JPH0321153B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to novel actinobacterial plasmids useful for recombinant DNA genetic engineering. In recent years, various genes have been developed using genetic manipulation technology.
Analyzing the structure and function of DNA,
A specific useful structural gene is incorporated into a vector to create a heterologous DNA, which is then taken up into a host cell system or other microbial cell system to produce, for example, animal hormones such as insulin and somatostatin, antibiotics, enzymes, etc. Various attempts to produce useful metabolic products are being researched and developed. These recombinations
Many DNA techniques use Eschrichia coli as the host cell. On the other hand, actinomycetes, especially Streptomyces
The genus Actinomycetes is well known as a bacterial group that produces a wide variety of secondary metabolites including antibiotics, and it has recently been revealed that plasmid genes are involved in antibiotic production in these actinomycetes. Research on plasmids in actinomycetes is rapidly progressing. The present invention provides a new plasmid derived from this actinobacterium. The method for producing the plasmid of the present invention will be described in detail below. The plasmid of the present invention was newly isolated from soil in Ikeda Town, Tokushima Prefecture, and was
It can be obtained from a microorganism named OFR1290 (Streptomyces SP OFR1290). the microorganism
OFR1290 strain exhibits the following mycological properties. () Morphological characteristics This strain OFR1290 was grown on various agar plates at 28℃ for 2 hours.
When cultured for a week, the aerial mycelia form simple branches, and the spore chains form a spiral pattern with 2 to 3 turns or several turns. Depending on the culture medium, a hook or open loop shape may be observed. More than 10 spores form a chain, and the shape is oval to cylindrical and 0.6
It has a size of ~0.9 x 1.2 ~ 1.5 μm. The surface structure of the spores is thorn-like or hair-like. Flagellated spores and sporangia are not observed. () Growth status in various media The growth status in various media is shown in Table 1 below (28
℃, after 2 weeks of culture). For descriptions of color tones, please refer to the Color Harmony Manual.
Mannual) [Container Corporation of America Chicago
of America, Chicago)].

【table】

[Table] () Physiological properties Growth temperature range 9-38℃ (optimum growth temperature 33-36℃) Growth PH range 5-10℃ (optimum growth PH 6-7) Liquefaction of gelatin (glucose-peptone-gelatin medium Above) Positive Hydrolysis of starch (on starch/inorganic salt agar) Positive Coagulation/peptonization of skim milk No coagulation. Peptonize. Production of melanin-like pigment (tyrosine agar medium,
(on peptone-yeast-iron agar medium and in tryptone-yeast extract medium) Negative Nitrate reducing action Positive cellulose decomposition ability Negative ( ) Availability of carbon source (on Pridham-Godlieb agar medium) L-arabinose + D-xylose + D-gululose + D-Fructose ± Sucrose - Inositol - L-Rhamnose ++ Raffinose - D-Mannite + However, ++ means frequently used, + means used, ±
indicates that the use is questionable, and - indicates that it is not used. () Diaminopimelic acid in the cell wall LL-diaminopimelic acid To summarize the above, this bacterial strain OFR1290 belongs to the genus Streptomyces, and according to the method of the International Streptomyces Project (abbreviation ISP), spore-forming hyphae The form belongs to the section Spirales,
The spore surface is spiny or hair-like, and the color of the mature aerial mycelium is gray (Cray color series). The color of the basal hyphae is colorless to yellow to light brown, and the color of the underside is light yellow to light brown. Melanin-like pigments are not produced, and the only other soluble pigments are yellow pigments that are observed on yeast/malt agar medium or peptone/yeast/iron agar medium. As carbon sources, L-rhamnose, L-arabinose, D-xylose, D-glucose, and D-mannite are used, and sucrose, inositol, and raffinose are not used. The above properties and other properties are described in “Birzee’s Manual of Determinative
Bacteriology (Berqey's Manual of
Determinative Bacteriology)” 8th edition (1974
The
Actinomycetes)” Volume 2 (1961) and EB
International Journal of Systematic Bacteriology, ISP report by EBShirting and D. Gottieb.
of Systematic Bacteriology) Volume 18 No. 69-189
(1968), Vol. 18, pp. 279-392 (1968),
Vol. 19, pp. 391-512 (1969) and Vol. 22.
A search on pages 265-394 (1972) revealed that there were no known actinobacteria, and Streptomyces virideiviolaceus was found as a related bacterium.
viridiviolaceus) [Virsey's Manual of Determinative Bacteriology, 8th edition, pp. 782, 785 (1974), International Journal of Systematic Bacteriology, Vol. 22, No. 368 ~370 pages (1972
)]. In other words, the morphology of the spore-forming hyphae of this bacterial strain OFR1290 belongs to the section spirals, and the spore surface is spiny or hair-like, it adheres to gray aerial hyphae, and it does not produce melanin-like pigments. It is very similar to Mrs. Viridabiiolaseus. but,
While Streptomyces viridiviolaceus exhibits the characteristic red underside and soluble pigments, the present strain OFR1290 does not exhibit these. Therefore, the present inventor identified this bacterial strain OFR1290 as a new species, and Streptomyces sp. OFR1290
(Streptomyces SP OFR-1290) to distinguish it from known strains. The OFR1290 strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology.
It has been deposited as No. 6526 (FERMP-6526). The method of the present invention includes actinomycetes carrying the above-mentioned OFR1290 strain or other plasmids of the present invention, such as
It is carried out according to the usual method [J. Antibiotics, 33 , 88 (1980)] using an artificially altered strain of OFR1290. That is, the method basically involves culturing the above-mentioned microorganisms in an appropriate medium, lysing the collected cultured bacteria according to a conventional method, and then extracting the desired plasmid from the lysate by conventional physicochemical means. This is done by isolating. Cultivation of the microorganisms mentioned above is carried out in media containing conventional nutrients and additives. Examples of nitrogen sources commonly used as a culture medium include soybean flour, cottonseed flour, corn syrup, yeast extract, dried yeast, meat extract, casein hydrolyzate, etc. Carbon sources include, for example, glucose, glycerin, Examples include maltose, starch, lactose, and molasses. Examples of additives added to the medium include inorganic salts such as calcium carbonate, sodium chloride, magnesium sulfate, and phosphoric acid.
It may contain a small amount of metal salts such as manganese and zinc. Specific examples of preferred media are as shown in Examples below. Cultivation can be carried out in a normal aqueous medium containing the above-mentioned culture medium by surface culture or deep aeration agitation culture, but it is preferable to perform deep aeration agitation culture. The culture conditions are not particularly limited, and normal culture conditions for actinobacteria are employed. Specifically, for example, under normal aeration conditions, the liquid pH is PH6.0 to 8.0, preferably PH6.5 to 7.5, and the doubling temperature is 15 to 37°C, preferably 28 to 36°C, usually for 2 to 4 days. It can be carried out advantageously. After culturing, the cultured bacterial cells are collected according to conventional methods, such as centrifugation, and the lysis of the bacterial cells can also be carried out according to conventional methods, using various conventional physicochemical means. As a specific bacteriolysis method, for example, after homogenizing the above-mentioned bacterial cells in an appropriate buffer solution, the bacterial cells obtained by centrifuging the cells,
This is done by treatment with a solubilizing agent in an appropriate buffer. The solubilizers used include various enzymes that are generally known to be able to solubilize actinomycetes, such as lysozyme and proteinase K (Boehringer Mannheim Yamauchi Co., Ltd.) as cell wall lytic agents, and various surfactants such as: Sodium dodecyl sulfate (SDS), Triton-X100 (Wako Pure Chemical Industries, Ltd.), Sarcosyl (sodium lauryl sarcosinate), Brij58
(Wako Pure Chemical Industries, Ltd.), etc., and they may be used in appropriate combinations. Physicochemical means for isolating the desired plasmid from the lysate thus obtained include, for example, centrifugation (density gradient method), electrophoresis,
An appropriate combination of conventional separation methods based on the physical and chemical properties of the desired plasmid to be isolated, such as a treatment method using a precipitant such as polyethylene glycol or ethanol, may be employed. Prior to such isolation operations, enzymatic treatment with proteolytic enzymes, RNA degrading enzymes, etc., salting out with SDS-NaCl, etc., extraction with phenol, etc. can be performed according to conventional methods. More preferably, the isolation of said plasmid comprises
First, the above-mentioned lysate was centrifuged to obtain a supernatant (cleared lysate) containing the desired plasmid, which was then deproteinized by a salting-out method. Mixed due to proteolytic enzyme treatment such as
RNA and protein are degraded, then a precipitant such as polyethylene glycol or ethanol is added thereto and centrifuged to precipitate the desired plasmid, which is then subjected to, for example, cesium chloride equilibrium density gradient centrifugation. This is carried out by isolating and purifying the desired plasmid, thus obtaining the plasmid pOF041 of the present invention. Details of this method are as shown in the examples described later. The plasmid pOF041 of the present invention is characterized based on the points obtained from Streptomyces sp. OFR1290 as described above and the results of the following tests. 1. Measurement of Molecular Weight by Electrophoresis Plasmid pOF041 of the present invention was electrophoresed using E-buffer having the following composition, and its molecular weight was measured as follows. <E-buffer composition> Tris [tris(hydroxymethyl)aminomethane] 40mM Sodium acetate 20mM EDTA・2Na 2mM PH=7.4 In other words, various known plasmid DNAs that serve as molecular weight markers were used as controls; ) and the logarithms of their molecular weights to prepare a standard curve in advance, and perform the same test to determine whether the plasmid of the present invention
The molecular weight of the plasmid was determined by determining the mobility of pOF041 and plotting its logarithm on the above standard curve. All electrophoresis was performed using a horizontal device (Wakenyaku, TWF-1 model).
Detection of each plasmid DNA was carried out on 0.7-1.0% agarose (Dotite, type, manufactured by Dojindo Chemical Co., Ltd.) containing 1 μq/ml of ethidium bromide (EtBr) in E-buffer with the above composition at 80-100 V.
After 4 to 6 hours of electrophoresis, it was determined by ultraviolet irradiation. Also, plasmid DNA used as a molecular weight marker
and its molecular weight are as follows. pBR322 2.7×10 ⁶ Daltons pBR328 (Pellinger Mannheim)
3.2×10 ⁶ Daltons Col El DNA (Takara Shuzo) 4.2×10 ⁶ Daltons RSF1010 5.5×10 ⁶ Daltons pN2DNA 6.4×10 ⁶ Daltons The molecular weight of the pOF041 of the present invention determined as above was 2.85 megadaltons. 2. Molecular weight measurement using an electron microscope A sample was prepared according to Yamagishi's method [1975, published by Tokyo Kagaku Dojinsha, "Biochemical Experimental Methods," Vol. 2, Chemistry of the Acid, p. 348] and used as a control sample.
pM2DNA (molecular weight 6.4 megadaltons) was examined and photographed using a transmission electron microscope (Hitachi H-500 electron microscope), and the length was measured using a Kirbymeter from the positive image (enlarged 5 to 10 times). was measured, and the molecular weight of the plasmid pOF041 of the present invention was calculated based on that of the control sample. As a result, the plasmid has a molecular weight of approximately 2.9 megadaltons,
It was almost the same as that obtained by electrophoresis in 1 above. 3 Reaction solution for cleavage of plasmid DNA with restriction enzymes (according to the manual of Takara Shuzo Co., Ltd.)
To 50 μl, 1 μg of the plasmid pOF041 of the present invention and an excess amount of each restriction enzyme solution (both manufactured by Takara Shuzo Co., Ltd.) were added and reacted at 37°C for 1 hour to perform a plasmid DNA cleavage test. I did this.
In addition, a double digestion test was conducted as follows using two or more different restriction enzyme solutions. That is, first, an active restriction enzyme is reacted under conditions of low salt concentration, and then the reaction is stopped by heat treatment at 65°C for 5 minutes.
Then, after increasing the salt concentration so that the other restriction enzyme can react, the other restriction enzyme is added and reacted in the same manner, or after the reaction using one restriction enzyme,
Phenol treatment was performed to precipitate the DNA in ethanol, and after dissolving this precipitate under conditions that allowed the other restriction enzyme to react, the other restriction enzyme was added and reacted in the same manner. After the cleavage reaction with each restriction enzyme, the reaction solution was subjected to agarose gel electrophoresis in the same manner as in 1 above to detect the cut plasmid DNA (molecular weight measurement). In addition, in this molecular weight measurement
DNA molecular weight markers (Hind digest of λDNA, Boehringer
Mannheim GmbH (Boehringer Mannhein
GmbH), Piochemica) and a Hae digest of Bacteriophage φ×174RF DNA (New England Bioland) were used. As a result of the above cleavage test, the plasmid of the present invention
The number of cuts of pOF041 by each restriction enzyme is as follows.
It was as shown in the table. Table 2 Restriction enzyme cleavage number BamH 0 Bql 0 EcoR 0 Hind 0 Hpa 0 Kpn 3 Mlu 2 Pst 0 Pvu 0 Sal 1 Sma 1 Xho 0 From the above table, the plasmid pOR041 of the present invention has Sal
, and Sma are cut at one place, Mlu is cut at two places, and Kpn is cut at three places. Furthermore, the results of a double digestion test conducted using two of the above-mentioned restriction enzymes in appropriate combinations revealed that the plasmid pOR041 of the present invention exhibits the following restriction enzyme map. As mentioned above, according to the present invention, the origin of Streptomyces sp.
A new plasmid is established with a size of 2.9 Md and a structure specified by the restriction enzyme cleavage map described above. The plasmid pOF041 established according to the present invention is
In the field of recombinant DNA genetic engineering, for example, a desired gene can be incorporated into a plasmid to create a recombinant plasmid, and such a recombinant plasmid can be transformed into a suitable host for cloning in DNA research. It is useful as a vector. The above method is well known in the art, and for example, the plasmid of the present invention can be cleaved with restriction enzymes such as Sal, Sma, Mlu, etc. at specific limited positions depending on the type of restriction enzyme. In addition, the flocculent DNA molecules (floc-like vector) obtained by this cleavage can be combined with other non-vector DNA molecules cleaved in the same way, as well as known
Covalently linked by DNA ligase into a single DNA
A ring may be formed. As other non-vector DNA molecules mentioned above, desired genetic elements such as genes encoding various animal hormones such as somatostatin and insulin, enzymes, and useful metabolites such as antibiotics can be used. In particular, the plasmid of the present invention can contain genetic information from actinomycetes, such as information regarding the production of useful antibiotics, such as genetic information for the entire biosynthetic pathway of the antibiotic, genetic information regulating the biosynthesis of the antibiotic, and genetic information for the production of the above-mentioned antibiotics. It is suitable for cloning genetic information about enzymes that determine the rate of synthesis. The recombinant plasmid containing the desired genetic element obtained above can also be transformed by conventional methods, such as transformation techniques typically using protoplasts, for expression of the genetic element. It can be introduced into a suitable host organism, thereby providing, for example, a host organism with the ability to produce a useful antibiotic, and by culturing the organism, it becomes possible to collect the useful antibiotic. Although the plasmid pOF041 of the present invention was not accepted for deposit at the Institute of Microbial Technology of the Agency of Industrial Science and Technology, it has been preserved by the present inventors in a state where it can be distributed at all times. In order to explain the present invention in more detail, an example of the production of the plasmid pOF041 of the present invention will be given below as an example. Example 1 Streptomyces sp. OFR1290
(Streptomyces SP OFR1290) spores in 20ml
GGCY medium (glucose 1.0%, glycine 0.1%, casamino acids (manufactured by Difco) 0.4%, yeast extract (manufactured by Difco) 0.05%, magnesium sulfate 7
Water salt 0.1%, calcium chloride dihydrate 0.01%, potassium dihydrogen phosphate 0.2%, disodium monohydrogen phosphate dodecahydrate 0.8%, trace metal solution (ferrous sulfate 7% per 100ml deionized water) water salt 0.1g, manganese chloride tetrahydrate 0.1g, zinc sulfate heptahydrate 0.1g)
4 ml/) was inoculated into a 100 ml Erlenmeyer flask, and cultured at 30°C for 2 days in a rotary shaking incubator (200 rpm). This was used as a seed culture. %, glycine 0.1%, casamino acid (manufactured by Difco) 0.4%,
Magnesium sulfate / heptahydrate 0.1%, calcium chloride / dihydrogen 0.01%, potassium dihydrogen phosphate 0.2%,
Inoculate 1 ml of the seed culture into a 500 ml Erlenmeyer flask containing 0.8% disodium monohydrogen phosphate dodecahydrate and 4 ml of trace metal solution, and incubate at 30℃ for 2 hours.
Culture in a rotating shaker incubator (150 rpm) for days. After culturing, the mycelium was centrifuged at low speed (4700 x g, 20 min, 4
Collect at ℃). For about 1ml of this wet bacterial body, 10ml
2x TES buffer (TES: Tris (hydroxymethylaminomethane) 25mM, EDTA disodium salt 25mM, NaCl 25mM), homogenized with a Polytron (Kinematica, Switzerland), and centrifuged (7200xg, Collect mycelium (10 min, 4°C), suspend it in 20 ml of 0.1N ammonia water containing 10mM MEDTA disodium salt,
Leave at 37°C for 20 minutes. Then, centrifuge at 7200 x g.
After collecting bacteria for 10 min at 4°C, suspend them in 19 ml of 2x TES buffer and add to 0.25 M Tris monohydrochloric acid (PH8.0).
Lysozyme solution dissolved to 20mg/ml
Add 2.2 ml and leave at 37°C for 1.5 hours. and 10%
Add 2.6 ml of SDS, let stand at room temperature for 20 minutes, centrifuge (34000 x g, 30 minutes, 4°C), and separate the supernatant.
Add 0.25 volume of 5M NaCl to this supernatant and leave it at 0°C overnight. After centrifugation (10,000 x g, 20 minutes, 4°C),
Separate the supernatant, add 10 mg/ml ribonuclease A to it to a concentration of 40 μg/ml, and incubate at 37°C.
After incubating for 30 minutes, add 100μ of 10mg/ml pronase E.
Add the solution to a concentration of g/ml, and further react at 37°C for 30 minutes. Then polyethylene glycol 6000
Add to a concentration of 10%, leave at 4°C for at least 4 hours, and centrifuge (650 x g, 20 minutes, 4°C). Transfer the precipitate to TE buffer (10mM Tris monohydrochloride, 1mM
Dissolve in EDT A2 sodium, PH8.0),
This is mixed with cesium chloride and ethidium bromide to a density of 1.57-1.58. This solution is subjected to equilibrium density gradient centrifugation (86000 x g/40 h, 15°C). After centrifugation, the band of the covalently bound closed circular plasmid DNA portion was fractionated under long-wavelength ultraviolet irradiation, ethidium bromide was removed with isopropanol saturated with cesium chloride, and the aqueous phase was dialyzed against TE buffer at 4°C to remove the essence. Obtain a completely pure plasmid pOF041.

Claims (1)

[Claims] 1 A plasmid originating from actiobacteria, with a molecular weight of approx.
A plasmid characterized by being 2.8 to 2.9 megadaltons and having the following restriction enzyme cleavage map:
pOF041.