JPH0446560B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a plasmid and a bacterium having the same, and more particularly to a plasmid vector that can proliferate in a coryneform bacterium cell and a coryneform bacterium having the same. (Prior Art) Coryneform bacteria include those such as Brevibacterium flavum that produce large amounts of amino acids such as glutamic acid, and are 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 (European patent application publication). 0 093 611). All of these plasmid vectors attempt to insert a marker gene for plasmid selection into a Cryptic plasmid that can proliferate in a coryneform bacterial vesicle, and therefore, the marker gene is derived from the genus Escherichia or Bacillus. This gene was inserted into a coryneform bacterium plasmid. Therefore, for coryneform bacteria, heterologous DNA has been integrated, and the use of such plasmid vectors therefore belongs to a recombination experiment with heterologous DNA. (Problems to be Solved by the Invention) Therefore, an object of the present invention is to provide 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 Problems) In order to solve the above problems, the present inventor constructed a plasmid that proliferates in coryneform bacterial cells and expresses trimethoprim resistance when introduced into the cells. . The gene expressing trimethoprim resistance here is derived from coryneform bacteria. Trimethoprim is a structural analog of dihydrofolic acid;
It is an inhibitor of dihydrofolate reductase. A gene that expresses trimethoprim resistance when introduced into coryneform bacterial cells can be obtained as follows. First, select a strain that is resistant to trimethoprim from coryneform bacteria. Wild strains of coryneform bacteria are 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 were created by irradiating the parent strain, which is a coryneform bacterium, with X-rays, γ-rays, ultraviolet light, etc., or by exposing it to mutagenic agents such as N-methyl-N'-nitro-N-nitrosoguanidine. After that, strains that have acquired trimethoprim resistance can be selected. Strains that have acquired trimethoprim resistance are, for example, treated with minimal medium (glucose 2 g/dl, ammonium sulfate 1 g/dl, urea 0.25
g/dl, KH ₂ PO ₄ 0.1g/dl, MgSO ₄・7H ₂ O,
0.04 g/dl, thiamine hydrochloride 200 μg/dl, piotin 50 μg/dl, agar 1.5 g/dl, adjusted to pH 7.0), etc., and add 100 μg/dl to this.
It can be obtained by adding trimethoprim at a concentration of more than 200 μg/ml, preferably 200 μg/ml, and isolating strains that grow on this medium. The method for isolating the gene expressing trimethoprim resistance is to first extract the chromosomal gene from a strain of coryneform bacteria that has the gene expressing trimethoprim resistance (for example, H. Saito and K. Miura,
The method of Biochem Biophys Acta 72 619 (1963) can be used), which is then cut 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 genes cut with restriction enzymes were then connected to a plasmid vector capable of propagating in coryneform bacteria, and the resulting recombinant DNA was used to transform coryneform bacteria, resulting in trimethoprim resistance. The ivy strain was isolated, and from it the gene expressing trimethoprim resistance could be isolated. 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. Coryneform bacteria are aerobic, gram-positive rod strains, non-acid-fast and classified as
It is described in Determinative Bacteriology, 8th edition, page 599 (1974). Among these, those that produce large amounts of L-glutamic acid are particularly known as shown below, and all of these are considered to belong to the same genus.

[Table] In addition to those having glutamic acid productivity as described above, coryneform bacteria also include mutant strains that have lost glutamic acid productivity and other mutant strains. In order to create a plasmid capable of propagating in coryneform bacterial cells, the gene expressing trimethoprim resistance is connected to the replication initiation region DNA of the plasmid capable of propagating in coryneform bacterial cells. As the replication initiation region 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. In addition, these wild-type plasmids or DNA fragments containing their replication initiation regions are derived from coryneform bacteria.
Connected DNAs can also be used as replication initiation region DNAs. Wild-type plasmids that can proliferate in coryneform bacterial cells include pAM330 and pAM286 described in JP-A-58-67699, pHM1519 described in JP-A-58-77895, and pHM1519 described in JP-A-58-134500. pCG1 described in JP-A-58-35197, pCG2 described in JP-A-57-183799
pCG4, pCG11, etc. are known. The plasmid containing the replication initiation region DNA is cut with the same restriction enzyme used to cut the chromosomal gene in order to connect with the DNA expressing trimethoprim resistance. The chromosomal DNA cut fragment and the cut plasmid DNA can be used as is, or if necessary, oligonucleotides having complementary base sequences can be connected to both ends of each, and then the plasmid DNA and the chromosomal DNA can be combined.
It is subjected to a ligation reaction with the fragment. In order to introduce the thus obtained recombinant DNA of chromosomal DNA and plasmid DNA into a recipient bacterium belonging to coryneform bacteria, the method described for Escherichia coli K-12 (Mandel,
M. and Higa, A., J. Mol. Biol., 53 159 (1970))
Treatment of recipient bacterial cells with calcium chloride to increase DNA permeability, and as reported for Bacillus subtilis (Duncan, CH.
Wilson, GA and Young, FE, Gene, 1 ,
153 (1977)) by introducing cells into a proliferation stage (so-called compatible cells) in which they become capable of taking up DNA. Or, Bacillus
As is known for S. subtilis, actinomycetes and yeasts (Chang, S. and Choen, SN,
Molec.Gen.Genet., 168 , 111 (1979); Bibb, M.
J., Ward, JMand Hopwood, OA, Nature,
274, 398 (1978)); Hinnen, A., Hicks, J.B.
and Fink, GR, Proc. Natl. Acad. Sci. USA, 75
1929 (1978)), DNA recipient bacteria, plasmid
It is also possible to introduce the plasmid into a DNA recipient bacterium in the form of protoplasts or spheroplasts, which readily take up DNA. In the protoplast method, a sufficiently high frequency can be obtained using the method used for Bacillus subtilis mentioned above, and polyethylene glycol is Alternatively, a method of incorporating DNA in the presence of polyvinyl alcohol and divalent metal ions can also be used. By adding carboxymethylcellulose, dextran, Ficoll, Pluronic F68 (Selva), etc. instead of polyethylene glycol or polyvinyl alcohol.
Similar results can be obtained by methods that promote DNA uptake. 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 incorporated. To isolate recombinant DNA, for example, a transformed strain is lysed by lysozyme/SDS treatment, treated with phenol, and then 2 volumes of ethanol are added to collect the DNA by precipitation. (Effect) The thus obtained plasmid, which grows in coryneform bacterial cells and expresses trimethoprim resistance when introduced into the same cells, does not contain heterologous DNA; are particularly suitable for insertion and expression by coryneform bacteria. Examples of genes to be inserted and expressed include genes involved in amino acid biosynthesis. <Example 1> Brevibacterium lactofamentum
AJ12036 was cultured on a yeast broth agar slant, and the grown cells were collected and suspended in 1/50M phosphate buffer (PH7.0) (10 ⁹ to 10 ⁹ cells [containing ml of cells). N-methyl-N'-nitro-N-nitrosoguanidine (abbreviated as NG) was added to the mixture (NG concentration was 200 μg/ml), and the mixture was kept at room temperature for 30 minutes. After thoroughly washing the NG-treated bacterial cells with the same phosphate buffer, a minimal medium containing 200 μg/ml trimethoprim (glucose 2 g/dl, ammonium sulfate 1 g/dl) was used.
dl, urea 0.25g/dl, KH ₂ PO ₄ 0.1g/dl,
MgSO ₄・7H ₂ O0.04g/dl, thiamine hydrochloride
200μg/, piotin 50μg/, agar 1.5g/dl
(containing 100% phthalate and adjusted to pH 7.0). A trimethoprim-resistant strain growing on this medium was isolated and named AJ12146 (FERM-P7672). <Example 2> (1) Brevibacterium lactofamentum
AJ12036 (FERM-P7559, trimethoprim-resistant mutant derived from FERM-BP734)
AJ12146 (FERM P-7673, FERM BP-785)
1 of CMG medium (peptone 1g/dl, yeast extract 1g/dl, glucose 0.5g/dl, and
Contains 0.5g/dl of NaCl and adjusted to PH7.2)
Inoculate the cells and culture with shaking at 30℃ for about 3 hours.
Bacterial cells in the logarithmic growth phase were collected. The cells were lysed with lysozyme/SDS, and the chromophore DNA was extracted and purified using the usual phenol treatment method.
Finally, 3.0 mg of DNA was obtained. (2) pAM330 was used as a vector. pAM330
was prepared as follows. First, Brevibacterium lactofamentum harboring pAM33 as a plasmid.
Inoculate ATCC13869 into 100 ml of CMG medium and
After culturing at °C until the late logarithmic growth phase, the cells were lysed by lysozyme SDS treatment, and the supernatant was obtained by ultracentrifugation at 30,000 x 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 Tris-HCl, 20mM NaCl, 1mM
After dissolving in EDTA (PH8.0), it was subjected to agarose gel electrophoresis for separation, and then excised to obtain about 15 μg of pAM330 plasmid DNA. (3) 20 μg of the chromosomal DNA obtained in (1) and 10 μg of the plasmid DNA obtained in (2) were each treated with restriction endonuclease MboI at 37° C. for 30 minutes to partially cleave them. After heat treatment at 65°C for 10 minutes, both reaction solutions were mixed and ligated with _T4 phage-derived DNA ligase in the presence of ATP and dithiothreitol.
DNA strand ligation reaction was performed at 10°C for 24 hours. 65
After heat treatment at ℃ for 5 minutes, 2 volumes of ethanol were added to the reaction solution to precipitate and collect the DNA after the completion of the ligation reaction. (4) Brevibacterium lactofamentum AJ12036, which is sensitive to trimethoprim, was used as the recipient bacterium. The protoplast transformation method was used as the transformation method. First, the strain was cultured in 5 ml of CSG liquid medium until the early logarithmic phase, and penicillin G was added at 0.6 units/ml.
After the addition, the cells were cultured for another 1.5 hours with shaking, and the cells were collected by centrifugation.
Washed with 0.5 ml of SMMP medium (PH6.5). then
The cells were suspended in SMMP medium containing 10 mg/ml lysozyme and incubated at 30°C for 20 hours to form protoplasts.
After centrifugation at 6000×g for 10 minutes, the protoplasts were washed with SMMP and resuspended in 0.5 ml of SMMP. Protoplasts obtained in this way and (3)
10 μg of the DNA prepared in was mixed in the presence of 5mM EDTA, and polyethylene glycol was added to the final concentration.
After adding the DNA to a concentration of 30%, it was left at room temperature for 2 minutes to incorporate the DNA into protoplasts. The protoplasts were washed with 1 ml of SMMP medium, resuspended in 1 ml of SMMP medium, and cultured at 30°C for 2 hours for expression. This culture solution
Plated onto protoplast regeneration medium at pH 7.0.
Protoplast regeneration medium contains 12 g of tris(hydroxymethyl)aminomethane per 1 ounce of distilled water.
KCl0.5g, _glucose 10g, _{MgCl2.6H2O8.1}
g, _CaCl2・_2H2O2.2g , peptone 4g, powdered yeast extract 4g, casamino acid (Difco) 1
g, _{K2HPO4 0.2g} , sodium succinate 135
g, agar 8g and trimethoprim (Sigma)
Contains 25 μg/ml. After culturing for one week at 30°C, about 100 colonies appeared, so we added a minimal medium containing trimethoprim (2% glucose, 1% ammonium sulfate, 0.25% urea, 0.1% potassium dihydrogen phosphate, 0.04% magnesium sulfate heptahydrate, 2ppm
Iron ion, 2ppm manganese ion, 200μg/
Thiamine hydrochloride, 50μg/biotin, PH
0.7, agar 1.8%, trimethoprim 50μg/ml)
One trimethoprim-resistant strain was obtained. (5) When a lysate was prepared from these strains by the method described in (2) and plasmid DNA was detected by agarose gel electrophoresis, a plasmid clearly larger than the vector pAM330 was detected. This stock is AJ12147 (FERM P
-7673, FERM BP-786). (6) In order to confirm the presence of the trimethoprim resistance gene on the plasmid (pAJ288) possessed by AJ12147, we used this plasmid DNA to test Brevibacterium lactofamentum.
AJ12036 was transformed again. When 10 of the resulting trimethoprim-resistant colonies were picked and plasmid DNA was detected by agarose electrophoresis, a plasmid of the same size as pAJ228 was present in all of them. It was revealed that a gene expressing trimethoprim resistance was present on the above recombinant plasmid. Brevibacterium lactofamentum
AJ12146 is produced by mutating Brevibacterium lactofamentum AJ12036 by contacting it with 1000 μg/ml N-methyl-N'-nitro-N-nitrosoguanidine at 0°C for 20 minutes, and then mutating Brevibacterium lactofamentum AJ12036 in minimal medium (glucose 2 g/dl). , ammonium sulfate 1g/dl, urea 0.25g/dl, KH ₂ PO ₄ 0.1g/dl, MgSO ₄ .
7H ₂ O0.04g/dl, saaamine hydrochloride 200μg/
, biotin 50μg/, 2ppm iron ion,
Contains 2ppm manganese ion, agar 1.5g/dl,
(adjusted to PH7.0) and trimethoprim 100μ
This strain was isolated as a strain that can grow in a medium containing 1.5 g/ml of the microorganism. AJ12036 can be easily obtained from AJ12147 by removing the complex plasmid in the host cell without damaging the host cell. That is, the plasmid can be naturally lost from the host, or it can be removed by a "removal" operation (Bact.
Rev., 36 , p361-405 (1975)). Removal operation 1
An example is as follows: a small amount of the bacterial strain is inoculated to approximately 10 ⁴ cells per ml into a medium containing acridine orange at a concentration (2-50 μg/ml) that completely inhibits host growth. After completely inhibiting the growth of the host bacterium, it is cultured overnight at 27-37°C (J. Bacteriol., 88 , 261 (1964)). Spread the culture solution on an agar medium and culture at 27-37°C overnight. Among the colonies that appeared on the medium, the strain showing sensitivity to trimethoprim (50 μg/ml) was a strain from which the plasmid had been removed, ie, AJ12036. (7) Properties of pAJ228DNA (a) The molecular weight of pAJ228 was determined by agarose gel electrophoresis. Agarose gel electrophoresis was performed using Sharp (PA)
Sharp et al.'s method (Biochemistry12, 3055
(1973)) using 0.8% gel, the gel length was
Electrophoresis was performed at a constant voltage of 5 V per cm for 15 hours. The molecular weight is Cla, a restriction enzyme that cuts pAJ228 at one site.
Calculated by reacting 0.5 units with 0.5 μg of pAJ228 at 37°C for 1 hour, cutting it, and directly cutting it into a shape. Calculated by comparing the mobility with a molecular weight marker of known molecular weight, the Hind fragment of λ phage (BRL). It was calculated to be 5.1Md. In addition, Table 1 shows the cleavage sites using various commercially available restriction enzymes using the same method. Table 1 Restriction enzymes Number of cutting sites Ava 5 BamH 0 Bcl 4 Bgl 0 BatE 3 Cla 1 EcoR 0 Hae 4 Hind 2 Hpa 1 Kpa 0 Mln 2 Mst 1 Pst 0 Pvu 0 Sal 0 Sca 2 Sma 1 Sph 1 Sat 0 Sat ０ Xba １ Xma １ The experiments were carried out under the conditions specified for each enzyme. When plasmid DNA is cut with one or more restriction enzymes to create a restriction map, the first restriction enzyme cut fragment is separated from a separation agarose gel using the method of Tanaka et al.
B. Weisblum. J. Bacteriol., 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. (c) Measurement of copy number of pAJ228 Brevibacterium harboring pAJ228.
Lactofermentum AJ12147 was inoculated into 5 ml of CMG liquid medium containing 50 μg/ml of trimethoprim, and after culturing at 30° C. overnight, 0.1 ml thereof was inoculated again into 5 ml of CMG liquid medium containing 50 μg/ml of trimethoprim. Culture at 30°C until early logarithmic growth phase, add ampicillin to 1000 μg/ml, and culture for additional 2 hours.
Collect bacterial cells by centrifugation, suspend in 1.5 ml of Tris/EDTA/NaCl buffer containing 10 mg/ml of lysozyme, incubate at 37°C for 2 hours, add SDS (final concentration 4%), and incubate at 65°C.
Lysed for 20 minutes. After confirming that the protoplasts were completely lysed, they were extracted with phenol, and then 2 volumes of ethanol were added at -20°C.
DNA was precipitated with a small amount of Tris.
It was suspended in EDTA/NaCl buffer. this
After treating the DNA solution with ribonuclease (reacting with 50 μg/ml of ribonuclease at 37°C for 60 minutes), phenol extraction is performed again, and then 2 volumes of ethanol are added to precipitate the DNA at -20°C. EDTA・NaCl
After suspending in buffer, it was subjected to 0.8% agarose gel electrophoresis, and the ^{electrophoresis} negative film was run on a densitometer to measure the ratio of chromosomal DNA to plasmid DNA. When the copy number was calculated using ⁶ Daltons, it was found that there were 16 copies per chromosome. The copy number of pAM330 determined by the same method was also 15 copies. Example 3 Corynebacterium glutamicum (ATCC 13060) was transformed with pAJ228 using the protoplast transformation method shown in Example 2, and then selected for trimethoprim resistance to obtain a transformed strain. Furthermore, it was confirmed by agarose electrophoresis that the transformed strain had pAJ228.

[Brief explanation of the drawing]

Figure 1 is a restriction enzyme cleavage map of plasmid pAJ228.

Claims (1)

[Claims] 1. A restriction enzyme cleavage pattern having the following restriction enzyme cleavage pattern and about
A DNA fragment derived from coryneform bacteria that is 3.2 Kb long and encodes a gene that confers trimethoprim resistance to coryneform bacteria. (For MboI, cleavage sites other than both ends may exist.) 2 Among plasmids derived from coryneform bacteria and capable of proliferating within coryneform bacterial cells, a plasmid of approximately 3.2 Kb with the following restriction enzyme cleavage pattern of coryneform bacteria that encodes a gene that confers trimethoprim resistance to coryneform bacteria.
Contains DNA fragments (For MboI, cleavage sites other than both ends may exist.) 3 Coryneform bacteria that contain a plasmid derived from coryneform bacteria and capable of proliferating within coryneform bacterial cells (however, the above plasmids are It has a restriction enzyme cleavage pattern like approx.
It is 3.2 Kb long and contains a DNA fragment derived from a coryneform bacterium that encodes a gene that confers trimethoprim resistance on coryneform bacteria) (For MboI, cleavage sites other than both ends may exist)