JPH07203977A - Gene recombination of coryneform bacteria - Google Patents

Abstract

PURPOSE:To provide a process for preparing a Coryneform bacterium where a specific DNA fragment in incorporated into the chromosome. CONSTITUTION:The pireparation process comprises (a) the formation of a recombinant plasmid where a plasmid which has a mutation in the replication origin of wild type having a specific base sequence and can effect autonomous replication in the Coryneform bacterium cells at a culture temperature lower than 31 deg.C, but not lower than 31 deg.C is connected to a DNA fragment having the homologous sequence to the gene existing on the chromosome of the Coryneform bacterium, (b) the introduction of the recombinant plasmid into the Coryneform bacterium cells, (c) the culture of the bacterium at a temperature higher than 31 deg., (d) the homologous recombination between the DNA fragment and the gene existing on the chromosome of the Coryneform bacterium having the sequence homologous to the fragment and (e) the selection of the Coryneform bacterium into which the DNA fragment is incorporated on its chromosome.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for modifying a chromosomal gene of a coryneform bacterium used for fermentative production of a useful substance such as an amino acid and converting its genetic trait. It can be used for breeding microorganisms useful for production and the like.

[0002]

2. Description of the Related Art Many attempts have been made to modify the genetic traits of coryneform bacteria by genetic engineering techniques to utilize them for the fermentation production of useful substances such as amino acids. But,
All of them utilize a plasmid that can autonomously propagate in coryneform bacteria as a vector. (JP-A-58-19
2900, JP-A-58-216199, etc.)

[0003]

[Problems to be Solved by the Invention] Since a plasmid proliferates and functions extrachromosomally, when the genetic properties of a bacterium are modified using plasmid DNA, the modification cannot be exerted on the chromosome of the bacterium which is the host. In particular, when the characteristics of the DNA of the host bacterium, such as deletion or mutation, pose a problem, no planned measures were taken. Until now, there has been used a method of treating a host bacterium with a mutating agent or the like and selecting a strain which has been modified as desired from randomly mutated bacteria, but it involved a great deal of labor and difficulty. Also, the plasmid
When DNA is used, the plasmid is unstable and drops out, and sufficient expression cannot be obtained, or due to the existence of multiple copies of the plasmid containing the target gene, the expression becomes excessive, resulting in bacterial growth and substance production. There were often problems such as adversely affecting. The present invention intends to solve these problems by intentionally modifying a specific gene on a chromosome and stably fixing a gene having a fixed copy number on the chromosome.

[0004]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have found that the origin of replication of a plasmid DNA capable of autonomous growth in coryneform bacteria is increased in culture temperature. The replication origin was changed to a temperature-sensitive mutant that rendered replication incompetent. The target gene is connected to a plasmid having this temperature-sensitive replication origin, introduced into a coryneform bacterium, and the strain holding the plasmid is cultured at 10 to 27 ° C, preferably 20 to 25 ° C, and then 31 to 37 ° C, preferably Is 33 to 36
The plasmid is removed by culturing at ℃, and the strain in which the gene is integrated into the chromosome is selected. At this time, a marker gene such as a drug resistance gene on the plasmid is integrated on the chromosome, and expression of the marker gene shows traits such as drug resistance without the plasmid being retained, so that the integrated strain can be selected. At this stage, the vector plasmid DNA and the target gene are simultaneously integrated on the chromosome, but the homologous recombination is caused again using the removal of the trait of the marker gene such as drug resistance from the integrated strain as an index, and the vector It is possible to select a strain in which a part has been removed from the chromosome. At this time, the chromosomal gene of the host corresponding to the target gene is removed at the same time, and gene replacement that replaces the gene introduced in the plasmid can occur.

As a method for gene integration into a chromosome, there is a method of introducing a replication-incompetent DNA and selecting a recombinant strain, as well as the method shown here, but a method using a temperature-sensitive plasmid. Since the target gene can be propagated in the form of a plasmid in cells and also in strains carrying the plasmid, recombination at a much higher frequency than the method of introducing non-replicating DNA to cause recombination. Is possible. Therefore, the transformation frequency of the host and DN
The effect of degradation of A hardly affects the appearance frequency of recombinants, and is characterized by being a highly general method in which gene recombination can be similarly performed in a wide range of hosts.

[0006] Examples of wild strains of coryneform glutamic acid-producing bacteria include the following. In addition to these, if the temperature-sensitive replication origin constructed here is functional and the plasmid is replicable, , All can be used as hosts. Brevibacterium divaricatum ATCC 14020 Brevibacterium saccharolyticum ATCC 14066 Brevibacterium inmario film ATCC 14068 Brevibacterium lactofermentum ATCC 13869 Brevibacterium roseum ATCC 13825 Brevibacterium flavum ATCC 13826 Brevi Corynebacterium acetoacetate film ATCC 13870 Corynebacterium acetoglutamicum ATCC 15806 Corynebacterium carnae ATCC 15991 Corynebacterium glutamicum ATCC 13032,13060 Corynebacterium lilium ATCC 15990 Corynebacterium・ Meracecola ATCC 17965 Microbacterium ・ Ammonia Philum ATCC 15354

[0007]

EXAMPLES The contents of the invention will be described in detail below based on examples.

Example 1 Acquisition of temperature-sensitive replication origin and construction of plasmid vector for gene integration

<Construction of pHK4> First, as a material for obtaining a temperature-sensitive replication origin, a plasmid vector, pHK4, which is capable of autonomous growth in both Escherichia coli and coryneform bacterium, was prepared. There are several reports of plasmid vectors capable of autonomous growth both in Escherichia coli and in coryneform bacteria. Here, pAJ1844 (JP-A-58-21619)
9) and pHSG298 (S. Takeshita et al : Gene 61,63-7).
4 (1987)), a new shuttle vector pHK4 was constructed. pAJ1844 was partially digested with the restriction enzyme Sau3AI and ligated with pHSG298 completely digested with the restriction enzyme BamHI. DN after connection
Brevibacterium lactofermentum AJ12036
(FERM-P7559). The electric pulse method (see JP-A-2-207791) was used for the transformation method. Transformants were selected on M-CM2G plates containing 25 μg / ml kanamycin.
(Glucose 5 g, polypeptone 10 g, yeast extract 10 g, NaCl5
g, DL-methionine 0.2 g and agar 15 g are included in 1 l of pure water. (pH7.2)
I went there. Prepare plasmid DNA from the transformant,
The smallest size was selected and named pHK4. This plasmid is capable of autonomous growth in Escherichia coli and in coryneform bacteria and confers kanamycin resistance on the host.

<Of a plasmid having a temperature-sensitive origin of replication
Acquisition and construction of plasmid vector for gene integration> pH
K4 was treated with hydroxylamine in vitro. Hydroxy
Silamine treatment is a known method (G.O.Humpheryset al
 : Molec.gen.Genet. 145, 101-108 (1976) etc.)
According to The treated DNA is recovered and then Brevibacterium
・ Lactofermentum AJ12036 strain was transformed. form
The transformants were M-CM2G plates containing 25 μg / ml kanamycin.
On low temperature (20 ° C). Appearing transformants
On a similar selection plate at high temperature (34 ° C)
Cultured. On selection plates containing kanamycin at elevated temperature
We acquired 3 strains that cannot grow. From this strain,
Were collected and designated as pHS4, pHS22, and pHS23. Add pHS4
Escherichia coli AJ12570 (FERM
-BP3523) and Escherichia holding pHS22 and pHS23 respectively
・ Recover the plasmid from E. coli and recover it in coryneform bacteria.
The gene fragment derived from pAJ1844, which is the starting point, was digested with the restriction enzyme Bam
Cut out with HI and KpnI and use for Escherichia coli
PHSG398 (S. Takeshitaet al : Gene 61,63-7
4 (see 1987)). This replication origin from pHS4
Has a replication origin derived from pHSC4 and pHS22
Has a replication origin derived from pHSC22 and pHS23
The plasmid was named pHSC23. Also, in the same way, pH
From K4, transfer the origin of replication in coryneform bacteria to pHSG398,
HS4 was produced. pHC4, pHSC4, pHSC22 and pHSC23 are
Proliferating autonomously in a necrobacterium and Escherichia coli,
Confer chloramphenicol resistance to the host. Also, pH
SC4, pHSC22, and pHSC23 replication origins that function in coryneform bacteria
It was confirmed that the points are temperature sensitive mutants. pHSC4
Escherichia coli AJ12571 as a plasmid
(FERM-BP3524) and pHSC22 are retained as plasmids
Escherichia coli AJ12615 (FERM-BP3530), pHSC23
Escherichia coli AJ12616 retained as a plasmid
(FERM-BP3531), which retains pHC4 as a plasmid
From E. coli AJ12617 (FERM-BP3532), plasmid
To prepare Brevibacterium lactofermentum
Introduced into AJ12036 and cultured at high temperature (34 ℃) in non-selective medium.
The change in the retention of the plasmid was observed. pHSC4, pHSC2
2.Reproduction of pHSC23 in AJ12036 was almost complete by high temperature culture.
All blocked. (Fig. 1-1,2,3,4)

<Determination of base sequence of origin of temperature-sensitive replication>
Plasmid pHC4 with a wild-type origin of replication and plasmids pHSC4, pHSC22, pHS with a temperature-sensitive origin of replication
The nucleotide sequence of the origin of replication of C23 in coryneform bacteria was determined. The nucleotide sequencing method is the method of Sanger et al. (FS
anger et al:. Proc.Natl.Acad.Sci 74, was by 5463 (1977), and the like). As a result, it was revealed that there were 2 to 4 base substitutions between the wild-type origin of replication and the temperature-sensitive mutant origin of replication. The nucleotide sequence of the temperature-sensitive origin of replication contained in pHSC4 and functioning in coryneform bacteria is shown in Sequence Listing 1.
2, the nucleotide sequence of the temperature-sensitive replication origin portion functioning in the coryneform bacterium contained in pHSC22 is shown in Sequence Listing 2, and the nucleotide sequence of the temperature-sensitive replication origin portion functioning in the coryneform bacterium contained in pHSC23 is shown in Sequence Listing 3. Table 4 shows the nucleotide sequence of the origin of replication of the wild type which is contained in pHC4 and functions in coryneform bacteria. The mutation points of the origin of temperature-sensitive replication are shown below. For pHSC4, 1542 on the sequence
The G at position 15 is mutated to A, and the G at position 1545 is mutated to A. Regarding pHSC22, the 683rd C in the sequence is mutated to T, the 1172nd C is mutated to T, and
The 1614th C is mutated to T. For pHSC23,
The 756th G in the sequence is mutated to A, the 1560th G is mutated to A, the 1667th C is mutated to T, and the 1684th C is mutated to T. .

Example 2 Incorporation of DNA on plasmid into Brevibacterium lactofermentum chromosomal gene using plasmid vector for gene integration and gene integration on chromosome Chromosome feedback inhibition by tyrosine and phenylalanine Brevibacterium lactofermentum released in 3
-Deoxy-D-arabinoheptulosonate-7-phosphate synthase gene (hereinafter, referred to as "DS gene") is a wild-type strain of Brevibacterium lactofermentum and is an analog of phenylalanine m-.
It is known to impart resistance to fluorophenylalanine and the ability to produce phenylalanine and tyrosine (see JP-A-61-124375). Using this property as an index, the DS gene was introduced into the chromosome of the Brevibacterium lactofermentum wild-type strain by homologous recombination.

<Preparation of plasmid for gene integration> pAR-1 (see JP-A-61-124375), which is a plasmid containing DS gene in which feedback inhibition by tyrosine and phenylalanine is substantially released, is partially decomposed with Sau3AI. , Connect with pHK4 completely digested with restriction enzyme BamHI.
Brevibacterium lactofermentum AJ12
It was introduced into strain 036 by the electric pulse method. Minimal medium plate containing 500 μg / ml of m-fluorophenylalanine (glucose 20 g, (NH ₄ ) ₂ SO ₄ 5 g, urea 2 g, KH ₂ PO ₄ 1 g, MgSO ₄ .7H ₂ O0.
5 g, FeSO ₄ .7H ₂ O 10 mg, MnSO ₄ .4H ₂ O 10 mg, biotin 50 μg, thiamine hydrochloride 2000 μg, and agar 15 g are included in 1 l of water. (pH 6.6)
Transformants were selected above. A plasmid was prepared from the transformant and the one having the smallest size was selected. This plasmid was digested with restriction enzymes BamHI and SalI to recover a 1.9-kilobase fragment containing the DS gene. This fragment was ligated with pHam4 cut with BamHI and SalI. The prepared plasmid was named pHSC4D.

<Introduction of integration plasmid into Brevibacterium lactofermentum and selection of integration strain into chromosome> From Escherichia coli AJ12572 (FERM-BP3525) containing pHSC4D as a plasmid, plasmid pHSC4D
To prepare Brevibacterium lactofermentum
It was introduced into the AJ12036 strain by the electric pulse method. Selection of transformants was performed at 20 ° C. on M-CM2G plates containing 5 μg / ml chloramphenicol. After introduction, the obtained strain is M-CM
After culturing in 2G plate medium, add 10 μg / ml to M-CM2G plate containing 5 μg / ml chloramphenicol.
^{It was} diluted to ³ to 10 ⁵ cfu and applied. Plate 34
Incubated at ° C. Strains harboring temperature-sensitive plasmids inhibit plasmid replication at this temperature,
Although it becomes chloramphenicol sensitive and cannot form colonies, strains that have integrated plasmid DNA into their chromosomes
They could be selected to form colonies. By homologous recombination, near the DS gene of the host chromosome,
Incorporation of the mutant DS gene derived from the vector plasmid was confirmed by Southern hybridization and resistance of the integrating bacterium to m-fluorophenylalanine.

(Example 3 Acquisition of a gene-replacement strain from a chromosome-integrating bacterium) D on the chromosome by homologous recombination
First, a chloramphenicol-sensitive strain was obtained from the AJ12573 strain (FERM-BP3526) into which the S gene had been incorporated. The integrated strain is diluted and spread on an M-CM2G plate, and cultured at 34 ° C. After colony formation, replica on M-CM2G plate containing 5 μg / ml chloramphenicol and culture at 34 ° C.
At this time, a strain that became susceptible to chloramphenicol was obtained. From the strain that became chloramphenicol sensitive,
A strain was selected which was sensitive to chloramphenicol and resistant to m-fluorophenylalanine. Chloramphenicol sensitivity means that the vector plasmid was recombinationally shed from the chromosome, and m-fluorophenylalanine resistance means that the mutant DS gene introduced from the plasmid still remained on the chromosome. That is, when a gene is shed from a chromosome by homologous recombination, the mutant-type gene introduced by the plasmid remains on the chromosome, and the wild-type gene originally on the chromosome is shed along with the vector plasmid. Means that a replacement has occurred.

Example 4 Production of Tyrosine and Phenylalanine by a Mutant-Type DS Gene-Incorporating Bacterium AJ12573 strain (FERM-BP3526) in which a mutant-type DS gene was integrated into a chromosome using a temperature-sensitive vector, and gene replacement Caused the mutant DS gene to be fixed on the chromosome
Tyrosine and phenylalanine were produced by the AJ12574 strain (FERM-BP3527). After refreshing each strain in M-CM2G plate medium, phenylalanine production medium (glucose 130 g, (NH ₄ ) ₂ SO ₄ 10 g, KH ₂ PO ₄ 1 g, MgSO ₄ .7H ₂ O
1g, fumaric acid 12g, acetic 3 ml, soybean protein acid hydrolyzate "Taste liquid" _{50ml, FeSO 4 · 7H 2 O10mg} , MnSO 4 · 4H 2 O 10mg, biotin 50
1 μl of pure water contains μg, 2000 μg of thiamine hydrochloride and 50 g of CaCO ₃ . The cells were inoculated to pH 7.0) and cultured at 31.5 ° C for 48 hours. Table 1 shows the amounts of tyrosine and phenylalanine produced in the culture broth after culturing.

[0017]

[Table 1]

Example 5 Brevibacterium lactofermentum wild-type strain integration of feedback inhibition-released aspartate kinase gene into chromosome and lysine production using the integrated strain

<Preparation of Plasmid for Gene Integration> Aspartokinase gene (hereinafter referred to as “AK gene”) in which feedback inhibition by lysine and threonine is substantially released is used as Corynebacterium glutamicum, lysine-producing bacterium AJ3463 (FERM- (P1987). The method is
The chromosomal DNA of AJ3463 was partially digested with the restriction enzyme Sau3AI and ligated with pHSC4 completely digested with BamHI. This DNA
Was transformed into AJ12036 strain. Selection of transformants is 5
Chloramphenicol at μg / ml, L-threonine at 100 mg / dl, and S- (2-aminoethyl) -L- at 3000 μg / ml.
Selection was performed at 20 ° C. in a minimal medium containing cysteine (hereinafter referred to as “AEC”). A plasmid was prepared from the transformant and named pHSC4AE.

<Incorporation of deblocking AK gene into Brevibacterium lactofermentum chromosome and acquisition of gene replacement strain> Brevibacterium lactofermentum ATCC13869 strain was transformed with plasmid pHSC4AE. Selection of transformants was performed at 20 ° C. The transformant was diluted with sterilized water and applied to an M-CM2G plate at 10 ³ to 10 ⁵ cfu per plate. Those which cultivated at 34 ° C. and formed colonies were selected as gene-integrated strains. A chloramphenicol-sensitive strain was obtained again from the gene-integrated strain, and a strain showing chloramphenicol-sensitive and AEC-resistant was selected from among the strains as a gene-substituted strain.

<Production of lysine using gene-replacement strain> Brevibacterium lactofermentum AJ1257 in which a derepression-type AK gene was integrated on the chromosome by gene replacement
5 strains (FERM-BP3528) and its parent strain ATCC13869, lysine production medium (glucose 100 g, (NH ₄ ) ₂ SO ₄ 55 g, KH _{2
PO 4 1g, MgSO 4 · 7H} 2 O1g, soybean protein acid hydrolyzate "Mameko" 50m
_{l, FeSO 4 · 7H 2 O10mg} , MnSO 4 · 4H 2 O 10mg, nicotinamide 5
1g of pure water contains mg and 50g of CaCO ₃ . pH 8.0) and inoculate 3
It was cultured at 1.5 ° C for 48 hours. The amount of lysine produced in the culture medium after culturing is as shown in Table 2.

[0022]

[Table 2]

[0023]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, the origin of replication of a plasmid DNA capable of autonomous growth in a coryneform bacterium is made incapable of replicating by raising the culture temperature, and thus a specific chromosome It is possible to obtain the effect of systematically modifying the gene and stably fixing a gene having a fixed copy number on the chromosome.

[0024]

[Sequence listing] SEQ ID NO: 1 Sequence length: 2959 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Origin Biologic name: Corynebacterium glutamicum
um) strain name: ATCC 13058 SEQ GGATCCGGTG TATGCCGCAG CAGGCATGAG CAGCCCGAAT ATGCGCCTGC TGGCTGCAAC 60 GACCGAGGAA ATGACCCGCG TTTTCGGCGC TGACCAGGCT TTTTCACATA GGCTGAGCCG 120 GTGGCCACTG CACGTCTCCG ACGATCCCAC CGCGTACCGC TGGCATGCCC AGCACAATCG 180 CGTGGATCGC CTAGCTGATC CCGAAAAAGT TTTTGCCTTT TGTAAAAAAC TTCTCGGTCG 240 CCCCGCAAAT TTTCGATTCC AGATTTTTTA AAAACCAAGC CAGAAATACG ACACACCGTT 300 TGCAGATAAT CTGTCTTTCG GAAAAATCAA GTGCGATACA AAATTTTTAG CACCCCTGAC 360 GTGCGCAAAG TCCCGCTTCG TGAAAATTTT CGTGCCGCGT GATTTTCCGC CAAAAACTTT 420 AACGAACGTT CGTTATAATG GTGTCATGAC CTTCACGACG AAGTACCAAA ATTGGCCCGA 480 ATCATCAGCT ATGGATCTCT CTGATGTCGC GCTGGAGTCC GACGCGCTCG ATGCTGCCGT 540 CGATTTAAAA ACGGTGATCG GATTTTTCCG AGCTCTCGAT ACGACGGACG CGCCAGCATC 600 ACGAGACTGG GCCAGTGCCG CGAGCGACCT AGAAACTCTC GTGGCGGATC TTGAGGAGCT 660 GGCTGACGAG CTGCGTGCTC GGCAGCGCCA GGAGGACGCA CAGTAGTGGA GGATCGAATC 720 AGTTGCGCCT ACTGCGGTGG CCTGATTCCT CCCCGGCCTG ACCCGCGAGG ACGGCGCGCA 780 AAATATTGCT CAGATGCGTG TCGTGCCGCA GCCAGCCGCG AGCGCGCCAA CAAAC GCCAC 840 GCCGAGGAGC TGGAGGCGGC TAGGTCGCAA ATGGCGCTGG AAGTGCGTCC CCCGAGCGAA 900 ATTTTGGCCA TGGTCGTCAC AGAGCTGGAA GCGGCAGCGA GAATTATCCG CGATCGTGGC 960 GCGGTGCCCG CAGGCATGAC AAACATCGTA AATGCCGCGT TTCGTGTGGC CGTGGCCGCC 1020 CAGGACGTGT CAGCGCCGCC ACCACCTGCA CCGAATCGGC AGCAGCGTCG CGCGTCGAAA 1080 AAGCGCACAG GCGGCAAGAA GCGATAAGCT GCACGAATAC CTGAAAAATG TTGAACGCCC 1140 CGTGAGCGGT AACTCACAGG GCGTCGGCTA ACCCCCAGTC CAAACCTGGG AGAAAGCGCT 1200 CAAAAATGAC TCTAGCGGAT TCACGAGACA TTGACACACC GGCCTGGAAA TTTTCCGCTG 1260 ATCTGTTCGA CACCCATCCC GAGCTCGCGC TGCGATCACG TGGCTGGACG AGCGAAGACC 1320 GCCGCGAATT CCTCGCTCAC CTGGGCAGAG AAAATTTCCA GGGCAGCAAG ACCCGCGACT 1380 TCGCCAGCGC TTGGATCAAA GACCCGGACA CGGGAGAAAC ACAGCCGAAG TTATACCGAG 1440 TTGGTTCAAA ATCGCTTGCC CGGTGCCAGT ATGTTGCTCT GACGCACGCG CAGCACGCAG 1500 CCGTGCTTGT CCTGGACATT GATGTGCCGA GCCACCAGGC CAGCAGGAAA ATCGAGCACG 1560 TAAACCCCGA GGTCTACGCG ATTTTGGAGC GCTGGGCACG CCTGGAAAAA GCGCCAGCTT 1620 GGATCGGCGT GAATCCACTG AGCGGGAAAT GCCAGCTCAT CTGGCTCATT GATCCGGTGT 168 0 ATGCCGCAGC AGGCATGAGC AGCCCGAATA TGCGCCTGCT GGCTGCAACG ACCGAGGAAA 1740 TGACCCGCGT TTTCGGCGCT GACCAGGCTT TTTCACATAG GCTGAGCCGG TGGCCACTGC 1800 ACGTCTCCGA CGATCCCACC GCGTACCGCT GGCATGCCCA GCACAATCGC GTGGATCGCC 1860 TAGCTGATCT TATGGAGGTT GCTCGCATGA TCTCAGGCAC AGAAAAACCT AAAAAACGCT 1920 ATGAGCAGGA GTTTTCTAGC GGACGGGCAC GTATCGAAGC GGCAAGAAAA GCCACTGCGG 1980 AAGCAAAAGC ACTTGCCACG CTTGAAGCAA GCCTGCCGAG CGCCGCTGAA GCGTCTGGAG 2040 AGCTGATCGA CGGCGTCCGT GTCCTCTGGA CTGCTCCAGG GCGTGCCGCC CGTGATGAGA 2100 CGGCTTTTCG CCACGCTTTG ACTGTGGGAT ACCAGTTAAA AGCGGCTGGT GAGCGCCTAA 2160 AAGACACCAA GATCATCGAC GCCTACGAGC GTGCCTACAC CGTCGCTCAG GCGGTCGGAG 2220 CAGACGGCCG TGAGCCTGAT CTGCCGCCGA TGCGTGACCG CCAGACGATG GCGCGACGTG 2280 TGCGCGGCTA CGTCGCTAAA GGCCAGCCAG TCGTCCCTGC TCGTCAGACA GAGACGCAGA 2340 GCAGCCGAGG GCGAAAAGCT CTGGCCACTA TGGGAAGACG TGGCGGTAAA AAGGCCGCAG 2400 AACGCTGGAA AGACCCAAAC AGTGAGTACG CCCGAGCACA GCGAGAAAAA CTAGCTAAGT 2460 CCAGTCAACG ACAAGCTAGG AAAGCTAAAG GAAATCGCTT GACCATTGCA GGTTGGTTTA 2520 TGAC TGTTGA GGGAGAGACT GGCTCGTGGC CGACAATCAA TGAAGCTATG TCTGAATTTA 2580 GCGTGTCACG TCAGACCGTG AATAGAGCAC TTAAGTCTGC GGGCATTGAA CTTCCACGAG 2640 GACGCCGTAA AGCTTCCCAG TAAATGTGCC ATCTCGTAGG CAGAAAACGG TTCCCCCCGT 2700 AGGGGTCTCT CTCTTGGCCT CCTTTCTAGG TCGGGCTGAT TGCTCTTGAA GCTCTCTAGG 2760 GGGGCTCACA CCATAGGCAG ATAACGGTTC CCCACCGGCT CACCTCGTAA GCGCACAAGG 2820 ACTGCTCCCA AAGATCGCCT AGCTGATCTT ATGGAGGTTG CTCGCATGAT CCTTTTTGAT 2880 AATCTCATGA CCAAAATCCC TTAACGTGAG TTTTCGTTCC ACTGAGCGTC AGACCCCGTA 2940 GAAAAGATCC CCGGGTACC 2959

SEQ ID NO: 2 Sequence length: 2959 Sequence type: Nucleic acid Number of strands: Double strands Topology: Linear origin Origin organism name: Corynebacterium glutamicum (Corynebacterium glutamic
um) strain name: ATCC 13058 SEQ GGATCCGGTG TATGCCGCAG CAGGCATGAG CAGCCCGAAT ATGCGCCTGC TGGCTGCAAC 60 GACCGAGGAA ATGACCCGCG TTTTCGGCGC TGACCAGGCT TTTTCACATA GGCTGAGCCG 120 GTGGCCACTG CACGTCTCCG ACGATCCCAC CGCGTACCGC TGGCATGCCC AGCACAATCG 180 CGTGGATCGC CTAGCTGATC CCGAAAAAGT TTTTGCCTTT TGTAAAAAAC TTCTCGGTCG 240 CCCCGCAAAT TTTCGATTCC AGATTTTTTA AAAACCAAGC CAGAAATACG ACACACCGTT 300 TGCAGATAAT CTGTCTTTCG GAAAAATCAA GTGCGATACA AAATTTTTAG CACCCCTGAC 360 GTGCGCAAAG TCCCGCTTCG TGAAAATTTT CGTGCCGCGT GATTTTCCGC CAAAAACTTT 420 AACGAACGTT CGTTATAATG GTGTCATGAC CTTCACGACG AAGTACCAAA ATTGGCCCGA 480 ATCATCAGCT ATGGATCTCT CTGATGTCGC GCTGGAGTCC GACGCGCTCG ATGCTGCCGT 540 CGATTTAAAA ACGGTGATCG GATTTTTCCG AGCTCTCGAT ACGACGGACG CGCCAGCATC 600 ACGAGACTGG GCCAGTGCCG CGAGCGACCT AGAAACTCTC GTGGCGGATC TTGAGGAGCT 660 GGCTGACGAG CTGCGTGCTC GGTAGCGCCA GGAGGACGCA CAGTAGTGGA GGATCGAATC 720 AGTTGCGCCT ACTGCGGTGG CCTGATTCCT CCCCGGCCTG ACCCGCGAGG ACGGCGCGCA 780 AAATATTGCT CAGATGCGTG TCGTGCCGCA GCCAGCCGCG AGCGCGCCAA CAAAC GCCAC 840 GCCGAGGAGC TGGAGGCGGC TAGGTCGCAA ATGGCGCTGG AAGTGCGTCC CCCGAGCGAA 900 ATTTTGGCCA TGGTCGTCAC AGAGCTGGAA GCGGCAGCGA GAATTATCCG CGATCGTGGC 960 GCGGTGCCCG CAGGCATGAC AAACATCGTA AATGCCGCGT TTCGTGTGGC CGTGGCCGCC 1020 CAGGACGTGT CAGCGCCGCC ACCACCTGCA CCGAATCGGC AGCAGCGTCG CGCGTCGAAA 1080 AAGCGCACAG GCGGCAAGAA GCGATAAGCT GCACGAATAC CTGAAAAATG TTGAACGCCC 1140 CGTGAGCGGT AACTCACAGG GCGTCGGCTA ATCCCCAGTC CAAACCTGGG AGAAAGCGCT 1200 CAAAAATGAC TCTAGCGGAT TCACGAGACA TTGACACACC GGCCTGGAAA TTTTCCGCTG 1260 ATCTGTTCGA CACCCATCCC GAGCTCGCGC TGCGATCACG TGGCTGGACG AGCGAAGACC 1320 GCCGCGAATT CCTCGCTCAC CTGGGCAGAG AAAATTTCCA GGGCAGCAAG ACCCGCGACT 1380 TCGCCAGCGC TTGGATCAAA GACCCGGACA CGGGAGAAAC ACAGCCGAAG TTATACCGAG 1440 TTGGTTCAAA ATCGCTTGCC CGGTGCCAGT ATGTTGCTCT GACGCACGCG CAGCACGCAG 1500 CCGTGCTTGT CCTGGACATT GATGTGCCGA GCCACCAGGC CGGCGGGAAA ATCGAGCACG 1560 TAAACCCCGA GGTCTACGCG ATTTTGGAGC GCTGGGCACG CCTGGAAAAA GCGTCAGCTT 1620 GGATCGGCGT GAATCCACTG AGCGGGAAAT GCCAGCTCAT CTGGCTCATT GATCCGGTGT 168 0 ATGCCGCAGC AGGCATGAGC AGCCCGAATA TGCGCCTGCT GGCTGCAACG ACCGAGGAAA 1740 TGACCCGCGT TTTCGGCGCT GACCAGGCTT TTTCACATAG GCTGAGCCGG TGGCCACTGC 1800 ACGTCTCCGA CGATCCCACC GCGTACCGCT GGCATGCCCA GCACAATCGC GTGGATCGCC 1860 TAGCTGATCT TATGGAGGTT GCTCGCATGA TCTCAGGCAC AGAAAAACCT AAAAAACGCT 1920 ATGAGCAGGA GTTTTCTAGC GGACGGGCAC GTATCGAAGC GGCAAGAAAA GCCACTGCGG 1980 AAGCAAAAGC ACTTGCCACG CTTGAAGCAA GCCTGCCGAG CGCCGCTGAA GCGTCTGGAG 2040 AGCTGATCGA CGGCGTCCGT GTCCTCTGGA CTGCTCCAGG GCGTGCCGCC CGTGATGAGA 2100 CGGCTTTTCG CCACGCTTTG ACTGTGGGAT ACCAGTTAAA AGCGGCTGGT GAGCGCCTAA 2160 AAGACACCAA GATCATCGAC GCCTACGAGC GTGCCTACAC CGTCGCTCAG GCGGTCGGAG 2220 CAGACGGCCG TGAGCCTGAT CTGCCGCCGA TGCGTGACCG CCAGACGATG GCGCGACGTG 2280 TGCGCGGCTA CGTCGCTAAA GGCCAGCCAG TCGTCCCTGC TCGTCAGACA GAGACGCAGA 2340 GCAGCCGAGG GCGAAAAGCT CTGGCCACTA TGGGAAGACG TGGCGGTAAA AAGGCCGCAG 2400 AACGCTGGAA AGACCCAAAC AGTGAGTACG CCCGAGCACA GCGAGAAAAA CTAGCTAAGT 2460 CCAGTCAACG ACAAGCTAGG AAAGCTAAAG GAAATCGCTT GACCATTGCA GGTTGGTTTA 2520 TGAC TGTTGA GGGAGAGACT GGCTCGTGGC CGACAATCAA TGAAGCTATG TCTGAATTTA 2580 GCGTGTCACG TCAGACCGTG AATAGAGCAC TTAAGTCTGC GGGCATTGAA CTTCCACGAG 2640 GACGCCGTAA AGCTTCCCAG TAAATGTGCC ATCTCGTAGG CAGAAAACGG TTCCCCCCGT 2700 AGGGGTCTCT CTCTTGGCCT CCTTTCTAGG TCGGGCTGAT TGCTCTTGAA GCTCTCTAGG 2760 GGGGCTCACA CCATAGGCAG ATAACGGTTC CCCACCGGCT CACCTCGTAA GCGCACAAGG 2820 ACTGCTCCCA AAGATCGCCT AGCTGATCTT ATGGAGGTTG CTCGCATGAT CCTTTTTGAT 2880 AATCTCATGA CCAAAATCCC TTAACGTGAG TTTTCGTTCC ACTGAGCGTC AGACCCCGTA 2940 GAAAAGATCC CCGGGTACC 2959

SEQ ID NO: 3 Sequence length: 2959 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear origin Origin organism name: Corynebacterium glutamicum
um) strain name: ATCC 13058 sequence GGATCCGGTG TATGCCGCAG CAGGCATGAG CAG
CCCGAAT ATGCGCCTGC TGGCTGCAAC 60 GACCGAGGAA ATGACCCGCG TTTTTCGGCGC TGA
CCAGGCT TTTTCACATA GGCTGAGCCG 120 GTGGCCACTG CACGTCTCCG ACGATCCCAC CGC
GTACCGC TGGCATGCCC CCAGCACAATCG 180 CGTGGATCGC CTAGCTGATC CCGAAAAAGT TTT
TGCCTTT TGTAAAAAAC TTCTCGGGTCG 240 CCCCGCAAAT TTTCGATTCC AGATTTTTTTA AAA
ACCAAGC CAGAAATACG ACACACCGTT 300 TGCAGAATAAT CTGTCTTTCG GAAAAAATCAA GTG
CGATACA AAATTTTTAG CACCCCCTGAC 360 GTGCGCAAAG TCCCGCTTCG TGAAAATTTT CGT
GCCGCGT GATTTTCCGC CAAAAACTTT 420 AACGAACGTT CGTTAATAATG GTGTCATGAC CTT
CACGACG AAGTACCAAA ATTGGCCCCGA 480 ATCATCAGCT ATGGATCTCT CTGATGTCGGC GCT
GGAGTCC GACGCGCTCG ATGCTGCCGT 540 CGATTTAAAA ACGGGTGATCG GATTTTTCCG AGC
TCTCGAT ACGACGGACG CGCCAGCATC 600 ACGAGACTGG GCCATGGCCG CGAGCGACCT AGA
AACTCTC GTGGCGGATTC TTGAGGAGCT 660 GGCTGACGAG CTCGCGTGTCC GGCAGCGCCA GGA
GGACGCA CATAGTAGTGGA GGATCGAATC 720 AGTTGCGCCT ACTGCGGGTGG CCTGATTCCT CCC
CGACCTG ACCCGCGAGG ACGGCGCGCA 780 AAAATTGCT CAGATGCGTG TCGTGCCGCA GCC
AGCCGCG AGCGCGCCAA CAAACGCCAC 840 GCCGAGGAGC TGGAGGGCGGC TAGGTCGCAA ATG
GCGCTGG AAGTGCGTCC CCCGAGCGAA 900 ATTTTGGCCA TGGTCGTCCAC AGAGCTGGAA GCG
GCAGCGA GAATTATCCG CGATCGTGGC 960 GCGGTGCCCCG CAGGCATGAC AAAACCATCGTA AAT
GCCGCGT TTCGTGTGGC CGTGGCCGCC 1020 CAGGACGTGT CACGCGCCGCCC ACCACCTGCA CCG
AATCGGC AGCAGCGTCG CGCGTCGCAAAA 1080 AAGCGCACAG GCGGCAAGAA GCCGATAAGCT GCA
CGAATAC CTGAAAAATG TTGAACGCCC 1140 CGTGAGCGGGT AACTCACAGG GCGTGCGGCTA ACC
CCCAGTC CAAACCTGGG AGAAAGCGCT 1200 CAAAAATGAC TCTAGCGGAT TCACGAGACA TTG
ACACACC GGCCTGGAAA TTTTCCGCTG 1260 ATCTGTTCGA CACCCATCCC GAGCTCGCGCGCGC
GATCACTG TGGCTGGACCG ACGGAAGACC 1320 GCCGCGAATT CCTCGCTCACCTGGGGCAGAG AAA
ATTTCCA GGGCAGCAAG ACCCGCGACT 1380 TCGCCAGCGC TTGGATCAAA GACCCGGACA CGG
GAGAAAC ACAGCCGAAG TTATACCGAG 1440 TTGGTTCAAA ATCGCTTGCC CGGTGCCAGT ATG
TTGCTCT GACGCACGCG CACGCACGCAG 1500 CCGTGCTTGT CCTGGACATT GATGTGCCGA GCC
ACCAGGC CGGCGGGAAA ATCGAGCACA 1560 TAAACCCCGA GGTCTACCGCG ATTTTGGGAGC GCT
GGGCACG CCTGGAAAA GCGCCAGCTT 1620 GGATCGGCGT GAATCCACTG AGCGGGAAAT GCC
AGCTCAT CTGGCTTTATT GATCCGGGTGT 1680 ATGTCGCAGC AGGCATGAGC AGCCCGAATA TGC
GCCTGCT GGCTGCAACG ACCGAGGAAA 1740 TGACCCGCGT TTTCGGCGCGCT GACCAGGCTT TTT
CACATAG GCTGA GCCGG TGGCCACTGC 1800 ACGTCTCCGA CGATCCCACC GCCGTACCGCT GGC
ATGCCCA GCACAATCGC GTGGATCCGCC 1860 TAGCTGATCT TATGGAGGTT GCTCGCATGA TCT
CAGGCAC AGAAAAACCT AAAAAACGCT 1920 ATGAGCAGGA GTTTTCTAGC GGACGGGGCAC GTA
TCGAAGC GGCAAGAAAA GCCACTGCGG 1980 AAGCAAAAGC ACTTGCCACG CTTGAAGCAA GCC
TGCCGAG CGCCGCTGAA GCGTCTGGAG 2040 AGCTGATCGA CGGCGTCCGT GTCCCTCTGGA CTG
CTCCAGG GCGTGCCGCC CGTGATGAGA 2100 CGGCTTTCG CCACGCTTTG ACTGTGGGGAT ACC
AGTTAAAA AGCGCGTGGT GAGCCGCCTAA 2160 AAGACACCCAA GATCATCGAC GCCTACGAGC GTG
CCTACAC CGTCGCTCAG GCGGTCGGGAG 2220 CAGACGGCCG TGAGCCTGAT CTGCCGCCGA TGC
GTGACCG CCAGACGATG GCGCGAGCGTG 2280 TGCGCGGCTACGTCGCTAAA GGCCAGCCAG TCG
TCCCTGC TCGTCAGACA GAGACGCAGA 2340 GCAGCCGAGG GCGAAAAGCT CTGGCCACTA TGG
GAAGACG TGGCGGTAAA AAGGCCGCAG 2400 AACGCTGGAA AGACCCAAAAC AGTGAGGTAG CCC
GAGCACA GCGAGAAAA CTAGCTAAGT 2460 CCAGTCAACG ACAAGCTAGG AAAGCTAAAG GAA
ATCGCTT GACCATTGCA GGTTGGTTTA 2520 TGACTGTTTGA GGGAGAGACT GGCTCGTGGGC CGA
CAATCAA TGAAGCTATG TCTGAATTTA 2580 GCGTGTCACG TCAGACCGTG AATAGAGAGCAC TTA
AGTCTGC GGGCATTGAA CTTCCCAGAG 2640 GACGCCGTAA AGCTTCCCAG TAAATGTGCC ATC
TCGTAGG CAGAAAACGG TTCCCCCCGT 2700 AGGGGTCTCT CTCTTGGCCT CCTTTCTAGGG TCG
GGCTGAT TGCTCTTGAA GCTCTCTAGG 2760 GGGGCTCACA CCATAGGCAG ATAACGGGTTC CCC
ACCGGCT CACCTGTAA GCGCACAAGG 2820 ACTGCTCCCA AAGATCGCCT AGCTGATCTT ATG
GAGGTTG CTCGCATGAT CCTTTTTGAT 2880 AATCTCATGA CCAAAATCCC TTAACGTGAG TTT
TCGTTCC ACTGAGCGTC AGACCCCGTA 2940 GAAAAGATCC CCGGGTACC
2959

SEQ ID NO: 4 Sequence length: 2959 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear origin Origin organism name: Corynebacterium glutamicum (Corynebacterium glutamic
um) strain name: ATCC 13058 SEQ GGATCCGGTG TATGCCGCAG CAGGCATGAG CAGCCCGAAT ATGCGCCTGC TGGCTGCAAC 60 GACCGAGGAA ATGACCCGCG TTTTCGGCGC TGACCAGGCT TTTTCACATA GGCTGAGCCG 120 GTGGCCACTG CACGTCTCCG ACGATCCCAC CGCGTACCGC TGGCATGCCC AGCACAATCG 180 CGTGGATCGC CTAGCTGATC CCGAAAAAGT TTTTGCCTTT TGTAAAAAAC TTCTCGGTCG 240 CCCCGCAAAT TTTCGATTCC AGATTTTTTA AAAACCAAGC CAGAAATACG ACACACCGTT 300 TGCAGATAAT CTGTCTTTCG GAAAAATCAA GTGCGATACA AAATTTTTAG CACCCCTGAC 360 GTGCGCAAAG TCCCGCTTCG TGAAAATTTT CGTGCCGCGT GATTTTCCGC CAAAAACTTT 420 AACGAACGTT CGTTATAATG GTGTCATGAC CTTCACGACG AAGTACCAAA ATTGGCCCGA 480 ATCATCAGCT ATGGATCTCT CTGATGTCGC GCTGGAGTCC GACGCGCTCG ATGCTGCCGT 540 CGATTTAAAA ACGGTGATCG GATTTTTCCG AGCTCTCGAT ACGACGGACG CGCCAGCATC 600 ACGAGACTGG GCCAGTGCCG CGAGCGACCT AGAAACTCTC GTGGCGGATC TTGAGGAGCT 660 GGCTGACGAG CTGCGTGCTC GGCAGCGCCA GGAGGACGCA CAGTAGTGGA GGATCGAATC 720 AGTTGCGCCT ACTGCGGTGG CCTGATTCCT CCCCGACCTG ACCCGCGAGG ACGGCGCGCA 780 AAATATTGCT CAGATGCGTG TCGTGCCGCA GCCAGCCGCG AGCGCGCCAA CAAAC GCCAC 840 GCCGAGGAGC TGGAGGCGGC TAGGTCGCAA ATGGCGCTGG AAGTGCGTCC CCCGAGCGAA 900 ATTTTGGCCA TGGTCGTCAC AGAGCTGGAA GCGGCAGCGA GAATTATCCG CGATCGTGGC 960 GCGGTGCCCG CAGGCATGAC AAACATCGTA AATGCCGCGT TTCGTGTGGC CGTGGCCGCC 1020 CAGGACGTGT CAGCGCCGCC ACCACCTGCA CCGAATCGGC AGCAGCGTCG CGCGTCGAAA 1080 AAGCGCACAG GCGGCAAGAA GCGATAAGCT GCACGAATAC CTGAAAAATG TTGAACGCCC 1140 CGTGAGCGGT AACTCACAGG GCGTCGGCTA ACCCCCAGTC CAAACCTGGG AGAAAGCGCT 1200 CAAAAATGAC TCTAGCGGAT TCACGAGACA TTGACACACC GGCCTGGAAA TTTTCCGCTG 1260 ATCTGTTCGA CACCCATCCC GAGCTCGCGC TGCGATCACG TGGCTGGACG AGCGAAGACC 1320 GCCGCGAATT CCTCGCTCAC CTGGGCAGAG AAAATTTCCA GGGCAGCAAG ACCCGCGACT 1380 TCGCCAGCGC TTGGATCAAA GACCCGGACA CGGGAGAAAC ACAGCCGAAG TTATACCGAG 1440 TTGGTTCAAA ATCGCTTGCC CGGTGCCAGT ATGTTGCTCT GACGCACGCG CAGCACGCAG 1500 CCGTGCTTGT CCTGGACATT GATGTGCCGA GCCACCAGGC CGGCGGGAAA ATCGAGCACA 1560 TAAACCCCGA GGTCTACGCG ATTTTGGAGC GCTGGGCACG CCTGGAAAAA GCGCCAGCTT 1620 GGATCGGCGT GAATCCACTG AGCGGGAAAT GCCAGCTCAT CTGGCTTATT GATCCGGTGT 168 0 ATGTCGCAGC AGGCATGAGC AGCCCGAATA TGCGCCTGCT GGCTGCAACG ACCGAGGAAA 1740 TGACCCGCGT TTTCGGCGCT GACCAGGCTT TTTCACATAG GCTGAGCCGG TGGCCACTGC 1800 ACGTCTCCGA CGATCCCACC GCGTACCGCT GGCATGCCCA GCACAATCGC GTGGATCGCC 1860 TAGCTGATCT TATGGAGGTT GCTCGCATGA TCTCAGGCAC AGAAAAACCT AAAAAACGCT 1920 ATGAGCAGGA GTTTTCTAGC GGACGGGCAC GTATCGAAGC GGCAAGAAAA GCCACTGCGG 1980 AAGCAAAAGC ACTTGCCACG CTTGAAGCAA GCCTGCCGAG CGCCGCTGAA GCGTCTGGAG 2040 AGCTGATCGA CGGCGTCCGT GTCCTCTGGA CTGCTCCAGG GCGTGCCGCC CGTGATGAGA 2100 CGGCTTTTCG CCACGCTTTG ACTGTGGGAT ACCAGTTAAA AGCGGCTGGT GAGCGCCTAA 2160 AAGACACCAA GATCATCGAC GCCTACGAGC GTGCCTACAC CGTCGCTCAG GCGGTCGGAG 2220 CAGACGGCCG TGAGCCTGAT CTGCCGCCGA TGCGTGACCG CCAGACGATG GCGCGACGTG 2280 TGCGCGGCTA CGTCGCTAAA GGCCAGCCAG TCGTCCCTGC TCGTCAGACA GAGACGCAGA 2340 GCAGCCGAGG GCGAAAAGCT CTGGCCACTA TGGGAAGACG TGGCGGTAAA AAGGCCGCAG 2400 AACGCTGGAA AGACCCAAAC AGTGAGTACG CCCGAGCACA GCGAGAAAAA CTAGCTAAGT 2460 CCAGTCAACG ACAAGCTAGG AAAGCTAAAG GAAATCGCTT GACCATTGCA GGTTGGTTTA 2520 TGAC TGTTGA GGGAGAGACT GGCTCGTGGC CGACAATCAA TGAAGCTATG TCTGAATTTA 2580 GCGTGTCACG TCAGACCGTG AATAGAGCAC TTAAGTCTGC GGGCATTGAA CTTCCACGAG 2640 GACGCCGTAA AGCTTCCCAG TAAATGTGCC ATCTCGTAGG CAGAAAACGG TTCCCCCCGT 2700 AGGGGTCTCT CTCTTGGCCT CCTTTCTAGG TCGGGCTGAT TGCTCTTGAA GCTCTCTAGG 2760 GGGGCTCACA CCATAGGCAG ATAACGGTTC CCCACCGGCT CACCTCGTAA GCGCACAAGG 2820 ACTGCTCCCA AAGATCGCCT AGCTGATCTT ATGGAGGTTG CTCGCATGAT CCTTTTTGAT 2880 AATCTCATGA CCAAAATCCC TTAACGTGAG TTTTCGTTCC ACTGAGCGTC AGACCCCGTA 2940 GAAAAGATCC CCGGGTACC 2959

[Brief description of drawings]

[Fig. 1] High temperature (34%) in drug-free M-CM2G liquid medium
FIG. 3 is a comparison of changes in the plasmid pHSC4 retention of the AJ12036 strain having a wild-type origin of replication and a temperature-sensitive mutant origin of replication when cultured at (° C.). The plasmid retention was determined by diluting the liquid culture and then adding M-containing chloramphenicol.
It was measured by comparing the number of colonies formed on a CM2G plate and a drug-free M-CM2G plate and measuring the number of colonies formed at low temperature (20 ° C) with the number of viable cells.

[FIG. 2] Similar to FIG. 1, a plasmid of the AJ12036 strain having a wild-type replication origin and a temperature-sensitive mutant replication origin when cultured at high temperature (34 ° C.) in a drug-free M-CM2G liquid medium. This is a comparison of changes in pHSC22 retention.

[Fig. 3] Similar to Fig. 1, a plasmid of the AJ12036 strain having a wild-type replication origin and a temperature-sensitive mutant replication origin when cultured at high temperature (34 ° C) in a drug-free M-CM2G liquid medium. It is a comparison of changes in pHSC23 retention.

[Fig. 4] In a M-CM2G liquid medium containing no drug, high temperature (34
AJ12036 with a wild-type origin of replication when cultured at
It is a comparison of changes in the plasmid pHC4 retention of the strains.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12P 13/22 B 2121-4B C 2121-4B // C12N 9/00 9/12 (C12N 15 / 09 ZNA C12R 1:13) (C12N 1/21 C12R 1:13) (C12P 13/08 A C12R 1:15) (C12P 13/22 B C12R 1:15) (C12P 13/22 C C12R 1:15) C72R 1:13) (72) Yutaka Matsui 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Ajinomoto Co., Inc. Central Research Laboratory (72) Katsuaki Sato 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Central Research Institute, Nomoto Co. (72) Wataru Nakamatsu 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Ajinomoto Co., Central Research Lab

Claims (7)

[Claims] 1. (a) It has a mutation in the origin of replication of a wild type having the nucleotide sequence represented by SEQ ID NO: 4 in the sequence listing, and autonomously replicates in coryneform bacterial cells at a culture temperature lower than 31 ° C. D, which has a sequence homologous to a gene present on the chromosome of coryneform bacteria, in a plasmid that can be formed but cannot autonomously replicate in the same cell at a temperature of 31 ° C or higher
The NA fragment is ligated to obtain a recombinant plasmid, (b) the recombinant plasmid is introduced into a coryneform bacterial cell, (c) the bacterium is cultured at a temperature of 31 ° C. or higher, and (d) the same DNA fragment. , Causing homologous recombination with a gene existing on the chromosome of a coryneform bacterium having a sequence homologous to the same DNA fragment, and (e) selecting a coryneform bacterium in which the same DNA fragment is integrated on the chromosome. A method for preparing a coryneform bacterium in which a DNA fragment is integrated on a chromosome, comprising: 2. A method for preparing a coryneform bacterium in which the DNA fragment according to claim 2 is integrated on a chromosome, wherein the DN
After selecting the coryneform bacterium in which the A fragment has been integrated on the chromosome, further between (a) the same DNA fragment and a gene existing on the chromosome of the coryneform bacterium having a sequence homologous to the same DNA fragment. (B) a gene having a sequence homologous to the DNA fragment originally present on the chromosome of the coryneform bacterium, which caused homologous recombination in the bacterium, and SEQ ID NO: in the sequence listing integrated on the chromosome of the bacterium. 4 has a mutation in the origin of replication of the wild type having the nucleotide sequence shown in 4, and the culture temperature is 3
At a temperature lower than 1 ° C., DNA derived from a plasmid capable of autonomous replication in coryneform bacterial cells but not at the temperature of 31 ° C. or higher is eliminated, and (c) the same DNA fragment is Characterized by selecting a coryneform bacterium which has replaced a gene existing on the chromosome of the coryneform bacterium having a sequence homologous to the DNA fragment,
A method for preparing a coryneform bacterium in which a DNA fragment is integrated on a chromosome. 3. The origin of replication of the plasmid has the nucleotide sequences shown in SEQ ID NOs: 1 to 3 in the sequence listing.
Or the method described in 2. 4. 3-deoxy-D- whose gene has been desensitized to feedback inhibition by tyrosine and phenylalanine derived from Brevibacterium lactofermentum.
The method according to claim 1 or 2, wherein the gene is an arabinoheptulosonate-7-phosphate synthase gene. 5. The method according to claim 1, wherein the gene is an aspartokinase gene in which feedback inhibition by lysine and threonine derived from Brevibacterium lactofermentum is canceled. 6. A coryneform bacterium prepared by the method according to claim 4 or 5. 7. The bacterium according to claim 6 is cultivated in a medium, tyrosine, phenylalanine or lysine is produced and accumulated in the medium, and the bacterium is collected, which is characterized by a fermentation method. Manufacturing method.