JPS63237788A - Novel multi-copying plasmid - Google Patents

Abstract

PURPOSE:To do precise replication in the host cells by preparing plasmid pNB-701 which has about 10.2 kilobase molecular weight and a specific incision point of the restriction enzyme as shown in the restriction map. CONSTITUTION:A strain in Streptomyces violaceoniger, bearing p-NB-701, such as KSA-6233 strain is cultured. Then, the cell bodies are collected, subjected to lysis with a lysin and then RNA, protein and other contaminants are removed from the solution. The product is subjected to density gradient ultra- centrifugation of electrophoresis-agarose gel column cutting to isolate the objective pNB-701. The plasmid is multicopying and a useful vector in cloning of the objective DNA.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a novel multicopy plasmid contained in an actinomycete belonging to the genus Streptomyces and a recombinant product incorporating foreign DNA such as various drug resistance genes into this plasmid. It concerns plasmids.

[Prior art]

Many of the various physiologically active substances such as antibiotics produced by microorganisms are produced from cultures of strains belonging to the genus Streptomyces among actinomycetes, and the genus Streptomyces is positioned as a useful microorganism. In recent years, with the development of biotechnology, genetic recombination research on the genus Streptomyces has been actively conducted, but the development of plasmid vectors that can be used for this has not been sufficient, which has hindered the progress of research. In particular, so-called multicopy plasmids, which have excellent replication properties within host cells, are essential for substance production, and their development is eagerly awaited.

Actinomycete plasmids known so far include Mo,
1Ecular and General Genet
ics 185+ 223-238 (1982).

Journal or General Microb
iology 129+ 2703-2714 (198
p I J -702 (5,8 kilobase) described in 3).

Journal of General Microb
iology 132+ 2071-2078 (198
ρJ V −1 (10,8 kilobases) described in 6).

pSF588(3) described in JP-A-57-188600
, 0 megadalton), ps V H1 (12.6 kilobase) described in JP-A-58-24594, JP-A-58-24594
pSLA3 described in No.-43997 (5, 9 to 6.
pS A N181 (5,0 megadaltons) described in JP-A No. 58-157800 (5,0 megadaltons), po FOII (5,2 to 5.3 megadaltons) described in JP-A-59-74989, pOAll (10.8 kilobase) described in No. 59-143588, JP-A-59
pN Mloo (9,1 kilobase) described in No. 466088, ps N2 described in JP-A-60-207584
2 (11.5 kilobase), JP-A-60-21488
pF J 301 (13.7 kilobase) described in No. 5, ps G 5 described in JP-A-60-256384
(12.7 kilobase) and JP-A-61-124
Examples include pBTl (5,6 kilohose) described in No. 386, but it cannot be said that the copy number of the plasmid is satisfactory for industrial use.

[Problem that the invention seeks to solve]

The copy number of general actinomycete plasmids is said to be several tens of copies, and those known as multicopy plasmids include the aforementioned pI J-101 (40 to 300 copies) and pI J-702 (40 to 300 copies). 300 copies).

and p, y V -1 (150 copies), but these copy numbers cannot necessarily be called sufficient copy performance for the purpose of substance production. Therefore, the present inventors aimed at developing a new plasmid that uses Streptomyces sp. actinomycetes as a host and has multicopy properties.

[Means for solving problems]

As a result of searching for promising plasmids from the many strains belonging to the genus Streptomyces, the present inventors succeeded in selecting a strain with a specifically large amount of plasmid, isolated the plasmid of the strain, and conducted an investigation. As a result, it was confirmed that this plasmid had extremely high copying performance, leading to the completion of the present invention.

The present inventors have discovered that KSA, -6, which belongs to the genus Streptomyces,
233 strains of Streptomyces violaceoniger (St
reptomyces violaceoniger)
The plasmid isolated from the cultured bacterial cells was identified as pN.
It was named B701. pN8701 is a novel plasmid with a molecular weight of approximately 10.2 kilobases, and is a so-called multicopy plasmid that has many cleavage points for restriction enzymes that recognize six bases and has high replication ability in host cells.

In addition, in the plasmid pNB701, a general
By incorporating foreign genes through manipulation techniques, multicopy recombinant plasmids can be obtained.

Specifically, the present invention relates to plasmid pNB701, which has a molecular weight of about 10.2 kilobases and has the restriction enzyme cleavage point shown in FIG. 1, and a recombinant plasmid obtained by incorporating a foreign gene into this plasmid It is something.

The contents of the present invention will be explained in detail below, including examples.

NB701 Tree-〇Convex 2 The mycological properties of actinomycete KSA6233 harboring pNB701 are as follows.

(11 Morphology Basal hyphae exhibit a zigzag shape with good branching, and no divisions are observed. Aerial hyphae are abundantly attached to starch inorganic salt agar, tyrosine agar, yeast malt agar, oatmeal agar, etc., and spores are formed. Although the formation is good, a moist black area called hygroscopic area appears on the aerial mycelia after !tIl (14 to 21 days of culture).

Aerial hyphae are axle-branched, and the spore chain is often spiral-shaped with a compact spiral tip, and consists of 10 to 50 or more spores. The spore surface is smooth and the size is 0.
6-0.8 x 1. It has an oval shape of 0 to 1.4 microns. Special forms such as sclerotia, spore lights, and motile spores are not observed. The whole bacterial cell hydrolyzate contains LL-diaminopimelic acid.

(3) Growth status in various media The growth status after 3 weeks of culture is as shown in Table 1.

(Leaving space below next summer) Table 1 The color harmony manual color codes are shown in brackets (2) Physiological properties ■ Growth temperature range 20 to 40℃ Optimum temperature range 25 to 35℃ ■ Liquefaction of gelatin
10 ■ Hydrolysis of starch 10 ■ Coagulation of skimmed milk - peptonization 10 ■ Production of melanin-like pigment Tyrosine agar medium 10 Peptone, yeast &'N heaven medium + tryptone yeast liquid medium 10 (4) Assimilation of each carbon source Assimilable carbon source L-7 rapinose (on Britham GoFIJ-P agar)
D-ki river-su.

D-glucose, D-fructose sucrose, L-rhamnose.

Raffinose, D-Mannit.

Non-assimilable carbonized source ■-Inositol The above mycological properties obtained are reported in the International Journal.
l of Systematic Bacterto,
1ogy+ Vol. 22, pp. 364-366 (1972), KSA-6233 was identified as Streptomyces violaceoniger.
Vtolaceoniger) and there are two slight differences, namely the production of melanin-like pigments and the availability of I-inositol, but other than these, they are considered to be the most closely related.

Therefore, this strain is Streptomyces violaceoniger K.
S A-6233 (Streptomyces vi
olaceonigerKSA-6233).

In addition, this strain, Streptomyces violaceoniger K.
S A-6233 has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiological Research Institute Deposit No. 9233.

"Σ1 sword lΔ" Keji describes the preparation method of multi-copy plasmid pNB701. Belongs to Streptomyces violaceoniger p.
A bacterial strain containing NB701, such as the KSA-6233 strain, is cultured, the bacterial cells are collected, lysed with a lytic enzyme, etc., and contaminants such as RNA and proteins are removed from the lysate, followed by density gradient ultracentrifugation or electrophoresis. The target plasmid pNB701 can be isolated by a separation operation such as an agarose gel excision method. This operation is a standard method for collecting plasmids from microorganisms. Also,
Nucleic Ac1ds Research,
9, No. 20, pp. 5493-5504 (1981), it is also possible to employ a simple method of insolubilizing and recovering double-stranded DNA by treatment with spermine. The details of each operation for preparing ρNB701 will be described below.

(1) Cultivation of KSA-6233 strain A general liquid culture method for actinomycetes is applied to the cultivation of Mokkan. That is, carbohydrates such as glucose and sucrose, peptone, yeast extract,
Organic nitrogen sources such as meat extract or inorganic nitrogen sources such as ammonium sulfate, urea, and sodium nitrate are used alone or in combination, and potassium phosphate salts, magnesium sulfate, sodium chloride, calcium carbonate, and ferrous sulfate are used. Culture in a liquid medium supplemented with inorganic salts such as If necessary, it is also effective to add amino acids such as glycine to increase the sensitivity of the lytic enzyme. A chemically defined medium such as TSBG medium or GPYG medium may also be used. Culture is 25 to 35
Preferably, the reaction is carried out at a temperature of 12 to 48 hours.

The cultured bacterial cells are collected by centrifugation and washed.

(2) Preparation of crude plasmid The washed bacterial cells were made into protoplasts using a lytic enzyme such as lysozyme, and sodium N-lauroylsarcosinate (S
After lysis by adding LS) or sodium lauryl sulfate (SDS), etc., remove chromosomal DNA, residual bacterial components, proteins, etc. by operations such as alkaline denaturation, salting out, and phenol denaturation, and then add cold alcohol to form a precipitate. is recovered as a crude plasmid. RN for removal of coexisting RNA
It is also possible to add an ase reaction during the recovery process.

(Isolation of 31pNB701 The target plasmid pNB701 can be isolated and recovered from the crude plasmid described above by DNA fractionation methods such as agarose electrophoresis or density gradient ultracentrifugation using cesium chloride.Generally, a trace amount is recovered. The former is used for sterile recovery, and the latter is used for star recovery, but DNA fractionation means such as preparative liquid chromatography may also be used, without being limited thereto.

"ΣB701Δ Notification" Analysis of the obtained pNB701 can be performed according to the conventional method of creating a restriction enzyme breakpoint map. That is, the number of cleavage sites for various restriction enzymes and the size of the DNA fragments can be determined from agarose gel electrophoresis analysis of DNA fragments obtained by digestion of pNB701 with restriction enzymes. Furthermore, by combining analysis information on the size of DNA fragments obtained from combinations of restriction enzymes, it is possible to determine a restriction enzyme breakpoint map. The copy number can also be determined using the conventional method by staining an agarose electrophoresis gel of the lysate with ethidium bromide, developing the color by irradiating it with ultraviolet rays, transferring it to a negative film, measuring the same film with a densitometer, and comparing it with the chromosomal DNA fi. This can be done by Furthermore, chromosome D
NA is calculated based on 104 km base.

The analysis results obtained using these methods are summarized as follows.

('') Size and molecular weight of plasmid pNB701: approximately 10.
It is a 2 kilobase deoxyribonucleic acid.

(2) Number of restriction enzyme cleavage sites Each restriction enzyme has the following number of cleavage sites.

Bcl I 2M1u I
2Stu 1
1 Pvu II 4H4nd m
I Bgl II 2 Apa I 2 (3) Restriction enzyme breakpoint map As shown in FIG.

(4) Copy number Next, the multicopy nature of the plasmid of the present invention will be demonstrated using test results.

pNB701 400 to 750 coby pl
J702 (control) 50 to 100 copies and GINB
It is possible to insert a foreign gene into 701 and create a recombinant plasmid using general NA manipulation techniques. That is, a part of the plasmid is opened using the above-mentioned restriction enzymes, and if necessary, it is treated with alkaline phosphatase to suppress self-repair, and then foreign DNA such as a drug resistance gene is repaired using ligase. By doing this, foreign DNA can be integrated or a combinant plasmid can be obtained.

These recombinant plasmids can be selected using the function of the foreign DNA as a marker. If the plasmid is cut into multiple parts during restriction enzyme treatment, the DNA of pN8701
A recombinant plasmid may be obtained in which foreign DNA is integrated into a portion of the A fragment. These recombinant plasmids depend on pNB701 for their replication function and inherit the multicopy property.

Example 1 Preparation of pNB701 Streptomyces violaceoniger KSA6233
Plasmid pNB701 was prepared from the following method.

KSA6233 was prepared using 5d TSBG medium (3% 7ry
pticSoy Broth (Dirco), 1%
A shaking culture was performed in a 21-mark test tube containing glucose (glucose) and glass peas, and the seeds were cultured at 28°C for 1 to 2 days. The main culture was carried out using a 500 rnl Erlenmeyer flask in 100% TSBG medium supplemented with 0.2% glycine.
The shaking culture of d was performed at 28°C for 36 hours. The culture solution was applied to a refrigerated centrifugal bone N machine to collect bacterial cells (wet weight: about 3 g), and then added to pH 8,07 EG buffer (25 mM Tris-H
The bacterial cells were washed with Cl, 16mM EDTA-2Na) and 50mM glucose. After suspending the bacterial cells in 10 ml of TEG buffer again, 2 d of Norizozyme solution (30 μ/-) was added and kept at 37° C. to form protoplasts. Protoplasts were prepared using 2 ml of 25% SDS solution and 0.36 N-N
10 ml of aOH solution was added to lyse the bacteria. Next, 6.5rnl of 5M lithium chloride was added to this lysate for salting out, and the supernatant obtained by centrifugation was further denatured and extracted with phenol and chloroform to remove impurities, and then cold ethanol was added. In addition, a precipitate was collected. 0 sediment
．． 10 μβ of a 1% RN asc solution was added to a plasmid solution dissolved in 5 mff of TE buffer, and the mixture was reacted at 37° C. for 15 minutes to degrade the RNA. After treatment with phenol and chloroform, the RN asc was denatured and removed. In order to recover the desired plasmid, the remaining plasmid solution was applied to a 0.7% agarose gel and subjected to slab electrophoresis, and the electrophoresis gel was stained with ethidium bromide and observed under UV light. The pNB701 band was cut out with a cutter. This gel section was sealed in a dialysis membrane along with a buffer solution and submerged in a submarine-type electrophoresis tank for electrophoresis.The plasmid was eluted into the buffer solution, impurities were removed by treatment with phenol and chloroform, and then collected by adding cold ethanol. .

The obtained plasmid weighed 35 to 40 μg and was electrophoretically confirmed to be single.

〔Effect of the invention〕

As described above, the novel plasmid pNB7 of the present invention
01 is a multicopy plasmid that replicates at high frequency in host cells of the genus Streptomyces, and is useful for cloning of target DNA in the genus Streptomyces,
It is expected to play an extremely important role as a vector for producing substances using recombinants of substance-producing genes and plasmids.

Furthermore, the recombinant plasmid of the present invention obtained from the plasmid pNB701 is the plasmid pNB701.
It maintains the multi-copy property of , and is extremely useful from the viewpoint of material productivity.

[Brief explanation of drawings]

FIG. 1 is a restriction enzyme cleavage map of pNB701. Applicant Agrochemical Biotechnology Development Technology Research Association

Claims (1)

[Scope of Claims] 1. Plasmid pNB-701, which has a molecular weight of about 10.2 kilobases and has a restriction enzyme cleavage point shown in the restriction enzyme map below. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ 2. A recombinant plasmid made by incorporating a foreign gene into plasmid pNB-701. 3. The recombinant plasmid according to claim 2, wherein the foreign gene is a drug resistance gene.