CCAGGATCCG
ATCCTTTTTA ACCCCATC-3 '(b-1) 5'-CCCGGATCCCA AAGGT
TAGG AATACGGGTT-3 ′ (A in the above sequence is adenine, G is guanine, C is cytosine, and T is thymine.)

When PCR was carried out using this primer and the chromosomal DNA prepared in (A) above as a template, sac
About 2.0 as long as the B and sacR gene regions are present
A kb reaction product is obtained.

(C) PCR reaction (sacB and sac
Amplification of R gene region) An actual PCR reaction was performed under the following conditions using a DNA thermal cycler manufactured by Perkin Elmer Cetus. Reaction solution: 50 mM KCl 10 mM Tris-HCl (pH 8.4) 1.5 mM MgCl ₂ template DNA 5 μl Primer prepared in the above (B) 0.25 μM dNTPs 200 μM Taq DNA polymerase (Takara Shuzo) 2.5un
Its and above were mixed to make 100 μl. PCR cycle: Denaturation process: 94 ° C. for 60 seconds Annealing process: 37 ° C. for 120 seconds Extension process: 72 ° C. for 180 seconds 30 cycles were carried out with the above as one cycle.

(D) Detection of reaction product 10 μl of the reaction solution generated in the above (C) was electrophoresed on a 2% agarose gel to detect an amplified DNA fragment having a size of about 2.0 kb.

(E) Construction of pCRY30 The plasmid vector pCRY30 is described in JP-A-3-210.
It was prepared by the method described in Japanese Patent No. 184.

(F) Construction of pCRY30-sacB 10 μl of a reaction solution containing the amplified DNA fragment of about 2.0 kb obtained in (C) above and pCRY3 obtained in (E) above.
0 0.5 μg was mixed and the restriction enzyme BamHI5uni was mixed.
The ts were mixed and reacted at 37 ° C. for 1 hour for complete digestion. After inactivating the enzyme by treating it at 65 ° C for 15 minutes, 5
0 mM Tris buffer (pH 7.6), 10 mM dithiothreitol, 1 mM ATP, 10 mM MgCl
Each component of ₂ and T4 DNA ligase 1 unit was added (concentration of each component is the final concentration), reacted at 4 ° C. for 15 hours to bind.

Using the obtained plasmid mixture, the calcium chloride method [J. Mol. Biol. , Vol. 53,1
59 (1970)] by Escherichia coli JM10
9 (manufactured by Takara Shuzo) was transformed with 50 mg of kanamycin.
Was dissolved in a medium containing 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl and 16 g of agar dissolved in 1 liter of distilled water.

The strain grown on this medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture, and the plasmid was cleaved with the restriction enzyme BamHI to confirm the inserted fragment. As a result, the length of the plasmid pCRY30 was 8.6.
In addition to the kb DNA fragment, an insert fragment having a size of about 2.0 kb was observed. This recombinant plasmid was designated as pCRY3
It was named 0-sacB. Plasmid pCRY30-s
A map of restriction enzyme cleavage points of acB is shown in FIG.

(2) Isolation of Insertion Sequence Derived from Corynebacterium glutamicum (A) Isolation of SacB-Resistant Strain The plasmid DNA prepared in (1) above was introduced into a coryneform bacterium to transform the cell. . The host coryneform bacterium used was Corynebacterium glutamicum ATCC3.
1831, Brevibacterium flavum MJ-233
(FERM BP-1497) plasmid pBY50
2 removed strain.

The electric pulse method was used for transformation. The coryneform bacterium was added to 100 ml of A medium [composition: urea 2
g, ammonium sulfate 7 g, monopotassium phosphate 0.
5g, dipotassium phosphate 0.5g, MgSO ₄ · 7H
₂ O 0.5 g, MnSO ₄ .4-6H ₂ O 6 mg,
FeSO ₄ .7H ₂ O 6 mg, yeast extract 1 g, casamino acid 1 g, biotin 200 μg, thiamine hydrochloride 100 μg was dissolved in deionized water to make 1 liter (pH 7.4)], and cultured until the initial logarithmic growth. G was added at 1 unit / ml, and the mixture was further shake-cultured for 2 hours. Collect the cultured cells by centrifugation,
20 ml pulse solution [composition: 272 mM sucrose, 7 mM KH ₂ PO ₄ , 1 mM MgCl ₂ ;
pH 7.4]. Again, the cells were collected by centrifugation, suspended in 5 ml of the pulse solution, mixed with 0.75 ml of the cells and 50 μl of the plasmid solution obtained in (A) above, and allowed to stand on ice for 20 minutes. did. 2500 Volt 25μF using Gene Pulser (BioRad)
After applying the pulse, the plate was allowed to stand in ice for 20 minutes. After culturing the whole amount for 1 hour at 30 ° C., it was smeared on A agar medium containing 10% sucrose and 50 μg / ml kanamycin. The cells were cultured at 30 ° C for 2 days.

(B) Isolation of Insertion Sequence A plasmid was prepared from the emerged sucrose-resistant and kanamycin-resistant strains by the method described in JP-A-1-95785. When this plasmid was cleaved with various restriction enzymes and its size was confirmed, there was a plasmid that was larger than the plasmid used for transformation by about 1.5 kb in two strains. In addition, as a result of analysis with a restriction enzyme, the inserted sequence having a size of about 1.5 kb was found to be
It was found to be present in the gene and inactivates the sacB gene product.

(3) Determination of the nucleotide sequence of the insertion sequence derived from Corynebacterium glutamicum In order to identify the insertion sequence having a size of about 1.5 kb obtained in the above (2), its nucleotide sequence was changed to pUC118 or It was determined by the dideoxynucleotide method using pUC119 (Takara Shuzo) according to the strategy diagram shown in FIG.

As a result, the sequence described in SEQ ID NO: 1 was revealed. An open reading frame (ORF) consisting of 436 amino acids was present on this sequence. At both ends of this insertion sequence, an inverted repeat of 24 bp and a direct repeat of 8 bp, which is the target sequence, were present. This size is about 1.5 kb
The number of recognition sites and the size of the cleaved fragment when the insert sequence was cleaved with various restriction enzymes were as shown in Table 1 above. The restriction enzyme cleavage point map is shown in FIG.

Example 2 Preparation of recA gene-deficient mutant strain The recA gene derived from Escherichia coli and Bacillus subtilis has been well studied, and its nucleotide sequence has been determined. The region conserved between these two types of microorganisms, particularly the region particularly conserved in the DNA base sequence, is examined, the following pair of primers are selected, and Applied Biosystems (Applied Biosystems), Model 394 DNA / RNA Synthesizer (synthe
It was synthesized using a sizer. A recognition site for the restriction enzyme BamHI is added to the ends of these primers.

(A-1) 5'-AAT GGA TCC
TTY ATI GAI GCIGAR CAY G
CI YT-3 '(b-1) 5'-AAT GGA TCC CCI C
CI GTI GTIGTI TCI GG-3 '(in the above sequence, R represents A or G, Y represents C or T, where A is adenine, G is guanine, C is cytosine, T is thymine, and I is deoxyinosine. Show.)

PCR was carried out using this primer using the Brevibacterium flavum MJ-233 chromosome as a template, so long as the recA gene was present, about 0.3 kb.
It is expected that the reaction product of PCR reaction was performed under the conditions described in Example 1 above, and 10 μl of the reaction solution was electrophoresed on a 2% agarose gel to confirm amplification of a fragment of about 0.3 kb.

Amplified DN having a size of about 0.3 kb obtained
The nucleotide sequence of the A fragment was determined by the dideoxynucleotide enzyme method (dideoxychain terminal method).
method) (Sanger, F. et al., Proceeding of National Academy of Sciences of United States of
America (Proc. Nat. Acad. Sci. US
A), Vol. 74, 5463, 1977).

As a result of the nucleotide sequence determination, the sequence shown in SEQ ID NO: 5 in the Sequence Listing below became clear. The inserted DNA fragment shows a high homology with a part of the recA fragment derived from Escherichia coli and Bacillus subtilis (the region sandwiched by the primers used for PCR), and Brevibacterium flavum MJ-2.
It was confirmed that a part of the recA gene DNA fragment derived from 33 was cloned.

10 μl of 3M Na was added to the PCR reaction completion solution.
Add OAc (pH 5.5) and add 250 μl of 99.5%
After adding ethanol and leaving it at -20 ° C for 20 minutes, 1
Centrifuge at 2,000 rpm for 15 minutes and precipitate (Amplification D
NA fragment) was recovered. Suspend the precipitate in 50 μl of water,
It was cut with the restriction enzyme BamHI. This and the plasmid p
HSG398 (manufactured by Takara Shuzo) is cut with the restriction enzyme BamHI
After cutting, mix the dephosphorylated ones and mix to 50 mM
 Tris buffer (pH 7.6), 10 mM dithio thread
Ititol, 1 mM ATP, 10 mM MgCl ₂And
And each component of T4 DNA ligase 1 unit
(The concentration of each component is the final concentration) 15 hours at 4 ℃
And then combined.

Using the plasmid mixture obtained above, the calcium chloride method (Journalof Molecule) was used.
ar Biology, Vol. 53,159,197
0) Escherichia coli JM109 (Takara Shuzo) was transformed with a medium containing 50 mg of chloramphenicol [trypton 10 g, yeast extract 5 g, NaCl 5 g and agar 16 g, and distilled water 1
Dissolved in liter].

The strain grown on this medium is liquid-cultured by a conventional method, and the plasmid DNA is extracted from the culture medium to obtain the restriction enzyme B.
The plasmid pHSG3 was digested with amHI.
It was confirmed that a DNA fragment of 0.3 kb was inserted into 98. Next, this plasmid was used to transform Brevibacterium flavum MJ-233.

The electric pulse method was used for transformation. The early coryneform bacterium was added to 100 ml of A medium [composition: urea 2
g, ammonium sulfate 7 g, phosphoric acid-potassium 0.
5g, dipotassium phosphate 0.5g, MgSO ₄ · 7H
_{2 O 0.5g, 4~6H 2 O6mg,} FeSO 4 · 7
H ₂ O 6 mg, yeast extract 1 g, casamino acid 1
g, biotin 200 μg, thiamine hydrochloride 100 μg
Is dissolved in deionized water to 1 liter (pH 7.
4)], the cells were cultured to the initial stage of logarithmic growth, and penicillin G was added thereto at 1 unit / ml, followed by further shaking culture for 2 hours. The cultured bacterial cells were collected by centrifugation, and 20 ml of a pulse solution [composition: 272 mM sucrose, 7
mM KH ₂ PO ₄ , 1 mM MgCl ₂ ; pH 7.
4]. Again, collect the cells by centrifugation, and
The cells were suspended in a pulse solution (ml), 0.75 ml of the cells were mixed with 50 μl of the plasmid solution obtained above, and the mixture was allowed to stand on ice for 20 minutes. Using a Gene Pulser (manufactured by Bio-Rad), the voltage was set to 2500 V and 25 μF, and after applying a pulse, the plate was allowed to stand in ice for 20 minutes. After culturing the whole amount at 30 ° C for 1 hour, chloramphenicol 3 μg / ml
It was smeared on the agar medium containing A and cultured at 30 ° C. for 2 days.

The transformant thus obtained had a chloramphenicol resistance gene, and a plasmid that could not replicate in coryneform bacteria was recombined on the chromosome by recombination using the homology of the recA gene fragment on the plasmid. The transformant has a recA on the chromosome.
It is thought that the gene is destroyed. This is recA
It was named a gene-deficient strain.

Example 3 Preparation of Plasmid for Obtaining Mutant Strain 24 bp (SEQ ID NO: 2 or 3) of either one of the inverted repeats existing 5'-upstream and 3'-downstream of the isolated insertion sequence The inserted sequence was amplified by the PCR method described in Example 1 above using the primer having

10 μl of 3M Na was added to the PCR reaction completed solution.
Add OAc (pH 5.5) and add 250 μl of 99.5%
After adding ethanol and leaving it at -20 ° C for 20 minutes, 1
The precipitate was collected by centrifugation at 2,000 rpm for 15 minutes.
The precipitate was suspended in 50 μl of water to make the ends blunt. This was mixed with a cloning vector pHSG398 (Takara Shuzo Co., Ltd.) digested with a restriction enzyme SmaI and then dephosphorylated, and mixed to obtain a 50 mM Tris buffer (pH 7.
6) 10 mM dithiothreitol, 1 mM AT
P, 10 mM MgCl ₂ and T4 DNA ligase 1
Each component of unit was added (the concentration of each component is the final concentration), and the mixture was reacted at 4 ° C. for 15 hours to be bound.

Using the obtained plasmid mixture, the calcium chloride method (Journal of Molecular) was used.
Biology, Vol. 53, 159, 1970) to transform Escherichia coli JM109 (Takara Shuzo), and a medium containing 50 mg of chloramphenicol [tryptone 10 g, yeast extract 5].
g, NaCl 5 g and agar 16 g were dissolved in distilled water 1 liter].

The strain grown on this medium is liquid-cultured by a conventional method, and the plasmid DNA is extracted from the culture broth to obtain the restriction enzyme E.
By cutting with coRI and HindIII, p
It was confirmed that the insertion sequence was inserted into HSG398. This plasmid was designated as pMV10. Using the restriction enzymes NheI and NaeI, this plasmid pMV
10 on the inserted sequence present on the plasmid
RF was deleted with the restriction enzymes NheI and NaeI. A kanamycin resistance gene cassette (Pharmacia) was added to this, and 50 mM Tris buffer (pH 7.
6) 10 mM dithiothreitol, 1 mM AT
P, 10 mM MgCl ₂ and T4 DNA ligase
1 unit of each component was added (the concentration of each component is the final concentration), and the mixture was reacted at 4 ° C. for 15 hours to be bound.

Using the obtained plasmid mixture, the calcium chloride method (Journal of Molecular) was used.
Biology, Vol. 53, 159, 1970) to transform Escherichia coli JM109 (Takara Shuzo), and a medium containing 50 mg of kanamycin [tryptone 10 g, yeast extract 5 g, NaCl.
Dissolve 5 g and agar 16 g in 1 liter of distilled water]
Smeared on

The strain grown on this medium was liquid-cultured by a conventional method, and plasmid DNA was extracted from the culture medium to obtain the restriction enzyme E.
It was confirmed that the kanamycin resistance gene cassette had been inserted by cutting with coRI and HindIII. This plasmid was designated as pMV12. On the other hand, a portion containing the ORF (SEQ ID NO: 4) on the inserted sequence and inside the inverted repeat was amplified by PCR, and was added to the SmaI site of the plasmid pHSG398.
A plasmid inserted in the same direction as the ac promoter was prepared. This plasmid was designated as pMV17.

This plasmid pMV17 was digested with the restriction enzyme Xb
The XbaI-SacI fragment derived from the above-mentioned plasmid pMV12 was cut out into the blunt-ended one after cleaving with aI, and the blunt-ended one was inserted into the plasmid pMV2.
3 was produced. As a result, as shown in FIG.
A plasmid pMV23 in which the ORF on the inserted sequence and the kanamycin resistance gene having inverted repeats at both ends were inserted in the same direction downstream of the c promoter could be constructed.

The number of recognition sites and the size of the cleaved fragment when this plasmid was cleaved with various restriction enzymes are shown in the second section below.
Shown in the table.

[0081]

[Table 2] Table 2 Restriction enzyme number of recognition sites Size of cleavage fragment (kb) HindIII 2 4.8, 0.4 SmaI 1 5.2 NaeI 1 5.2 NheI 1 5.2 KpnI 1 5.2 EcoRI 1 5.2

When transformation is carried out using this plasmid pMV23, the function of the protein encoded by the ORF allows the kanamycin resistance gene to be transposed and inserted at various positions on the chromosome in the same manner as the insertion sequence. Furthermore, this artificial mini-insert sequence is inserted into the chromosome and then OR
Since it does not have F, it is considered that the dislocation insertion cannot be repeated any more and that it is stably retained.

Example 4 Preparation of Mutant Strain Using the prepared plasmid pMV23, the wild strain Brevibacterium flavum MJ-233 and the recA gene-deficient strain prepared in Example 2 were transformed.

The electric pulse method was used for transformation. These coryneform bacteria were each added to 100 ml of A medium [composition: urea 2 g, ammonium sulfate 7 g, phosphate-potassium 0.5 g, dipotassium phosphate 0.5 g, MgSO _{4
· 7H 2 O 0.5g, 4~6H 2} O 6mg, FeS
O ₄ · 7H ₂ O 6mg, yeast extract 1g, casamino acid 1g, biotin 200 [mu] g, thiamine hydrochloride 10
Dissolve 0 μg in deionized water to 1 liter (pH
7.4)], the cells were cultured until the initial logarithmic growth, and then penicillin G was added at 1 unit / ml to further add 2
Culture was performed with shaking for an hour. Collect the cultured cells by centrifugation and
ml pulse solution [composition: 272 mM sucrose, 7 mM KH ₂ PO ₄ , 1 mM MgCl ₂ ; pH
7.4]. The bacterial cells were collected again by centrifugation, suspended in 5 ml of the pulse solution, mixed with 0.75 ml of the cells and 50 μl of the plasmid solution obtained in Example 3 above, and allowed to stand in ice for 20 minutes. did. 2500 Volt 25μF using Gene Pulser (BioRad)
After applying the pulse, the plate was allowed to stand in ice for 20 minutes. After culturing the whole amount at 30 ℃ for 1 hour, kanamycin 50μ
It was smeared on A agar medium containing g / ml and cultured at 30 ° C. for 2 days.

The obtained transformant has a kanamycin resistance gene. The kanamycin resistance gene on this plasmid, which is not able to replicate in coryneform bacteria, has various functions on the chromosome like the insertion sequence due to the function of the protein encoded by the ORF derived from the insertion sequence present on this plasmid. It has been dislocated to the position.

As a result, as shown in Table 2 below, the recA gene-deficient strain was 10 times more efficient than the wild-type strain (MJ-233), and a kanamycin-resistant strain was obtained. It was shown to be more useful in strain production.

[0087]

[Table 3] Table 3 Host host transformation rate (pieces / μg DNA) Wild strain (MJ-233) 8.2 × 10 ³ recA gene deficient strain 9.2 × 10 ⁴

[0088]

[Sequence list]

SEQ ID NO: 1 Sequence Length: 1469 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: Genomic DNA Origin Biological Name: Corynebacterium glutamicum
terium glutamicum) Strain name: ATCC 31831 Sequence features Characteristic symbols: Insertion Seq Location: 1-1469 Method of determining features: E

Sequence CCTTTTGCGG CCCTTCCGGT TTTGGGGTAC ATCACAGAAC CTGGGCTAGC GGTGTAGACC 60 CGAAAATAAA CGAGCCTTTT GTCAGGGTTA AGGTTTAGGT ATCTAAGCTA ACCAAACACC 120 AACAAAAGGC TCTACCC ATG AAC ATC ATC ATC ATC ATC ATC ATC ATC ATC GLA GCG GAA CTA GGA CTC ACC ATC ACC GGC GCT TCC GAT 218 Ile Cys Arg Thr Ala Glu Leu Gly Leu Thr Ile Thr Gly Ala Ser Asp 15 20 25 GCA GGT GAT TAC ACC CTG ATC GAA GCA GAC GCA CTC GAC TAC ACC TCC 266 Ala Gly Asp Tyr Thr Leu Ile Glu Ala Asp Ala Leu Asp Tyr Thr Ser 30 35 40 ACC TGC CCA GAA TGC TCC CAA CCT GGG GTG TTT CGT AAT CAC ACC CAC 314 Thr Cys Pro Glu Cys Ser Gln Pro Gly Val Phe Arg Asn His Thr His 45 50 55 CGG ATG CTC ATT GAT TTA CCC ATC GTC GGG TTT CCC ACC AAA CTG TTT 362 Arg Met Leu Ile Asp Leu Pro Ile Val Gly Phe Pro Thr Lys Leu Phe 60 65 70 75 ATC CGT CTA CCT CGC TAC CGC TGC ACC AAC CCC ACA TGT AAG CAA AAG 410 Ile Arg Leu Pro Arg Tyr Arg Cys Thr Asn Pro Thr Cys Lys Gln Lys 80 85 90 TAT TTC C AA GCA GAA CTA AGC TGC GCT GAC CAC GGT AAA AAG GTC ACC 458 Tyr Phe Gln Ala Glu Leu Ser Cys Ala Asp His Gly Lys Lys Val Thr 95 100 105 CAC CGG GTC ACC CGC TGG ATT TTA CAA CGC CTT GCT ATT GAC CGG ATG 506 His Arg Val Thr Arg Trp Ile Leu Gln Arg Leu Ala Ile Asp Arg Met 110 115 120 AGT GTT CAC GCA ACC GCG AAA GCA CTT GGG CTA GGG TGG GAT TTA ACC 554 Ser Val His Ala Thr Ala Lys Ala Leu Gly Leu Gly Trp Asp Leu Thr 125 130 135 TGC CAA CTA GCC CTC GAT ATG TGC CGT GAG CTG GTC TAT AAC GAT CCT 602 Cys Gln Leu Ala Leu Asp Met Cys Arg Glu Leu Val Tyr Asn Asp Pro 140 145 150 155 CAC CAT CTT GAT GGA GTG TAT GTC ATT GGG GTG GAT GAG CAT AAG TGG 650 His His Leu Asp Gly Val Tyr Val Ile Gly Val Asp Glu His Lys Trp 160 165 170 TCA CAT AAT AGG GCT AAG CAT GGT GAT GGG TTT GTC ACC GTG ATT GTC 698 Ser His Asn Arg Ala Lys His Gly Asp Gly Phe Val Thr Val Ile Val 175 180 185 GAT ATG ACC GGG CAT CGG TAT GAC TCA CGG TGT CCT GCC CGG TTA TTA 746 Asp Met Thr Gly His Arg Tyr Asp Ser Arg Cys Pro Ala Arg Leu Leu 190 195 2 00 GAT GTC GTC CCA GGT CGT AGT GCT GAT GCT TTA CGG TCT TGG CTT GGC 794 Asp Val Val Pro Gly Arg Ser Ala Asp Ala Leu Arg Ser Trp Leu Gly 205 210 215 TCC CGC GGT GAA CAG TTC CGC AAT CAG ATA CGG ATC GTG TCC ATG GAT 842 Ser Arg Gly Glu Gln Phe Arg Asn Gln Ile Arg Ile Val Ser Met Asp 220 225 230 235 GGA TTC CAA GGC TAC GCC ACA GCA AGT AAA GAA CTC ATT CCT TCT GCT 890 Gly Phe Gln Gly Tyr Ala Thr Ala Ser Lys Glu Leu Ile Pro Ser Ala 240 245 250 CGT CGC GTG ATG GAT CCA TTC CAT GTT GTG CGG CTT GCT GGT GAC AAG 938 Arg Arg Val Met Asp Pro Phe His Val Val Arg Leu Ala Gly Asp Lys 255 260 265 CTC ACC GCC TGC CGG CAA CGC CTC CAG CGG GAG AAA TAC CAG CGT CGT 986 Leu Thr Ala Cys Arg Gln Arg Leu Gln Arg Glu Lys Tyr Gln Arg Arg 270 275 280 GGT TTA AGC CAG GAT CCG TTG TAT AAA AAC CGG AAG GCT TTG TTG ACC 1034 Gly Leu Ser Gln Asp Pro Leu Tyr Lys Asn Arg Lys Ala Leu Leu Thr 285 290 295 ACG CAC AAG TGG TTG AGT CCT CGT CAG CAA GAA AGC TTG GAG CAG TTG 1082 Thr His Lys Trp Leu Ser Pro Arg Gln Gln Glu Ser Leu Glu Gln Leu 300 305 310 315 TGG GCG TAT GAC AAA GAC TAC GGG GTG TTA AAG CTT GCG TGG CTT GCG 1130 Trp Ala Tyr Asp Lys Asp Tyr Gly Val Leu Lys Leu Ala Trp Leu Ala 320 325 330 TAT CAG GCG ATT ATT GAT TGT TAT CAG ATG GGT AAT AAG CGT GAA GCG 1178 Tyr Gln Ala Ile Ile Asp Cys Tyr Gln Met Gly Asn Lys Arg Glu Ala 335 340 345 AAA AAG AAA ATG CGG ACC ATT ATT GAT CAG CTT CGG GTG TTG AAG GGG 1226 Lys Lys Lys Met Arg Thr Ile Ile Asp Gln Leu Arg Val Leu Lys Gly 350 355 360 CCG AAT AAG GAA CTC GCG CAG TTG GGT CGT AGT TTG TTT AAA CGA CTT 1274 Pro Asn Lys Glu Leu Ala Gln Leu Gly Arg Ser Leu Phe Lys Arg Leu 365 370 375 GGT GAT GTG TTG GCG TAT TTC GAT GTT GGT GTC TCC AAC GGT CCG GTC 1322 Gly Asp Val Leu Ala Tyr Phe Asp Val Gly Val Ser Asn Gly Pro Val 380 385 390 395 GAA GCG ATC AAC GGA CGG TTG GAG CAT TTG CGT GGG ATT GCT CTA GGT 1370 Glu Ala Ile Asn Gly Arg Leu Glu His Leu Arg Gly Ile Ala Leu Gly 400 405 410 TTC CGT AAT TTG AAC CAC TAC ATT CTG CGG TGC CTT ATC CAT TCA GGG 1418 Phe Arg Asn Leu Asn His Tyr Ile Leu Arg Cys Leu Ile His Ser Gly 415 420 425 CAG TTG GTC CAT AAG ATC AAT GCA CTC TAA AA CAGGAAGAGC CCCTTTTGC 1469 Gln Leu Val His Lys Ile Asn Ala Leu Stop 430 435

SEQ ID NO: 2 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Corynebacterium glutamicum
terium glutamicum) Strain name: ATCC 31831 Sequence features Characteristic symbols: Inverted Repeat Location: 1-24 Method of determining features: E sequence GGCCCTTCCG GTTTTGGGGT ACAT 24

SEQ ID NO: 3 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Double Strands Topology: Linear Sequence Type: Genomic DNA Origin Biological Name: Corynebacterium glutamicum
terium glutamicum) Strain name: ATCC 31831 Sequence features Symbols representing features: Inverted Repeat Location: 1-24 Method of determining features: E sequence GGCTCTTCCT GTTTTAGAGT GCAT 24

SEQ ID NO: 4 Sequence length: 1311 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Corynebacterium glutamicum
terium glutamicum) Strain name: ATCC 31831 Sequence features Characteristic symbols: CDS Location: 1-1311 Method of determining features: E

Sequence ATG AAG TCT ACC GGC AAC ATC ATC GCT GAC ACC ATC TGC CGC ACT GCG 48 Met Lys Ser Thr Gly Asn Ile Ile Ala Asp Thr Ile Cys Arg Thr Ala 1 5 10 15 GAA CTA GGA CTC ACC ATC ACC GGC GCT TCC GAT GCA GGT GAT TAC ACC 96 Glu Leu Gly Leu Thr Ile Thr Gly Ala Ser Asp Ala Gly Asp Tyr Thr 20 25 30 CTG ATC GAA GCA GAC GCA CTC GAC TAC ACC TCC ACC TGC CCA GAA TGC 144 Leu Ile Glu Ala Asp Ala Leu Asp Tyr Thr Ser Thr Cys Pro Glu Cys 35 40 45 TCC CAA CCT GGG GTG TTT CGT AAT CAC ACC CAC CGG ATG CTC ATT GAT 192 Ser Gln Pro Gly Val Phe Arg Asn His Thr His Arg Met Leu Ile Asp 50 55 60 TTA CCC ATC GTC GGG TTT CCC ACC AAA CTG TTT ATC CGT CTA CCT CGC 240 Leu Pro Ile Val Gly Phe Pro Thr Lys Leu Phe Ile Arg Leu Pro Arg 65 70 75 80 TAC CGC TGC ACC AAC CCC ACA TGT AAG CAA AAG TAT TTC CAA GCA GAA 288 Tyr Arg Cys Thr Asn Pro Thr Cys Lys Gln Lys Tyr Phe Gln Ala Glu 85 90 95 CTA AGC TGC GCT GAC CAC GGT AAA AAG GTC ACC CAC CGG GTC ACC CGC 336 Leu Ser Cys Ala Asp His Gly Lys Lys Val Thr His Arg Val Th r Arg 100 105 110 TGG ATT TTA CAA CGC CTT GCT ATT GAC CGG ATG AGT GTT CAC GCA ACC 384 Trp Ile Leu Gln Arg Leu Ala Ile Asp Arg Met Ser Val His Ala Thr 115 120 125 GCG AAA GCA CTT GGG CTA GGG TGG GAT TTA ACC TGC CAA CTA GCC CTC 432 Ala Lys Ala Leu Gly Leu Gly Trp Asp Leu Thr Cys Gln Leu Ala Leu 130 135 140 GAT ATG TGC CGT GAG CTG GTC TAT AAC GAT CCT CAC CAT CTT GAT GGA 480 Asp Met Cys Arg Glu Leu Val Tyr Asn Asp Pro His His Leu Asp Gly 145 150 155 160 GTG TAT GTC ATT GGG GTG GAT GAG CAT AAG TGG TCA CAT AAT AGG GCT 528 Val Tyr Val Ile Gly Val Asp Glu His Lys Trp Ser His Asn Arg Ala 165 170 175 AAG CAT GGT GAT GGG TTT GTC ACC GTG ATT GTC GAT ATG ACC GGG CAT 576 Lys His Gly Asp Gly Phe Val Thr Val Ile Val Asp Met Thr Gly His 180 185 190 CGG TAT GAC TCA CGG TGT CCT GCC CGG TTA TTA GAT GTC GTC CCA GGT 624 Arg Tyr Asp Ser Arg Cys Pro Ala Arg Leu Leu Asp Val Val Pro Gly 195 200 205 CGT AGT GCT GAT GCT TTA CGG TCT TGG CTT GGC TCC CGC GGT GAA CAG 672 Arg Ser Ala Asp Ala Leu Arg Ser Trp Leu Gly S er Arg Gly Glu Gln 210 215 220 TTC CGC AAT CAG ATA CGG ATC GTG TCC ATG GAT GGA TTC CAA GGC TAC 720 Phe Arg Asn Gln Ile Arg Ile Val Ser Met Asp Gly Phe Gln Gly Tyr 225 230 235 240 GCC ACA GCA AGT AAA GAA CTC ATT CCT TCT GCT CGT CGC GTG ATG GAT 768 Ala Thr Ala Ser Lys Glu Leu Ile Pro Ser Ala Arg Arg Val Met Asp 245 250 255 CCA TTC CAT GTT GTG CGG CTT GCT GGT GAC AAG CTC ACC GCC TGC CGG 816 Pro Phe His Val Val Arg Leu Ala Gly Asp Lys Leu Thr Ala Cys Arg 260 265 270 CAA CGC CTC CAG CGG GAG AAA TAC CAG CGT CGT GGT TTA AGC CAG GAT 864 Gln Arg Leu Gln Arg Glu Lys Tyr Gln Arg Arg Gly Leu Ser Gln Asp 275 280 285 CCG TTG TAT AAA AAC CGG AAG GCT TTG TTG ACC ACG CAC AAG TGG TTG 912 Pro Leu Tyr Lys Asn Arg Lys Ala Leu Leu Thr Thr His Lys Trp Leu 290 295 300 AGT CCT CGT CAG CAA GAA AGC TTG GAG CAG TTG TGG GCG TAT GAC AAA 960 Ser Pro Arg Gln Gln Glu Ser Leu Glu Gln Leu Trp Ala Tyr Asp Lys 305 310 315 320 GAC TAC GGG GTG TTA AAG CTT GCG TGG CTT GCG TAT CAG GCG ATT ATT 1008 Asp Tyr Gly Val Leu Lys LeuAla Trp Leu Ala Tyr Gln Ala Ile Ile 325 330 335 GAT TGT TAT CAG ATG GGT AAT AAG CGT GAA GCG AAA AAG AAA ATG CGG 1056 Asp Cys Tyr Gln Met Gly Asn Lys Arg Glu Ala Lys Lys Lys Met Arg 340 345 350 ACC ATT ATT GAT CAG CTT CGG GTG TTG AAG GGG CCG AAT AAG GAA CTC 1104 Thr Ile Ile Asp Gln Leu Arg Val Leu Lys Gly Pro Asn Lys Glu Leu 355 360 365 GCG CAG TTG GGT CGT AGT TTG TTT AAA CGA CTT GGT GAT GTG TTG GCG 1152 Ala Gln Leu Gly Arg Ser Leu Phe Lys Arg Leu Gly Asp Val Leu Ala 370 375 380 TAT TTC GAT GTT GGT GTC TCC AAC GGT CCG GTC GAA GCG ATC AAC GGA 1200 Tyr Phe Asp Val Gly Val Ser Asn Gly Pro Val Glu Ala Ile Asn Gly 385 390 395 400 CGG TTG GAG CAT TTG CGT GGG ATT GCT CTA GGT TTC CGT AAT TTG AAC 1248 Arg Leu Glu His Leu Arg Gly Ile Ala Leu Gly Phe Arg Asn Leu Asn 405 410 415 CAC TAC ATT CTG CGG TGC CTT ATC CAT TCA GGG CAG TTG GTC CAT AAG 1296 His Tyr Ile Leu Arg Cys Leu Ile His Ser Gly Gln Leu Val His Lys 420 425 430 ATC AAT GCA CTC TAA 1311 Ile Asn Ala Leu Stop 435

SEQ ID NO: 5 Sequence length: 318 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Brevibacterium flavum Strain name: MJ-233 Characteristic of sequence: Symbol representing the characteristic: CDS (peptide) Location: 1-318 Method of determining the characteristic: E

Sequence GAT CCA GAT TAT GCT CGC AAG CTT GGT GTA GAT ACT GAT GCG CTT CTG 48 Asp Pro Asp Tyr Ala Arg Lys Leu Gly Val Asp Thr Asp Ala Leu Leu 1 5 10 15 GTT TCG CAG CCA GAC ACT GGT GAG CAA GCA CTA GAA ATC GCC GAC ATG 96 Val Ser Gln Pro Asp Thr Gly Glu Gln Ala Leu Glu Ile Ala Asp Met 20 25 30 CTG GTT CGT TCC GGC GCA ATC GAC ATC ATC GTG ATT GAC TCG GTG GCT 144 Leu Val Arg Ser Gly Ala Ile Asp Ile Ile Val Ile Asp Ser Val Ala 35 40 45 GCG CTG ACA CCA AAG GCT GAA ATT GAA GGC GAA ATG GGC GAT AGC CAC 192 Ala Leu Thr Pro Lys Ala Glu Ile Glu Gly Glu Met Gly Asp Ser His 50 55 60 GTT GGT CTT CAG GCC CGC CTC ATG AGC CAG GCG CTT CGT AAG ATG ACA 240 Val Gly Leu Gln Ala Arg Leu Met Ser Gln Ala Leu Arg Lys Met Thr 65 70 75 80 GGT GCG CTG TAC AAC TCG GGT ACC ACC GCG ATC CTC ATT AAC CAG CTG 288 Gly Ala Leu Tyr Asn Ser Gly Thr Thr Ala Ile Leu Ile Asn Gln Leu 85 90 95 CGT GAA AAG ATC GGT GTG ATG TTC GGT TCC 318 Arg Glu Lys Ile Gly Val Met Phe Gly Ser 100 105

[Brief description of drawings]

FIG. 1 shows the plasmid p used in the present invention to isolate the insert sequence.
Restriction enzyme cleavage point map of CRY30-sacB.

FIG. 2 is a restriction map of the insertion sequence used in the present invention and a strategy diagram for determining the nucleotide sequence.

FIG. 3 is a restriction enzyme map of the plasmid pMV23 of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI technical display location (C12N 1/21 C12R 1:13) (72) Inventor Hideaki Yukawa 8 Central Ami-cho, Inashiki-gun, Ibaraki Prefecture 3-3-1 Tsukuba Research Institute, Mitsubishi Petrochemical Co., Ltd.

Claims (7)

[Claims] 1. (a) Incapable of autonomous replication in a coryneform bacterium, (b) 5'-upstream and 3'-of the indicator gene DNA.
A DNA region having at least one of the inverted repeats present upstream and 3'-downstream of the open reading frame in the insertion sequence derived from the coryneform bacterium in the downstream side; )
A plasmid having a DNA region containing an open reading frame in an insertion sequence derived from a coryneform bacterium. 2. The plasmid according to claim 1, wherein the indicator gene is a drug resistance gene. 3. The plasmid according to claim 1, wherein the inverted repeat is the nucleotide sequence represented by SEQ ID NO: 2 or 3. 4. The plasmid according to claim 1, wherein the open reading frame is the nucleotide sequence represented by SEQ ID NO: 4. 5. The method according to claim 1, wherein the indicator gene is a drug resistance gene, the inverted repeat is the nucleotide sequence represented by SEQ ID NO: 2 or 3, and the open reading frame is the nucleotide sequence represented by SEQ ID NO: 4. Plasmid. 6. A method for transforming a coryneform bacterium using the plasmid according to any one of claims 1 to 5 and isolating an indicator gene expression strain using the expression of the indicator gene of the transformant strain as an indicator. A method for obtaining a characteristic mutant strain. 7. The method according to claim 6, wherein the coryneform bacterium is a recombinant-deficient coryneform bacterium.