JPH0734744B2 - Transducible complex plasmid - Google Patents

Description

The present invention relates to a transducible complex plasmid, and more particularly to a transducible complex plasmid using a coryneform glutamate-producing bacterium as a host.

Coryneform glutamic acid-producing bacteria are known to produce a large amount of L-glutamic acid, and mutants thereof are known to produce amino acids such as lysine and purine nucleotides such as inosine acid, and are industrially important microorganisms. . Recently, attempts have been made to breed and improve industrial microorganisms by DNA recombination technology, and recombinant DNA technology is being developed for coryneform glutamic acid-producing bacteria. For example, pp. 333 (198)
3), Proc. Of the Japan Fermentation Engineering Society Conference 1983, 283, 2
On page 84 (1983), respectively, the development of a host-vector system for Corynebacterium glutamicum, Brevibacterium
An example of breeding threonine-producing bacteria has been reported as the development of a lactofermentum host-vector system.

However, in the conventionally known coryneform glutamic acid-producing bacterium host-vector system, all use a plasmid as a vector.Therefore, in order to transfer this plasmid vector into a host,
First, this plasmid vector must be separated, and then a complicated transformation means such as a protoplast method and a calcium treatment method is required.

By the way, in Escherichia coli, a so-called cosmid, which is a recombinant plasmid having a DNA region containing a cohesive end (Cos) derived from lambda phage, has been constructed. (Fukumaki, Y., Shimada, K., Takagi, Y; Proc.Nat
l.Acad.Sci., 73 , 3238 (1976), Collins, J. Hohn, B: Proc.
Natl.Acad.Sci., 75 , 4242 (1978)) This cosmid is defined as a transfection method using a phage (here, the transduction method is defined as a phage-mediated gene transfer (Protein / Nucleic Acid Enzyme Separate Volume “Bacteria It is characterized in that it can be easily transferred to another strain from the retained strain by the phage genetic experiment method "P64 (1972)". That is, when the cosmid-retaining strain is infected with phage, it contains cosmid DNA at a certain ratio. Pseudophage particles are formed, and these particles have the activity of introducing cosmid DNA into a new host bacterium.With cosmid, the phage lysate of the donor bacterium is prepared and mixed with the recipient bacterium to infect it. It suffices to transfer the plasmid quickly and easily as compared with the case of using a conventional plasmid.

However, in the coryneform glutamic acid-producing bacterium, there is no example of such a vector that can be used for transduction, and the plasmid could not be transferred by the transduction method.

Under the above circumstances, the present inventors combine a plasmid hosting a coryneform glutamic acid-producing bacterium with a DNA fragment of a phage hosting a coryneform glutamic acid-producing bacterium to prepare a transducible complex plasmid. Was successful. That is, the present invention relates to (1) a DNA containing at least a replication origin of a plasmid that can grow in cells of coryneform glutamate-producing bacteria, and (2) a cohesive end (COS) derived from a phage that can infect coryneform glutamate-producing bacteria. Is a transducible composite vector consisting of DNA containing

This complex plasmid can be transferred into coryneform glutamate-producing bacterial cells very simply and rapidly by the transduction method, and is extremely advantageous for breeding useful strains by the recombinant DNA method.

Coryneform bacteria are aerobic, Gram-positive bacilli, non-acidic, and the Verge's Manual of Determinative Bacteriology, 8th Edition, page 599 (1974).
It is described in.

The coryneform glutamic acid-producing bacterium of the present invention is a mutant strain that loses glutamic acid productivity inducing a large amount of glutamic acid in these coryneform bacteria or derived therefrom, and examples of wild strains thereof are as follows. Something like this.

Brevibacterium divaricatum ATCC 14020 Brevibacterium saccharolyticum ATCC 1406
6 Brevibacterium Inmario Film ATCC 1406
8 Brevibacterium lactofermentum ATCC 13
869 Brevibacterium roseum ATCC 13825 Brevibacterium flavum ATCC 13826 Brevibacterium thiogenitalis ATCC 19240 Corynebacterium acetoside film ATCC 13
870 Corynebacterium acetoglutamicum ATCC 1580
6 Corynebacterium carnae ATCC 15991 Corynebacterium glutamicum ATCC 13032,1306
0 Corynebacterium Lilium ATCC 15990 Corynebacterium Melacecola ATCC 17965 Microbacterium Ammonia Philum ATCC 1535
4 Coryneform / glutamic acid-producing bacteria have mutants that have lost glutamate productivity, or amino acids such as lysine and arginine, purine nucleotides such as inosine,
Mutant strains that produce purine nucleotides such as inosine 5'-monophosphate or other products are also included.

Any plasmid may be used as a plasmid that can be propagated in the cells of these coryneform glutamate-producing bacteria. For example, pAM330, pAM286 (see JP-A-58-67699), pHM1
519 (see JP-A-58-77895), pAJ655, pAJ611, pAJ1844,
(See JP-A-58-192900), pCG1 (see JP-A-57-134500), pCG2 (see JP-A-58-35197), pCG4, pCG11 (see JP-A-57-183799), and the like.

Any phage that can grow in the cells of coryneform glutamate-producing bacteria may also be used, such as CP-
2, CP-3, CP-5, CP-7 etc. J.Gen.Appl.Microbiol., 22 1
ΦCG1 and φCG found using the phage Corynebacterium glutamicum described in 19 (1976) as a host.
2, φCG3, φCG4, φCG5, etc. (Agricultural Chemical Society of Japan 1983 Annual Meeting P332) “Fermentation and Industry” 35, 198, 294, 3
There are phages described on pages 92 and 473 (1977).

The composite plasmid of the present invention may contain all of the above plasmid DNA as a part of the composite plasmid, but it is required that it contains at least an origin of replication. Further, the above-mentioned phage may contain all of its DNA as a part of the composite plasmid of the present invention, but at least when coryneform glutamate-producing bacteria are infected by this phage, it is incorporated into the phage particle. It must contain at least the DNA region described above. In the present invention, such a DNA region is a DNA containing a cohesive end (COS) derived from a phage that can infect a coryneform glutamate-producing bacterium.

Plasmid DNA and phage DNA can be separated by a conventional method. Then, the phage DNA and the plasmid DNA are cleaved with restriction enzymes, respectively. Phage D
DNA containing a phage-derived sticky end (COS) that cleaves NA and can infect coryneform glutamate-producing bacteria
To prepare. It is known that the cohesive ends of λ phage DNA are usually single-stranded complementary ends, and the cohesive ends are paired by hydrogen bonding, but easily separated by heating and quenching. Therefore, if the DNA fragment before and after the heating and quenching operation is analyzed by agarose gel electrophoresis, the DNA fragment having a sticky end can be easily identified. DNA containing a cohesive end (COS) derived from a phage that can infect coryneform glutamate-producing bacteria can also be identified by the same method as in the case of λ phage. Once a DNA fragment having sticky ends is identified, this fragment can be extracted and purified from the agarose gel after electrophoresis by a conventional method.

As a method for ligating the thus obtained phage DNA fragment and the cleaved plasmid DNA, an ordinary method using ligase can be used.

On the other hand, using terminal transferase, deoxyadenylic acid and deoxythymidylic acid, or deoxyguanylic acid and deoxycytidylic acid were respectively added to the phage DNA fragment and the cleaved plasmid DNA, mixed, and then annealed. It is also possible to use a method of connecting by connecting.

Thus obtained phage DNA and plasmid DNA
The introduction of the ligation product with Escherichia coli K-12 into a recipient bacterium belonging to a coryneform glutamate-producing bacterium has been reported (Mandel, M. and Higa, A., J. Mo).
l.Biol., 53 .159 (1970) ) method treating recipient cells with calcium chloride to increase permeability for DNA or Bacillus,
As reported for subtilis (Duncan, CH,
Wilson, GA and Young, FE, Gene, 1 , 153 (1977)) 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 Choen, SN, Mol.
ec.Gen.Genet., 168 , 111 (1979); Bibb.MJ, Ward, JMa
nd Hopwood.OA, Nature, 274 , 398 (1978); Hinnen.A., H
icks, JBand Fink, GR, Proc.Natl.Acad.Sci.USA, 75 1
929 (1978)), it is also possible to introduce the plasmid into the DNA recipient bacterium by converting the DNA recipient bacterium into a protoplast or spheroplast that easily incorporates the plasmid DNA.

In the protoplast method, a sufficiently high frequency can be obtained by the method of Bacillus subtilis described above.
The method of incorporating DNA into protoplasts of the genus Corynebacterium or the genus Brevibacterium described in 183799 in the presence of polyethylene glycol or polyvinyl alcohol and a divalent metal ion can naturally be used. Instead of polyethylene glycol or polyvinyl alcohol, carboxymethyl cellulose, dextran,
Similar effects can be obtained by a method of promoting DNA uptake by adding FICOL, Pluronic F68 (Selva) or the like.

Selection of strains carrying a transducible recombinant plasmid can be achieved by the following method. After transformation, colonies are once formed on an appropriate medium, and then these colonies are mixed and infected with phage to lyse them to prepare a phage solution. After infecting this with a new host bacterium that does not contain a plasmid, the strain into which the plasmid has been introduced may be searched. When a drug resistance gene or the like is present on the plasmid, this trait can be selectively used, and a strain carrying the desired plasmid can be easily obtained.

Plasmid from strains carrying the desired recombinant plasmid
The method for isolating DNA can be accomplished by a conventional method such as lysing the cells by lysozyme / SDS treatment, phenol treatment, and then adding 2 volumes of ethanol to precipitate and recover the DNA.

Example (1) Preparation of F ₁ phage DNA Phage F ₁ used in the construction of a composite plasmid was newly isolated using Brevibacterium lactofermentum ATCC19869 as a host bacterium.

F ₁ phage DNA was prepared by the following method.

Brevibacterium lactofermentum ATCC13869
1 CMG medium (peptone 1 g / dl, yeast extract 1 g / dl,
Include glucose 0.5d / dl, and NaCl0.5d / dl, was inoculated into one) that was adjusted to pH 7.2, then was subjected to about 1 hour shaking culture at 30 ° C., multiplicity of infection of F ₁ phage (moi ) Was added to about 0.1, and shaking culture was continued for another 5 hours, whereby lysis by the phage was caused. The lysate was filtered through Hyflo Supercell (Junsei Kagaku) to remove cell residue, and then 10 μg / ml of deoxyribonuclease I and Ribonuclease A (Sigma) were added to the filtrate, and the filtrate was kept at 30 ° C for 20 minutes. I kept it. Pancreatin 200 is added to the filtrate.
After adding μg / ml and keeping it at 30 ℃ for 20 minutes, add sodium chloride to the final concentration of 0.5M and polyethylene glycol 6000 to the final concentration of 10%, and leave it at 4 ℃ for 1 day. , F ₁ phage particles were precipitated. 5000
After collecting the precipitate by centrifugation at rpm for 10 minutes, TE containing 1% SDS
N buffer (20 mM Tris hydrochloride, 20 mM NaCl, 1 mM EDTA (pH
8.0)) It was dissolved in 10 ml. From this, F ₁ phage DNA was extracted and purified by an ordinary phenol treatment method to finally obtain about 5 mg of DNA.

(2) Preparation of DNA Fragment Containing F ₁ Phage Cohesive End (COS) The restriction endonuclease Hind III was added to a buffer solution containing about 5 μg of F ₁ phage DNA and the DNA chain was completely cleaved at 37 ° C. for 2 hours. A part of the reaction solution was taken, heated at 70 ° C. for 10 minutes and then rapidly cooled, and subjected to 0.8% agarose gel electrophoresis simultaneously with the reaction solution which was not subjected to heat treatment. As a result of electrophoresis, Hind III
Of the F ₁ phage DNA fragments generated by the treatment, a fragment of about 2.1 kb was separated by heat treatment into fragments of about 1.6 kb and about 0.5 kb,
It was revealed that a sticky end (COS) was present in this 2.1 kb fragment. Therefore, this 2.1 kb fragment was extracted and purified from an agarose gel to obtain about 0.4 μg of DNA.

(3) Preparation of plasmid DNA As a vector, pAJ43 (deposited as Brevibacterium lactofermentum AJ11997 (FERM P-6857.FERM BP-591)) (molecular weight 3.4 megadalton) was used.

From pAJ655 (see JP-A-58-192900), pA
Created J43.

Brevibacterium lactofermentum No. 64 holding pAJ655 is a CM containing 100 μg / ml chloramphenicol
It cannot grow on G agar medium (containing peptone 10 g /, yeast extract 10 g /, glucose 5 g /, NaCl 5 g /, agar 20 g / and adjusted to pH 7.2), but this bacterium was cultured in CMG medium. , Chloramphenicol 100μg / ml in CMG liquid medium at 30 ℃, after culturing overnight at 30 ℃ CMG medium containing chloramphenicol at the same concentration, and cultivated at 30 ℃ 1-2 days One strain showing resistance to phenicol 100 μg / ml was obtained. When the chloramphenicol resistance of this strain was examined in CMG medium, it showed resistance up to 200 μg / ml.

As a result of the above, pAJ43 was prepared from the resulting chloramphenicol high-concentration resistant strain as follows. First, this strain was inoculated into 1 CMG liquid medium containing 10 μg / ml of chloramphenicol, cultured at 30 ° C. until the late logarithmic growth phase, and the cells were collected. After lysing the lysate with lysozyme and SDS by a conventional method, the supernatant was obtained by ultracentrifugation at 30,000 × g for 30 minutes. Add polyethylene glycol (final concentration 10%) to the DN
Precipitate A, concentrate it, and precipitate the precipitate with Tris-EDTA.
-Dissolved in 10 ml of NaCl buffer (pH 8.0). Treat the DNA with ribonuclease (ribonuclease 150 μg / ml
After reacting at ℃ for 30 minutes), extract with phenol, add 2 volumes of ethanol and precipitate the DNA at -20 ℃.
It was dissolved in ml of Tris-EDTA-NaCl buffer. This DN
Solution A was applied to agarose gel electrophoresis (5 cm per 1 cm of voltage gel).
The final 150 μg of pure pAJ43 plasmid DNA was fractionated by (V, 15 h).

The molecular weight of pAJ43 was determined by agarose gel electrophoresis.

The agarose gel electrophoresis was carried out by the method of PASharp et al. (Biochemistry 12, 3055 (1973)) using 0.8% gel, and gel electrophoresis was performed at a constant voltage of 5 V for 15 hours per cm of gel length. The molecular weight is Hind II, a restriction enzyme that cuts pAJ43 at one site.
I 0.5 unit was reacted with 0.5 μg of pAJ43 for 1 hour at 37 ° C, cleaved and linearized, and then the mobility was compared with that of Hind III fragment of λ phage, a molecular weight marker with known molecular weight (purchased from BRL). Calculated to be 3.4 Md.

As a result of agarose gel electrophoresis analysis of the DNA fragment after restriction enzyme digestion, pAJ43 was found to be an indispensable region for maintaining replication of pAM330 and about 1 Md containing the chloramphenicol resistance gene region of pBR325 generated by in vivo deletion from pAJ655. It was found to be a small plasmid consisting of a fragment of about 2.4 Md containing

(4) Insertion of F ₁ phage into COS-containing DNA fragment vector About 1 μg of the plasmid pAJ43 obtained in (3) was kept at 37 ° C. for 2 hours with the restriction endonuclease Hind III and completely cleaved. 2.1 kb DNA obtained in (2) after heat treatment at 65 ℃ for 10 minutes
The fragment was added, and the DNA chain ligation reaction was carried out at 10 ° C. for 24 hours with T ₄ phage-derived DNA ligase in the presence of ATP and dithiothreitol. Two volumes of ethanol was added to the reaction solution to precipitate and collect the DNA after the ligation reaction was completed.

The obtained DNA was dissolved in a small amount of TEN buffer and used for the following transformation.

(5) Transformation The protoplast transformation method was used as the transformation method. Brevibacterium lactofermentum AJ12036 (FERM P-755
9. FERM BP-734) was isolated from Brevibacterium lactofermentum ATCC 13869 strain as a streptomycin resistant strain. First, the recipient bacteria were cultured in 5 ml of CMG liquid medium until the beginning of the logarithmic growth phase, 0.6 unit / ml of penicillin G was added to the medium, and the mixture was further shake-cultured for 1.5 hours, and the cells were collected by centrifugation. 0.5M sucrose, 20mM
The cells were washed with 0.5 ml of SMMP medium (pH 6.5) consisting of maleic acid, 20 mM magnesium chloride and 3.5% Penassay broth (Difco). Then, the cells were suspended in SMMP medium containing 10 mg / ml lysozyme and protoplasted at 30 ° C. for 20 hours. 6000 ×
After centrifugation for 10 minutes at 10g, the protoplasts were washed with SMMP and resuspended in 0.5 ml SMMP. The protoplast thus obtained and about 1 μg of the DNA prepared in (4) were added to 5 mM.
After mixing in the presence of EDTA and adding polyethylene glycol to a final concentration of 30%, the mixture was allowed to stand at room temperature for 2 minutes to incorporate DNA into protoplasts. The protoplasts were washed with 1 ml of SMMP medium, resuspended in 1 ml of SMMP medium, and cultured at 30 ° C. for 2 hours for expression of traits. This culture solution was applied on a protoplast regeneration medium having a pH of 7.0. Protoplast regeneration medium is tris (hydroxymethyl) aminomethane 12 g, KCl 0.5 g, glucose 10 g, MgCl ₂ .6H ₂ O 8.1 g, CaCl ₂ .2H ₂ O 2.2 g, peptone 4 g, powdered yeast extract 4 g per 1 distilled water. Casamino acid (Difco) 1g, K
₂ HPO ₂ 0.2 g, sodium succinate 135 g, agar 8 g and chloramphenicol 3 μg / ml.

After culturing at 30 ° C for 10 days, about 1,000 chloramphenicol resistant colonies appeared.

(6) Transduction All of these colonies were scraped together, suspended in physiological saline, and a part of the colony was added to CMG medium plate 1 containing about 10 ⁵ F ₁ phage, 2% agar, and 10 μg / ml chloramphenicol. It was applied to one sheet and cultured at 30 ° C. for 1 day. Since the lysis phenomenon by F ₁ phage was confirmed on the plate, 5 ml of physiological saline was added to the plate to suspend the phage particles. 15000rpm, 1
After removing the bacterial cell residue by centrifugation for 0 minute, the viable bacteria were removed through a bacteria removal filter (pore size 0.22μ).

Brevibacterium lactofermentum AJ12036 was added to 0.1 ml of the above phage solution to about 10 ° of cells in the logarithmic growth phase, gently shaken at 30 ° C for 90 minutes, and centrifuged at 3000 rpm for 10 minutes to recover the cells. Then, it was washed twice with physiological saline. The washed cells were spread on a CMG medium plate containing 2% agar and 10 μg / ml chloramphenicol. Culturing for 2 days at 30 ° C produced 3 colonies. Each of these three strains was suspended in physiological saline, and then cultured on an agar plate together with F ₁ phage in the same manner as above to prepare a phage solution and transduce the AJ12036 strain.

As a result, no transducing ability was detected in the phage solution obtained from the strains carrying the vector pAJ43, but in the above 3 strains, chloramphenicol resistant colonies were frequently produced by the phage solutions prepared from any of the three strains.
About 10 phages obtained from 6 (FERM P-7560.FERM BP-735)
^When 10 was infected with about 10 ⁹ of the recipient strain AJ12036, about 10 ³ chloramphenicol-resistant colonies were formed. A plasmid of approximately 4.8 Md was detected in these resistant colonies and the AJ12136 strain, and this plasmid was used as the vector pAJ43 (3.4 Md).
d) and a DNA fragment (1.4 Md) containing the Cos site of F ₁ phage, which is clearly a transducible cosmid. The cosmid found from the AJ12136 strain was named pAJ667. The phage solution obtained from AJ12136 was used to introduce pAJ667 into Corynebacterium glutamicum ATCC13060 strain.

Forty ^nine chloramphenicol resistant strains were obtained when using approximately 10 ⁹ phage particles. From now on, pAJ667 plasmid DNA of 4.8 megadalton was detected and pAJ667 was transformed into Corynebacterium glutamicum ATCC1306 by transduction.
It is clear that it was introduced into 0 strain.

As a plasmid vector, pAJ1844 (JP-A-58-1)
92900), CP-23 phage (J. Gen. Appl.
Microbiol., 22 119 (1976)) was used to obtain a transducible recombinant plasmid pAJ668 in the same manner.

The F ₁ phage used in the present invention was lysogenized in the AJ12136 strain, and spontaneous induction or mitomycin C was 0.05 to 0.2 μg / m 2.
It is released into the medium by shaking culture in a CMG liquid medium containing l. If this is infected with, for example, Brevibacterium lactofermentum ATCC13869 strain, phage particles can be easily obtained in high yield. Strain Brevibacterium lactofermentum AJ1199 containing pAJ43
7 has been deposited as FERM-P6857.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12R 1:13) (C12N 15/09 C12R 1:15) C12R 1:13) (C12N 15/00 A C12R 1:15)

Claims (1)

[Claims] 1. A DNA containing at least a replication origin of a plasmid capable of proliferating in the cells of a coryneform glutamate-producing bacterium, and (2) a cohesive end (CO) derived from a phage capable of infecting a coryneform glutamate-producing bacterium.
A transducible composite vector consisting of DNA containing S).