JPH0739383A - Plasmid vector - Google Patents

Abstract

PURPOSE:To obtain a plasmid vector, replicable and proliferative in a coryneform bacterial cell and stably held in the same cell. CONSTITUTION:This plasmid vector holds (a) a DNA fragment containing a gene participating in the autonomous replication of a plasmid in a coryneform bacterium derived from a plasmid pBY503, (b) a DNA fragment capable of stably holding the plasmid, replicable and proliferative in the coryneform bacterial cell derived from the plasmid pBY503 in the bacterium, (c) a DNA fragment containing a gene having the function of the autonomous replication in Escherichia coli derived from ColEl-based plasmid, (d) a DNA fragment containing a gene capable of coding a beta-galactosidase derived from the Escherichia coli and (e) a DNA fragment capable of providing a plasmid marker in the coryneform bacterial cell and Escherichia coli.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a plasmid vector which replicates and proliferates in coryneform bacterial cells, is present in the same cells at a high copy number, and is stably retained.

[0002]

2. Description of the Related Art In gene recombination technology, a plasmid vector is a gene DNA encoding useful genetic information.
It is extremely important as a carrier of a useful gene for introducing the fragment into a host cell, stably replicating it in the host cell, and expressing the genetic information encoded by the useful gene.

Coryneform bacteria are known as Brevibacterium flavu.
m ) and the like that produce a large amount of amino acids such as aspartic acid, and are a group of microorganisms that are extremely important industrially. Using such coryneform bacteria as a host, several plasmid vectors have already been developed in order to enhance the productivity of useful substances of these coryneform bacteria by gene recombination technology (see Japanese Patent Laid-Open No. 3-210184). ).

Although all of these plasmid vectors replicate and grow in coryneform bacteria, they have a low plasmid copy number and it is difficult to distinguish directly inserted recombinant DNA when a useful gene is to be inserted. Moreover, since the size of the plasmid vector itself is large, the size of the inserted gene is limited. Therefore, there is a demand for the development of a small-sized plasmid vector which is present in a coryneform bacterium at a high copy number, is stably retained in the host, and is capable of easily discriminating gene insertion.

[0005]

Therefore, an object of the present invention is to enable replication and growth in coryneform bacterial cells, which has a high copy number in host cells and is stably retained. The purpose is to create a small-sized plasmid vector in which the selection of recombinant DNA by insertion is easy, and there are a plurality of insertion sites of useful genes.

[0006]

[Means for Solving the Problems] In order to solve the above-mentioned problems, the present inventors have earnestly studied and as a result, Brevibacterium statinis ( Bre) capable of replicative growth in coryneform bacteria has been developed.
vibrator stationis ) IFO
By modifying the DNA region involved in the function of controlling autonomous replication of the plasmid derived from the plasmid pBY503 (see JP-A-1-95785) carried by 12144 (FERM BP-2515), the A function capable of obtaining a DNA fragment capable of causing an increase in copy number, and further stably retaining a plasmid capable of replicative growth in a coryneform bacterium derived from the plasmid pBY503 proposed by the present inventors in the bacterium Fragment containing a gene involved in Escherichia coli (see JP-A-2-276579), a DNA fragment containing a gene having an autonomous replication function in Escherichia coli derived from a Col E1 plasmid, and an Escherichia coli derived gene having a plurality of cloning sites. a DNA fragment containing a gene encoding β-galactosidase By combining the DNA fragment containing a drug resistance gene that can be a plasmid markers,
The inventors have found that a plasmid vector which exists in a coryneform bacterium in a large number of copies and is stably retained in a host and which allows easy selection of gene insertion in Escherichia coli can be constructed, and completed the present invention.

Thus, according to the present invention, (a) a DNA fragment containing a gene involved in the function of autonomous replication of a plasmid in a coryneform bacterium derived from plasmid pBY503, which mutation is capable of increasing the copy number of the plasmid. A DNA fragment in which points are present in at least one location within the gene,
(B) a DNA fragment containing a gene involved in the function of stably retaining a plasmid capable of replicative growth in a coryneform bacterium derived from the plasmid pBY503 in the bacterium,
(C) a DNA fragment containing a gene responsible for the autonomous replication function in E. coli derived from the Col E1 plasmid, and (d) a D containing a gene encoding β-galactosidase derived from E. coli.
DN containing NA fragment and (e) drug resistance gene that can be a plasmid marker in coryneform bacterium and in Escherichia coli
Provided is a plasmid vector characterized in that it carries an A fragment.

The above plasmid vector of the present invention has a plurality of cloning sites, has a large number of copies in coryneform bacteria, and stably replicates and grows in the bacteria. By introducing a useful gene into this plasmid vector and transforming a coryneform bacterium, it is possible to improve breeding of an industrially useful coryneform bacterium. Hereinafter, the plasmid vector of the present invention will be described in more detail.

A DNA containing a gene involved in the function of controlling autonomous replication of a plasmid in a coryneform bacterium (hereinafter referred to as "autonomous replication gene"), which is used for constructing the plasmid vector of the present invention. Fragments,
A DNA fragment having a mutation point capable of increasing the copy number of a plasmid at at least one site in the gene (hereinafter, this may be referred to as "multi-copy number autonomously replicating DNA fragment") is a DNA fragment used herein. And a copy number of a plasmid vector created by using a DNA fragment containing an autonomously replicating gene that does not have a mutation point, which means a DNA fragment having an action capable of increasing It is a thing.

In the present specification, the term copy number means the number of plasmid molecules per cell (the number of plasmids present), and the term multi-copy number is larger than the copy number of the plasmid pBY503 or pCRY3. It means the number of copies. The above-mentioned high copy number autonomous replication DN
Although the A fragment can be synthesized after its nucleotide sequence is determined, it is usually a Brevibacterium stionis ( Brevibacterium st).
ationis ) IFO12144 (FERM BP-
2515) holds a plasmid p of about 15 kb in size.
After the gene involved in the function of controlling autonomous replication in coryneform bacteria present on BY503 (see Japanese Patent Laid-Open No. 1-95785) is modified by mutagenesis, a plasmid having an increased copy number is obtained. It can be separated and obtained from the digested fragments obtained by digestion with an appropriate restriction enzyme.

That is, a plasmid p containing an autonomously replicating gene
DNA obtained by cutting BY503 with an appropriate restriction enzyme
The fragment is a vector plasmid having a drug resistance gene that can serve as a marker, for example, pHSG398 having a chloramphenicol resistance gene (Takara Shuzo), pHSG298 having a kanamycin resistance gene (Takara Shuzo), or pBR having a tetracycline resistance gene.
322 (manufactured by Takara Shuzo Co., Ltd.) by a conventional method known per se, and this vector plasmid was introduced into a host coryneform bacterium by transformation by the electric pulse method (note that the host coryneform bacterium and the transformation method are described later. , And plasmid DNA from the transformant obtained,
A DNA fragment containing the inserted pBY503-derived autonomously replicating gene can be confirmed by extraction by a conventional method known per se, for example, the alkali-SDS method, etc., cutting with an appropriate restriction enzyme, and analysis.

This plasmid-holding strain is irradiated with X-rays, γ-rays, ultraviolet rays, or the like, or N-methyl-N '
-A strain that has been exposed to a mutagenic agent such as nitro-N-nitrosoguanidine and then cultured under selective pressure with various concentrations of a marker drug to show a gene amplification effect by increasing the plasmid copy number of a strain having an increased drug resistance concentration Therefore, the selected strain is selected and the copy number of the plasmid carried by the selected strain is analyzed. A multi-copy number autonomously replicating DNA fragment can be obtained by extracting a recombinant plasmid from the thus obtained strain having drug resistance and plasmid copy number higher than that of the parent strain and cleaving the plasmid with an appropriate restriction enzyme.

Here, the copy number is analyzed by Journa.
l of Bacteriology, 152 , p72
2 (1982) according to the method described above.
It can be carried out by extracting NA and plasmid DNA and determining the ratio of the numbers of both molecules. Examples of the high copy number autonomously replicating DNA fragment obtained by the above method include Brevibacterium flavum MJ233 GE102 (FERM BP- which carries the plasmid pCRY3.
2513) is treated with N-methyl-N'-nitro-N-nitrosoguanidine, and is a strain of Brevibacteria which has an increased chloramphenicol resistance concentration and a plasmid copy number increased about 2-fold. Umm flavum MJ233 cop1 (FERM P-1361
9) and MJ233 cop2 (FERM P-1).
3620) respectively obtained plasmid pCOP1
And pCOP2 obtained by cleaving with restriction enzymes Kpn I and include DNA fragment of a size of approximately 6.0 kb.

In the present specification, the term "the number of recognition sites" by a restriction enzyme means that a degradation product obtained by completely decomposing a DNA fragment or a plasmid in the presence of an excessive restriction enzyme is a method known per se. It means the value determined from the number of separable fragments when subjected to 1% agarose gel electrophoresis and 4% polyacrylamide gel electrophoresis.

The term "size of the cleaved fragment" means that when 1% agarose gel electrophoresis is used, the DNA of λ phage of Escherichia coli is a restriction enzyme Hin.
Based on a standard line obtained by running a DNA fragment of known molecular weight obtained by cleaving with dIII at the same time as the sample,
When 4% polyacrylamide gel electrophoresis is used, D of known molecular weight obtained by digesting DNA of φx174 phage of Escherichia coli with a restriction enzyme Hae III
The size of each cleaved DNA fragment can be calculated and determined based on the standard line obtained by running the NA fragment at the same time as the sample. The "size of the plasmid" can be determined by determining the size of each DNA fragment of the plasmid by the method of determining the "size of the cleaved fragment" and adding them. In the determination of the size of each DNA fragment, the result of 1% agarose gel electrophoresis was used for the size of the fragment of 1 kb or more, and the size of the fragment of less than 1 kb was determined by the electrophoresis of 4% polyacrylamide gel. Adopted the result.

DNA whose size has been determined in this way
A DNA fragment having a specific size can be obtained by extracting the fragment from the electrophoresis gel. The above plasmids pCOP1 and pCOP2 were digested with the restriction enzyme Kp
The number of recognition sites and the size of the cleaved fragment obtained by cleaving the multicopy number autonomously replicating DNA fragment of about 6.0 kb, which was obtained by cleaving with n I, with each other were the same in both plasmids. The values are shown in Table 1 below.

[0017]

[Table 1] Table 1 Number of restriction enzyme recognition sites Size of cleaved fragment (kb) Sma I 1 1.3, 4.7 Xho I 2 1.6, 4.0, 0.4 Sph I 2 3.6 1.7, 0.7

The size shown in Table 1 above is about 6.0 k.
The Kpn I-cleaved fragment of b was cleaved with Xho I to obtain a DNA fragment of about 4.0 kb in size, and the fragment of Hha I obtained to cleave to a DNA fragment of about 1.8 kb both had a high copy number. It has been confirmed to have an autonomous replication function, and therefore these DNA fragments can also be used as a high copy number autonomous replication DNA fragment.

As described above, the autonomously replicating gene or the multicopy number autonomously replicating gene is the plasmid pBY503, pC
RY3, pCOP1 or pCOP2 is a restriction enzyme Xho
It is considered to be contained in a DNA fragment of about 4.0 kb obtained by cleaving with I and a DNA fragment of about 1.8 kb obtained by cleaving the plasmid with a restriction enzyme Hha I. To be

The above plasmids pCOP1 and pCOP
The size obtained by cleaving 2 with the restriction enzyme Xho I is about 4.
The number of recognition sites and the size of the cleaved fragment when the 0 kb DNA fragment was further cleaved with various restriction enzymes were the same in both plasmids, and the values are shown in Table 2 below.

[0021]

Table 2 Table 2 Number of restriction enzyme recognition sites Size of cleaved fragment (kb) Pst I 2 0.9,0.8,2.3 Sph I 2 2.0,1.7,0.3

The size obtained by cleaving the above pCOP1 and pCOP2 with the restriction enzyme Hha I is about 1.8 k.
The number of recognition sites and the size of the cleaved fragment when the DNA fragment of b was further cleaved with various restriction enzymes were the same in both plasmids, and the values are shown in Table 3 below.

[0023]

[Table 3] Table 3 Number of restriction enzyme recognition sites Size of cleaved fragment (kb) Pst I 1 0.4,1.4 Sph I 1 0.7,1.1 Spe I 1 1.3,0.5

A map of restriction enzyme cleavage points of these high copy number autonomously replicating DNA fragments is shown in FIG. On the other hand, plasmid pC
A DNA fragment containing a large copy number autonomously replicating gene of about 1.8 kb obtained by cleaving OP1 and pCOP2 with a restriction enzyme Hha I, and a plasmid pBY503.
And a DNA fragment containing an autonomously replicating gene having a size of about 1.8 kb obtained by cleaving pCRY3 with a restriction enzyme Hha I, the nucleotide sequence was determined by the dideoxy nucleotide enzyme method [dideoxy chain termination] using plasmid pUC18 or pUC19.
n method; Sanger, F.N. Et al., Proc.
Natl. Acad. Sci. USA, 74 , p546
3 (1977)].

The thus obtained plasmid pB
1.8 kb DN containing an autonomously replicating gene derived from Y503 and pCRY3 or a high copy number autonomously replicating gene derived from plasmids pCOP1 and pCOP2
The base sequence of the A fragment is composed of 1897 base pairs, in which an open reading frame (OR) consisting of 783 base pairs encoding 261 amino acids is formed.
F) was present. The size obtained from the plasmids pBY503 and pCRY3 is about 1.8 kb.
Had an identical base sequence (hereinafter this may be referred to as "wild-type sequence").

On the other hand, in the sequence of the DNA fragment obtained from the plasmid pCOP1, the amino acid coded by mutating the guanine (G) at position 1307 of the wild type sequence to alanine (A) changed the amino acid encoded from arginine (Arg) to histidine (His). ), And the sequence of the DNA fragment obtained from the plasmid pCOP2 was 157 of the wild type sequence.
The amino acid coded by mutating the 7th G to A changes from glycine (Gly) to aspartic acid (Asp)
It became clear that it was converted into.

Thus, the base sequence shown in SEQ ID NO: 1 in the Sequence Listing below can also be used as the high copy number autonomously replicating DNA fragment used in the present invention. In addition, in the sequence obtained from pCOP1 in the sequence shown in SEQ ID NO: 1, R at the 1307th base sequence is A, R at the 1577th base sequence is G, and Xxx at the 129th amino acid sequence is Hi.
s, the 219th Zzz is Gly. Also pCOP
In the sequence obtained from 2, the R of the base sequence 1307 is G, the R of the base 1577 is A, and the amino acid sequence 129
Xxx is Arg, and 219th Zzz is Asp.
Furthermore, the wild-type sequence has nucleotide sequences 1307 and 1
The Rs at the 577th are both G, and the amino acid sequence 219
The th xxx is Arg and the 219 th Zzz is Gly.

The multicopy number autonomously replicating DNA fragment shown in SEQ ID NO: 1 in the Sequence Listing mentioned above is a plasmid pCO.
Not only those isolated from P1 and pCOP2,
It may be one synthesized using a commonly used DNA synthesizer, for example, Model 373A manufactured by Applied Biosystems. In addition, the multicopy number autonomously replicating DNA fragment having the sequence shown in SEQ ID NO: 1 has a part of the base sequence replaced by another base, as long as it does not substantially impair the multicopy number autonomously replicating function. May have been
Alternatively, a new base may be inserted or a part of the bases may be deleted, and any of these derivatives can be used as a high copy number autonomously replicating DNA fragment.

Next, (b) a DNA containing a gene involved in the function of stably retaining a plasmid capable of replicative growth in a coryneform bacterium derived from the plasmid pBY503, which is used for constructing the plasmid vector of the present invention.
The base sequence of the fragment (hereinafter, sometimes referred to as "stabilized DNA fragment") has been determined (Japanese Patent Laid-Open No. H2-1990).
It is possible to publication reference) Synthesis No. 276,579, usually present on Brevibacterium Sutachionisu IFO12144 (FERM BP-2515) plasmid pBY503 that held mentioned above, the plasmid pBY503 with restriction enzymes Kpn I After digestion, the digestion product is separated by 0.8% agarose gel electrophoresis, and a Kpn I fragment of about 7.4 kb in size is extracted from the gel.
The obtained DNA fragment was digested with restriction enzymes Xho I and Hind.
It was cleaved with III and separated by 0.8% agarose gel electrophoresis to obtain a DNA fragment ( Xho I
- a Hin dIII cleavage) and extracted from the gel, this can be used as a stabilizing DNA fragment.

Further, (c) a DNA fragment containing a gene having an autonomous replication function in Escherichia coli derived from a Col E1 plasmid used for constructing the plasmid vector of the present invention (hereinafter referred to as "E. coli autonomously replicating DNA fragment"). In some cases, for example, a plasmid pBR322 (manufactured by Takara Shuzo) is obtained from a plasmid pBR325 (manufactured by Takara Shuzo), which is a DNA fragment of about 0.8 kb obtained by cleaving plasmid pBR322 (manufactured by Takara Shuzo) with restriction enzymes Bst YI and Pvu II. The DNA fragment etc. which are mentioned can be mentioned.

Next, (d) a DNA fragment containing a gene encoding β-galactosidase derived from Escherichia coli (hereinafter sometimes referred to as "β-galactosidase gene DNA fragment") encodes β-galactosidase. Which has an appropriate cloning site in the gene DNA, and the function of encoding β-galactosidase is destroyed by inserting a gene DNA fragment into the cloning site, and β-galactosidase gene-deficient Escherichia coli is expressed using the expression of this β-galactosidase as an index. It means a DNA fragment derived from Escherichia coli in which the insertion of the gene DNA fragment into the cloning site can be directly selected in the mutant strain. Specifically, for example, a plasmid pBlue having a gene encoding β-galactosidase having a multi-cloning site
escriptII SK ⁺ (manufactured by Stratagene),
From pUC118 (manufactured by Takara Shuzo), pHSG398 (manufactured by Takara Shuzo), pHSG298 (manufactured by Takara Shuzo), etc., a DNA fragment containing a gene encoding β-galactosidase is cut out with an appropriate restriction enzyme, or a DNA fragment containing the gene. A DNA fragment obtained by amplifying DNA by the PCR method can be mentioned.

Furthermore, (e) a DNA containing a drug resistance gene that can be used as a plasmid marker in coryneform bacteria and Escherichia coli, which is used for constructing the plasmid vector of the present invention.
The fragment (hereinafter sometimes referred to as “drug resistance gene DNA fragment”) is expressed in the host coryneform bacterium and the host Escherichia coli, and can impart drug resistance to the host. There is no particular limitation as long as the presence of the plasmid can be detected depending on the performance, and one kind or a plurality of kinds may be present in the plasmid vector. Specific examples of the drug resistance gene DNA fragment include pHSG398 having a chloramphenicol resistance gene (Takara Shuzo) and pHSG29 having a kanamycin resistance gene.
8 (manufactured by Takara Shuzo), pBR322 having a tetracycline resistance gene (manufactured by Takara Shuzo) and the like can be exemplified by a DNA fragment obtained by cutting with an appropriate restriction enzyme.

The plasmid vector newly provided in the present invention includes (a) a multicopy number autonomously replicating DNA fragment, (b) a stabilized DNA fragment, (c) an Escherichia coli autonomously replicating DNA fragment, and ( It possesses five essential DNA fragments of d) β-galactosidase gene DNA fragment and (e) drug resistance gene DNA fragment. Construction of the plasmid vector of the present invention is carried out by the above-mentioned five essential DNs.
The A fragment was prepared by the method detailed above and used to prepare T4 DNA.
This can be easily carried out by allowing the ligase to act and sequentially binding the DNA fragments.

A preferred specific example of the plasmid vector provided by the present invention is a plasmid substantially consisting of the above-mentioned 5 DNA fragments and having a size of about 5.4 kb,
The present inventors have established that pKPRL-1 and pKPRL, respectively.
The plasmid designated as -2 can be mentioned. The number of recognition sites and the size of the cleaved fragment when the plasmids pKPRL-1 and pKPRL-2 were cleaved with various restriction enzymes were the same in both plasmids, and the values are shown in Table 4 and the restriction enzyme cleavage point map is shown in FIG. 2 shows.

[0035]

[Table 4 restriction enzyme recognition sites number cutting size of fragments (kb) Sac II 1 5.4 Not I 1 5.4 Eco RI 1 5.4 Eco RV 1 5.4 Apa I 1 5.4 Kpn I 1 5.4 Xho I 3 3.8, 1.3, 0.3 Sac I 2 4.6, 0.8

The plasmid vectors pKPRL-1 and pKPRL-2 of the present invention described above can be prepared, for example, by the method shown below. Each of the above-mentioned plasmids pCOP1 and pCOP2, which had a size of about 1.8 kb and had a high copy number, was cloned into a restriction enzyme Kpn.
PCR method using a primer having a cleavage site of I and a primer having a cleavage site of restriction enzyme Xho I [Nat
ure, 324 , p163 (1986)] and prepared as a fragment of Kpn I- Xho I. On the other hand, a stabilized D of about 1.1 kb in size prepared as described above.
NA fragment (Xho I- Hin dIII fragment) and the T4 D
Vectors ligated with NA ligase and having appropriate cloning sites, such as E. coli plasmid pBlue
scriptII K of SK ⁺ (Stratagene)
pn I, is introduced into the Hin dIII site. A kanamycin resistance gene (Pharmacia) is blunt-ended and introduced into the Sma I site of the prepared plasmid.
From the constructed plasmid, a high copy number autonomously replicating DNA fragment, a stabilizing DNA fragment and a kanamycin resistance gene DN
The A fragment is obtained by cutting with the restriction enzymes Kpn I and Bam HI. The Col E1 plasmid pBR322 was cleaved with restriction enzymes Bst I and Pvu II to obtain a 0.8 kb Escherichia coli replication-replicating DNA fragment, and the fragment and the above Kpn I-
The Bam HI fragment is treated with blunt ends to prepare a ligated plasmid. The prepared plasmid is a restriction enzyme Eco R
Cleavage with I and Eco RV is performed for blunt end treatment. Further, pBluescript II SK ⁺ containing the Escherichia coli β-galactosidase gene was amplified by the PCR method using a primer having a restriction enzyme Sma I site as a template DNA to prepare an Sma I fragment,
PCOP1 was inserted into a blunt-ended plasmid.
It is possible to prepare plasmid vectors pKPR-1 and pKPR-2 having multicopy number autonomously replicating DNA fragments derived from pCOP2 and pCOP2, respectively.

The plasmid vector p thus constructed
KPRL-1 and pKPRL-2 have Eco RI site, Eco RV site, Sac II site, Not I site, Apa I site, Kpn I site, etc. in the β-galactosidase gene as cloning sites for useful genes. It is possible to clone various target genes, and it is possible to easily discriminate gene insertion.

The plasmid vector of the present invention, which is capable of stably replicating at a high copy number in a coryneform bacterium having a high copy number autonomously replicating DNA fragment and a stabilizing DNA fragment, comprises:
A DNA fragment containing a structural gene or the like encoding various industrially useful substances can be incorporated. Therefore, by introducing a plasmid in which the plasmid vector of the present invention has been recombined with a useful gene or the like into a host microorganism and culturing it, it becomes possible to efficiently and industrially produce a useful substance encoded by the useful gene. .

The DNA containing a useful structural gene or the like which can be incorporated into the plasmid vector of the present invention includes, for example,
DNA fragment containing a gene encoding aspartase (EC 4.3.1.1); genes encoding at least a tryptophan synthase (EC 4.2.1.20) gene (trpA and trpB) And a promoter and an operator that control the expression of the gene
NA fragment: a DNA fragment containing an amino acid biosynthesis gene such as a threonine biosynthesis gene or an isoleucine biosynthesis gene, and the like.

As a host microorganism which can be transformed with the plasmid vector of the present invention into which the above-mentioned useful gene has been incorporated, coryneform bacteria such as Brevibacterium flavum MJ233 (FERM BP) can be used.
-1497), Brevibacterium flavum MJ23
3-AB-41 (FERM BP-1498), Brevibacterium flavum MJ233-ABT-11 (F
ERM BP-1500), Brevibacterium flavum MJ233-ABD-21 (FERM BP-14)
99) and the like.

The strain of FERM BP-1498 is DL using the strain of FERM BP-1497 as a parent strain.
It is an ethanol-assimilating microorganism positively imparted with -α-aminobutyric acid resistance (see Japanese Patent Publication No. 59-28398). In addition, the strain of FERM BP-1500 is FER
This is a highly active mutant of L-α-aminobutyric acid transaminase using the MBP-1497 strain as a parent strain (Japanese Patent Application Laid-Open No. 6-58242).
2-51998). Furthermore, FERM BP
The strain -1499 is a D-α-aminobutyric acid deaminase highly active mutant strain having the strain FERM BP-1497 as a parent strain (see Japanese Patent Laid-Open No. 61-177993).

In addition to the above microorganisms, Brevibacterium
Ammonia genez ( Brevibacterium a
mmoniagenes ) ATCC 6871, ATC
C13745, ATCC13746, Brevibacterium d.
ivaricatum) ATCC4020, Brevibacterium lactofermentum (Brevibact
erium lactofermentum) ATCC
13869, Corynebacterium glutamicum ( Co
rynebacterium glutamicum )
ATCC31830 etc. can also be used as a host microorganism.

When a strain derived from Brevibacterium flavum MJ233 is used as a host, transformation may be difficult due to the plasmid pBY502 carried by this strain. In such a case, this strain can be used. It is more desirable to remove the plasmid pBY502.
As a method of removing the plasmid pBY502, for example, it is possible to spontaneously remove it by repeating subculture, or it is possible to remove it artificially [Bact. Rev. 36 , p361-405 (197)
2)].

Transformation of the above-mentioned host microorganism with the above-mentioned recombinant plasmid vector can be performed by a method known per se, for example, Satoh, Y. et al. Et al, Journal of In
Dustrial Microbiology, 5 , p
159 (1990) (a method of energizing a host microorganism with a pulse wave); Calvin, N. et al. M. And Hanawal
t, P. C. , Journal of Bacteri
, 170, 2796 (1988); Ioo,
K. , Nishida, T .; And Izaki, K .; ，
Agricultural and biologic
al Chemistry, 52 , p293 (198).
The method described in the literature such as 8) can be used.

Next, the present invention will be described more specifically by way of examples. As a matter of course, the following examples are given only as an aid to gain specific recognition of the present invention, and do not limit the scope of the present invention in any way.

[0046]

EXAMPLES Reference Example 1 Plasmid pBY503 and plasmid pHSG398
Construction of a composite plasmid pCRY3 consisting of (A) Preparation of plasmid pBY503 Plasmid pBY503 is used for Brevibacterium statinis.
onis) IFO12144 (FERM BP-251
Molecular weight of about 10 megadalton (about 15)
kb) plasmid, the details of which are described in JP-A-1-95.
No. 785. Plasmid pBY50
3 was prepared by the following method.

Semi-synthetic medium A medium [Composition: urea 2 g, ammonium sulfate 7 g, monopotassium phosphate 0.5 g, dipotassium phosphate 0.5 g, MgSO ₄ 0.5 g, MnSO ₄
・ 4-6H ₂ O 6mg, FeSO ₄・ 7H ₂ O 6m
g, yeast extract 2.5g, casamino acid 5g, biotin 2
00 μg, thiamine hydrochloride 200 μg, glucose 20 g
To 1 liter by dissolving in distilled water (pH 7.4)]
Brevibacterium stationis was cultured in 1 liter until the late logarithmic growth phase to collect bacterial cells. Buffer containing the obtained bacterial cells at a concentration of 10 mg / ml of lysozyme [composition: 25 mM tris (hydroxymethyl) aminomethane, 10 mM EDTA, 50 mM glucose] 20 m
It was suspended in 1 and reacted at 37 ° C. for 1 hour. Alkali-SDS solution [composition: 0.2N NaOH, 1% (W
/ V) SDS] 40 ml was added, mixed gently and allowed to stand at room temperature for 15 minutes.

Next, an aqueous potassium acetate solution [composition: 5M potassium acetate solution 60 ml, acetic acid 11.5
30 ml of a mixed solution of 1 ml of distilled water and 28.5 ml of distilled water was added and mixed sufficiently, and then allowed to stand in ice water for 15 minutes. Transfer the total amount of lysate to a centrifuge tube and store at 15,000 for 1 minute at 4 ℃.
The supernatant was obtained by centrifugation at 0 × g.

To this, an equal volume of a phenol / chloroform (1: 1, v / v) mixture was added and suspended, and then the whole volume was transferred to a centrifuge tube, and the mixture was placed at 15,000 × g for 5 minutes at room temperature. It was subjected to centrifugation to obtain an aqueous layer. To the aqueous layer was added twice the amount of ethanol, the mixture was allowed to stand at -20 ° C for 1 hour and then at 4 ° C for 1 hour.
The precipitate was collected by centrifugation at 15,000 × g for 0 minutes.

The obtained precipitate was dried under reduced pressure, and then the pH was adjusted with TE buffer [10 mM Tris, 1 mM EDTA; hydrochloric acid].
Adjusted to 8.0] Dissolved in 2 ml. Cesium chloride solution [composition: a solution prepared by dissolving 170 g of cesium chloride in 100 ml of TE buffer having a concentration of 5 times] 15 ml and 10
1 ml of a mg / ml ethidium bromide solution was added to adjust the density of the liquid to 1.392 g / ml. This solution was centrifuged at 116,000 xg for 42 hours at 12 ° C. After centrifugation, the plasmid pBY503 is found by UV irradiation as a lower band in the centrifuge tube. The band was extracted from the side of the centrifuge tube with a sterilized plastic syringe to obtain a fractionated solution containing plasmid DNA.

Next, this fraction was treated with an equal amount of isoamyl alcohol four times to extract and remove ethidium bromide, and then dialyzed against TE buffer. To the dialysate containing the plasmid pBY503 thus obtained, a 3M sodium acetate solution was added so that the final concentration was 30 mM, and then a double amount of ethanol was added.
It was left still at 20 ° C. for 1 hour. 15,000 x g of this solution
DNA is precipitated by centrifugation of the plasmid pB
About 50 μg of Y503 DNA was recovered.

(B) Preparation of plasmid pHSG398 Plasmid pHSG398 (manufactured by Takara Shuzo) does not replicate in coryneform bacteria but replicates in Escherichia coli and expresses chloramphenicol resistance, and has a size of about 2.2 kb. And can be purchased from Takara Shuzo as a commercial product.

(C) Construction of composite plasmid pCRY3 0.5 μ of the above plasmid pHSG398 (Takara Shuzo)
g was reacted with a restriction enzyme Kpn I5 unit at 37 ° C. for 1 hour to completely decompose the plasmid DNA.

Plasmid pBY5 prepared in (A) above
2 μg of 03DNA was reacted with a restriction enzyme Kpn I 1 unit at 37 ° C. for 30 minutes to partially decompose the plasmid DNA. Mix both plasmid DNA digests,
After heat treatment at 5 ° C. for 10 minutes to inactivate the restriction enzyme, the final concentration of the mixture was 50 mM Tris buffer (pH 7.6), 10 mM MgCl ₂ , 10 mM.
Dithiothreitol, 1 mM ATP, and T4 DN
Each component was added and strengthened so that A ligase was 1 unit, and incubated at 16 ° C. for about 15 hours. Using this solution, Escherichia coli JM109 competent cell (Takara Shuzo) was transformed according to the manufacturer's manual.

The transformant obtained was 30 μg / ml.
(Final concentration) Chloramphenicol, 100 μg / ml
(Final concentration) Isopropyl-β-D-thiogalactopyranoside (IPTG), 100 μg / ml (Final concentration) 5
-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) in L medium [composition: tryptone 10 g, yeast extract 5 g, sodium chloride 5 g
Was dissolved in distilled water to make 1 liter (pH 7.
2)] at 37 ° C. for 24 hours, and the strains grown as white colonies on the medium were independently selected, and each plasmid was treated with the alkali-SDS method [T. Maniati
S.E. F. Fritsch, J. et al. Sambrook,
"Molecular cloning", p90-9
1 (1982)].

(D) Transformation of composite plasmid into coryneform bacterium The electric pulse method was used for transformation. Brevibacterium flavum MJ233 (FERM BP-1497)
After culturing the plasmid-depleted strain in 100 ml of the above medium A until the early logarithmic growth phase, penicillin G was added at 1 unit / m 2.
It was added so that the amount became 1 and further permeated and cultured for 2 hours. The cultured cells were collected by centrifugation and 20 ml of a pulse solution [composition: 272 mM sucrose, 7 mM KH ₂ P
It was washed with O ₄ , 1 mM MgCl ₂ ; pH 7.4]. The cells were collected again by centrifugation, suspended in 5 ml of the pulse solution, mixed with 0.75 ml of the cells and 50 ml of the DNA solution obtained in (C) above, and allowed to stand in ice for 20 minutes. did. Using Gene Pulser (manufactured by Bio-Rad), the voltage was set to 2500 V and 25 μFD, and the sample was left standing for 20 minutes after the pulse was applied. The whole amount was transferred to 3 ml of the A medium, cultured at 30 ° C. for 1 hour, inoculated into the A agar medium containing 3 μg / ml (final concentration) of chloramphenicol, and cultured at 30 ° C. for 2 to 3 days. The chloramphenicol resistant strain that emerged from Brevibacterium flavum MJ2
33 GE102, and a plasmid was obtained from this transformant using the method described in (A) above. This plasmid was cleaved with various restriction enzymes, and the size of the resulting cleaved fragment was measured. The results are shown in Table 5 below.

[0057]

[Table 5] Table 5 Number of restriction enzyme recognition sites Cleavage fragment size (kb) Kpn I 2 6.0, 2.2 Sau I 1 8.2 Bam HI 1 8.2 Pst I 2 5.7, 2. 5

The plasmid characterized by the above cleavage fragment of the restriction enzyme was designated as "pCRY3". Table 5 above
As is clear from the above, the plasmid pCRY3 was cleaved with the restriction enzyme Kpn I to give the plasmid pHSG.
In addition to the 2.2 kb size DNA fragment derived from 398, the size 6.0 k derived from the plasmid pBY503.
A DNA fragment containing the autonomously replicating gene of b was confirmed. Brevibacterium flavum MJ233 GE102 transformed with the composite plasmid pCRY3.
Is entrusted with the contract number: FERM BP-2 to the Institute of Biotechnology, Institute of Industrial Science and Technology, 1-3-1 Higashi, Tsukuba, Ibaraki Prefecture.
513 has been internationally deposited under the Budapest Treaty.

Example 1Mutant high copy number plasmid from plasmid pCRY3
Acquisition of code (A) Acquisition of chloramphenicol high-concentration resistant strain Brevibacterium transformed with plasmid pCRY3
Umm flavum MJ233 GE102 (FERM B
P-2513) strain at 300 μg / ml of N-methyl-
N'-nitro-N-nitrosoguanidine at 30 ° C for 30
A mutagenesis treatment was carried out by exposing for a minute. Next, semi-synthetic A medium [pair
Composition: Urea 2g, (NH_Four)₂SO_Four7g, K₂HPO_Four
0.5g, KH₂PO_Four0.5 g, MgSO_Four0.5
g, FeSO _Four・ 7H₂O 6mg, MnSO_Four・ 4〜
6H₂O 6mg, yeast extract 2.5g, casamino acid 5
g, biotin 200 μg, thiamine hydrochloride 200 μg,
Dissolve 20 g of Lucose in distilled water to make 1 liter]
To the medium containing 6 μg / ml of chloramphenicol
It was used and cultured at 33 ° C. for 5 hours. The obtained cultured cells are
Chloramphenicol 36 μg / ml, agar 15 g
Chloram)
Two chloramfu strains were selected by selecting highly resistant phenicol strains.
A highly resistant strain of henicol was obtained.

The mutant strains capable of growing on the above-mentioned medium containing a high concentration of chloramphenicol were prepared as Brevibacterium flavum MJ233 cop1 and MJ233 co, respectively.
It was named p2.

(B) Confirmation of plasmid copy number Brevibacterium flavum MJ obtained in (A) above
233 cop1 and MJ233 cop2, and MJ233 G harboring the plasmid pCRY3.
E102 (FERM BP-2513) was cultured in the semi-synthetic A medium until the late logarithmic growth phase, and the cells were collected. The obtained cells were suspended in 20 ml of a buffer solution containing 10 mg / ml of lysozyme [composition: 25 mM tris (hydroxymethyl) aminomethane, 10 mM EDTA, 50 mM glucose] and reacted at 37 ° C. for 1 hour. Alkali-SDS solution [Composition: 0.2N NaO]
40 ml of H, 1% (W / V) SDS] was added, gently mixed and allowed to stand at room temperature for 15 minutes. Next, 30 ml of an aqueous potassium acetate solution [composition: a mixed solution of 60 ml of 5M potassium acetate solution, 11.5 ml of acetic acid and 28.5 ml of distilled water] was added to this reaction solution, and the mixture was sufficiently mixed and then allowed to stand in ice water for 15 minutes. I put it.

The total amount of the lysate was transferred to a centrifuge tube and centrifuged at 4 ° C. for 10 minutes at 15,000 × g to obtain a supernatant. Add an equal amount of phenol / chloroform (1: 1,
(v / v) After adding and suspending the mixed solution, the whole amount was transferred to a centrifuge tube and centrifuged at room temperature for 5 minutes at 15,000 × g to obtain an aqueous layer. To the aqueous layer was added twice the amount of ethanol, and the mixture was allowed to stand at -20 ° C for 1 hour and then at 4 ° C for 10 minutes, 1
The precipitate of DNA was recovered by centrifugation at 5,000 xg.

The obtained precipitate was dried under reduced pressure and then dissolved in 2 ml of a TE buffer solution (composition: 10 mM Tris, 1 mM EDTA; adjusted to pH 8.0 with hydrochloric acid). The plasmid and chromosomal DNA extracted as described above were separated by 0.8% agarose gel electrophoresis. Next, the electrophoretic negative film is put on a densitometer to measure the ratio of the chromosomal DNA and the plasmid DNA.
The plasmid copy number was calculated assuming that the molecular weight of NA is 3.0 × 10 ⁹ daltons and the molecular weight of plasmids is 5.4 × 10 ³ daltons (8.2 kb).

As a result, 10 to 12 copies of plasmid were present in Brevibacterium flavum MJ233 cop1 and MJ233 cop2.
3GE102 contains 5-6 copies of plasmid (pCRY
3) was found to exist.

(C) Acquisition of a high copy number autonomously replicating DNA fragment of about 6.0 kb In the same manner as in Reference Example 1 (A), Brevibacterium flavum MJ233 cop1 and Extract the plasmid from MJ233 cop2,
This was cleaved with various restriction enzymes and the size of the cleaved fragment was measured. As a result, the number of sites recognized by restriction enzymes and the size of the cleaved fragments of the plasmids obtained from both strains were the same, which was in agreement with the value of the plasmid pCRY3 shown in Table 5 above.

As described above, it is characterized in that it is present in a copy number of 10 to 12 per bacterium, and has the number of recognition sites by restriction enzymes and the size of cleaved fragments shown in Table 5. Flavum MJ233 cop
The plasmid obtained from No. 1 is "pCOP-1", and the plasmid obtained from MJ233 cop2 is "pCOP-1".
OP-2 "was named respectively.

Next, plasmids pCOP-1 and pC
The size of OP-2 is about 6.0 kb with the restriction enzyme Kpn I.
This DNA was cut out from a high copy number autonomously replicating DNA fragment of
The A fragment was cleaved with various restriction enzymes, and the size of the cleaved fragment was determined. As a result, the number of restriction enzyme recognition sites of the DNA fragments obtained from both plasmids and the size of the cleaved fragments were the same, as shown in Table 1 above.

Brevibacterium flavum MJ233 cop1, which carries the plasmid pCOP-1,
And the same MJ233 carrying the plasmid pCOP-2
cop2 is located at 1-3-1 Higashi 1-3-2, Tsukuba-shi, Ibaraki Prefecture, and is registered by the Institute of Biotechnology, Institute of Industrial Science and Technology, on April 27, 1993, under the consignment number: FERM P-13619 and the consignment number: FERM P-13620. Has been deposited as.

Example 2 of a multi-copy number autonomously replicating DNA fragment of about 4.0 kb in size
Cloning plasmids pCOP-1 and pC prepared in Example 1
In order to specify the gene region responsible for the function of OP-2 capable of autonomously replicating in a coryneform bacterium, an approximately 6.0 kb autonomously replicating DNA fragment possessed by both plasmids was further reduced. All of the following operations were independently performed for both plasmids.

1 μg of the plasmid was digested with the restriction enzyme Xho I 1
Plasmid DNA by reacting with unit for 1 hour at 37 ℃
Was completely disassembled. The resulting degradation product was separated by 0.8% agarose gel electrophoresis to obtain a DNA of about 4.0 kb in size.
Fragment fractions were excised from the gel. DNA was extracted and purified from this fraction to obtain about 0.5 μg of DNA. Next, the E. coli plasmid pHSG398 1 μ described in Reference Example 1 was used.
After reacting g with the restriction enzyme Sal I1 unit at 37 ° C. for 1 hour, the reaction solution was heated at 70 ° C. for 10 minutes to inactivate the enzyme. This quenching solution and the size of about 4.0 obtained above.
kb DNA was mixed, and the final concentration of each mixture was 50 mM Tris buffer (pH 7.6), 10 mM.
Dithiothreitol, 1 mM ATP, 10 mM Mg
Each component was added so that Cl ₂ and T4 DNA ligase 1 unit were added, and this was reacted at 12 ° C. for 15 hours to bind the DNA fragment.

The ligated DNA was introduced into Brevibacterium flavum MJ233 in the same manner as in Reference Example 1 (D) to transform the bacterium. The transformant strain was cultured for 2-3 days on a solid medium prepared by adding 15 g / l of agar to a semi-synthetic A medium containing 36 μg / ml of chloramphenicol to obtain a chloramphenicol high-concentration resistant strain. The strain carrying the plasmid into which the DNA fragment of about 4.0 kb in size derived from the plasmid pCOP-1 was introduced, which was obtained by the above operation, was designated as "Brevibacterium flavum MJ.
233cop1-4.0 ", and plasmid pCOP
The strains harboring the plasmid into which the DNA fragment of about -2 and having a size of about 4.0 kb was introduced were designated as "Brevibacterium flavum MJ233 cop-4.0".

The plasmid copy number of the above-mentioned strain was calculated using the same method as in Example 1 (B), with the molecular weight of the plasmid being 4.0 × 10 ³ Daltons (6.2 kb). As a result, it was revealed that the plasmid copy number retained by each transformant was 10-12. Plasmids were extracted from the obtained two kinds of transformants by the same method as in Reference Example 1 (A), and the plasmids were each cleaved with various restriction enzymes to determine the size of the cleaved fragments. As a result, the number of restriction enzyme recognition sites and the size of the cleaved fragments of the DNA fragments obtained from both plasmids were the same. The values are shown in Table 6.

[0073]

[Table 6] Table 6 Number of restriction enzyme recognition sites Cleavage fragment size (kb) Eco RI 1 6.2 Bam HI 1 6.2 Kpn I 1 6.2 Sph I 3 4.2, 1.7, 0.0. Three

As described above, it is characterized in that it is present in a copy number of 10 to 12 per cell, and has the number of recognition sites by restriction enzymes and the size of cleaved fragments shown in Table 6. Flavum MJ233 cop1
The plasmid harbored by -4.0 is "pCOP1-4.
0 ", and the plasmids carried by the same MJ233cop2-4.0 were named" pCOP2-4.0 ".

Next, the plasmids pCOP-1 and pC
The size of the cleaved fragment was determined by cleaving the above-mentioned multi-copy number autonomously replicating DNA fragment having a size of about 4.0 kb obtained from OP-2 with various restriction enzymes. As a result, the number of restriction enzyme recognition sites of the DNA fragments obtained from both plasmids and the size of the cleaved fragments were the same, as shown in Table 2 above.

Example 3 Preparation of a high copy number autonomously replicating DNA fragment of about 1.8 kb
1 μg each of the plasmid pCRY3 obtained in Cloning Reference Example 1 and the plasmids pCOP-1 and pCOP-2 obtained in Example 1 were taken, and each of them was used as a restriction enzyme Kpn I1uni.
It was reacted with t for 1 hour to be completely decomposed. Then, the degradation product was separated by 0.8% agarose gel electrophoresis.
At that time, a digestion product of Escherichia coli λ phage with a restriction enzyme Hin dIII (λ Hin dIII digestion product) was run at the same time as the sample as a size marker. Size of about 6.0, which is involved in the function of autonomous replication of plasmid from the gel used for electrophoresis
An agarose fraction containing a kb DNA fragment was cut out, and a DNA fragment having a size of about 6.0 kb was extracted from the fraction.

0.8 μg of the obtained DNA fragment of about 6.0 kb was added to the restriction enzyme Hha I 0.1 unit and 37.
The reaction was carried out at 30 ° C. for 30 minutes for incomplete decomposition to obtain decomposed products of various sizes, and then the reaction solution was heated at 70 ° C. for 10 minutes to inactivate the enzyme. Next, 0.5 μg of the above-mentioned E. coli plasmid pHSG398 was added to the restriction enzyme Acc I 0.
After completely reacting with 5 units at 37 ° C. for 1 hour, the reaction solution was also heat-treated at 70 ° C. for 10 minutes to inactivate the enzyme.

Both the obtained degradation products were mixed, and the final concentration of each mixture was 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 was added so that it became unit, and this was reacted at 12 ° C. for 15 hours to bind the DNA fragment. The plasmid DNA obtained by ligation was introduced into a coryneform bacterium by the electric pulse method in the same manner as in Reference Example 1 (D) to transform the bacterium, and the cells were cultured in the semi-synthetic A medium. further,
DN from plasmids pCOP-1 and pCOP-2
The agar of the A medium containing 30 μg / ml (final concentration) of the cells transformed with A and 5 μg / ml (final concentration) of the chloramphenylchol for cells transformed with the DNA derived from pCRY3. Inoculate on medium and
The cells were cultured at 0 ° C for 2 to 3 days to obtain chloramphenicol resistant strains.

The emerged chloramphenicol-resistant strains were independently cultured in 1 liter of the semi-synthetic A medium until the late logarithmic growth phase, and then the cells were recovered. A buffer containing the obtained bacterial cells at a concentration of 10 mg / ml [composition: 25 mM tris (hydroxymethyl) aminomethane, 10 mM EDTA, 50 mM glucose]
It was suspended in 20 ml and reacted at 37 ° C. for 1 hour. Alkali-SDS solution [composition: 0.2N NaO]
H, 1% (W / V) SDS] 40 ml was added, mixed gently and allowed to stand at room temperature for 15 minutes. Next, a potassium acetate solution [composition: 5M potassium acetate solution 60] was added to the reaction solution.
ml, acetic acid 11.5 ml, distilled water 28.5 ml] 30 ml was added, and the mixture was thoroughly mixed and allowed to stand in ice water for 15 minutes.

The whole lysate of lysozyme was transferred to a centrifuge tube and centrifuged at 4 ° C. for 10 minutes at 15,000 × g to obtain a supernatant. An equal amount of a phenol / chloroform solution (1: 1, v / v) was added to the supernatant to suspend it, and the suspension was transferred to a centrifuge tube and centrifuged at room temperature for 5 minutes at 15,000 × g to obtain the water. The layers were collected. To the aqueous layer was added twice the amount of ethanol, and the mixture was allowed to stand at -20 ° C for 1 hour and then at 4 ° C.
The precipitate was recovered by centrifugation at 15,000 xg for 10 minutes at room temperature. The obtained precipitate contains chromosomal DNA and plasmid DNA. After drying this precipitate under reduced pressure,
TE buffer [Composition: 10 mM Tris, 1 mM EDT
A: pH adjusted to 8.0 with hydrochloric acid] Dissolved in 2 ml to prepare a sample.

Extracted plasmid DN obtained by the above procedure
2. Each sample containing A was subjected to electrophoresis using 0.8% agarose gel to measure its molecular weight.
A band of 0 × 10 ⁹ Dalton chromosomal DNA and a band of 2.6 × 10 ³ Dalton (4.0 kb) plasmid DNA were confirmed. Therefore, in the obtained plasmid, the plasmids pCRY3, pCOP-1 and pC were added to the 2.2 kb DNA fragment of the plasmid pHSG398.
It was confirmed that a DNA fragment of about 1.8 kb in size ( multicopy number autonomously replicating DNA fragment) obtained by cutting OP-2 with each of the restriction enzymes Hha I was inserted.

The three sizes obtained above were about 1.8 k.
The size of the cleaved fragment was determined by cleaving the plasmid into which the restriction enzyme Hha I cleaved DNA fragment of b was introduced with various restriction enzymes. As a result, the number of restriction enzyme recognition sites and the sizes of the cleaved fragments of the three plasmids were the same, and the values are shown in Table 7 below.

[0083]

[Table 7] Table 7 Number of restriction enzyme recognition sites Number of cleaved fragments (kb) Eco RI 1 4.0 Kpn I 1 4.0 Bam HI 1 4.0 Sph I 2 0.7, 3.3 Pst I 2 0.4, 3.6

Plasmid p, obtained as described above,
Restriction enzyme H of about 1.8 kb in size derived from COP-1
A plasmid having a ha I digested DNA fragment and characterized by the digested fragments shown in Table 7 above was designated as "pCOP1-
1.8 "having a restriction enzyme Hha I digested DNA fragment of about 1.8 kb in size derived from the plasmid pCOP-2,
The plasmids characterized by the cleavage fragments shown in Table 7 above were designated as "pCOP2-1.8" and plasmid pCRY.
Plasmids having a restriction enzyme Hha I digested DNA fragment of about 1.8 kb in size from 3 and characterized by the digested fragments shown in Table 7 above were designated as "pS-1".

The plasmid and chromosomal DNA obtained by the same procedure as in Example 1 (B) were subjected to agarose gel electrophoresis to give a chromosomal DNA having a molecular weight of 3.0.
× 10 ⁹ Dalton, the molecular weight of the plasmid is 2.6 × 10
^The plasmid copy number was calculated as ³ Daltons (4.0 kb). As a result, the plasmid copy number was pS-1.
5 to 6, pCOP1-1.8 and pCOP2-1.
It was found that 8 was 10 to 12.

In addition, the plasmids pCOP-1 and p
A large copy number autonomously replicating DNA fragment of about 1.8 kb obtained by cleaving COP-2 with a restriction enzyme Hha I was cleaved with various restriction enzymes to determine the size of the cleaved fragment. As a result, the number of restriction enzyme recognition sites of the DNA fragments obtained from both plasmids and the size of the cleaved fragments were the same, as shown in Table 3 above.

The Brevibacterium flavum COP1-1. 8 carrying the plasmid pCOP1-1.
And Brevibacterium flavum COP2-1.8, which retains pCOP2-1.8, are east 1 in Tsukuba City, Ibaraki Prefecture.
At the Institute of Biotechnology, Institute of Biotechnology, 1st-3rd,
Contract number on March 4, 1993: FERM P-135
06 and FERM P-13507.

Example 4 Determination of Mutation Position in Multicopy Number Mutant Plasmid The nucleotide sequence of a DNA fragment of about 1.8 kb obtained by cleaving with the restriction enzyme Hha I obtained in Example 3 was used as the plasmid pUC18 or Using the plasmid pUC19, the dideoxynucleotide enzymatic method [dideoxy cha]
in termination method, San
ger, F.G. Et al., Proc. Natl. Acad. Sc
i. USA, 74 , p5463 (1977)].

The sequence of each DNA fragment consists of 1897 base pairs, in which an open reading frame encoding 261 amino acids is present, which encodes a replication protein essential for plasmid replication. It is presumed that. Plasmids pCOP-1 and pCOP
-2 was compared with the sequence obtained from pS-1, and in the open reading region in the sequence, in the sequence obtained from pCOP-1, the 1307th base G was mutated to A The amino acid encoded by Arg is converted from His to pCOP-2
In the sequence obtained from, the 1577th G was mutated to A so that the amino acid encoded by the site was changed from Gly to A.
It was found to be converted to sp.

From these sequences, the size is 1.8k.
It was confirmed that the multicopy number autonomously replicating DNA fragment of b had the sequence represented by SEQ ID NO: 1 in the sequence listing below.
In the sequence shown in SEQ ID NO: 1, in the sequence obtained from pCOP1, R at the 1307th base sequence is A, 1
The R at the 577th position is G, the Xxx at the 129th amino acid sequence is His, and the Zzz at the 219th amino acid sequence is Gly.
The sequence obtained from pCOP2 has a nucleotide sequence of 1307
The R at the th position is G, the R at the 1577th position is A, and the 129th Xxx is Arg and the 219th Zzz.
Is Asp. Furthermore, the wild type sequence has a nucleotide sequence of 13
The 07th and 1577th Rs are both G, and the amino acid sequence 219th Xxx is Arg and the 219th Zzz is Gly.

Example 5 Plasmid vectors pKPRL-1 and pKPRL-2
(A) Preparation of high copy number autonomously replicating DNA From the plasmids pCOP1-1.8 and pCOP2-1.8 obtained in Example 3, the following primer templates were used,
A DNA thermal cycler type 480 (manufactured by Takara Shuzo Co., Ltd.) was used to amplify a multicopy number autonomously replicating DNA fragment having a size of about 1.8 kb by the PCR method. Amplification of the DNA fragment was performed using the following primers to which Xho I and Kpn I restriction enzyme sites were added, respectively.

Primer 1 ( Xh is underlined)
o I)): TC CTCGAG GCGCAGCCTGACCATG
(SEQ ID NO: 2) Primer 2 (underlined portion is Kpn I site): TC GGTACC GCGCAGCAAAGCCTCG
(SEQ ID NO: 3) 0.5 μg of the amplified DNA fragment was added to the restriction enzyme Xho I
It was digested with (0.5 unit) and Kpn I (0.5 unit) and prepared as an Xho I- Kpn I fragment. These fragments are hereinafter referred to as cop1 fragment and cop2 fragment, respectively.

(B) Preparation of Stabilized DNA Fragment A DNA fragment containing a gene involved in the function of stably retaining a plasmid in coryneform bacteria (stabilized DNA fragment)
Examples thereof include the DNA fragment derived from plasmid pBY503 described in JP-A-2-276579. The stabilized DNA fragment was prepared as follows.

PBY503 (5 μm) obtained in Reference Example 1
g) was completely digested with a restriction enzyme Kpn I (5 unit) at 37 ° C. for 1 hour. The degradation products were separated by 0.8% agarose gel electrophoresis, and Kpn I having a size of about 7.4 kb was separated.
The fragment was extracted from the gel and purified. The obtained DNA 1μ
g for restriction enzymes Xho I (1 unit) and Hin dII
It was cleaved with I (1 unit), separated by 0.8% agarose gel electrophoresis, and a DNA fragment of about 1.1 kb in size was extracted from the agarose gel to obtain a stabilized DNA fragment.

(C) Preparation of DNA fragment containing gene responsible for autonomous replication function in Escherichia coli Plasmid pBR322 (1 μg) was digested with restriction enzyme Bst Y
37 I (1unit) and Pvu II (1unit)
Complete decomposition occurred at 1 ° C for 1 hour. Degradation products were separated by 0.8% agarose gel electrophoresis, and the size of D was about 0.8 kb.
The NA fragment was extracted from an agarose gel to prepare an E. coli autonomously replicating DNA fragment.

(D) Preparation of β-galactosidase gene Plasmid pBluescriptII having a multicloning site in the β-galactosidase gene.
Β- using KS ⁺ (manufactured by Stratagene) as a template DNA
The galactosidase gene fraction 0.5 kb was amplified by the PCR method using the following primers.

Primer 1 (underlined part is Sm
a I site): TT CCCGGG TGCGCGTAACCACCACA
C (SEQ ID NO: 4) Primer 2 (underlined portion is Sma I site): TT CCCGGG ATACGCAACCGCCCTCTC
C (SEQ ID NO: 5) 0.5 μg of the amplified DNA fragment was added to the restriction enzyme Sma I (0.
5 unit) and prepared as an Sma I fragment.

(E) Plasmid vector pKPRL-1
Preparation of pKPRL-2 and pKPRL-2 0.5 μg of plasmid pBluescript II KS ⁺ (manufactured by Stratagene) was digested with restriction enzymes Kpn I and H.
It was digested with indIII at 37 ° C. for 1 hour and completely decomposed.
Next, the multicopy number autonomously replicating DNA fragment prepared in (A) and (B) above and the stabilized DNA fragment are mixed, heated at 70 ° C. for 10 minutes to inactivate the restriction enzyme, and then 50 mM Tris buffer is added. Solution (pH 7.6) 10 mM dithiothreitol, 1 mM ATP 10 mM MgCl ₂ and T4D
Each component of NA ligase 1 unit was added (concentration of each component is the final concentration) and reacted at 12 ° C. for 15 hours for binding. E. coli JM using ligated plasmid DNA
Transformed 109 competent cells (Takara Shuzo),
It was obtained as an ampicillin resistant strain. Plasmid DNA was extracted from the transformant strain in the same manner as described above and
g was cleaved with the restriction enzyme Sma I. The kanamycin resistance gene (Pharmacia) was cleaved with the restriction enzyme Pst I, blunt-ended, mixed with the plasmid cleaved with Sma I, and ligated. Escherichia coli JM109 competent cells were transformed with the ligated DNA and cultured in the above L medium containing kanamycin at 37 ° C. for 24 hours to obtain a growing strain. Plasmids were extracted from these grown strains in the same manner as above. From the plasmid thus constructed, a high copy number autonomously replicating DNA fragment,
Three kinds of DNA fragments, a stabilized DNA fragment and a kanamycin resistance gene DNA fragment, were digested with restriction enzymes Kpn I and Bam H.
It was cleaved with I and separated by agarose gel electrophoresis, and DNA was extracted and recovered from the gel.

Next, E. coli autonomously replicating DN prepared in (C)
The A fragment and the three DNA fragments obtained above were blunt-ended and ligated to transform Escherichia coli JM109 competent cells and cultured in the above L medium containing kanamycin for 24 hours at 37 ° C. Was obtained as. A plasmid DNA was prepared from the obtained growth strain, 1 μg thereof was cleaved with restriction enzymes Eco RI and Eco RV, blunt-ended, and then mixed with the β-galactosidase gene DNA fragment prepared in (D), Binding was performed under the above conditions. The ligated DNA was transformed into Escherichia coli JM109 competent cell which is a β-galactosidase deficient strain, and 50 μg
/ Ml (final concentration) kanamycin, 100 μg / ml
(Final concentration) Isopropyl-β-D-thiogalactopyranoside (IPTG), 100 μg / ml (Final concentration) 5
-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) was cultured in an L medium at 37 ° C., and X-g was expressed by β-galactosidase.
Colonies that became blue due to the decomposition of al were obtained.

A plasmid was extracted from this transformant, and Brevibacterium flavum MJ233 was transformed with the plasmid by the electric pulse method as follows. Brevibacterium flavum MJ233
(FERM BP-1497) plasmid pBY502
The removed strain was cultivated in 100 ml of the medium A until the initial logarithmic growth, penicillin G was added at 1 unit / ml, and the mixture was further cultivated with shaking for 2 hours, and the cells were collected by centrifugation to obtain 20 ml of cells. Pulse solution (272mMS
ucrose, 7 mM KH ₂ PO ₄ , 1 mM MgC
l _2: pH7.4) was washed with. The cells were further collected by centrifugation and suspended in 5 ml of the pulse solution to give 0.75
ml of cells and the plasmid DNA solution obtained above 5
0 μl was mixed and left standing in water for 20 minutes. Using a Gene Pulser (manufactured by Bio-Rad), the voltage was set to 2500 V and 25 μFD, and after applying a pulse, the plate was allowed to stand in ice for 20 minutes. The whole amount was transferred to 3 ml of the A medium and cultured at 30 ° C. for 1 hour, then inoculated into the A agar medium containing 15 μg / ml (final concentration) of kanamycin, and cultured at 30 ° C. for 2 to 3 days. From the emerged kanamycin resistant strain, reference example 1
A plasmid was obtained by the method described in. This plasmid was cleaved with various restriction enzymes and the size of the cleaved fragment was measured. The results were the same for both plasmids and were as shown in Table 4 above.

The plasmid having the DNA fragment obtained from the plasmid pCOP1-1.8 and characterized by the cleavage fragment shown in Table 4 above was "pKPRL-1", and the DNA fragment obtained from the plasmid pCOP2-1.8. And a plasmid characterized by the cleavage fragment shown in Table 4 above was designated as "pKPRL-2". The restriction enzyme map of these plasmids is shown in FIG.

Example 6 Plasmid Vectors pKPRL-1 and pKPRL-2
Copy number and stability of the plasmid vector pKPRL- obtained in Example 4
1, pKPRL-2 and pS-obtained in Example 3
Brevibacterium flavum M containing 1 each
Chromosomal DNA and plasmid were obtained from J233 by the same procedure as in Example 1 (B), and subjected to agarose gel electrophoresis to obtain a chromosomal DNA having a molecular weight of 3.0 × 10 ⁹ daltons, pKPRL-1 and pKPRL-2. Was determined to be 3.5 × 10 ³ Dalton (5.4 kb) pS-1 with a molecular weight of 2.6 × 10 ³ Dalton (4.0 kb), and the plasmid copy number was calculated. As a result, the plasmid copy number was 5-6 for pS-1, pKPRL-1 and pK-1.
It was found to be 10-12 for PRL-2.

The stability of the plasmid was confirmed as follows. The above-mentioned pKPRL-1, pKPRL-2 and p
Brevibacterium flavum MJ233-derived strains each containing S-1 are inoculated into the A medium containing kanamycin 50 μg / ml (final concentration), and precultured at 30 ° C. for 15 hours. Next, in A medium, 10 ⁴ cells per 1 ml
The cells were subcultured so that the ratio became s, and the cells were cultured for about 200 generations. Table 8 below shows the results in which the ratio of the number of kanamycin resistant strains in the culture solution, that is, the number of plasmid-carrying strains to the total number of bacteria, is shown in percentage.

[0104]

[Table 8] Table 8 Strains (retention plasmid name) Plasmid retention rate (%) pS-1 18 pKPRL-1 98 pKPRL-2 98

[0105]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1897 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: Plasmid DNA Origin Biological name: Brevibacterium flavum Strain name: MJ233 cop1 or MJ233 cop2 Sequence Characteristic of the symbol: peptide Location: 922-1704 Method of determining the characteristic: E Other information In the following nucleotide sequence, R at the 1307th and 1577th is G or A, but both are G at the same time. Not,
In addition, 129th Xxx in the following amino acid sequence is Arg
Or His, 219th Zzz is Gly or Asp
, Respectively, but when the 129th Xxx is Arg, the 219th Zzz is never Gly.

[0106] SEQ GCGCAGCCTG ACCATGGACA TGCTTCTACG TTCTTCCACA CTGGTATCTT TTGGTTGCAA 60 TATATATTGC AACCATGCTG GTAAAACAAC TTTTTAGCGT GTCTTGCCCA AAATTACAGT 120 CATGTGATTT ACAGTTGAAA GATACTGAAA ATCAAAGAAG GCCACTCGGC GGCAACCGAA 180 TGACCTTCGC TTATCACCCA GTCCTACCTG GGAGAAAGCA TGTACATGAT TATACCCAAT 240 AGTACGCCCG CGTACCCCAT TTCCGGTTCG CGTGTTAACA CGCCATCACG TTTGCGCCCT 300 GACTGCGCAG GCCTTGATGA CACCCCAGCG AGCACCGTTG ACCGGGACAA GCTGCTCGCA 360 CATCTTGGCC GTACAGCACT GCACGGGTCT ATAAGTCGAA ACTTCAAAGG CGCTTATCGC 420 CTCGTTGTGG ATAAGGAAAC GGGGGAAAAG CGAAGCGTTC CGAACCTTTA CCGCATTGAT 480 TCCGAAAAAC TCGGTCGCTG CGAATACGTC ATGCTGACTA GTAAGCAATA TGCTTCGGTC 540 ATGGTCATAG ACGTTGACCA GATAGGAGAG GCTGGAGGAC ATCCAGAAAA CCTCAACTCC 600 TATGTCAAAG GCGTTATCTG GGTACTTGTG CAGCACGGAA TTGGACCAGC ATGGGCAGGC 660 ATTAATCCGA TTAGTGGTAA AGCGCAGTTT ATTTGGCTTA TTGACCCAGT TTATGCAGGA 720 AAGAATCGTG CGTCCCGGAA TATGGAGCTA CTCAAAGCCA CAAGTCACGA GTTGGGTGAG 780 TTACTGGATC ATGATCCACA TTTTGCGCAT CGGTTTAGCC GGAGTCCTTT TTATACTGGA 840 AAGTCACCGG AGGCTTATCG CTGGTATTGC CAGCATGACC GGGTTATACG CCTCCAAGAC 900 TTTCTAAGGC AGGTGCGCGA G ATG GCG GGA CAA TCC CAG CAC ATT AAA AAC 951 Met Ala Gly Gln Ser Gln His Ile Lys Asn 1 5 10 AAG CGC CAGT AGT CGC CAG AGT GTT AAA 999 Lys Arg Gln Gln Phe Asn Ser Gly Arg Glu Leu Ile Asn Ala Val Lys 15 20 25 ACT CGC CGT GAA GAA GCC CAA GCC TTT AAA GCA CTT GCT GAG GAT GTC 1047 Thr Arg Arg Glu Glu Ala Gln Ala Phe Lys Ala Leu Ala Glu Asp Val 30 35 40 GAA AAC GAG ATA AGT GAA GAA ATC GAT CAA TAC GAC CCG GAA CTA ATC 1095 Glu Asn Glu Ile Ser Glu Glu Ile Asp Gln Tyr Asp Pro Glu Leu Ile 45 50 55 GAC GGG GTG CGC GTG CGC TGG ATT AGC CAA GGG GTC GCA GCA CGC GAC 1143 Asp Gly Val Arg Val Arg Trp Ile Ser Gln Gly Val Ala Ala Arg Asp 60 65 70 GAA ACA GCG TTT AGC CAC GCA CTA AAA ATT GGT CAC CGC CTA CGC AAA 1191 Glu Thr Ala Phe Ser His Ala Leu Lys Ile Gly His Arg Leu Arg Lys 75 80 85 90 GCA GGA CAA CGA CTC ACA GAC GCC GCC GTT ATC GAT GCC TAC GAG CAT 1239 Ala Gly Gln Arg Leu Thr Asp A la Ala Val Ile Asp Ala Tyr Glu His 95 100 105 GCC TAT AAC GTT GCA CAA CAA CAA GGC TCA GCA GGC CGA GAC AGC GAA 1287 Ala Tyr Asn Val Ala Gln Gln Gln Gly Ser Ala Gly Arg Asp Ser Glu 110 115 120 ATG CCA CCC ATG CGT GAC CRC CAG ACC ATG GCA CGA CGC GTA CGC GGC 1335 Met Pro Pro Met Arg Asp Xxx Gln Thr Met Ala Arg Arg Val Arg Gly 125 130 135 TAT GTC ACC CAA TCC AAA ACC AAC ACA AGT CTA GGA GCT AGC GCT CCC 1383 Tyr Val Thr Gln Ser Lys Thr Asn Thr Ser Leu Gly Ala Ser Ala Pro 140 145 150 GGA CGC GTT ACC AGC AGC GAA CGC AAA GCA CTG GCC ACC ATG GGG CGA 1431 Gly Arg Val Thr Ser Ser Glu Arg Lys Ala Leu Ala Thr Met Gly Arg 155 160 165 170 AAA GGC GGA CAG AAA GCA GCA CAA CGC TGG AAA ACC GAT CCA GAA GGT 1479 Lys Gly Gly Gln Lys Ala Ala Gln Arg Trp Lys Thr Asp Pro Glu Gly 175 180 185 CAA TAC GCA CAA AAT CAG CTG CAG AAA CTA AAG AAA ACG CAC CGG AAG 1527 Gln Tyr Ala Gln Asn Gln Leu Gln Lys Leu Lys Lys Thr His Arg Lys 190 195 200 AAG CGG GTG GAA GGA CAG ACC ACG CGT GCG AAG ATT CAA GCC TTA ATT 1575 Lys Arg Val Glu Gly Gln Thr Thr Arg Ala Lys Ile Gln Ala Leu Ile 205 210 215 GRT GAA GCT TAC GTG CAA ACA GGC GAG GTA CTT ACC CGC AAA CAG ATT 1623 Zzz Glu Ala Tyr Val Gln Thr Gly Glu Val Leu Thr Arg Lys Gln Ile 220 225 230 GTG GAT GAG ACA GGA CTA TCT AGA GCT ACA GTG ACA CGG CAT TTG GCG 1671 Val Asp Glu Thr Gly Leu Ser Arg Ala Thr Val Thr Arg His Leu Ala 235 240 245 250 GCA CTT CGA GAA CAG GGA GCA CTT CCG GAA ACG TAGGGGCTCATACCGTAAGCA 1725 Ala Leu Arg Glu Gln Gly Ala Leu Pro Glu Thr 255 260 ATATACGGTT CCCCTGCCGG TAGGAATGTA GTAATAACCT CTCTTGAAGA AAACCTTGTA 1785 GGGCAAGGCT ACTTATGCTT CCGGGGTTAG TCGTTCTTCT ATTGCGGTGAGTGAGTGA TGAGTGATCGACTGA

SEQ ID NO: 2 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence TCCTCGAGG CGCAGCCTGA CCATG 24

SEQ ID NO: 3 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence TCGGTACCGC GCAGCAAAGC CTCG 24

SEQ ID NO: 4 Sequence Length: 26 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence TTCCCGGGTG CGCGTAACCA CCACAC 26

SEQ ID NO: 5 Sequence Length: 26 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence TTCCCGGGAT ACGCAACCGC CTCTCC 26

[Brief description of drawings]

FIG. 1 is a map of restriction enzyme cleavage points of a high copy number autonomously replicating DNA fragment used for constructing the plasmid vector of the present invention.

FIG. 2 is a restriction enzyme cleavage point map of the plasmid vectors pKPRL-1 and pKPRL-2 of the present invention.

Claims (1)

[Claims] 1. A DNA fragment containing a gene involved in a function of controlling autonomous replication of a plasmid in a coryneform bacterium derived from plasmid pBY503, wherein a mutation point capable of increasing the copy number of the plasmid is within the gene. At least one of
DNA fragment present in a place, (b) plasmid pBY5
A DNA fragment containing a gene involved in the function of stably retaining a 03-derived coryneform bacterium in the coryneform bacterium, (c) a gene having an autonomous replication function in Escherichia coli derived from the Col E1 plasmid DN including
A fragment, (d) a DNA fragment containing a gene encoding β-galactosidase derived from Escherichia coli, and (e) a DNA fragment containing a drug resistance gene that can serve as a plasmid marker in coryneform bacteria and in Escherichia coli. Plasmid vector.