JPS61124387A - Vector for detection of promoter - Google Patents

Abstract

PURPOSE:To provide a vector for the detection of a promoter, having a replication initiation point functioning in the cell of a coryneform bacterium, an expressed drug-resistant gene, and a structural gene region of a gene having a selectable phenotypic transformation different from the above drug-resistant gene. CONSTITUTION:The objective promoter-detecting vector can be prepared by constructing the plasmid vector having a replication initiation point functioning in the cell of a coryneform bacterium, a drug-resistant gene expressed in said bacterial cell, and a structural gene region of a gene having a selectable phenotypic transformation different from the above drug-resistant gene and free from the promoter sequence at the part incised with a restriction enzyme and its downstream side. The figure is an explanation of the process for forming the promoter-detection vector pEB001.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a vector for detecting a promoter, and more particularly to a vector for detecting a promoter that functions in coryneform bacterial cells.

Coryneform bacteria are known to produce large amounts of L-glutamic acid, and their mutant strains are known to produce amino acids such as lysine, purine nucleotides such as inosinic acid, etc. Ah W #I:I:1
4 a & 4/Ill +lTS &! −＋
JL -e v-s%t&bes Jaa.

Breeding and improvement of industrial microorganisms using
However, for coryneform bacteria, vectors suitable for using these microorganisms as hosts have not been developed, and this has hindered the breeding and improvement of coryneform/glutamic acid producing plants using recombinant DNA K. was.

Several vectors are known that propagate using coryneform bacteria as a host and express drug resistance as a marker (for example, European Patent Application Publication No. 93611),
The promoter was not placed at an appropriate location so that the inserted foreign gene 1 would be expressed, and it was not designed to be used as an expression vector9.

Problems to be Solved by the Invention The present inventors believe that in order to develop an expression vector that uses coryneform bacterial cells as a host, it is first necessary to obtain a promoter that functions within coryneform bacterial cells. Friends started developing vectors to detect such promoters.

Means for Solving the Problems The present inventors succeeded in obtaining a vector for promoter detection by constructing a Grasmid vector as described below. That is, the vector for promoter detection of the present invention has a replication origin that functions in coryneform bacterial cells, a drug resistance gene expressed in the bacterial cells, a site that is cleaved by a restriction enzyme, and no promoter sequence located downstream thereof. This is a promoter detection vector having a structural gene region of a gene with a selectable phenotypic trait different from the drug resistance gene described above.

A vector having a replication origin that functions within the cells of coryneform bacteria is one that has at least a DNA region that controls the function of replicating within the cells of coryneform bacteria. For example, the following wild-type plasmids having such a replication origin are known.

(1) pAM 330 JP-A-58-6769
9 reference (2) pAM 1519 JP-A-58-7
7895 reference (3) pAJ 655 % open/close 5
Reference 8-192900 (4) pAJ 611
See JP-A-58-192900 (5) pAJ 1
844 Same as above (6) pCG 1
% open/close 57-134500 reference (7) p
CG2 See JP-A-58-35197 (8)
pCG 4 See JP-A-57-183799 (9) pcc 11 Same
The vector of the Josui invention only needs to have a replication origin, so there is no D in the vector other than the replication origin.
Even if there is NA, there is no particular problem.

Coryneform bacteria are aerobic, Gram-positive rods;
Non-acid-fast, Purdes Manual of Determinative Pacteriology, M8 edition, page 599 (1974)
It is described in. Among the coryneform bacteria that can be used as host bacteria of the present invention, the following are examples of wild-tasting coryneform bacteria that have glutamic acid productivity.

Brevibacterium diparicatum ATC
C14020 Pleuttherium salacalolyticum
ATCC14066 Plecdutherium inmariophyllum ATCC14068 Breviducterium lactofermentum ATCC13869 Flevipecterium roserum ATCo
13825 pre ez4) Thillium flavum
ATCC13826 Previpacterium thiochetalis ATCC19240 Corynebacterium acetoacitophy #A ATCC13870
Corynenoduterium acetoglutamicum ATC
o 15806 Corynepacterium garnae
ATCC15991 Corynebacterium glutamicum ATCC13032,130
60 Corynebacterium Lilium A
TCC15990 Corynebacterium mellana cola
ATCC17965 Microbacterium ammoniaphyllum ATCC15354 Coryneform bacteria are derived from these glutamic acid producing bacteria,
Also includes mutant strains that have lost glutamic acid productivity, or mutant strains that produce amino acids such as lysine and arginine, purine nucleosides such as inosine, purine nucleotides such as inosine 5'-monophosphate, or other products. It will be done.

The following drug resistance genes are known to be expressed in coryneform bacterial cells.

(1) Chloramphenicol resistance gene (US application 3)
86,98057.6,10)/9)-? 7 + square η so 1 pestle Koban Hayabusa / I?
l-% (3) Tet juice icrin resistance gene (% open/close 58
-126789) (4) Spectinomycin resistance gene (see JP-A-57-183799) (5) Streptomycin resistance gene (same as above)
These drug resistance genes are carried by the following plasmids.

(1) Chloramphenicol resistance gene: pBR325
(See G@ns, 20, 305 (1982)) (2) Kanamycin resistance gene: ptyc4K (
Gone, 19, 259 (1982)) pUB 1
10 (Proc Na$t, Aead, Sei,.

to, 1423C19η))Pν) (3) Tetracycline resistance gene: pBR322(G
on@, 2.95 (1977)) (4) Spectinomycin resistance gene: pCG4 (Hand open/close 57-183
(Refer to 799) (5) Streptomycin resistance gene: pCG4 (same as above) It is cut by restriction enzyme.
HI.

These include C2aI, Hindl, HpaI, and ECORI. The site that is cut by the restriction enzyme is in the vector,
Although it is most desirable that there is only one location, even if there are multiple locations, it can be used as a vector for promoter detection.

The structural gene region of a gene with a selectable phenotype different from the drug resistance gene is placed in the vector so that no promoter sequence is present downstream of the site that is cleaved by the restriction enzyme.

The selectable phenotypic gene may be any type of gene as long as it can be expressed in coryneform bacterial cells and has a selectable trait.For example, in addition to the drug resistance genes mentioned above, 5-fluorotryptophan, meta Genes encoding resistance to amino acid analogs such as fluorophenylalanine, α-amino-hydroxyvaleric acid, and ethionine, or genes that can complement the auxotrophy of various auxotrophic strains can be used. Structural gene region is a gene region of these selectable phenotypic genes excluding the promoter sequence necessary for the expression of that gene, and is a gene region in which trait expression occurs only by incorporating a promoter upstream of the gene region. It is about. Such a structural gene region is a DNA containing a gene for a selectable phenotypic trait as described above.
? It can be extracted by cutting with an appropriate restriction enzyme.

The site that is cut by the restriction enzyme and the structural gene region of the gene for the selectable phenotypic trait are arranged so that when the promoter is inserted into the site that is cleaved by the restriction enzyme, the phenotypic trait in question is expressed. It would be fine if it had been done.

While the invention has been generally described above, it is only further listed and is not intended to limit the scope of the claims.

Example 1 Construction of pBB O01, capable of replicating in the cells of coryneform bacteria, expressing kanamycin resistance, lacking the promoter sequence, and containing the lola1 phenicol resistance gene, with C1* r and Hlnd m as promoter insertion sites. A promoter detection vector pEB O01 having a cleavage site was constructed by the method shown in FIG. That is, plasmid pCM containing the structural gene region of the chloramphenicol resistance gene.
7 (Gono, 20, 305-316 (19
In order to confer kanamycin resistance, which is a marker that can be expressed in the cells of coryneform bacteria, to (see 82)), we first created a plasmid pU04K (G@n@,
19.

259-268 (1982), J. Mot. Bi.
ot, 147, 217-226 (1981))
About 1 cut with PstI and containing the kanamycin resistance gene
．． A 4 kilobase DNA fragment was taken out, cut with P+stI, mixed with pCM7 DNA, and then converted into T, DNA.
They were bonded using lysate. The obtained recombinant DNA was introduced into Escherichia coli) In 101 strain, and a strain having kanamycin resistance was selected. Grasmid DNA was obtained from the isolated strain, and it was determined that the kanamycin resistance gene had been inserted correctly by determining the size of the plasmid and cutting with PatI.
Named it A. Next, pAJ2401. A cilasmid pHM15 capable of replicating in the cells of coryneform bacteria in order to impart the ability to replicate within the cells of coryneform bacteria.
19 (see JP-A-58-77895) DNA f
pAJ24 cut with B5tI and then with BamHI
01A (mix with D DNA, T 4DNA IJ
It was bound using case. The obtained recombinant DNA was introduced into Escherichia coli HBIOI, and a strain having kanamycin resistance was selected. Grasmid DN from the isolated strain
After confirming that the plasmid is the desired grasmid by the size of the plasmid and the fact that it cannot be cut with BamHl and BgtII, this plasmid was
It was named EBOOl. , EBOO1t Grepipacteri, Rum lactofermentum AJ12036 (F]
iILM-P7.5'''') and Corynebacterium glutamicum ATCC 13060, and strains with kanamycin resistance were selected. Plasmid DNA was obtained from the isolated strain and confirmed to be pEBOOl DNA by size and PstI, BamHI
Confirmed by restriction enzyme cleavage pattern. Brevibacterium lactofermentum, in which a more distinct pEBOOl DNA band was detected than in these strains.
It is a shuttle vector that can be replicated in Lactofermentum and Corynebacterium glutamicum and fully expresses kanamycin resistance. It has the structural gene region of the chloramphenicol resistance gene with the bromota part deleted, and C2a can be used as the promoter insertion site. I, Hln
d m has a cleavage site.

The 90M7 DNA used in the experiment was prepared from Escherichia coli ATCC37173 and was added to pUC4.
NA was purchased from Pharmacia (Japan).

In addition, the DNA of pHM 1519 was prepared from its own strain, Corynebacterium glutamicum ATCC13058.

Example 2 Construction of pEB O03 It has the structural gene region of the chloramphenicol resistance gene which is capable of replicating in the cells of coryneform bacteria and expresses kanamicun resistance, and the promoter sequence has been deleted, and PskI is used as the site into which the promoter can be inserted.

Hpa I, BamHI, Cta I, H
A promoter detection vector pEB 003 having five types of restriction enzyme cleavage sites for lnd m was constructed by the method shown in FIG.

First, 7'j pAJ2401 was created using the method of Example 1.
AO DNA was partially digested with PstI, and the DNA fragment formed by cutting at the - site was collected from agarose glue and the DNA
After cutting off the protruding single-stranded portions at both ends of the DNA fragment to blunt ends using A polymerase (Kl@nov 7 fragment), 7. DNA! Next, bind with J gauze. The obtained recombinant DNA 'i Escherichia HB 101
Next, select strains with kanamycin resistance.

Obtain plasmid DNA from the isolated strain and select the DNA that can be cut at one site with PitI.

These glasmids contain the 5′ of the kanamycin resistance gene.
There are nine plasmids with deletion of the PatI cleavage site on the side and grasmid with deletion of the P and tI cleavage sites on the 31st side. Next, the DNA of pEB O01 constructed by the method of Example 1 was cut with CtaI and 5atI to obtain DNA fragment A containing the structural gene region of the chloramphenicol resistance gene and the origin of replication in coryneform bacterial cells. Ta. On the other hand, pEBOO2A was cut with Ps tI and 5atI to obtain DNA fragment B containing the kanamycin resistance gene. In addition, oligonucleotides as shown in Figure 3 were synthesized using the phosphite method, and the 5'Song-II section had a P-co-tI section, and the 3'-terminus had a C/a
A DNA fragment C was prepared which had the r cleavage plane and HpaI and BamHI cleavage sites in the middle. After mixing DNA fragments A, B and Cf, they were ligated with T4 DNA ligase,
The obtained recombinant DNA' was introduced into Brevibacterium lactofermentum human J 12036 (FERMP-7ritq) to select a strain having kanamycin resistance.

Plasmid DNA was obtained from the isolated strain, and plasmid size, PstI, HpaI, BamHI
Confirm that it is the desired grasmid DNA by cutting with a restriction enzyme such as
It was named BOO3. pEBOO3 has Ps tI, HpaI, Bam)I as promoter insertion sites.
I, CtaI, Hlnd[l +05 types of restriction enzyme cleavage sites, among which Pg-4I, Hpa
The cleavage site for BamHI and BamHI is found only in the vector, and is extremely useful for promoter insertion.

Brevibacterium lactofermentum AJ12036 introduced with pEB OOa (FERMP 'i!;
DAQ) was deposited as AJ12178 (F 訝圓PqQTO3).

Example 3 Construction of pEB OO3Cmr It is known that pEB OO3 constructed in Example 2 is expressed in coryneform bacterial cells at the bromoter insertion site (see % opening/closing 58-192900).

Plasmid pEBOO3Cm' incorporating the bromotor sequence of the chloramphenicol resistance gene was constructed by the method shown in FIG. That is, pBR325(G@1
1@, 14, 289-299 (1981))
The DNA fragment Di containing the bromotor sequence of the chloramphenicol resistance gene was cut with tI and Pvgl, and pEB0 was cut with Pgtl and HpaI.
03 DNA and ligated with T and DNA ligase. The obtained recombinant DNA was transformed into Brevibacterium
Lactofermentum AJ 12036 (FIRM-P
'Bj de), and strains showing resistance to chloram 7 alcohol were selected. Plasmid DNA was obtained from the isolated strain, and the size of cilasmid, PstI, BamHI
, Hpa I.

We confirmed that it was the desired plasmid DNA from the cleavage pattern with restriction enzymes such as Pvun, and named this plasmid pEB 0030m'. Table 1 shows pEn
OO3 or pEBOO3cm”! (Introduced Brevibacterium lactofermentum AJ12036 (
FERM-P'l! ;! ;De) showed susceptibility to chloramphenicol and kanamycin. As seen in these results,<pgnooa expresses kanamycin resistance within coryneform bacterial cells, and by inserting a DNA fragment containing a promoter sequence that functions within coryneform bacterial cells into its promoter insertion site, chloramphenicol resistance can be achieved. is a bromotor detection vector expressed in coryneform bacterial cells.

Table 1 Susceptibility of P1/Pipacterium lactofermentum harboring pEBOO3 or pEBOO3cmr9 to chloramphenicol and kanamycin Chloramphenicol kanamycin host fungus plasmid 30d74tl 25 Growth in a 25-year-old culture medium Growth in a medium AJ12036 Shi ---
AJ12036 pggooa
+AJ12036 pEBO03cm'
+ +In addition, pBR32 used in the experiment
The DNA of 5 is produced by Bethesda Research Uga Laboratory (BR
Purchased from Company L.

[Brief explanation of drawings]

Figure 1 shows promoter detection vector pEB O01.
It is an explanatory diagram of the creation progress. P: PstT C2C1aI H: Hindll [B: BamHI Km': Kanamycin resistance gene Cm': Structural gene region of chloramphenicol resistance gene Figure 2 shows promoter detection vector pEB O03
It is an explanatory diagram of the creation progress. P: F'it I C: C1aI H: Hlndnl B: BamHI S: Sal I Hp: Hpa I Kmr: Kanamycin resistance gene Cm3: Structural gene region of chloramphenicol resistance gene Figure 3 shows promoter detection vector pEB O
FIG. 4, which shows the structure of DNA fragment C used in the construction of O3, is an explanatory diagram showing an example of how the promoter detection vector is used. Ps: Pstl Pv: Pvull B: BamH[ C: C1m1 H: Hlndn[ Hp: Hpa I Kmr: Kanamycin resistance gene CmP: Promoter region of chloramphenicol resistance gene Cm': Structural gene region of chloramphenicol resistance gene

Claims (1)

[Claims] A replication origin that functions within coryneform bacterial cells, a drug resistance gene expressed within the same bacterial cell, a site that is cleaved by restriction enzymes, and a promoter sequence downstream thereof that can be selected to be different from the above drug resistance genes. A promoter detection vector having a structural gene region of a gene with a specific phenotypic trait.