JPS58216199A - Hybrid plasmid - Google Patents

Abstract

PURPOSE:A hybrid plasmid, prepared by integrating a chemical-resistant gene into a natural plasmid separated from coryneform bacteria capable of producing glutamic acid, and useful for breeding and improving industrial microorganisms by using the coryneform bacteria capable of producing the glutamic acid. CONSTITUTION:A hybrid plasmid, e.g. pAJ655, consisting of (A) a drive unit region of a plasmid (A) multipliable in cells of coryneform microorganisms capable of producing glutamic acid and a genetic range containing a chemical-resistant genetic part of a plasmid (B) multipliable in cells of Escherichia coli or Bacillus subtilis, preferably further containing drive units of the plasmid (B). pAM330, pAM286, etc. may be cited as the plasmid (A), and pT127, pBR325, etc. may be cited as the plasmid (B). The coryneform bacteria, having the above- mentioned integrated hybrid plasmid, and capable of producing the glutamic acid can be easily transformed into chemical-resistant microorganisms and identified. The plasmid containing the drive units of the plasmid (B) is usable as a shuttle vector.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a complex plasmid, particularly a complex plasmid capable of replicating in coryneform glutamate producing bacteria.

Coryneform glutamic acid-producing bacteria are known to produce a large amount of L-glutamic acid, and their mutant strains are known to produce amino acids such as lysine, purine nucleotides of inosinic acid, etc., and are used industrially. It is a useful microorganism. On the other hand, recent attempts have been made to breed and improve industrial microorganisms using recombinant DNA technology, including Escherichia coli, but it is difficult to find vectors suitable for using these microorganisms as hosts for coryneform-glutamate-producing bacteria. Coryneform, which has not yet been developed, is produced by DNA recombination.
This has hindered the breeding and improvement of glutamic acid-producing bacteria.

Several plasmids that replicate within coryneform-glutamate-producing bacteria have been found in coryneform-glutamate-producing bacteria, but the genetic properties of these that can be easily identified as markers have not been clarified. Not yet.

There are examples of multicopy plasmids isolated from coryneform-glutamate-producing bacteria being used as vectors, but because they do not have easily distinguishable genetic markers, they are more susceptible to industrial microorganisms than coryneform-glutamate-producing bacteria. Applying recombinant DNA technology for breeding remained not easy.

Therefore, there was still a need for the development of new plasmids and vectors for breeding and improving industrial microorganisms from coryneform-glutamate producing bacteria.

In this context, the purpose of the present invention is to construct a novel complex plasmid useful for breeding and improving industrial microorganisms using coryneform-glutamate-producing bacteria, and to construct a novel complex plasmid useful for breeding and improving industrial microorganisms using coryneform-glutamate-producing bacteria. The objective is to improve the isolated natural plasmid and make it suitable for breeding and improving microorganisms for industrial production such as coryneform glutamic acid producing bacteria.

These objectives were achieved as described below.

a drive unit (A) derived from a plasmid (a) capable of replicating in a coryneform glutamate producing bacterium;
derived from a drug resistance plasmid (b) capable of replicating in Escherichia koiha or Bacillus subtilis;
A complex plasmid consisting of a gene fragment (B) containing at least one drug resistance site.

If the gene fragment further contains the drive unit site of plasmid (b), this complex plasmid can be propagated in Escherichia coli and Bacillus subtilis, and can be used to reproduce the gene using Escherichia coli or Bacillus subtilis. It becomes possible to select or amplify complex plasmids.

The attached drawings are as follows.

Figure 1: Restriction enzyme cleavage map of pAM330 Figure 2:
Restriction enzyme cleavage map of pAM286 Figure 3: p@M15
Restriction enzyme cleavage map of 19 Figure 4: Composite plasmid pAJ
Restriction enzyme cleavage map of 655 Figure 5: Restriction enzyme cleavage map of composite plasmid pAJ61.1 Figure 6: Restriction enzyme cleavage map of composite plasmid pAJ1844 Figure 7: Restriction enzyme cleavage map of composite plasmid pAJ440 Figure 8: Restriction enzyme cleavage map of composite plasmid pAJ440 Restriction enzyme cleavage map of pAJ3148 "Multi-copy plasmid" refers to a plasmid that exists in multiple copies in one cell, as commonly defined.

"Drive unit" refers to the smallest genetic region necessary for gene replication within a host cell.

[Drug resistance-1, as commonly defined, refers to resistance to drugs such as antibiotics that inhibit host cell growth.

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

Coryneform bacteria are aerobic, gram-positive bacilli, non-acid-fast and described in Birdes Manual of Determinative Bacteriology, 8th edition, p. 599 (19
74).

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

Brevibacterium divaricatum ATCC
14020 Brevibacterium zanocalolittum
ATCC14066 Previbacterium inmariophyllum ATCC140689- Brevibacterium lactofermentum ATC
C13869 Frivibacterium e roseum
ATOO13825 Brevibacterium flavum ATCC13826 Previbacterium thiogenitalis ATCC19240 Corynebacterium acetoacidophilum ATCC
13870 Corynebacterium acetoglutamicum
ATOC! 15806 Corynebacterium e carnae ATCO15991 Corynebacterium glutamicum ATOC! 13
032.13060 Corynebacterium jar Lilium
ATCC15990 Corynebacterium melasecola ATOO1,7965 Microbacterium ammoniaphyllum ATOC!
15354 coryneform glutamate producing bacteria include mutant strains that have lost glutamate production, or amino acids such as lysine and arginine, purine nucleotides such as inosine, purine nucleotides such as inosine 5'-monophosphate, or other production. This also includes mutant strains that produce products.

When using the above-mentioned una coryneform glutamate-producing bacterium as a host for the complex plasmid of the present invention, a mutation that reduces restriction enzyme activity should be given to the host by a conventional method. Even better results can be obtained.

If such a coryneform glutamic acid producing bacterium is used to introduce a composite plasmid into which nine foreign genes have been inserted, it will be more advantageous to express the genetic information carried by the foreign genes.

Examples of multicopy plasmids capable of propagating within the cells of coryneform-glutamate producing bacteria include the following.

(1) pAM330: (a) Separation source: Brevibacterium lactofermentum ATCC13869 (b) Molecular weight: 30 megadaltons (calculated from migration distance on agarose gel electrophoresis and DNA chain length under electron microscopy) ( C) Restriction enzyme cleavage site: see Table 1 (,i)
Restriction enzyme cleavage map: See Figure 1 No. 1
Table Limit Enzyme Number of cutting tubes Alu I
Arthrobacter luteus ≧
4Ava I Anabaena paribilis
≧2BCI I Bacillus
Cartolyticus 1BamH
(Bacillus amyloliquefaciens HOBgl
l Bacillus globigii
0BstEll Bacillus stearothermophilus ET ≧4EcoRl
Escherichia coli R engineering +
OHaθ ■ Haemophilus aegygutius
lHg1A I Herpetosiphon giganteus ≧4Hin
cl 17 Haemophilus influenzae
≧4Hind l Haemophilus
influenzae IHpa
l Haemophilus parainfluenzae
≧4Kpnl Klebsiella pneumoniae 0Pvu II Proteus vulgaris 0s
ac I Streptomyces achromogenes 08al I Streptomyces albus 08au3
A Staphylococcus aureus
≧48ma l Serratia marcescens 1Sst l
Streptomyces Stanford
0Xba l Xanthomonas badri
1xho I Xanthomonas horicola IX
ma I Xanthomonas malvasarum
IXor ■ Xanthomonas oryzae 0 (2)
pAM 286: (a) Isolation source: Corynebacterium glutamicum A
, T 11560 (FERM-P 5485) (phrase
Molecular weight: 30 megadaltons (calculated from migration distance on agarose gel electrophoresis and DNA chain length under electron microscopy) (C) Cutting site by restriction enzyme: See Table 2 (d)
Restriction enzyme cleavage map: See Figure 2 13- Table 2 Restriction enzyme Number of cleavage sites Alu I
≧4 Ava l
≧2BamHl
0BCI l
IBgl l 0
BstE II ≧4EcoR
l OHa
θ■l Hg1A l ≧4Hind
ll ≧4Hind l
IHpa ll
≧4Kpn l
0Pvu■0 8ac ■Q Sal l
QSau 3A
≧ 4Sma (l 5st l 0xba H
I XhOl IXma 11 Eh. rH0 14- (3) pHM15]9: (a) Separation source: Corynebacterium glutamicum A
TCO13058 (b) Molecular weight: 18 megadaltons (C) Restriction enzyme cleavage site: See Table 3 (,i)
Restriction enzyme cleavage map: See Figure 3.
Table Restriction enzyme Number of cutting sitesAva
l ≧4BamHlo Bcl l IBg
l l IEcoR
f IHae l
≧7HgiA
(≧4 Hind l 2Kp
n T
.

Small
06ac (2 Xba l
OXh, o l
OXma l
Other examples of multi-copy plasmids that can proliferate in O-coryneform glutamate producing bacterial cells include Agric, Biol, Chemi.
, 43, 867 (1979).
This plasmid is also the multicopy plasmid of the present invention (a
)include. This plasmid was isolated from Brevibacterium lactofermentum and has a molecular weight of 37 megadaltons.

Plasmid (b), which can be propagated in Escherichia coli and has genetic information for drug resistance, is a multicopy plasmid. For example pAo 105. R8F2124゜pcRl, pMB 9. pBR313, pBR3
22, pBR324.

pBR325, pBR327, pBR328, pKY2
289. pKY2700, pKN80. pKO
7, pKB15B, pKM2004゜pACYC!
177, and pAC! There is YC184.

The plasmid (b) that can be propagated in Bacillus subtilis and has genetic information for drug resistance is multicopy, for example, pT 127. pC194, p0221゜pc 223. pUB112. pUB110 psA, 0501. psA2100, pE
194, pTP4 and pTP5.

Conventional methods can be used to construct the composite plasmid of the present invention from plasmids (a) and (b). For example, both plasmids (a) and (b) are cut with one or more restriction enzymes, ie, restriction enzymes that produce complementary single-stranded oligonucleotides at the cut points or that produce flush end ends, and Plasmids (a) and (b) cut after
) are ligated using ligase. Alternatively, both plasmids (a) and (b) can be cut with one or more restriction enzymes or shear forces, treated with an exonuclease if necessary, and then a terminal transferase can be used to transfer the plasmid at the break point. After addition of complementary single-stranded oligonucleotides, these plasmids (a) are finally
and (b) derivatives are annealed (annealin).
g) A complex plasmid can also be prepared by

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

-17= It is most desirable that the gene fragment derived from plasmid (b) has both a drug-depleting gene region and a drive unit region so that the composite plasmid becomes a shuttle vector.

After the ligation reaction, the desired complex plasmid can be selected as follows. A complex plasmid with a drive unit region derived from niblasmid (a) and a gene fragment containing a drug resistance gene derived from the plasmid (a) can be selected using coryneform. It is obtained as a plasmid that can proliferate in glutamic acid-producing bacteria and can transform coryneform glutamic acid-producing bacteria into drug resistance.Furthermore, plasmid (a)
A plasmid that has both the drive unit region derived from plasmid (b), the drive unit region derived from plasmid (b), and the drug resistance gene derived from plasmid fb) is a plasmid that has a drive unit region derived from plasmid (b), and a drug resistance gene derived from plasmid fb).
c can be grown in Bacillus subtilis and can be grown in (1) coryneform glutamate producing bacteria or (2) Escherichia coli (if the plasmid used can be grown in Escherichia coli) or Bacillus subtilis (if the plasmid used is capable of growing in Escherichia coli). If the plasmid can be propagated in Bacillus subtilis, a plasmid can be obtained that can transform Bacillus subtilis to become drug resistant.

The preferred strain for such experiments with Escherichia coli and Bacillus subtilis is -12
and Bacillus subtilis FIM125.

Introduction of plasmid DNA into coryneform glutamate producing bacteria was carried out as reported for Escherichia coIJK-12 (Mandel, M. and Hi.
Ga, A,.

J, Mo1. Biol,, sa, 159 (19
79)) DN by treating recipient bacterial cells with calcium chloride.
A method to increase the permeability of Bacillus subtilis is yet to be reported (Duncan, C, H,
, Wilson, G.A., and Young.

F.E. , Gene, 1.153 (1977)) by a method in which cells are introduced into a proliferative stage (so-called competent cells) so that they can take up DNA. Alternatively, as is known for Bacillus subtilis, actinomycetes and yeasts (Chang, S. and
Choen.

S,N,,Mo1ec,Gen,Genet,,
] 68. l l l (l 979) i Bibb
, M.J.

Ward, J. M., and Hopwood, O. A.
Nature, 27;, 398 (1978):
Hinnen, A., Hicks, J.B.a.
nd Fink, G.R., Proc.

Natl, Acad, Sci, USA, 75. 192
9 (1978)), convert the DNA recipient bacteria into plasmid DNA.
It is also possible to make protoplasts or spheroplasts that easily incorporate the plasmid into DNA recipient bacteria.

Since the complex plasmid obtained in the present invention can transform coryneform glutamate-producing bacteria into drug-resistant bacteria, coryneform glutamate-producing bacteria having this complex plasmid or a complex plasmid incorporating a foreign gene can be investigated for drug resistance. Easy to identify.

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

A host carrying the complex plasmid of the present invention has been deposited as follows.

pAJ655: Escherichia coli AJ11882F
ERM-p 6517 = FERM-BP 136 Corynebacterium glutamicum 5R8201ATC!
0 39135 pAJ6] 1: Escherichia coli AJ 1]
884FERM-p 6519=FERM-BP
138pAJ 1844: Escherichia coli
AJl”1883FERM-p 6518=FERM
-BP137 Corynepacterium glutamicum 5R8
202ATCO'39136 pAJ 440: Bacillus subtilis AJ
11901FERM-BP 140 pAJ 3148: Corynebacterium glutamicum 5R8203ATCC39137 The complex plasmid of the present invention grows deposited microbial cells to the late logarithmic phase, and then grows lysozyme and S
Elute with DS, centrifuge at 30,000Xg, add polyethylene glycol to the resulting supernatant, and purify the precipitated DNA by fractionating it with cesium chloride ethidium bromide equilibrium density gradient centrifugation. be able to.

In order to obtain a 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 from the host. It can also be removed by the “remove” operation (Bact, Rev, 3
6, p361-405 (1972)).

An example of a removal procedure is as follows: A small amount of the bacterial strain, approximately 104 cells per ml, is added to a medium containing acridine orange at a concentration (2-50 μf/me) that completely inhibits host growth. After inoculating and completely inhibiting the growth of host bacteria, culture at 27-35C overnight (J, Ba
cteriol, 88.261 (1964)).

The culture solution is applied to an agar medium and cultured overnight at 27-42r.

22- It is highly likely that the plasmid has been removed from many of the colonies that appear on the medium.

Although the present invention has been described generally, the same spirit will be more easily understood by referring to the Examples. The examples in this patent are included only as an aid to specific understanding and are not intended to limit the scope of the claims.

Example 1 In this example, plasmids pBR325 and pAM33
The complex plasmid pAJ655 consists of 0 and can be replicated in Escherichia coli in 12 cells.

(1) Plasmid pBR325 is a vector plasmid with a molecular weight of 36 megadaltons that expresses ampicillin, chloramphenicol, and tetracycline resistance in Escherichia coli (Boliver).

F. Gene, 4, 1.21 (1978)). Plasmid pBR325 used in the experiment was purchased from Bethesda Research Laboratories (BRL).

(2) Plasmid pAM330 is Brevibacterium
23 - This is a plasmid with a molecular weight of 30 megadaltons that we newly isolated from Lactofermentum ATOO13869. Figure 1 shows its restriction enzyme cleavage map. Plasmid pAM330 DNA was prepared as follows.

109 peptone, 10 powdered yeast extract per liter of distilled water
9. OMG containing 52 thorium chloride and 5 g glucose
Brevibacterium lactofermentum ATCC 13869 was cultured in medium (pH 7.2) Ii at a temperature of 30'C until the late logarithmic growth phase, and the bacterial cells were collected. After lysing the obtained bacterial cells by a simple method of lysing them with lysozyme and SDS, 30. ,O'O,TI Ik,!
J centrifugation for 30 minutes to obtain 64 mg of supernatant. Plasmid DNA in the supernatant was precipitated by adding polyethylene glycol (final concentration 10%) to the supernatant.
It was dissolved in TEN buffer of rrtl.

After the DNA was treated with liphonuclease (reacted at 37c for 30 min at 50 pg/vtl! of liphonuclease), it was extracted with phenol and then extracted with 2 volumes of ethanol 2.
4- was added to precipitate the DNA at -2 Orr, and the precipitate was added to 1 ml of T.
Dissolved in EN buffer. This DNA solution was subjected to agarose gel electrophoresis, and approximately 74 μ2 of pure plasmid DNA was separated from the gel.

(3) Construction, isolation, and identification of complex plasmids (i) Plasmid pBR3250, 1 unit of restriction enzyme per 2 μg
The DNA was sufficiently degraded by reacting with BamHI (purchased from BRL) at 370 for 60 minutes.

(11) Add 02 units of restriction enzyme M'b OI to plasmid pAM330 (1, 2a, ?) at 370 and 15
The reaction was allowed to proceed for a minute to partially degrade the DNA.

0ii) After mixing the DNA obtained in (i) and (11) and heat-treating at 65R for 10 minutes to inactivate the restriction enzyme,
0.2 hours at 22r in the presence of ATP and dithiothreitol.
01 unit of T4 DNA IJ gauze was applied. T4 DNA ligase was inactivated by treatment at 65r for 10 minutes, and 15,000 ml of ethanol was added.
DNA was collected by centrifugation for 15 minutes.

The composite plasmid thus obtained was transduced into It was used for sformation θV).

lv) Escherichia coli O-600 (thr-, 1e
u-.

thiamine-, r-, m) (Mesels
on, M., and Yuan, R.

Nature, 217. ] 110 (] 968
)) was cultured in 20 ml of OMG medium at 30 U in late logarithmic growth phase 1, and the bacterial cells were collected. Kushner et al.
Geneted Engineering ll.

p, 17 (1978), Elsevier/N
orthHo1land Biomedical
D obtained in (iii) according to the method of Press)
0-600 were transformed using NA.

The transformed strain was selected by culturing in CMG medium containing 20 μm and 17 ml of chloramphenicol at 37'C for 24 hours. AJI, 1882 was selected from the transformed strains and used in the next experiment.

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

After culturing AJI 1882 in CMG medium, a simple method (Ta
Naka et al., J. Bacteriol.
, Shagen, 354 (1975)).
The lysate was applied to an agarose gel and subjected to electrophoresis (S
harp et al., Biochemistry
Tsubo, 3055 (1973)). The molecular weight of the pl plasmid was calculated to be 6.6M (1) by comparison with the molecular weight marker. The restriction enzyme cleavage map of the plasmid is shown in Figure 4. The plasmid consists of fragments of pBR325 and pAM330. , K.J.Danna's Methods in Enzymology 6
5.449. Academic Press (198
0) ).

Example 2 This example uses the composite plasmid pAJ65 obtained in Example 1.
5 replicates in Brevibacterium lactofermentum and expresses an antibiotic resistance marker derived from pBR322 on pA, T655.

Brevibacterium lactofermentum ATCC1
Brevibacterium lactofermentum NO,64, a triple auxotrophic (lysine, methionine, threonine) mutant strain Brevibacterium lactofermentum NO.64 whose parent strain is 3869, was used in the experiment as a DNA recipient.

(1) Preparation of protoplasts Brevibacterium lactofermentum No. 64
(A'rcC39134) was cultured in 5me CMG medium until the early logarithmic growth phase, and after adding penicillin GO, 6 units/m13, it was further cultured with shaking for 15 hours, and the bacterial cells were collected by centrifugation. The cells were washed with 0.5 ml of SMMP medium (1) H6,5 consisting of .5M sucrose, 20mM maleic acid, 20mM magnesium chloride, and 3.5% Penancei broth (Difco). SMM containing 10” fl/ml of lysozyme with
The cells were suspended in P medium and incubated at 30C for 20 hours to form protoplasts. After centrifugation at 6000g for 10 minutes, wash the protoplasts with 5ml of SMMP! resuspended in SMMP.

(2) Transformation, selection of transformants,
Identification of plasmid The obtained protoplasts were transformed into pAJ prepared in Example 1.
After mixing with 655 DNA 2 af and adding polyethylene glycol to a final concentration of 30%,
Bring the DNA to room temperature for 2 hours to incorporate the DNA into the protoplasts.
Leave it for 1 minute. After washing the cells with 1 rnl of SMMP medium, the protoplasts loaded with DNA were resuspended in 1 ml of SMMP medium, and in order to express drug resistance,
Cultured at 30C for 3 hours. This culture solution was spread on a protoplast regeneration medium at pH 7.0. Protoplast regeneration medium contains 1 liter of distilled water and Tris (hydroxymethyl)
Aminomethane 129, KOI O, 5,? .

Glucose 10y% MgC1□・6H208,1y.

Ca1l□-2H202, 2f/1 peptone 4y, powdered yeast extract 42, casamino acid (Difco) 121
2HPO, 0,2H succinate 135#.

H Ten 1sg and 0: Chloramphenicol 5μm 11ml
including.

After culturing at 30'C for 3 to 10 days, 5 strains among the colonies that appeared were selected and cultured in CMG medium containing 20 μm and 17 tug of chloramphenicol, and it was confirmed that they were drug (chloramphenicol) resistant strains. .

Furthermore, to confirm the presence of the plasmid, the chloramphenicol-resistant strain grown here was lysed, and the lysate was subjected to agarose gel electrophoresis in the same manner as in Example 1. A plasmid with an enzyme cleavage map was observed.

Example 3 This example uses the composite plasmid pA made in Example 4.
Figure 3 shows that J611 replicates in C. glutamicum and expresses an antibiotic resistance marker derived from pBR325.

Corynebacterium glutamicum ATCO13060
was used as the recipient strain, Escherichia coli AJ11
Protoplast transformation was performed as described in Example 2 using pAJ 611 DNA extracted from 884.

The transformed strain named 5R-8201 showed chloramphenicol resistance in CMG medium, and the molecular weight and restriction enzyme cleavage map of the plasmid it contained revealed that this plasmid was the same as pAJ 611. did.

Example 4 Where! f, shows composite plasmid PAJ611.

pAJ61] is a 30-complex plasmid composed of pBR325 and pAM286 and can be propagated in Escherichia coli.

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

(2i pAM286 is Corynebacterium glutamicum) A, T11560 (FERM-P548
5) newly isolated molecule t3. The restriction enzyme map of the O megadalton plasmid is shown in FIG. About 20a9 of pAM28 was obtained by the same method as in Example 1.
6 were isolated.

(3) The construction, isolation, and identification of composite plasmid pAJ 611 were carried out in accordance with the method of Example 1, except that pAM 286 was used instead of pAM 330. T 611 was selected in the same manner as in Example 1 using Escherichia coli 0-600 as a host. The DNA of pAJ611 was isolated from Escherichia co1 JAJ11884 strain (pA
strain containing J611) according to the method of Example 1.

Example 5 Here, composite plasmid pAJ440 is shown.

pA, :T440 is a composite plasmid consisting of pUBllo and pAM330, B, 5ubsti
can be propagated in lis.

(t) pUB 110 is a plasmid with a molecular weight of 30 megadaltons derived from Staphylococcus aureus that grows in the ant Bacillus subtilis and exhibits kanamycin resistance. (xeggins, K, M, LoV
ett, p, s.

and Duval, E. J., Proc, Na.
tl, Acad, 5ci-+75.1423 (197
8) ], pUB 110 is available from Bethesda
Re5earch Laboratory (B R
It was purchased from L.L. and used as a marker for complex plasmids.

(2) I) The method for preparing AM330 is shown in Example 1.

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

(i) pUBllo (0,2μ#) is restriction enzyme B
37C6 using 1 unit of am H engineering (manufactured by BRL)
It was completely cut in 0 minutes.

(ii) pAM 330 (1,24?) was partially cleaved using the restriction enzyme Mbo 0232-unisoto at 37tl' for 15 minutes.

(iii) mixing both enzymatically cut DNAs;
Bonding was performed using the method of Example 1. The combined DNA is 0.
Electrophoresis was performed using a Section 8 agarose gel in Tris-acetate buffer at 5v/ctn for 12 hours. pU
A DNA fraction with lower migration mobility than Blo was extracted from the gel, treated with phenol, precipitated with ethanol, and isolated.

Gv) Bacillus subtilis RM125 (Rainbow "4.

ET,! ! ! 7) (Vozumi, T.
et, al,, Mo1ec, Gen, Gene
t.

152, 65 (1975)) used the DNA mixture obtained in (::+) using the method 1 of Dabnau et al.

Small, J, Mo1. Biol, 56, 20
9 (1971)). Transformants were selected as those that grew on OMG agar plates containing 5 of 1 ml of kanamycin and cultured at 37C for 24 hours.

Thirty-three to twenty transformants grown on CMG agar plates containing 5 μm7/lnl of kanamycin were selected, and the presence of plasmids in their cells was examined. One of the bacteria in which a clear plasmid band was detected was named KS440 and deposited as ATCIC3913917. The plasmid contained in KS440 is pAJ
It was named 440. pAJ440 was extracted from the gel and cut with several restriction enzymes. Since the molecular weight is 60 megadaltons and the restriction endonuclease map shown in Figure 7 was obtained, pAJ440 is pUB
It is a complex plasmid consisting of pAM330 and pAM330.

In this example, composite plasmid pAJ440 is grown in Brevibacterium lactofermentum and pAJ440 is grown in Brevibacterium lactofermentum.
40 shows that antibiotic resistance markers derived from pUB 110 on pUB 110 can be expressed.

As a host, Brevibacterium lact
64 was used. pAJ440
The protoplast transformation method of the A64 strain is described in Example 2.
This was done using the method described in. However, this time, 1
pAJ440 of Bg 34- was used for transformation, and the protoplast regeneration medium contained ]00μ9 to select transformed strains.
/lne of kanamycin was added. Two of the transformed strains that had grown in 3 to 10 days were cultured in CMG medium containing 20 μt/ml kanamycin, and in order to examine the plasmid, they were lysed using the same method as in the previous example. The lysate was subjected to agarose gel electrophoresis. The molecular weight of the plasmid and its restriction enzyme cleavage map were the same as pAJ440 in Example 4.

Example 7 Here, composite plasmid pAJ1844 is shown.

pAJ 1844 is a composite plasmid consisting of pBR325 and pHM1519 and can be propagated in Escherichia coli.

(1) 1) The same BR325 as in Example 1 was used.

(2) pHM1519 is a plasmid with a molecular weight of 18 megadaltons newly isolated from Corynebacterium glutamicum ATCC13058, and its restriction enzyme cleavage map is shown in FIG. Example 35- Approximately 24 μg of pHM 151.9 was prepared in the same manner as in (1).
was isolated.

(3) Construction, isolation and identification of complex plasmid pAJ 1844.

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

(ii) 1. oμ2 pHM15], 9 is the restriction enzyme Bgl (Boehringer Manheim
It was cut in 37μ60 minutes using 5 units (manufactured by GmbH).

(iii) A transformed strain was obtained by introducing the complex plasmid into Escherichia coli C-600 according to the method shown in Example 1. A complex plasmid named pAJ1844 was isolated from one of the transformed strains, Escherichia coli AJ 11883. pA,T
The molecular weight of 1844 is 54 megadaltons, and its restriction enzyme cleavage map is shown in Figure 6.

Example 8 Here, the composite plasmid AJ1 produced in Example 7
844 grows in Corynebacterium glucumin 36- humans and expresses the antibiotic resistance marker derived from pBR325 on pAJ1844.

Corynebacterium glutamicum AT as a recipient bacterium
OC! 13032 was used. According to the protoplast transformation method of Example 2, Escherichia coli AJ1188
pAJ1844 obtained from the three strains could be introduced into Corynebacterium glutamicum ATOC13032. Furthermore, it was confirmed that the plasmid in this transformed strain was the same plasmid as pAJ1844 derived from Escherichia coli AJ11883. 5R8202 is a Corynebacterium strain harboring pAJ1844.
This is a typical transformed strain of Glutamicum.

Example 9 In this example, plasmids pUB110 and pHM15
The composite plasmid pAJ3148 constructed from No. 19 is shown below.

(1) Plasmid pUB used] 10 is the same preparation as in Example 5, and the restriction enzyme BamHI was added using the method of Example 5.
I cut it with.

37-'2) The plasmid pHM1519 used was the same preparation as in Example 7, and the restriction enzyme Bgll was added using the method of Example 7.
I cut it with.

(3) The cut DNAs obtained in (1) and (2) were mixed and ligated. The ligated D1 (A was subjected to electrophoresis. DNA with a larger molecular weight than the pUB110 monomer was collected, and according to the method of Example 5, Bacillus subtilis Fj
It was introduced into M125. A large molecular weight plasmid, similar to pUB 110, was identified in transformant A1129. This plasmid pAJ3148 has a molecular weight of 66
The cut map of the megadalton is shown in Figure 8. 8th
As shown in the figure, this plasmid contains one molecule of pUB1
10 and two molecules of pHM1519.

Example 10 This example shows that the composite plasmid pAJ3148 constructed in Example 9 replicates in Corynebacterium glutamicum and expresses the pUB 110-derived drug ffj.

Corynebacterium glutamicum ATC! 01303
238- was used as the DNA recipient bacterium and the protoplast transformation method described in Example 2 was used to transform Bacillus subtilis A1.
The pAJ 3148 DNA obtained from 129 was
It can be introduced into ATCC13032.

pAJ 3148 replicated and expressed kanamycin resistance. Molecular weight and restriction enzyme cleavage map is pAJ3148
It was the same.

Example 11 This example shows an example of a cloning experiment.

A new composite plasmid pslR8204 was constructed from the vector plasmid pAJ1844 described in Example 7 and a chromosomal DNA fragment carrying at least the gene for m-fluoro-phenylalanine resistance.

(1) Plasmid pAJ1844 is the complex plasmid constructed in Example 7 and was replicated in Escherichia coli AJ11883 as described in Example 7.

pAJ 1844 was used as a vector and the restriction enzyme p
Cut at st:r.

(2) Brevibacterium lactofermentum FE, RM p-1914 was selected as the DNA fragment to be inserted, and chromosomal DNA was extracted from this and cut with PstI. FERM p-1914 is resistant to m-fluoro-phenylalanine and produces phenylalanine in the medium.

(3) The vector plasmid cut with PstI and the chromosomal DNA cut with the same enzyme were mixed and ligated.

(4) The ligated DNA preparation was brought into contact with protoplasts of Corynebacterium glutamicum ATCC 13060, and transformed strains were first selected for resistance to chloramphenicol. Next, they were screened for resistance to m-fluoro-phenylalanine, and the productivity of phenyl-alanine was examined. Plasmid DNA analysis in the lysed supernatant of the transformed strain was performed by agarose gel electrophoresis.

(i) Vector DNA; composite plasmid pA, T
1844 was isolated from Escherichia coli AJ1188:3. A'J1'1883 was cultured in 20 y, ql of CMG medium, chloramphenicol was added to the culture solution at the late stage of logarithmic growth to a final concentration of 170 ay/rrtl, and the culture was further cultured for 18 hours. From the lysed supernatant of chloramphenicol-treated bacterial cells, 10 tJ of plasmid DNA was obtained. 005μg of the obtained DNA was heated at 37C.
Complete cutting was performed using a PST machine for 60 minutes.

(11) Chromosomal DNA; Brevibacterium lactofermentum AJ34: U7 (F'E RIV4
3071 pieces of chromosomal DNA of 1914 p.p.

10g of real DNA! F for 3705 minutes to 60 minutes 10
It was partially digested with unit PstI.

(Face ligation; mix the DNA of (1) and (II),
The mixture was ligated with 0.1 unit of T ligase for 4 hours at 100°C.

Other conditions were the same as in Example 1.

(iv) D tq A receptor; DNA ligated with protoplasts of Corynebacterium glutamicum ATOO13060 (used as a surface DNA receptor).

To prepare pro-plasts, add penicillin G to the cells in the early stage of logarithmic growth in CMG medium to a final concentration of 12 units/me, and after culturing for 15 hours, collect the bacteria and antibacterialize the cells for 10 days.
'''y After resuspending in 0.5 tul of 41-8M MP medium containing egg white lysozyme, 30r
I kept it there for 20 hours and went there.

(v) Transformation: Mix the ligated DNA Af surface and protoplast) (iv), and transform the D'NA of protoplast-\
Polyethylene glycol (final concentration of 30%) was added to promote the adsorption of . The DNA thus obtained-
Wash the protoplast complexes and use 5μ of chloramphenicol as the drug of choice. /rttl on regeneration medium plates (see Example 2).

(VD Transformants: Hundreds of chloramphenicol-resistant transformants were obtained, using a minimal medium containing 1"f/prl of m-fluorophenialanine (1 i of distilled water, 10 t of glucose, 2 f of ammonium sulfate, KH ,,PO41
y, Mg5O, 7H200, 4f, Fe 2pp
m1Mn-” 2 ppm, d-biotin 100 pg
, thiamine HCl1. "51' Agar 15 # (pH 7)
, 2) ), m-fluoro-phenylalanine resistance t: was investigated.

&il Plasmid analysis; The lysate supernatant of the transformed strain was
Molecular weight markers (pAJ 1844 and 1) AJ655)
and 42- were subjected to agarose gel electrophoresis.

The molecular weight of the plasmid in the transformant was approximately 6 megadaltons.

This transformant is 5R8204, ATCC391
38, the plasmid was named psR8204.

111 Phenylalanine productivity; the amount of phenylalanine produced in P-medium is 1f/111 in the case of 5R8204.
No, host ATCIC! 13060 was less than 01t/no. The amount of tyrosine produced in the same medium was 5R82
In the case of 04, it is 0.4f/i, and the host ATCIC! 1306
0 was 01#71 or less. The p-medium contained 1 l of distilled water and 100 g of glucose. ? .

Ammonium sulfate 40 #, KH2PO41, r, Mg5O,・
7H200,4? , Fe” 2 ppm, M
n"2 ppm, d-biotin 100 td, thiamine HCl 1"jil and (laco3501 (pH 7
,0).

Six strains described in the present invention (SR-8201, 5R-8
202,5R-8203,5R-8204, -■1■-
64 and KS440) were deposited with ATOO on June 3, 1982.

It will be appreciated that the same operations may be performed within a wide range of comparable conditions, parameters, etc.

[Brief explanation of the drawing]

FIG. 1 shows a restriction enzyme cleavage map of pAM330. FIG. 2 shows a restriction enzyme cleavage map of pAM286. FIG. 3 shows a restriction enzyme cleavage map of pHM1519. FIG. 4 shows a restriction enzyme cleavage map of the composite plasmid pAJ 655. FIG. 5 shows a restriction enzyme cleavage map of composite plasmid pAJ611. FIG. 6 shows a restriction enzyme cleavage map of composite plasmid pAJ1844. FIG. 7 shows a restriction enzyme cleavage map of the composite plasmid pAJ 440. The symbols indicate the following restriction enzymes. A: Aval II: HindIIB
c:Bcll Old: HindlBg: Bg
ll[X: Xba IE: EcoRl Figure 8 shows a restriction enzyme cleavage map of the composite plasmid pAJ3148. The symbols indicate the following restriction enzymes. Bc: Bcl I II: Hl
nallBg : Bgl El l :
HindlE: EcoRJX:
X'ba IH: Haell Patent applicant Ajinomoto Co., Inc. 45- Figure 1 Figure 2 Figure 3 Bcl E Figure 4 Figure 5 To OO Figure 7 Figure 8

Claims (1)

[Claims] 1. Drive unit region (A) of plasmid (a) capable of proliferating in cells of coryneform glutamate producing bacteria
) and a gene region tB) containing the drug resistance gene portion of plasmid (b) that can proliferate in Escherichia coli or Bacillus subtilis cells. 2. The complex plasmid according to claim 1, wherein the gene region (B) further contains a drive unit in addition to the drug resistance gene. 3 Plasmid (a) is pAM330, pAM28
6 or pHM1519. 4 Plasmid (b) is pT127, pC! 194,
PC! 221, p0223, pUB112, pUBll
o, psAO501, psA2100, pK194, p
Claim 1 which is TP4 or pTP5
1- Complex plasmid according to Section 1 or Section 2. 5 Plasmid (b) is pA0105, R8F'21
24, PC! R1, pMB9, pBR313, pBR3
22, pBR324, pBR325, pBR327, p
BR328, pKY2289, pKY2700. pKN
80. pKO7, pKB158, pKM2004, pA
The composite plasmid according to claim 1 or 2, which is CY0177 or pACYOI 84. 6 The coryneform/glutamic acid producing bacteria are Microbacterium ammoniaphyllum Previbacterium devaricum Previbacterium sasocalolyticum Previbacterium imariophyllum Flechhacterium Lactofermentum Brevibacterium Rose Claims that are: Lambrevibacterium, Flambrevibacterium, Thiogenitalis Corynebacterium, Acetoglutamicum Corynebacterium, Carnaecorynebacterium, Glutamicum Corynebacterium Lilium or Corynebacterium Melasecola. The composite plasmid according to item 1 or 2. 7. The complex plasmid according to claim 2, wherein the complex plasmid is pAJ655. 8. The complex plasmid according to claim 2, wherein the complex plasmid is pAJ 61 ]. 9. The complex plasmid according to claim 2, wherein the complex plasmid has pAJ 1.844. 10. The composite plasmid according to claim 2, wherein the composite plasmid is pAJ440. 11. The complex plasmid according to claim 2, wherein the complex plasmid is pAJ 3148. 12. The composite plasmid according to claims 1 and 2, wherein the composite plasmid further contains genes other than those derived from coryneform glutamic acid producing bacteria. 13 The composite plasmid is a chromosome-plasmid or phage DNA of a coryneform glutamate-producing bacterium.
Claims 1 and 2 further comprising an A fragment
Composite plasmid as described in section. 14. The composite plasmid according to claims 1 and 2, wherein the composite plasmid is housed in a cell of a coryneform glutamic acid producing bacterium. 15. The composite plasmid according to claims 1 and 2, wherein the composite plasmid is housed in a cell of Escherichia coli or Bacillus subtilis. 16 Coryneform glutamate producing bacteria include Brevibacterium devaricatum, Brevibacterium imariophilum, Brevibacterium lactofermentum, Brevibacterium roserum, Brevibacterium thiogenitalis, Corynebacterium acetoglutamicum, and Corynebacterium - The complex plasmid according to claim 14, which is C. carnae, Corynebacterium glutamicum, Corynebacterium rillium, Corynebacterium melasecola, or Microbacterium ammoniaphyllum. 17. Claim 14, wherein the coryneform glutamic acid producing bacterium is a mutant strain with low restriction enzyme activity.
The composite plasmid described in Sections 1 and 16. 18 Claim 1, in which the complex plasmid is incorporated into the cells of a coryneform glutamic acid producing bacterium.
The composite plasmid described in Items 2 and 13.