JPS61152289A - Plamid and bacterium containing same - Google Patents

Abstract

PURPOSE:A plasmid which proliferates in cell bodies of Corynebacterium and resists trimethoprim, when it is introduced into the cells is prepared to obtain a plasmid vector which can proliferate in cells of Corynebacterium and contains only a same kind of DNA. CONSTITUTION:A strain resisting to trimethoprim (trimethoprim-resistant mutant) is selected from a Cornebacterium, trimethoprim is added to the culture medium and only the growing bodies are separated to obtain a trimethoprim- resistant strain. The chromosomal genes are extracted therefrom, cleaved with restriction enzymes and connected to a plasmid vector which an proliferate in a Corynebacterium. The resultant recombinant DNA is used to transform a Corynebacterium and a strain resistant to trimethoprim is separated. Further, a gene expressing trimethoprim resistance is separated therefrom. The gene is connected to DNA in the replication initiation zone of a plasmid which can proliferate in cells of Corynebacterium.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) This invention relates to a plasmid and a bacterium containing the same,
Specifically, the present invention relates to a plasmid vector that can proliferate in coryneform bacteria cells and coryneform bacteria having the same.

(Prior art) Coryneform bacteria include those that produce large amounts of amino acids such as glutamic acid, such as Brevibacterium flavum.
It is an extremely important group of microorganisms industrially.

Several plasmid vectors have already been developed to utilize these coryneform bacteria as hosts and increase the productivity of fermentation products of these coryneform bacteria by recombinant DNA technology (in Europe). Patent application publication 009361
1). All of these plasmid vectors are cryptogenic vectors that can proliferate within coryneform bacterial cells.
ic) A marker gene for plasmid selection is intended to be inserted into a plasmid, and therefore a marker gene is inserted into a plasmid of a coryneform bacterium such as a gene derived from the genus Escherichia or Bacillus.

Therefore, for coryneform bacteria, foreign DNA has been integrated, and therefore, the use of such a plasmid vector belongs to a recombination experiment with foreign DNA.

(Problems to be Solved by the Invention) Therefore, an object of the present invention is to develop a plasmid vector that can proliferate in coryneform bacterial cells and is composed only of the same kind of DNA and has a marker gene derived from the same kind of bacteria. It's about getting.

(Means for solving the problem) In order to solve the problem of slanting, the present inventor created a cilasmid that proliferates within coryneform bacterial cells and exhibits trimethoprim resistance when introduced into the cells. . Here, the trimethoprim resistance '2 gene in Table 3J is derived from coryneform bacteria. Trimethoprim is a structural analog of dihydrofolate and an inhibitor of dihydrofolate reductase.

A gene that expresses trimetogerim resistance when introduced into coryneform bacterial cells can be obtained as follows. First of all, it is more resistant to trimethoprim than coryneform bacteria! The wild strain of the coryneform bacterium to be selected is usually sensitive to trimethoprim, and therefore, in order to obtain a trimethoprim-resistant strain, it is preferable to induce a mutant strain.

Trimethoprim-resistant mutant strains are produced by irradiating the parent strain, which is a coryneform bacterium, with X-rays, R-rays, ultraviolet rays, etc., or by exposing it to mutagenic agents such as N-methyl-N'-nitro-N-nitroguanidine. After that, strains that have acquired trimethoprim resistance can be selected. Strains that have acquired trimethoprim resistance can be grown, for example, on minimal medium (glucose 2 g/di, ammonium sulfate 1 g/di).
/di, urea 0.25 g/dl, K) T2PO40
,1jj/dl, MgSO44H200,04g/d
e, thiamine hydrochloride 200 μg/l, biotin 50
μg/l, agar 1,59/de, pH 7,0
100119 to this medium.
/fnl or more preferably by adding 200tt97fn1 or more of trimethoprim and isolating the strain that grows on this medium.

A method for isolating a gene expressing trimethoprim resistance involves first extracting a chromosomal gene from a strain of coryneform bacteria that has a gene expressing trimethoprim resistance.

Bjochem Rjophys Acta 7261
9 (1963)) and cleave it with an appropriate restriction enzyme.

To cleave chromosomal genes, a wide variety of restriction enzymes can be used by adjusting the degree of cleavage by adjusting the cleavage reaction time and the like. The chromosomal gene that has been cleaved with restriction enzymes is then connected to a plasmid vector that can be propagated in coryneform bacteria, and the resulting recombinant DNA is used to transform coryneform bacteria to maintain trimethoprim resistance. By isolating a single bacterial strain, it is possible to isolate the gene expressing trimethoprim resistance.

The coryneform bacteria used in this case are sensitive to trimethoprim, and therefore, a transformed strain having a gene expressing trimethoprim resistance can be obtained as a trimethoprim-resistant strain as described above.

Corynebacteria are aerobic, Gram-positive rods, non-acid fast, and are described in Purdes Manual of Determinogenic Pacteriology, 8th edition, page 599 (1974). Among these, those that produce large amounts of monoglutamic acid are particularly known as shown below, and all of these are considered to belong to the same genus.

Brevibacterium divaricatum ATCC
14020 Brevibacterium salacalolyticum
ATCC14066 Brevibacterium inmariophyllum ATCC14068 Brevibacterium lactofermentum ATCC13869 Brevibacterium roserum ATCC1382
5 Brevibacterium flavum ATC
C13826 Prepipacterium thiognitalis
ATCC19240 Corynebacterium acetoazidophyllum ATCC13870 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium carnae ATCC1599
1 Corynebacterium glutamicum ATC
C13032,13060 Corynebacterium Lilium ATCC15990 Corynebacterium Melasecola ATCC17965 Microbacterium Town Ammoniaphyllum ATCC153
In addition to those having glutamic acid productivity as described above, 54 coryneform bacteria also include mutant strains that have lost glutamic acid productivity and other mutant strains.

In order to obtain a plasmid that can proliferate in coryneform bacterial cells, the gene expressing trimethoprim resistance is connected to the replication initiation region DNA of the plasmid that can proliferate in coryneform bacterial cells. As the replication initiation region jd DNA, the entire wild-type plasmid capable of propagating in coryneform bacterial cells or a DNA fragment containing the replication initiation region can be used. Furthermore, DNA derived from coryneform bacteria is added to these wild-type plasmids or DNA fragments containing their replication initiation regions.
The DNA to which A is connected can also be used as a replication initiation region DNA.

As a wild-type plasmid that can proliferate in coryneform bacterial cells, pAM described in JP-A No. 58-67699 is used.
330, pAM286, pT described in JP-A-58-77895 (M 1519, JP-A-57-13)
pCG 1 described in 45nO, JP-A-58-
pcc 2 described in 35197, JP-A-57-
pCG 4, pcGll described in 183799
etc. are known.

The plasmid containing the replication initiation region DNA is cleaved with the same restriction enzyme used to cleave the chromosomal gene in order to connect with the DNA expressing trimethoprim resistance. The chromosomal DNA cleavage fragment and the cleaved plasmid DNA may be used as is, or if necessary, nucleotides having complementary base sequences at both ends of each are connected, and then the plasmid DNA and the chromosomal DNA are combined.
It is subjected to a ligation reaction with the A fragment.

Chromosomal DNA and cilasmid D thus obtained
In order to introduce recombinant DNA with NA into recipient bacteria belonging to coryneform bacteria, methods such as those reported for Escherichia coIJ K-12 (Mandel M, an
dHlga, A, , J, Mo1. Biol
, 53159 (1970)) to increase DNA permeability by treating recipient cells with calcium chloride, and as reported for Bacillus subtilis (D
unean, C.H., Wilson.

G, A, and Young, F, F2. ,
Gsn+s + 1, 153 (1977)) is possible by introducing the cells into a proliferation stage (so-called competent cells) in which they become capable of taking up DNA. Alternatively, as is known for Bacillus subtilis, actinomycetes and yeasts (Cbang*S, and
Choen, S.N., Molec, Ge.
n, Genet, +168, 111 (1979)
: Bibb, M.J., Ward, J.
M.

and Hopwood, O.A., Natu
re, 274.398 (1978): Hlnns
n * A, , Hicks r J, B, and
Flnk + G, R,, Proe.

Natl, Acnd, Sc1. USA, 75 1
929 (197B)), it is also possible to transform the DNA recipient bacteria into protoplasts or spheroplasts that readily take up plasmid DNA, and to introduce cilasmids into the DNA recipient bacteria.

In the protoplast method, a sufficiently high frequency can be obtained even with the method used for Bacillus subtilis mentioned above, and protocillasts of the genus Corynebacterium or Brevibacterium described in JP-A-57-183799 Naturally, a method in which DNA is incorporated in the presence of polyethylene glycol or/IJ vinyl alcohol and divalent metal ions can also be used. DNA can be prepared by adding carboxymethylcellulose, dextran, Ficoll, Naluronic F68 (Selva), etc. to polyethylene glycol or polyvinyl alcohol.
Equivalent results can also be obtained by methods that promote the uptake of

After transformation, a strain that has acquired trimethoprim resistance is isolated as a desired transformed strain. Such a transformed strain has a recombinant DNA into which a gene expressing trimethoprim resistance has been integrated. To isolate recombinant DNA, for example, a transformed strain is lysed by lysozyme/SDS treatment, and after phenol treatment, 2 volumes of ethanol are added to precipitate and recover the DNA.

(Effect) The thus obtained plasmid, which grows in coryneform bacteria cells and expresses trimethoprim resistance when introduced into the same cells, does not contain heterologous DNA, and therefore does not contain genes derived from coryneform bacteria. It is particularly suitable for insertion and expression by coryneform bacteria. Insertion 1 Expressed genes include genes involved in amino acid biosynthesis.

Example 1 Construction of pAJ 228 (1) Brevibacterium lactofermenta A
Trimethoprim-resistant mutant strain AJ 12146 (FERM-P 76) mutagenized from AJ 12036
72) to 11(1) CMG medium (peptone 1 fi/d
1, yeast extract 1 g/di, glucose 0.
59/dl, and Na CLO,' 5 g/dl
(adjusted to pH 7.2) and incubated at 30°C.
Shaking culture was carried out for about 3 hours, and bacterial cells in the logarithmic growth phase were collected. After lysing this bacterial body with lysozyme/SDS,
Chromosomal DNA was extracted and purified by a conventional phenol treatment method, and finally 3.0 m9 of T)NA was obtained.

(2) pAM 330 was used as the 4 vector. pAM
330 was prepared as follows.

First, Brevibacterium lactofermentum ATCC13869 carries pAM330 as a plasmid.
After inoculating 100 ml of I) CMG medium and culturing at 30°C until the late logarithmic growth phase, the cells were lysed by treatment with lysozyme 80S, and a supernatant was obtained by ultracentrifugation at 30,000×g for 30 minutes. After the phenol treatment, 2 volumes of ethanol were added to collect the DNA as a precipitate. Add this to a small amount of TEN buffer (20mM) IJS hydrochloride, 2
0mM NaCt, 1mM EDTA (p'
8.0), subjected to agarose gel electrophoresis for separation, and excised to obtain approximately 15 μs of pp, M330 plasmid DNA.

(3) 20 μg of chromosomal DNA obtained in (1) and (2
10 μl of the cilasmid DNA obtained in ) was treated with restriction endonuclease Mbo) at 37° C. for 30 minutes to partially cleave it. After heat treatment at 65°C for 10 minutes, both reaction solutions were mixed, and in the presence of ATP and dithiothreitol, T
4 phage-derived DNA II gauze at 10°C.

The DNA strand ligation reaction was carried out for 24 hours. After heat treatment at 65° C. for 5 minutes, twice the volume of ethanol was added to the reaction solution, and the DNA after the completion of the ligation reaction was collected by precipitation.

(4) Brevibacterium lactofermentum AJ, 12036, which is sensitive to trimethoprim, was used as the recipient bacterium.

The protocilast transformation method was used as the transformation method. First, add the bacterial strain to 5 ml of CM.
The cells were cultured in G liquid medium until the early logarithmic growth phase, and after adding 0.6 units/rnl of penicillin G, the cells were further cultured with shaking for 1.5 hours, and the cells were collected by centrifugation.
M Shake Rose, 20mM Maleic Acid, 20mMjJ
Magnesium chloride, 3.5% Penassai Broth (Dlf
SMVP medium (pi(6,5)0.5d
Washed with. Subsequently, the cells were suspended in SW medium containing 101n9/rnIl norizozyme to form protoplasts at 30°C for 20 hours. After centrifugation at 6000XP for 10 minutes, protoplasts were washed with SNMP and 0.5 ml of SMM.
resuspended in P. The protoplasts obtained in this way and 10μs of the DNA prepared in (3) were mixed with 5mM E.
After mixing in the presence of DTA and adding &IJ ethylene glycol to a final concentration of 30%, the mixture was left at room temperature for 2 minutes to allow DNA to be incorporated into protoplasts.

1m7 of SMMP medium for these protoplasts! After washing with
The cells were resuspended in SMMP medium]d and cultured at 30°C for 2 hours for expression. This culture solution was spread on a protoplast regeneration medium at pH 7.0. Protoplast regeneration medium contains 12 g of tris(hydroxymethyl)aminomethane, 60.5 g of KC, 10 I! of glucose per 11 I! of distilled water. ,
MgCl2・6H208,1g, CaCl2・2H
202,2,ji+, pezoton 4g, powdered yeast extract 4
g, casamino acid ([)Ifeo) 19, K2I
(PO40.2g, sodium succinate 1359.agar 8g and trimethoprim (Sigma) 25μ9/m
Contains l.

After culturing at 30°C for one week, approximately 100 colonies appeared, which were then transferred to a minimal medium containing trimethoprim (2
% glucose, 1% ammonium sulfate, 0.25% urea, 0.1% potassium dihydrogen sulfate, 0.04 magnesium sulfate heptahydrate, 2 ppm iron ion, 2 ppm manganese ion, 200 μg/e thiamine hydrochloride, 50μg
1/l biotin, PH 7.0, agar 1.8%, trimethoprim 50μ9/rldl') to obtain one strain resistant to trimethoprim.

(5) From these stocks, by the method described in (2),
A lysate was prepared and cilasmid DNA was detected by agarose del electrophoresis.
A cilasmid clearly larger than 330 was detected.

This strain was AJ 12147 (FERM-P 7673
) was named.

(6) Plasmid possessed by AJ 12147 (pAJ
228) Brevibacterium lactofermentum AJ 12036 was transformed again using this plasmid DNA to confirm the presence of the trimetogerim resistance gene.

When cilasmid DNA was detected in 10 of the resulting trimethoprim-resistant colonies (IA) by Tsuribe agarose electrophoresis, a plasmid of the same size as PAJ 228 was present in all of them. It was revealed that a gene expressing trimethoprim resistance was present on the above recombinant plasmid.

Brevibacterium lactofermentum AJ 12
146 was mutagenized by contacting Brevibacterium lactofermentum AJ 12036 with 1,000μ9/ml of N-methyl-N'-nitro N-nitrosoguanidine at 0°C for 20 minutes, and then using a minimal medium. (glucose 'l 9/dl, ammonium sulfate 1 j; i/de, urea 0
．． 25 jj/di, KH2PO40,111/di
1MgSO4.7H200,049/di, thiamine hydrochloride 200μm1/l, biotin 50μg/l, 2
ppm iron ion, 2 ppm manganese ion, agar 1.
The strain was isolated as a strain that can grow in a medium containing 597 dl (adjusted to pH 7.0) and 100 μf17fnl of trimethoprim.

AJ 12036 is easily obtained from AJ 12147 by removing the complex plasmid in the host cell without damaging the host cell. That is, grasmids can be naturally lost from the host, or they can be removed by a "removal" operation (Baet, Row, r.).

p361-405 <1972)). An example of the removal operation is as follows; a small amount of the bacterial strain is inoculated to about 104 cells per one medium containing acridine orange at a concentration (2-50 μg AfLl) that completely inhibits the growth of the host. After completely inhibiting the growth of the host bacteria, the host bacteria are cultured overnight at 27-37°C (J, Bacteri
ol, +88, 261 (1964)). The culture solution is spread on an agar medium and cultured overnight at 27-37°C.

Among the colonies that appeared on the medium, trimethoprim (5
This is a strain from which the plasmid has been removed, that is, AJ12036.

(7) pA, 7228 Properties of DNA (a) p
The molecular weight of AJ 228 was determined by agarose del electrophoresis.

Agarose del electrophoresis was performed using a Sharp (P, A-5har)
p) et al.'s method (Biochemistry 12.30
55 (1973)), 0.8% glue was used, the glue length was cfn, and electrophoresis was performed at a constant voltage of 5V for 15 hours. The molecular weight is restriction enzyme C that cuts pAJ 228 at one site.
1a IO, 5 units pAJ2280, 5 μg I
After reacting at 37°C for 1 hour, cutting and making it into a straight line,
Molecular weight marker with known molecular weight, Hlnd of λ phage
It was calculated by comparing the mobility with III fragment (BRI, Inc.) and was calculated to be 7.6 Kb.

In addition, Table 1 shows the cleavage sites using various commercially available restriction enzymes using the same method.

Table 1 Restriction enzyme Number of cutting sites Ava T 5 BamHT 0 Bcl I 4 Bgl l 0 BstE Tl 3C1a I
I EeoRI 0 Haen 4 Hlnd m 2 Hpa I I Kpn I 0 M1u I 2 M5t T I Pat I 0 Pvu II 08at I
, 08ea 1
2 Sma I l 5at I 0 8st II 0Xbal
, I Xma I I Xor It 0Xho I
, 1(b) Creation of a restriction enzyme cleavage map of pA'522B DNA Use commercially available products from BRL for restriction enzymes, and cut pAJ 228 DNA with restriction enzymes using at least a 3-fold excess of each enzyme. Each test was carried out under the specified conditions. When plasmid DNA is cut with one or more restriction enzymes to create a restriction enzyme map, the first restriction enzyme cut fragment is placed on a separation agarose gel using the method of Shitanaka et al.
,B.

Welsblum, J., Baeteriol, .
121.354 (1975)), concentrated by ethanol precipitation, and cleaved with a second restriction enzyme. The cut fragments were subjected to agarose gel electrophoresis, the molecular weight was calculated, and a restriction enzyme cleavage map was created.

(e) Measurement of copy number of pAJ 228 pAJ 2
Brevibacterium lacto7armentum AJ 12147 holding 28 was inoculated into 5 ml of CMG liquid medium containing 50μ of trimethoprim, and after culturing overnight at 30°C, 0.1- of the inoculated solution was inoculated into 5 ml of CMG containing 50μ of trimethoprim. The liquid medium was re-inoculated.

Cultivate at 30°C until early logarithmic growth phase and add 1000μfl/
After adding ampicillin to a volume of 1.5 ml, the cells were cultured for another 2 hours, collected by centrifugation, suspended in Tris-EDTA-NaCt buffer 1 and 5 ml K containing 10 mp/m/ml of lysozyme, and incubated at 37°C for 2 hours. After incubation for an hour, SDS (final concentration 4%) was added and
Lysed for minutes. After confirming that the protocilasts were completely lysed, the protocillasts were extracted with 1 to 2 times the amount of ethanol, then precipitated with NA at -20°C, and the precipitate was suspended in a small amount of Tris/EDTA and NaCt Pufferni. It got cloudy. This DNA solution was treated with ribonuclease (50 tt & Al) at 37°C, 6
After 0 minutes of reaction), phenol extraction was performed again, then twice the amount of ethanol was added, DNA was precipitated at -20°C, the precipitate was suspended in a small amount of Tris/EDTA-NaCt buffer, and 0.8% agarose dal was added. Before electrophoresis, the electrophoretic negative film was run on a densitometer, the ratio of chromosomal DNA to plasmid DNA was measured, and the molecular weight of chromosomal DNA was determined. OX 10'tal) y, pAJ228 to 5.
When the copy number was calculated using 0x10' Daltons, it was found that there were 16 copies per chromosome. The copy number of pAM330 determined by the same method was also 1.
There were 5 copies.

Example 2 Using the protocilast transformation method shown in Example 1,
Corynebacterium glutamicum (ATCC1306
0) was transformed using pAJ 228, and then selected for trimethoprim resistance to obtain a transformed strain.

In addition, agarose electrophoresis revealed that the transformed strain was pAJ
228e was confirmed.

Example 3 Miniaturization of 22 g of pAJ 5 μg of pAJ 228 DNA prepared in Example 1 was treated with restriction endonuclease Hae [l (5 units)) at 30°C.

It was treated for 30 minutes and then partially cut. Thereafter, in the same manner as in Example 1, the DNA strands were ligated using T4 phage-derived DNA IJ gauze. After reaction, 65°C.

After heat treatment for 10 minutes, the DNA was precipitated and collected by adding 2 volumes of ethanol, and the collected DNA was treated as a recipient strain of Citrimethoprim-susceptible Brevibacterium Lactofermentum AJ 12036'e21 in the same manner as in Example 1. ) After performing protoplast transformation, the cells were cultured in a protoplast regeneration medium containing 100 μg/m of trimedobub 11 (!). As a result, about 100 regenerated colonies appeared, so 910 of these strains were selected. A lysate was prepared by the method described in Example 1, and cilasmid DNA was detected by agarose gel electrophoresis.
- One strain of Shirasumi Y, which was clearly smaller than pAJ 228, was detected. pAJ'
It was named 225. The molecular weight of pAJ225 was determined to be 5.5 by the same agarose Dull electrophoresis analysis as shown in Example 1. It was calculated as I Kb. The second figure I wore is p.
A restriction enzyme cleavage map of AJ 225 is shown. pAJ 2
25 is a structure obtained by removing the approximately 2.5 Kb DNA fragment excised by the restriction enzyme HaeIl from pAJ 228, and by making it smaller, it is possible to incorporate a larger NA fragment compared to pAJ 228. It is a useful plasmid vector that can

Example 4 Introduction of Pstl cleavage site into pAJ 228 r99) pAJ 228 has restriction enzyme cleavage sites as shown in Table 1, but CtaT is a single cleavage site useful for gene insertion. + Hpa (+ Mstl +
Only SmaL) (bai, Xhol + Xmal) are known.

On the other hand, the restriction enzyme pgt
The cleavage site by l has been used as a useful gene insertion site (European Patent Application Publication No. 0093611).
. Therefore, we decided to create a new Pst cleavage site in pAJ228.

0.1 μg of pAJ 228 DNA was treated with restriction endonuclease HpaI at 37°C for 2 hours, and after complete cleavage, heat treatment was performed at 65°C for 10 minutes. Add Takara Shuzo's PstI linker-d (pG-C-T-G-
Add 100 pmole of C-A-G-C) and add ATP
and D from T4 phage in the presence of dithiothreitol.
DNA strand ligation reaction was carried out using NAU gauze at 10°C for 24 hours.

After heat treatment at 65° C. for 5 minutes, twice the volume of ethanol was added to the reaction solution, and the DNA after the completion of the ligation reaction was collected by precipitation. The obtained DNA was transformed into trimethoprim-sensitive Brevibacterium lactofermentum AJ in the same manner as in Example 1.
12036 by the protoplast transformation method, 100 μg of trimetogerim
As a result of culturing in protoplast regeneration medium containing l.
Since 0 regenerated colonies appeared, 10 strains were selected from these strains, a lysate was prepared by the method described in Example 1, and plasmid DNA was detected by agarose/R-luminescence electrophoresis. In both cases, cilasmids of approximately the same size as pAJ 228 were detected. These plasmids were digested with restriction enzymes Pstl or Hpa (37
After complete cleavage by treatment at ℃ for 2 hours, siblasmid DNA was detected by agarose/R-luminescence electrophoresis.
A plasmid that was cut by tl but not Hpal was detected. This plasmid was transformed into pAJ226
It was named. FIG. 3 shows a restriction enzyme cleavage map of pAJ226. 1) AJ 226 is pAJ 228) Tp
As a result of insertion of a Pstl'J linker into the aI cleavage site, the )Tpal cleavage site disappeared and a PstI cleavage site was formed.

Example 5 Construction of expression vector from pAJ 22B When genetic DNA is integrated into a plasmid vector, if a promoter that controls the expression of the gene is present on the integrated genetic DNA, gene expression is performed by the action of the promoter. However, if a promoter does not exist on the inserted gene DNA, it is necessary to express the gene using the promoter in the vector. Therefore, a vector in which a restriction enzyme cleavage site for inserting genetic DNA is placed downstream of the promoter region of the vector is extremely useful as an expression vector. Examples 1 and 3
．． and the Plasmid vector pAJ 2 shown in 4.
28, pAJ 225, and pAJ 226
The restriction enzyme cleavage site for gene DNA insertion was not necessarily located downstream of the promoter region, and its usefulness as an expression vector was low. Therefore, in this embodiment, p
A method for constructing an expression vector in which a restriction enzyme cleavage site is placed downstream of the promoter region present in AJ228 will be described.

To search for the promoter region in pAJ 228, we used as a probe an NA fragment that contains the promoter region and, when integrated downstream of the promoter region, expresses a detectable phenotype in coryneform bacterial cells. The method is convenient. Brevibacterium lactofermentum AJ 111 as a NA fragment like this
88 (FKRM-P 4190) (% Kosho 56-3
A 2.9 Kb DNA fragment encoding homoserine kinase ()IK) derived from 038) was used. This DNA fragment is Brevibacterium lactofermentum AJ 1
Recombinant cilasmid pAJ into which the HK gene present in 2079 (FERM-P 7237) was inserted 21.
1 can be excised by partial cleavage using the restriction enzyme PstI. The absence of a promoter region in this DNA fragment was confirmed by the fact that when it was inserted into the Pst cleavage site of pAJ226 shown in Example YiIJ4, no expression of the HK gene was observed regardless of the direction of insertion.

Next, using this DNA fragment containing the HK gene, pAJ
A method for constructing a 228 Yoshi expression vector will be described. An outline of the method is shown in FIG.

Bam was inserted at both ends of the 1 and 9 Md DNA fragment excised from pAJ 211 by partial cleavage with the restriction enzyme PstI.
HI r 5 alt.

A synthetic oligonucleotide linker as shown in Figure 4 with a PstI cleavage site was added to the T4 phage-derived DNA.
After ligation using A IJ gauze, restriction enzyme Bam
Cut with HI. The resulting DNA mixture was placed in agarose.
A DNA fragment of approximately 2.9 Kb was separated and extracted by gel electrophoresis, and the DNA fragment was recovered by ethanol precipitation.

The DNA fragment thus obtained is a 2.9 Kb I (K gene) with Pstl l 5ail IB at both ends.
It has a structure in which amHl cleavage sites are lined up (Figure 4).

On the other hand, pAJ 228 was partially digested with the restriction enzyme Mbol and then heat-treated at 65° C. for 10 minutes. The DNA fragment containing the HK gene obtained by the method described above was added to this reaction solution, and T4
DNA strand ligation reaction was performed using DNA IJ gauze.

After the reaction, the DNA was heat-treated at 65°C for 5 minutes and precipitated by adding 2 times the volume of ethanol. The DNA was collected in the same manner as in Example 1. After protoplast transformation was performed using Brevibacterium lactofermentum AJ 12078 as a recipient bacterium, approximately 200 regenerated colonies appeared as a result of culturing in a protocylast regeneration medium containing 100 μW of trimethoprim.

This was replicated to a minimal medium containing 200μ97 trimethoprim and not containing threonine, which is a substance required by the recipient bacteria, to obtain four strains that were resistant to trimethoprim and had lost their threonine requirement. A lysate was prepared from these strains by the method shown in Example 1, and plasmid DNA was detected by agarose gel electrophoresis.
Kb, 12.2Kb.

Cilasmids of 9.6 Kb and 6.5 Kb were detected. Among these, the plasmid with the smallest molecular weight of 6.5 Kb was named pAJ 224 HK.

Next, after completely cleaving pAJ 224 HK with the restriction enzyme Pstl, vector pA,
A 3 to 6 Kb fragment, which is thought to be a smaller version of 1228, was collected, and the DNA strands were ligated using T4 DNA IJ gauze in the same manner as in Example 1, and trimethoprim-susceptible Brevibacterium lactofermentum AJ12036 was transformed into a recipient strain. Protoplast transformation was performed as follows. Trimethoprim 1. OO
After culturing in a protoplast regeneration medium containing buds in μ, 910 strains were selected from among the approximately 100 regenerated colonies obtained, a lysate was prepared in the same manner as in Example 1, and siblasmid was analyzed by agarose gel electrophoresis. When DNA was detected, f
lAJ 224) 2.9K containing HK gene from TK
3.6 Kb of cilasmid DNA was detected, which is thought to be the result of removal of the DNA of b. This plasmid is pAJ
The strain carrying this pAJ224 was named Brevibacterium lactofermentum AJ 121.
96 (FERM-P gel left). pAJ
A restriction enzyme cleavage map of 224 is shown in FIG.

The fact that pA, T224 has the function as an expression vector is that, as mentioned above, when a DNA fragment of the HK gene that does not have a promoter region is inserted into the cleavage site of the restriction enzyme Pstl, the threonine Although this can be confirmed by the disappearance of the requirement, the following experiment was conducted to further investigate the strength of the expressed activity.

As an indicator of the intensity of expression activity, we decided to use the degree of resistance to loramphenicol, which is expressed in coryneform bacterial cells and can be easily measured. As shown in Figure 6, the structural gene region excluding the promoter region of the chloramphenicol resistance gene was transformed into cilasmid pCM4 (Gen
e, 20, 305-306 (1982)) using the restriction enzyme BamH1, mixed with the same DNA (pAJ 224, which had been completely cut with BamHl), and the DNA chains were ligated using T4 DNA IJ gauze. Heat denaturation and ethanol precipitation were performed in the same manner as in Example 1, and the resulting DNA was transformed into derotoplasts using chloramphenicol-sensitive Brevibacterium lactofermentum AJ 12036 as a recipient strain. After performing J/, the protoplasts were cultured in a protoplast regeneration medium containing 2 μg of chloramphenicol to obtain chloramphenicol-resistant regenerated colonies.Ten strains of thisella were selected from among the chloramphenicol-resistant strains. A lysate was prepared using the same method as in 1 C30), and plasmid DNA was detected by agarose gel electrophoresis.
A 4.4Kb DNA fragment containing the chloramphenicol resistance structural gene region derived from pCM4 was inserted into 224.
It was a plasmid. This plasmid was transformed into pAJ22
It was named 4 Cm'.

Next, Table 2 shows the results of comparing the degree of resistance to chloramphenicol between Brevibacterium lactofermentum AJ 12036 carrying pAJ 224 Cmr and other strains. As a representative strain of chloramphenicol resistant strain, plasmid pAJ 1844 (European Patent Application Publication No. 0093611) containing the entire region of chloramphenicol resistance gene was used as a control. As is clear from Table 2, the strain harboring pAJ 224 Cmr exhibits chloramphenicol resistance equivalent to that of the strain harboring pAJ 1844, confirming that pAJ224 is highly useful as an expression vector. Ta. In addition, as a result of analysis of the insertion direction of the structural gene region of the chloramphenicol resistance gene in pAJ 224 Cmr using the cleavage pattern using the restriction enzymes ' side) (pAJ 224 Cmr-A) and one inserted in the opposite direction (pAJ 224 Cm')
It has become clear that there are two types: -B).

This means that in pAJ 224, the restriction enzyme Ram
This shows that there are promoter regions on both sides of the cleavage site of 'HI+5all and Pstl, and that the gene inserted into this cleavage site can be expressed regardless of the direction of insertion.

Example 6 Construction of pAJ 223 After complete cleavage of pAJ 224 HK constructed as in Example 5 with restriction enzyme 5al, the DNA chains were ligated using T4 DNA ligase. Heat denaturation was performed in the same manner as in Example 1. , the DNA obtained by ethanol precipitation
was protoplast transformed into trimethoprim-sensitive Brevibacterium lactofermentum AJ 12036, and cultured in protoplast regeneration medium. The regenerated colonies that appeared were replicated onto a minimal medium containing trimethoprim, and trimethoprim-resistant strains were selected. Among these resistant strains, 20 strains were selected, a lysate was prepared in the same manner as in Example 1, and plasmid DNA was detected by agarose gel electrophoresis. A 3.6 Kb plasmid DNA was detected. This plasmid was transformed into pAJ2
Named 23. FIG. 7 shows a restriction enzyme cleavage map of pAJ223. pAJ 223 has a structure obtained by removing the restriction enzyme P+stI cleavage site from pAJ 224.

Table 2 Host bacteria Plasmid 0 10 20 4
0 80AJ 12036 None + -1--AJ
12036pAJ224 + ----A
J12036pAJ224Cm + +
+ + fAJ 12036 pAJ 1
844 + + + ten ±
+: Grows. ±: Slight growth. - Each strain that did not grow was inoculated onto an agar plate containing a complete medium containing chloramphenicol, and cultured at 30°C for 24 hours.

[Brief explanation of the drawing]

FIG. 1 is a restriction enzyme cleavage map of plus-9 mid pAJ 22B. FIG. 2 is a restriction enzyme cleavage map of plasmid pAJ225. FIG. 3 is a restriction enzyme cleavage map of plasmid pAJ226. FIG. 4 shows a method for constructing the expression cilasmid pAJ 224. FIG. 5 is a restriction enzyme cleavage map of plasmid pAJ224. FIG. 6 is a method for constructing cilasmid pAJ 224 Cmr. FIG. 7 is a restriction enzyme cleavage map of cilasmid pAJ 223.

Claims (2)

[Claims] (1) A plasmid that grows in coryneform bacterial cells and expresses trimethoprim resistance when introduced into the cells. (2) A coryneform bacterium that has a plasmid that proliferates within a coryneform bacterial cell and expresses trimethoprim resistance when introduced into the same cell.