JPS59120090A - Plasmid vector paj43 - Google Patents

Abstract

PURPOSE:To enable growth of strain of a bacterium capable of producing Coryneform glutamic acid, by using plasmid vector pAJ43 having 3.4 megadalton molecular weight and a specific scission map of restriction enzyme. CONSTITUTION:pAJ655 is formed from plasmid pBR325 and pAM330 and it is used as an intermediate to give pAJ43. This plasmid is propagated using a bacterium capable of producing Coryneform glutamic acid as a host, has 3.4 megadalton molecular weight, and has a scission map of restriction enzyme shown by the Fig. This plasmid vector has genetic information of chloramphenicol resistance, a large copy number, and is scissored at only position with restriction enzyme Xma III, Hind III BamH1, Xba, and HaeII. Since the scission position is not genetic information of chloramphenicol, so the plasmid vector is excellent as a vector.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the plasmid vector pAJ43. pAJ43 can be grown using coryneform glutamic acid producing bacteria as a host.

Free-form glutamate-producing bacteria produce a large amount of 1. - Known to produce glutamic acid. Moreover, its mutant strains are known to produce amino acids such as lysine, purine nucleotides such as ynone heptacid, etc., and are industrially useful microorganisms. On the other hand, recently attempts have been made to breed and improve industrial microorganisms using DNA engineering techniques, such as E. coli, but these microorganisms are not suitable for use as hosts. No vector has been developed for this purpose, which has hindered the breeding and improvement of coryneform-glutamic acid-producing bacteria through DNA recombination.

The present inventors have developed a plasmid vector pAJ43, which is a plasmid that grows in free-form glutamate-producing bacteria as a host, has a molecular weight of 3.4 megadaltons, and has a limiting oxygen map shown in Figure 1. succeeded in creating. This plasmid vector has genetic information for chloramphenicol resistance, has a large copy number, and contains the restriction enzyme χma m+Hi+1d [1+ Ba'mHl
＋

Plasmid vector pAJ43 was constructed by the method described in Example IP.

Coryneform bacteria are aerobic, Gram-positive bacilli, and are non-acid-fast.
74) This is described.

Examples of wild strains of Corynevorum glutamic acid-producing bacteria that can be used as host bacteria of the present invention include the following.

Brevibacterium divaricatum ATCC
I 4020 Brevibacterium sancalolyticum ATCC] 4066 Previbacterium immariophilum ATCC + 4068 Brevibacterium lactofermentum ATCC] ] 3869 Brevibacterium roseum ATCCI 3825 Brevibacterium flavum ATCC1
3826 Previbacterium thiogenitalis
ATCCl 9240 Corynebacterium acetoacidophilum ATCC 13870 Col) Subbacterium acetoglutamicum ATCCI 5806 Corynebacterium carnae ATCC] 59
91 Phrinebacterium glutamicum A
TCC] ]30323060 Corynebacterium Lilium ATCC
+ 5990 Corynebacterium melasecola ATC
CI 7965 Microbacterium ammoniaphyllum A'TCC15354 Coryneform glutamic acid producing bacteria include mutant strains that have lost glutamic acid productivity, or amino acids such as lysine and arginine, purine nucleosides such as inosine, and inosine 51-monophosphate. Also included are mutant strains that produce purine nucleotides such as or other products.

When using a coryneform-glutamate producing bacterium as described above as a host for the complex plasmid of the present invention or a complex plasmid into which a heterologous gene has been inserted, it is possible to introduce a mutation that reduces restriction enzyme activity into the host using a conventional method. Good results are obtained.

Using such coryneform-glutamic acid producing bacteria by introducing a composite plasmid into which a foreign gene has been inserted is more advantageous than expressing genetic information carried by a foreign gene.

Example 1 +11 pAJ65 was an intermediate for the construction of pAJ43
5 was constructed from plasmids pBR325 and pAM330.

Plasmid pBR325 is a vector plasmid with a molecular weight of 3.6 megadaltons that expresses resistance to ampicillin, chloramphenicol, and tetracycline in Escherichia (Boliver + F, Gene + 4.121
(1978)).

The plasmid pBR325 used in the experiment was (Bethesda)
Research Laboratory) (BRL).

Is plasmid pAM330 new from Brevibacterium lactofermentum ATCC13869? This is a plasmid with a molecular weight of 3.0 megadaltons that we isolated, and its restriction enzyme cleavage map is shown in Figure 1, and the cleavage sites determined by the restriction enzymes are shown in Table 1. Plasmid pAM
330 DNA was prepared by V straining as follows.

Table 1 Restrictions Enzyme Number of cutting sites Alu
l Arthrobacter luteus ≧
4Ava l Anabaena palabilis
≧2Bcl l Bacillus cultolicus IBamHI Bacillus amyloliquefaciens H, OBgl [Bacillus globigii 0BstE
11 Bacillus fist stearothermophilus ET
≧4EcoR,l Escherichia coli R10
〇Hae ■ Haemophilus aegyptius lHg1Al
Herpetosiphon giganteus ≧4H
ind l Haemophilus fistulae
≧4Hind III Haemophilus influenzae IHpa [Haemophilus bala influenzae ≧4Kpn I
Klebsiella pneumoniae 0
Pvu ll Proteus vulgaris
05ac l Streptomyces achromogenes 05al l Streptomyces albus 03au
3A Staphylococcus aureus
≧43ma l Serratia marsetuscens
l5st l Streptomyces Stanford 0Xba I
Xanthomonas Φ badri
1xho I Xanthomonas horicola
IXma l Xanthomonas φ malvacearum IXor [Xanthomonas oryzae 0 CMG medium (pH 7,
2) Brevibacterium lactofermentum ATCC] 3869 was cultured at a temperature of 30 C in mA until the late logarithmic growth phase, and the bacterial cells were collected. The obtained bacterial cells were lysed by a simple method of lysing lysozyme and 5DSr, and then centrifuged at 30,000Xr for 30 minutes to 64 m
A supernatant liquid of No. 1 was obtained. The plasmid DNA in the supernatant was precipitated by adding polyethylene glycol (final concentration 10%) to the supernatant, and then dissolved in IOme's TEN buffer.

After treating the DNA with ribonuclease,
Extract with phenol, then add twice the amount of ethanol, precipitate the DNA at -20'C, and transfer the precipitate to 1 ml of TEN.
Dissolved in buffer. 7 galose gel with this DNA solution?
Approximately 74 μ2 of pure plasmid DNA was separated from the gel.

Plasmid pBR3250, 2pWlc 1 unit of restriction enzyme BamHI (purchased from BRL) at 37C
The reaction was carried out for 600 minutes to fully degrade the DNA.

Plasmid pAM, 330 (1,2μ2) was reacted with 0.2 unit of restriction enzyme MbOI at 37tZ'' for 15 minutes to partially degrade the DNA.

The obtained DNA fragments were mixed and heat treated at 65C for 10 minutes to inactivate the restriction enzymes, and then incubated at 0.01 unit for 2 hours in the presence of ATP and dithiothreitol.
4 DNA ligase was allowed to act. T4 DNA ligase was inactivated by treatment with 65CS for 10 minutes, and after adding twice the amount of ethanol, the DNA was recovered by centrifugation at 15,000 r for 15 minutes.

The composite plasmid thus obtained was used as a transfer main.

Enoshierihia + Furi C-6,00 (thr.

leu'-+ thiamine-+ r + m-
) (Meselson+M, and Yuan+
R, Nature+ 217 + 1110(+
968)) 20 ml'(r) CM G medium) n3
The cells were cultured at 0 tl until the mid-logarithmic growth phase, and the bacterial cells were collected.

Kushner et al.
ering”+p, 17(+978)+
Elsevier / North Holland
Biomedical Press) method? Therefore, using the DNA obtained in (tii), C-600
was transformed.

Type 'Jl&i' transgenic strains were selected by culturing in CMG medium containing 20 μV of chloramphenicol at 37C for 24 hours. AJI] 882 (FERM
-BP136) was selected and used in the next experiment.

The composite plasmid pAJ655 was isolated from the lysate of Pkoyoya AJ11882 by the following method.

AJ ] +188 was cultured in CMG medium, and then the simple method (T
anaka eLal, + J, Bacterio
The bacterium was lysed using agaro-7, +I2] +354 (+975)), and the lysate was poured into agaro-7, gel, and electrophoresed (Harp et al, +
Biochemistry 12.

3055 (1973)). From the comparison with molecular weight markers, the molecular weight of the plasmid was calculated to be 6.6 Md. A restriction enzyme cleavage map of the plasmid is shown in Figure 2e. The plasmid was determined to consist of fragments of pBR325 and pAM330 using the method of K.J.Danna (
Methods in Enzymology65 +
449, Academic Press (
1980)) Confirmed by V.

pA'J655 was used as a triple trophic (lysine, methionine, threonine) mutant strain Brevibacterium lactofermentum ATCC13s 69 (
ATCC'39134) It was introduced as follows.

Brevibacterium lactofermentum ivy 64 5
ml of CMG medium until the early logarithmic growth phase, and after adding 0.6 units/me of Venicillin G, culture with shaking for 5 hours, and collect the bacterial cells by centrifugation. in SMVP medium (Dirco) consisting of 0.5M sucrose, 20mM maleic acid, 20mM magnesium chloride, and 3.5% Bennace broth (Dirco).
pH 6,5)) was washed with 0.5 ml. Then lOm
The cells were suspended in SMMP medium P containing g/ml of lysozyme and transformed into protoplasts in 301Z' for 20 hours. 6
0000? After centrifugation for 10 min, protoplasts were
It was washed with MMP and resuspended in 0.5 ml of SMI tube.

The obtained protoplasts were transformed into pAJ655DNA2μ2
and polyethylene glycol to a final concentration of 30
%, the DNA was added to protoplast p
The sample was left at room temperature for 2 minutes to allow it to be absorbed.

After washing the cells with one scrap of SMMP medium, resuspending the DNA-loaded protoplasts in SMMP medium I was incubated at 30°C for 3 hours to develop drug resistance.
Cultured for hours. This culture solution was applied on top of the protoplus regeneration medium at pH 7.0. Protoplus 1-Regeneration medium contains 129 Tris(hydroquinomethyl)amitumecun, 5y KCI O, 10 curcose per lt distilled water.
≦7, MgC1□・6H208, +5J, CaCl2−
2H, 02, 22, peptone 42, powdered yeast extract 4?
, casamino acid (Difco) ] f XK2HPO
40,27, Thorium phosphate 135? , agar 18
7 and chloramphenicol 5μf/dwo.

Select 5 strains from the 3. cultured colonies at 30C for 3 to 10 days and culture them in CMG medium containing 20μ2e of chloramphenicol to confirm that they are drug (chloramphenicol) resistant strains. confirmed.

In order to confirm the presence of the Saraguko plasmid, we cultured the chloramphenicol-resistant strain grown here, poured the lysate into acarose gelatin IJIP, and transferred it to P A J
A plasmid with the same molecular weight and restriction enzyme cleavage map as 655 was found.

(From 21p A J 655, pAJ4
Created 3. Brevibacterium lactofermentum flatus 64 carrying pAJ655 was grown on CMG agar medium (Pep) containing 100 μq/me of chloramphenicol.
・N1 o9/l, yeast extract 1 oy/l, t1 crucose 59/l, NaCl 5? /l, agar 20y
Although it was impossible to grow on CMG medium (containing 100 μy/ml of chloramphenicol at pH 7.21), it was cultured overnight in CMG liquid medium P containing 100 μy/ml of chloramphenicol. Apply an appropriate amount of 1 CMG medium containing the same concentration of phthalamphenicol and culture for ~2 days at 30C.
One strain showing resistance to rttJ was obtained. When the chloramphenicol resistance of the tree stock was examined using CMG medium, 2001
It showed resistance up to 1f/me.

From the chloramphenicol-resistant strain obtained as a result of the above, pAJ43 was prepared by straining the DNA as follows.

First, the tree stock was inoculated into IA's CIVi G liquid medium containing 10 μf of chloramphenicol, cultured at 30'C until the late logarithmic growth phase, and bacteria were collected. Conventional method (30.00 psr after bacteriolysis with Koyori Renozyme and 5 psr)
Ultracentrifuge at 0XP for 30 minutes to remove 2L kidneys for +4J.

Polyethylene glycol (@ final concentration IO%) was added to this to precipitate the DNA, and after concentrating it, the precipitate was transferred to a Tris-EDTA-NaCl buffer (pH 8,0).
10m1f was dissolved. Treat DNA with ribonuclease (ribonuclease 1 37C1 with 50 μ7/4
After reacting for 30 minutes, extract with phenol, add twice the volume of ethanol under a strainer, precipitate the DNA at -20'C, and collect 1 ml of the precipitate! The solution was dissolved in Tris-EDTA-NaCl Puffer V. This DNA solution was subjected to agarose gel electrophoresis (voltage: 5V per cm of gel, 15 hours) to obtain a final 150μ of pure pAJ43 plasmid DNA.
A fraction was collected.

[3] Properties of pAJ43 DNA The molecular weight of pAJ43 was determined by agarose gel electrophoresis.
It was scary.

Agarose gel electrophoresis was performed at Nyani 7' (P, A.

5harp) et al.'s method (Biochemistry
] 2 +3055 (, 1973)), 0.8
Using Tigel, electrophoresis was performed at constant voltage for 15 hours at 5 μm per meter of gel length. The molecular weight is determined by using the restriction enzyme HindllIO, which cuts pAJ43 at one site, and adding 5 units to pAJ.
430.5 pf IC37C, after reacting for 1 hour, cutting and linearizing, molecular weight Yakker of known molecular weight,
Comparison of mobility with Hind fragment of λ phage (purchased from BRL et al.), 3.4 M
It was calculated as d.

b pAJ L) Transformation of Brevibacterium lactofermentum A64 by NAV
ATCC3'9134) 5 me (7) CM G
Cultured in medium until early logarithmic growth phase, 0.6 units/
ml! After adding Beninrin G, add 1 more
, cultured with shaking for 5 hours, centrifuged at 1 tube to collect cells, and mixed with 0.5 M nuculose, 20 mM maleic acid,
SMMP medium (pH 6,
5) Washed with 0.5 ml. Then 10 m9 /
Suspended in SMMP medium containing Norizozyme for 30r
: Protoplast formation was attempted for 20 hours. 6000Xy
After centrifugation for 10 minutes at
and resuspended in 0.5 ml of SMMP. The protoplast obtained in this manner was converted into pAJ43 DNA/
Mix polyethylene/glycol with μ2 to a final concentration of 3.
After adding V to give a concentration of 0%, the mixture was left at room temperature for 2 minutes in order to incorporate the DNA into protoplasts. After washing the protoplasts with 1 ml of SMMP medium, the protoplasts were incubated again for 1 ml.
To express drug resistance by suspending 1 of SMMPf,
Cultured at 30C for 2 hours. This culture solution was spread on a protoplast regeneration medium at pH 7.0.

The protoplast regeneration medium contains 127 tris(hydroquinomethyl)amitsumecun and 0.0 KCI per ton of distilled water.
5f, gluco-7,1oy, MgC12・6H,,08
, 1f, CaCl2・2H2012, 2f, peptone 4
7. Yeast extract 42, [casamino acids] (Difco)
] ? , K, 2HP0. 0.2 f, sodium co/'phosphate 135? , agar 82 and chloramphenicol 3μ2e. After culturing at 30r for 10 days, 5 colonies were selected from among the colonies that appeared, and these were cultured in 10.0μf of CMG medium containing chloramphenicol.
It was confirmed that this strain exhibited higher chloramphenicol resistance than the J655 strain. In order to confirm the presence of this plasmid, we lysed the chloramphenicol-resistant strain grown here and subjected the lysate to 7-galose gel electrophoresis, and found that a plasmid with the same molecular weight as pAJ43 was present. .

Creation of restriction enzyme cleavage map of pAJ43 DNA Use commercially available products from BRL as restriction enzymes.
Cleavage of J't' and DN'A was carried out using at least a 3-fold excess of the enzyme under the specified conditions for each enzyme. When cutting plasmid DNA with one or more restriction enzymes to create a restriction enzyme cleavage map, the first restriction enzyme cleavage fragment was separated from a separation agarose gel using the method of Kunaka et al. , Weis
blum, J., Bacteriol, + start-up;
354(]ri75))? After isolation from this, it was concentrated using ethanol precipitate aRr and cleaved with a second restriction enzyme. The cut fragments were subjected to agarose gel electrophoresis according to the method of Example 3.
Then, the molecular weight was calculated and a restriction enzyme cleavage map was created. From this result, pAJ43 is derived from pAJ655.
pBR325 generated by deletion in vivo
Approximately IMd containing the chloramphenicol resistance gene region of
Approximately 2.0% of pAM330 contains this essential region for maintenance of replication.
It turned out to be a small plasmid consisting of a fragment of 4 Md.

Nilmentum Bian 64 (AJ 1 (%7, FER
M-p b'1jr7) Wokuro 7mu74=+-
It was inoculated into 5 ml of CMZG liquid medium containing 10 μy/ral of Opf/all and incubated overnight at 30C.
The CMZG liquid medium was inoculated again. Culture at 30C until early logarithmic growth phase, add ampicillin at 1000μy/ragr, further culture for 2 hours, collect cells by centrifugation. Tris-EDTA-NaCl containing 10my/me lysozyme. Buffer+
, sfig was suspended and incubated at 37C for 2 hours, 5DS (final concentration 4T) was added and the cells were lysed at 65R for 20 minutes. After confirming that the protoplasts were completely lysed, they were extracted with phenol, followed by the addition of twice the amount of ethanol for 20 minutes to precipitate pNA, and the precipitate was suspended in a small amount of Tris/EDTA-NaCl buffer.

This DNA solution was treated with ribonuclease (ribonuclease reaction for 150 μsec at 37C for 60 minutes), extracted with phenol again, added with twice the amount of ethanol, precipitated the DNA at -20°C, and added the precipitate with a small amount of Tris.・
After suspending in EDTA-NaCl buffer, perform electrophoresis on a 0.8% 7-gallose gel, place the electrophoresis negative film on a densitometer, and remove the chromosomal DNA and plasmid D.
Measure the proportion of NA and determine the molecular weight of chromosomal DNA. OX
] 0” Dalton, pAJ43 3.jX +06
When Dalton calculated the number of copies,
It was found that there are 24 hupies per chromosome. The copy number of pAJ 655 determined by the same method was 11 copies, and it was confirmed that the copy number doubled due to miniaturization.

[Brief explanation of drawings]

FIG. 1 is a restriction enzyme cleavage map of plasmid pAM330. FIG. 2 is a restriction enzyme cleavage map of plasmid pAJ655. FIG. 3 is a restriction enzyme cleavage map of plasmid pAJ43. Patent applicant: Ajinomoto Co., Inc. Figure 1 Figure 2

Claims (1)

[Claims] Plasmid vector pAJ with a molecule ff of 13.4 megadaltons and a restriction enzyme cleavage map shown in Figure 1.
43°