JPH0616713B2 - Complex plasmid - Google Patents

Description

The present invention relates to a composite plasmid, particularly a composite plasmid capable of replicating in coryneform glutamic acid-producing bacteria.

Coryneform / glutamic acid-producing bacteria are known to produce large amounts of L-glutamic acid, and mutants thereof are known to produce amino acids such as lysine and purine nucleotides of inosinic acid, which are industrially available. Is a useful microorganism. On the other hand, recently, breeding and improvement of industrial microorganisms by DNA recombination technology have been attempted by Escherichia coli and the like. For coryneform glutamic acid-producing bacteria, vectors suitable for using these microorganisms as hosts have not been developed. However, it has been an obstacle to breeding and improving coryneform / glutamic acid-producing bacteria by DNA recombination.

Several plasmids capable of replicating in the coryneform / glutamic acid-producing bacterium were found in the coryneform / glutamic acid-producing bacterium, but among these properties, the genetic traits that can be easily distinguished as a marker are clearly shown. It has not been. Although there are cases where multicopy plasmids isolated from coryneform / glutamic acid-producing bacteria are used as vectors, since these do not have easily identifiable gene markers, breeding industrial microorganisms from coryneform / glutamic acid-producing bacteria. Applying recombinant DNA technology to do so has not been easy.

Therefore, there is still a need to develop new plasmids and vectors for breeding and improving industrial microorganisms derived from coryneform glutamic acid-producing bacteria.

In such a background, the object of the present invention is to use coryneform glutamic acid-producing bacteria to construct a novel complex plasmid useful for improving breeding of industrial microorganisms, and also from glutamic acid-producing coryneform bacteria. The isolated natural type plasmid is modified so as to be suitable for improving breeding of microorganisms for industrial production from coryneform glutamic acid-producing bacteria.

These objectives have been achieved as described below.

Plasmids pAM330, pAM286, and pHM151 capable of growing in coryneform glutamic acid-producing bacteria
Proliferation in coryneform / glutamic acid-producing bacteria consisting of one selected from 9 (A) and a gene region (B) containing the drug resistance gene portion of a plasmid (b) capable of growing in Escherichia coli or Bacillus subtilis cells Possible composite plasmids.

When the drive unit site of the plasmid (b) is further included on the gene fragment, this composite plasmid can grow in Escherichia coli or Bacillus subtilis, and the composite plasmid can be obtained using Escherichia coli or Bacillus subtilis. It is possible to single out or widen.

The accompanying drawings are as follows:

Fig. 1: Restriction enzyme digestion map of pAM330 Fig. 2: Restriction enzyme digestion map of pAM286 Fig. 3: Restriction enzyme digestion map of pHM1519 Fig. 4: Restriction enzyme digestion map of complex plasmid pAJ655 Fig. 5: Complex plasmid pAJ611 Restriction enzyme digestion map Fig. 6: Restriction enzyme digestion map of composite plasmid pAJ1844 Fig. 7: Restriction enzyme digestion map of composite plasmid pAJ440 Fig. 8: Restriction enzyme digestion map of composite plasmid pAJ3148 "Multicopy plasmid" is the usual definition As described above, it means a plasmid that exists in multiple copies in one cell.

"Drive unit" refers to the minimum gene region required for gene replication in a host cell.

"Resistance to a drug" refers to resistance to a drug such as an antibiotic that inhibits the growth of host cells, according to the usual definition.

Such antibiotics include penicillin, kanamycin, chloramphenicol erythromycin, actinomycin and the like.

Coryneform bacteria is an aerobic, Gram-positive bacillus, non-acidic, and is the Verge's Manual of Determinative Bacteriology, 8th Edition, page 599 (19).
74).

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

Brevibacterium divaricatum ATCC 14020 Brevibacterium saccharolyticum ATCC 14066 Brevibacterium inmario film ATCC 14068 Brevibacterium lactofa-mentum ATCC 13869 Brevibacterium roseum ATCC 13825 Brevibacterium flavum ATCC 13826 Brevi Corynebacterium acetoacetate film ATCC 13870 Corynebacterium acetoglutamicum ATCC 15806 Corynebacterium karnae ATCC 15991 Corynebacterium glutamicum ATCC 13032,13060 Corynebacterium lycerium ATCC 15990 Corynebacterium CC Melaceco Microbacterium ammoniaphilum ATCC 15354 Coryneform glutamic acid-producing bacterium has a mutant strain that loses glutamic acid productivity Is lysine, amino acids such as arginine, Bryn nucleosides such as inosine, mutants are also included to produce Bryn nucleotides such as inosine 5'-monophosphate, or other products.

When the coryneform / glutamic acid-producing bacterium as described above is used as a host for the composite plasmid of the present invention or a composite plasmid having a heterologous gene inserted, it is even better if a mutation for reducing the restriction enzyme activity is given to the host by a conventional method. Results are obtained.

When such a coryneform / glutamic acid-producing bacterium is used to introduce a composite plasmid having a foreign gene inserted therein, it is more advantageous to express the genetic information carried by the foreign gene.

The following are examples of multicopy plasmids capable of growing in the cells of coryneform glutamic acid-producing bacteria.

(1) pAM330: (a) Separation source: Brevibacterium lactofermentum ATCC 13869 (b) Molecular weight: 3.0 megadalton (calculated from migration distance on agarose gel electrophoresis and DNA chain length under electron microscopy) (C) Cleavage site by restriction enzyme: See Table 1 (d) Restriction enzyme cleavage map: See Figure 1 (2) pAM286: (a) Separation source: Corynebacterium glutamicum AJ11560 (FERM-P5485) (b) Molecular weight: 3.0 megadalton (movement distance on agarose gel electrophoresis and DNA chain length under electron microscopy) (C) Cleavage site by restriction enzyme: See Table 2 (d) Restriction enzyme cleavage map: See Figure 2 (3) pAM1519: (a) Isolation source: Corynebacterium glutamicum ATCC
13058 (b) Molecular weight: 1.8 megadalton (c) Cleavage site by restriction enzyme: See Table 3 (d) Restriction enzyme cleavage map: See FIG. Another example of a multicopy plasmid capable of propagating in coryneform glutamate-producing cells is Agric.Biol.Che.
mi., 43, 867 (1979), and this plasmid is also included in the multicopy plasmid (a) of the present invention. This plasmid was isolated from Brevibacterium lactofermentum and has a molecular weight of 37 megadaltons.

The plasmid (b) that can propagate in Escherichia coli and has the genetic information of drug resistance is a multicopy plasmid. For example, PAC105, RSF2124, pCR1, pMB9, pBR313, pBR3
22, pBR324, pBR325, pBR327, pBR328, pKY2289, pKY2700, pKN
80, pKC7, pKB158, pKM2004, pACYC177, and pACYC184.

The plasmid (b), which can be propagated in Bacillus subtilis and has the genetic information for drug resistance, is a multicopy, for example, pT127, pC194, pC221, pC223, pUB112, pUB110, pS.
There are A0501, pSA2100, pE194, pTP4 and pTP5.

In order to construct the composite plasmid of the present invention from the plasmids (a) and (b), a usual method can be used. For example, both plasmids (a) and (b) are cleaved with a restriction enzyme that produces a complementary single-stranded oligonucleotide at one or more restriction enzyme cleavage points or produces a flush end. The subsequently cleaved plasmids (a) and (b) are ligated with ligase. Alternatively, both plasmids (a) and (b) are cleaved with one or more restriction enzymes or shearing forces and, if necessary, treated with exonuclease and then complementary to the breakpoint of the plasmid using terminal transferase. A complex plasmid can also be prepared by adding such a single-stranded oligonucleotide and finally annealing the derivatives of these plasmids (a) and (b).

As with the plasmid (a), it is desirable that the gene fragment derived from the plasmid (b) is cleaved so that the gene region expressing drug resistance and the region other than the gene region containing the drive unit are reduced.

Most preferably, the gene fragment derived from the plasmid (b) has a drug resistance gene region and a drive unit region so that the composite plasmid can be used as a shuttle vector.

After the ligation reaction, the desired composite plasmid can be selected as follows: A composite plasmid having a gene fragment containing a drive unit region derived from plasmid (a) and a drug resistance gene derived from plasmid (b) is It can be obtained as a plasmid which can grow in coryneform glutamic acid-producing bacteria and can transform coryneform glutamic acid-producing bacteria into drug resistance. Furthermore, the plasmid having the drive unit region derived from the plasmid (a), the drive unit region derived from the plasmid (b), and the drug resistance gene derived from the plasmid (b) is a coryneform glutamic acid-producing bacterium and Escherichia coli or Bacillus.・ Proliferation in subtilis and (1) Coryneform glutamic acid-producing bacterium or (2) Escherichia coli (when the used plasmid can be propagated in Escherichia coli)
Alternatively, it is obtained as a plasmid capable of transforming Bacillus subtilis (when the used plasmid can grow in Bacillus subtilis) into drug resistance.

Preferred strains for such experiments in Escherichia coli and Bacillus subtilis are Escherichia coli K-12.
And Bacillus subtilis RM125.

The introduction of plasmid DNA into coryneform glutamate-producing bacteria has been reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53,
159 (1979)) treatment of recipient cells with calcium chloride to increase DNA permeability, or as reported for Bacillus subtilis (Duncan, CH, wi).
lson, GAand Young, FE, Gene, 1,153 (197
7)) It is possible by a method of introducing into a growth stage (so-called competent cell) where cells can take up DNA. Alternatively, as is known for Bacillus subtilis, actinomycetes and yeast (Chang, S. and Ch.
oen, SN, Molec.Gen.Genet., 168.111 (197)
9); Bibb, MJ, ward, JMand Hopwood, OA, Nature,
274, 398 (1978); Hinnen, A., Hicks, JBan.
d Fink, GR, Proc.Natl.Acad.Sci.USA, 75,1929 (1
It is also possible to introduce the plasmid into the DNA recipient bacterium by converting the DNA recipient bacterium into a protoplast or spheroplast which easily incorporates the plasmid DNA.

Since the composite plasmid obtained in the present invention can transform a coryneform glutamic acid-producing bacterium into a drug-resistant bacterium, a coryneform glutamic acid-producing bacterium having this composite plasmid or a composite plasmid into which a foreign gene is incorporated can be analyzed by examining drug resistance. It can be easily identified.

When the composite plasmid has a gene fragment having the drive unit region of the plasmid (b), the composite plasmid can be propagated in Escherichia coli or Bacillus subtilis. In addition, this composite plasmid or a composite plasmid into which a foreign gene is incorporated can be amplified or cloned using Escherichia coli or Bacillus subtilis.

The host containing the composite plasmid of the present invention has been deposited as follows.

pAJ 655: Escherichia coli AJ 11882 FERM-p 6517 = FERM-BP136 pAJ 611: Escherichia coli AJ 11884 FERM-p 6519 = FERM-BP 138 Corynebacterium glutamicum SR8201 ATCC39135 pAJ 1844: Escherichia coli FER AJ FER AJ FER AJ FERA 6518 = FERM-BP137 Corynebacterium glutamicum SR8202 ATCC 39136 pAJ 440: Bacillus subtilis KS440 ATCC39139 pAJ 3148: Corynebacterium glutamicum SR8203 ATCC 39137 The composite plasmid of the present invention is used for depositing microbial cells at the late logarithmic growth phase. Lysozyme and S after being grown to
Lysis was performed by DS, and polyethylene glycol was added to the supernatant obtained by centrifugation at 30,000 xg to precipitate D
NA can be purified by fractionating by cesium chloride-ethidium bromide equilibrium density gradient centrifugation.

To obtain the host microorganism, it is possible to remove the complex plasmid in the host cell from the deposited microorganism without damaging the host cell as follows: the plasmid is naturally lost by the host. It can also be removed by a "removal" operation (Bact. Rev., 36, p361-405 (19
72)). An example of the removal procedure is as follows: A small amount of strain was added to a medium containing acridine orange at a concentration (2-50 μg / ml) that incompletely inhibits the growth of the host so that about 10 ⁴ cells per 1 m were obtained. And incompletely inhibit the growth of the host fungus, and then incubate overnight at 27-35 ° C (J. Bact
eriol., 88,261 (1964)). Apply the culture solution to the agar medium,
Incubate overnight at 27-42 ° C. Most of the colonies appearing on the medium are likely to have the plasmid removed.

Although the present invention has been generally described, further intent will be readily appreciated by reference to the examples. The examples in this patent are given only as an aid to gain a specific recognition, and are not intended to limit the scope of the claims.

Example 1 This example consists of plasmids pBR325 and pAM330,
The composite plasmid pAJ655 capable of replicating in Escherichia coli K-12 is shown.

(1) The plasmid pBR325 is a vector plasmid with a molecular weight of 3.6 megadalton that expresses resistance to ampicillin, chloramphenicol and tetracycline in Escherichia coli [Boliver, F. Gene, 4.121 (1971).
8)]. The plasmid pBR325 used in the experiment was purchased from (Bethesda Research Laboratory) (BRL).

(2) The plasmid pAM330 is a plasmid with a molecular weight of 3.0 megadalton newly isolated by us from Brevibacterium lactofermentum ATCC 13869, and its restriction enzyme cleavage map is shown in FIG. Plasmid pAM330DNA
Was prepared as follows.

10 g peptone / distilled water, 10 powdered yeast extract
C, containing 5 g, 5 g of sodium chloride and 5 g of glucose
Brevibacterium lactofermentum ATCC 13869 was cultured in medium (pH 7.2) 1 at a temperature of 30 ° C. until the late logarithmic growth phase, and the cells were collected. The obtained bacterial cells were lysed by a simple method of lysing with lysozyme and SDS, and then centrifuged at 30,000 × g for 30 minutes to obtain a supernatant of 64 m. The plasmid DNA in the supernatant was added to the supernatant by adding polyethylene glycol (final concentration 10%) and then precipitated, and then dissolved in 10 m of TEN buffer.

After treating the DNA with ribonuclease (ribonuclease I 50 μg / m, reaction at 37 ° C. for 30 minutes), phenol extraction was performed, and then 2 volumes of ethanol was added to give −20.
The DNA was precipitated at ° C and the precipitate was dissolved in 1m TEN buffer. The DNA solution was subjected to agarose gel electrophoresis, and about 74 μg of pure plasmid DNA was separated from the gel.

(3) Preparation, isolation and identification of complex plasmid (i) 1 unit of restriction enzyme per 0.2 μg of plasmid pBR325
BamHI (purchased from BRL) was reacted at 37 ° C. for 60 minutes to sufficiently decompose the DNA.

(ii) The plasmid pAM330 (1.2 μg) was reacted with 0.2 unit of the restriction enzyme MboI at 37 ° C. for 15 minutes to partially decompose the DNA.

(iii) The DNAs obtained in (i) and (ii) were mixed and heat-treated at 65 ° C for 10 minutes to inactivate the restriction enzyme, and then 0.01 units of 2 units at 22 ° C for 2 hours in the presence of ATP and dithiothreitol. T4 DNA ligase was allowed to act. T4 DNA ligase was inactivated by treatment at 65 ° C. for 10 minutes, 2 volumes of ethanol was added, and then D was obtained by centrifugation at 15,000 g for 15 minutes.
NA was recovered.

The composite plasmid thus obtained was used for transification (iv).

(iv) Escherichia coli ^{C-600 (thr -, leu} -,
thiamine ⁻ , r ⁻ , m ⁻ ) (Meselson, M.and Yuan, R.Natur
e, 217,1110 (1968)) was cultured in 20 m of CMG medium at 30 ° C. until the mid-logarithmic growth phase, and the cells were collected.
Kushner et al. (“Geneted Engineering”, p.17 (197)
8), C-60 using the DNA obtained in (iii) according to the method of Elsevier / North Holland Biomedical Press)
0 was transformed.

The transformant was selected by culturing at 37 ° C. for 24 hours in CMG medium containing 20 μg / m of chloramphenicol. AJ 11882 was selected from the transformants and used in the next experiment.

The composite plasmid pAJ655 was transformed into AJ1188 by the following method.
It was separated from the lysate of 2. After culturing AJ11882 in CMG medium, a simple method (Tanaka et al., J. Bacteriol., 121, 354 (197
Lysis was performed by 5)) and the lysate was applied to an agarose gel and electrophoresed (sharp et al., Biochemistry 1
2, 3055 (1973)). By comparison with the molecular weight marker, the molecular weight of the plasmid was calculated to be 6.6 Md. A restriction enzyme digestion map of the plasmid is shown in FIG. The plasmid is pBR3
KJDanna consisting of 25 and a fragment of pAM330
Method in Enzymology 65,449, Academic Pre
ss (1980)).

Example 2 In this example, the composite plasmid pAJ655 obtained in Example 1 replicated in Brevibacterium lactofermentum,
It shows that an antibiotic resistance marker derived from pBR322 on pAJ655 can be expressed.

Brevibacterium lactofermentum ATCC13869
A triple auxotrophic (lysine, methionine, threonine) mutant strain Brevibacterium lactofermentum No. 64, which is a parent strain, was used as a DNA recipient in the experiment.

(1) Preparation of protoplasts Brevibacterium lactofermentum No. 64
(ATCC39134) was cultivated in a CMG medium of 5 m until the beginning of the logarithmic growth phase, and after adding 0.6 unit / m of penicillin G, the cells were further cultivated with shaking for 1.5 hours, and the cells were collected by centrifugation to recover the cells. SMMP medium (pH 6.5) 0.5m consisting of 0.5M sucrose, 20mM maleic acid, 20mM magnesium chloride and 3.5% Penassay Broth (Difco)
Washed with. Next SMM containing 10 mg / m lysozyme
The cells were suspended in P medium and protoplastized at 30 ° C. for 20 hours. After centrifugation at 6000 g for 10 minutes, the protoplasts were washed with SMMP and resuspended in 0.5 m SMMP.

(2) Transformation, selection of transformants,
Identification of plasmid The obtained protoplasts were pAJ655 prepared in Example 1.
After mixing with 2 μg of DNA and adding polyethylene glycol to a final concentration of 30%, the mixture was allowed to stand at room temperature for 2 minutes to incorporate the DNA into protoplasts. After washing the cells with 1 m of SMMP medium, the protoplasts incorporating DNA were resuspended in 1 m of SMMP medium and cultured at 30 ° C. for 3 hours in order to develop drug resistance.
This culture solution was spread on a protoplast regeneration medium having a pH of 7.0. Protoplast regeneration medium contains 12 g of tris (hydroxymethyl) aminomethane and KCl 0.5 per distilled water.
g, glucose _{10g, MgCl 2 · 6H 2 O} 8.1g, CaCl 2 · 2H
₂ O 2.2 g, peptone 4 g, powdered yeast extract 4 g, casamino acid (Difco) 1 g, K ₂ HPO ₄ 0.2 g, sodium succinate 135 g, agar 18 g and chloramphenicol 5 μg / m.

5 out of colonies that appeared after 3-10 days of culture at 30 ° C
Strains were selected and cultured in a CMG medium containing 20 μg / m chloramphenicol, and it was confirmed that they were drug (chloramphenicol) resistant strains.

In order to further confirm the presence of the plasmid, the chloramphenicol resistant strain grown here was lysed, and the lysate was subjected to agarose gel electrophoresis by the same method as in Example 1 to give the same molecular weight and restriction enzyme digestion map as PAJ655. The carrying plasmid was recognized.

Example 3 This example is based on the composite plasmid pAJ6 made in Example 4.
11 replicates in Corynebacterium glutamicum, pB
It shows that an antibiotic resistance marker derived from R325 is expressed.

Protoplast transformation was performed as described in Example 2 using Corynebacterium glutamicum ATCC 13060 as the recipient and pAJ611 DNA extracted from Escherichia coli AJ 11884.

The transformant strain designated SR-8201 showed chloramphenicol resistance in CMG medium, and the plasmid had a molecular weight and a restriction enzyme cleavage map.
It turned out to be the same as pAJ611.

Example 4 Here, the composite plasmid PAJ611 is shown. pAJ611 is pBR325
It is a composite plasmid composed of pAM286 and pAM286 and can grow in Escherichia coli.

(1) The same pBR325 as in Example 1 was used.

(2) pAM286 is Corynebacterium glutamicum) A
Molecular weight of 3.0 newly separated from J11560 (FERM-P5485)
The restriction map of the megadalton plasmid
It is shown in the figure. About 20 μg by the same method as in Example 1.
Of pAM286 was isolated.

(3) The construction, isolation and identification of the composite plasmid pAJ611 were all carried out according to the method of Example 1, except that pAM286 was used instead of pAM330.

A composite plasmid pAJ611 having a molecular weight of 6.6 megadalton and having a restriction enzyme map as shown in FIG. 5 was selected by the same method as in Example 1 using Escherichia coli C-600 as a host. The pAJ611 DNA was isolated by the method of Example 1 from the Escherichia coli AJ11884 strain (a strain containing pAJ611).

Example 5 Here, the composite plasmid pAJ440 is shown. pAJ440 is pUB1
It is a composite plasmid composed of 10 and pAM330.
Can grow in bstilis.

(1) pUB110 is a Staphylococcus aureus-derived plasmid having a molecular weight of 3.0 megadalton, which grows in Bacillus subtilis and exhibits kanamycin resistance.
(Keggins, K, M, Lovett, P, S, and Duval, EJ, proc, Natl,
Acad, Sci., 75, 1423 (1978)], pUB110 is Bethesd
a Purchased from Research Laboratory (BRL) and used as a marker for complex plasmid.

(2) A method for preparing pAM330 is shown in Example 1.

(3) Construction, isolation and identification of complex plasmids.

(i) pUB110 (0.2 μg) was completely cleaved at 37 ° C. for 60 minutes using 1 unit of restriction enzyme Bam HI (manufactured by BRL).

(ii) pAM330 (1.2 μg) was partially digested with 0.2 unit of the restriction enzyme MboI at 37 ° C. for 15 minutes.

(iii) Both enzymatically cleaved DNAs were mixed and ligated by the method of Example 1. Bound DNA was electrophoresed in a Tris-acetate buffer at 5 V / cm for 2 hours using a 0.8% agarose gel. A DNA fraction having a lower mobility than pUB110 was extracted from the gel, treated with phenol, and precipitated by ethanol for isolation.

(iv) Bacillus subtilis RM125 (arg ^-, leu ^-,
r ^-, m ^-) [Uozumi T.et.al., Molec.Gen.Genet, 152,65
(1975)], using the DNA mixture obtained in (iii), the method of Dabnau et al. [Dabnau, D., et al., J. Mol. Biol., 56,
209 (1971)]. The transformant is
They were selected as those that grow on CMG agar plates containing 5 μg / m of kanamycin at 37 ° C. for 24 hours.

Twenty of the transformants grown on CMG agar plates containing 5 μg / m of kanamycin were selected, and the presence of the plasmid in those cells was examined. One of the bacteria in which a clear plasmid band was detected was named KS440 and was named ATC.
Deposited as C39139. The plasmid contained in KS440 was named pAJ440. pAJ440 was extracted from the gel and cut with several restriction enzymes. PAJ440 is a complex plasmid composed of pUB110 and pAM330, because the molecular weight is 6.0 megadalton and the restriction endonuclease map shown in FIG. 7 was obtained.

Example 6 Here, the composite plasmid pAJ440 grows in Brevibacterium lactofermentum and also pU on pAJ440.
It is shown that an antibiotic resistance marker derived from B110 can be expressed.

Brevibacterium lactofermentum No. 64 as a host
It was used. The protoplast transformation method of the No. 64 strain with pAJ440 was performed by the method described in Example 2. However, this time, 1 μg of pAJ440 was used for transformation, and 100 μg / m of kanamycin was added to the protoplast regeneration medium in order to select a transformant.
20 μ of 2 strains out of the transformed strains grown in 3 to 10 days
The cells were cultured in a CMG medium containing g / m of kanamycin, and in order to examine the plasmid, they were lysed by the same method as in the above-mentioned Example, and the lysate was subjected to agarose gel electrophoresis. The molecular weight of the plasmid and its restriction enzyme cleavage map were the same as in pAJ440 of Example 4.

Example 7 Here, the composite plasmid pAJ1844 is shown. pAJ1844 is
It is a complex plasmid composed of pBR325 and pHM1519 and can grow in Escherichia coli.

(1) The same pBR325 as in Example 1 was used.

(2) pHM1519 is Corynebacterium glutamicum ATCC
A newly isolated plasmid from 13058 having a molecular weight of 1.8 megadalton and its restriction enzyme digestion map is shown in FIG. About 24 μg of pHM151 was prepared in the same manner as in Example 1.
9 was isolated.

(3) Construction, isolation and identification of composite plasmid pAJ1844.

(i) 0.2 μg of pBR325 was cleaved by the method of Example 1.

(ii) 1.0 μg of pHM1519 is a restriction enzyme Bgl II (Boehring
er Manheim GmbH) 5 units at 37 ° C 60
Cut in minutes.

(iii) The transformant was obtained by introducing the composite plasmid into Escherichia coli 600 according to the method described in Example 1. The composite plasmid, named pAJ1844, is one of the transformants, Escherichia coli AJ 1
Separated from 1883. The molecular weight of pAJ1844 is 5.4 megadalton, and its restriction enzyme cleavage map is shown in FIG.

Example 8 Here, the composite plasmid AJ1844 prepared in Example 7 is used.
Grow in Corynebacterium glutamicum, and pAJ1
It shows that the antibiotic resistance marker from pBR325 on 844 is expressed. Corynebacterium glutamicum ATCC13032 was used as a recipient bacterium. According to the protoplast transformation method of Example 2, pAJ1844 obtained from the Escherichia coli AJ11883 strain could be introduced into Corynebacterium glutamicum ATCC13032. Furthermore, the plasmid in this transformant is Escherichia coli AJ11883.
It was confirmed that it was the same plasmid as pAJ1844 from which it was derived. SR8202 is a representative transformant of Corynebacterium glutamicum harboring pAJ1844.

Example 9 This example demonstrates the composite plasmid pAJ3148 constructed from plasmid pUB110 and pHM1519.

(1) The plasmid pUB110 used was the same preparation as in Example 5 and cleaved with the restriction enzyme BamHI by the method of Example 5.

(2) The plasmid pHM1519 used was the same preparation as in Example 7 and cleaved with the restriction enzyme BgIII by the method of Example 7.

(3) The cut DNAs obtained in (1) and (2) were mixed and ligated. The ligated DNA was electrophoresed. A DNA having a larger molecular weight than the pUB110 monomer was collected and introduced into Bacillus subtilis RM125 according to the method of Example 5. pUB11
For plasmids with a molecular weight higher than 0, transformant N
Confirmed in o.1129. This plasmid pAJ3148 has a molecular weight of 6.6 megadalton, and its cleavage map is shown in FIG. As shown in FIG. 8, this plasmid contains one molecule of pU.
It consists of B110 and two molecules of pHM1519.

Example 10 In this example, the composite plasmid pA constructed in Example 9 was used.
It is shown that J3148 replicates in Corynebacterium glutamicum and expresses a drug resistance marker derived from pUB110.

Corynebacterium glutamicum ATCC13032 DNA
PAJ3148DNA obtained from Bacillus subtilis No. 1129 could be introduced into ATCC13032 using the protoplast transformation method described in Example 2 as a recipient bacterium (Corynebacterium glutamicum SR8203 ATCC3
9137). pAJ3148 replicated and expressed kanamycin resistance. The molecular weight and restriction enzyme digestion map were the same as pAJ3148.

Example 11 This example shows an example of a cloning experiment. The new composite plasmid pSR8204 comprises the vector plasmid pAJ1844 described in Example 7 and chromosomal DNA carrying at least the gene for m-fluoro-phenylalanine resistance.
Created from fragments.

(1) Plasmid pAJ1844 is a composite plasmid constructed in Example 7 and was replicated in Escherichia coli AJ11883 as described in Example 7. pAJ1844 was used as a vector and cut with the restriction enzyme PstI.

(2) Brevibacterium lactofermentum FERMp-1914 was selected as the DNA fragment to be inserted.
Chromosomal DNA was extracted and cut with PstI. FERMp-1914
Is resistant to m-fluoro-phenylalanine and produces phenylalanine in the medium.

(3) A vector plasmid digested with PstI and a chromosomal DNA digested with the same enzyme were mixed and ligated.

(4) The ligated DNA preparation was brought into contact with the protoplast of Corynebacterium glutamicum ATCC13060, and the transformant was first selected by chloramphenicol resistance. Next, selection was performed by resistance to m-fluoro-phenylalanine, and the productivity of phenyl-alanine was examined. Analysis of plasmid DNA in the lysis supernatant of the transformant was performed by agarose gel electrophoresis.

(i) Vector DNA; The composite plasmid pAJ1844 was isolated from Escherichia coli AJ11883. 20 meters for AJ11883
CMG medium, and chloramphenicol was added to the culture medium at the late logarithmic growth stage to a final concentration of 170 μg / m, and the culture was continued for 18 hours. 10 μg of plasmid DNA was obtained from the lysis supernatant of the chloramphenicol treated cells. 0.05 µg of the obtained DNA was added to Ps for 60 minutes at 37 ℃.
Completely cut at tI.

(ii) Chromosomal DNA: Chromosomal DNA of Brevibacterium lactofermentum AJ3437 (FERMp-1914) was prepared from CMG culture medium 1 according to the conventional method using phenol by 30
mg was collected. 10 μg of this DNA at 37 ° C for 5 minutes to 60
Partially decomposed with 10 units of PstI per minute.

(iii) Ligation: The DNAs of (i) and (ii) were mixed and ligated with 0.1 unit of T ₄ ligase at 22 ° C. for 4 hours. Other conditions were the same as in Example 1.

(iv) DNA recipient bacterium: Used as a DNA recipient bacterium of DNA (iii) in which protoplasts of Corynebacterium glutamicum ATCC13060 were ligated. For protoplast preparation, cells in the early logarithmic growth in CMG medium were used at a final concentration of 1.
Penicillin G was added to 2 units / m, 1.5
After culturing for a time, the cells were collected and washed, resuspended in 0.5 m of SMMP medium containing 10 mg of egg white lysozyme, and kept at 30 ° C. for 20 hours.

(v) Transformation; ligated DNA (iii) and protoplast
(iv) was mixed, and polyethylene glycol (final concentration 30%) was added to promote adsorption of DNA to protoplasts. The DNA-protoplast complex thus obtained was washed and plated on a regeneration medium plate (see Example 2) containing chloramphenicol 5 μg / m as a selective drug.

(vi) Transformant: A few chloramphenicol-resistant transformants were obtained, and a minimum medium containing 1 mg / m of m-fluoro-phenialanine (10 g of glucose, 2 g of ammonium sulfate, KE ₂ per distilled water). PO ₄ 1 g, MgSO ₄ .7H ₂ O 0.4 g,
The resistance to m-fluoro-phenylalanine was investigated in Fe ⁺⁺ 2 ppm, Mn ⁺⁺ 2 ppm, d-piotine 100 μg, thiamine HCl 1 mg agar 15 g (pH 7.2)).

(vii) Plasmid analysis: The lysate supernatant of the transformed strain was subjected to agarose gel electrophoresis together with molecular weight markers (pAJ1844 and pAJ655). The molecular weight of the plasmid in the transformant was approximately 6 megadaltons.

This transformant was named SR8204 and ATCC39138, and the plasmid was named pSR8204.

(viii) Phenylalanine productivity; The amount of phenylalanine produced in P-medium is 1 g / SR8204,
Host ATCC13060 was less than 0.1 g /. In the case of SR8204, the amount of tyrosine produced in the same medium was 0.4 g /
Host ATCC13060 was less than 0.1 g /. The p-medium was 100 g glucose and 40 ammonium sulfate per distilled water.
g, KH ₂ PO ₄ 1 g, MgSO ₄ .7H ₂ O 0.4 g, Fe ⁺⁺ 2 ppm, Mn ⁺⁺ 2
ppm, 100 μg of d-biotin, 1 mg of thiamine HCl and Ca
Contains 50 g of CO ₃ (pH 7.0).

Six strains described in the present invention (SR-8201, SR-8202, SR-820
3, SR-8204, No. 64 and KS440) deposited with the ATCC on June 3, 1982.

As described above, since the present invention has been sufficiently described, it will be understood that the same operation can be carried out within a wide range of equivalent conditions and parameters without departing from the spirit and knowledge of the invention. .

[Brief description of drawings]

FIG. 1 shows a restriction map of pAM330. FIG. 2 shows a restriction map of pAM286. FIG. 3 shows a restriction enzyme digestion map of pHM1519. FIG. 4 shows a restriction enzyme digestion map of the composite plasmid pAJ655. FIG. 5 shows a restriction enzyme digestion map of the composite plasmid pAJ611. FIG. 6 shows a restriction enzyme digestion map of the composite plasmid pAJ1844. FIG. 7 shows a restriction enzyme digestion map of the composite plasmid pAJ440. The symbols indicate the following restriction enzymes. A: AvaI, II: HindII Bc: BclI, III: HindIII Bg: BglII, X: XbaI E: EcoRI FIG. 8 shows a restriction enzyme digestion map of the composite plasmid pAJ3148. The symbols indicate the following restriction enzymes. Bc: Bcl I, II: HindII Bg: Bgl II, III: HindIII E: EcoRI, X: Xba IH: Hae II

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location (C12N 1/21 C12R 1:13) (C12N 1/21 C12R 1:15) Examiner Yuko Saeki ( 56) References JP-A-56-148295 (JP, A) JP-A-56-148296 (JP, A) JP-A-56-109588 (JP, A) Mol, Gen, Genet. , 168 (1979) p. 111-115 Mol, Gen, Genet. , 174 (1979) p. 281-286 J. Bacteriol. , 146 (1) (1981) p. 360-368

Claims (13)

[Claims] 1. A plasmid that can grow in a coryneform glutamic acid-producing bacterium and is expressed by the following restriction enzyme cleavage map: pAM330, pAM286, and pHM1.
Proliferation in coryneform / glutamic acid-producing bacteria consisting of one selected from 519 (A) and a gene region (B) containing a drug resistance gene portion of a plasmid (b) capable of growing in Escherichia coli or Bacillus subtilis cells Possible composite plasmids. 2. The composite plasmid according to claim 1, wherein the gene region (B) further has a drive unit in addition to the drug resistance gene. 3. The composite plasmid according to claim 1 or 2, wherein the plasmid (b) is pUB110. 4. The composite plasmid according to claim 1 or 2, wherein the plasmid (b) is pBR325. 5. The coryneform glutamic acid-producing bacterium is Brevibacterium lactofermentum or Corynebacterium glutamicum.
Item 7. The composite plasmid according to any one of items 4 to 4. 6. The composite plasmid according to claim 2, wherein the composite plasmid is pAJ655. 7. The composite plasmid according to claim 2, wherein the composite plasmid is pAJ611. 8. The composite plasmid according to claim 2, wherein the composite plasmid is pAJ1844. 9. The composite plasmid according to claim 2, wherein the composite plasmid is pAJ440. 10. The composite plasmid according to claim 2, wherein the composite plasmid is pAJ3148. 11. The composite plasmid according to claim 1, further comprising a gene derived from coryneform glutamic acid-producing bacterium. 12. The composite plasmid according to any one of claims 1 to 11, wherein the composite plasmid is incorporated in the cells of a coryneform glutamic acid-producing bacterium. 13. A coryneform / glutamic acid-producing bacterium,
13. The composite plasmid according to claim 12, which is Brevibacterium lactofermentum or Corynebacterium glutamicum.