KR890004028B1 - Cloning vector for nocardia sp. - Google Patents

Abstract

The method of preparing recombinant cloning vector for Nocarding sp. comprises: (a) culturing N mediterranei in Yu medium and centrifuging culture broth; (b) washing the pellet with buffer solution containing 24mM tris-cl, 10mM EDTA-Na2 and 50mM Glucose; (c) suspending the pellet in 7ml of buffer solution and adding lysozyme to the suspension; (d) mixing 1.4ml of 25% SDS and 7ml of 0.36N NaOH with above solution; (e) centrifuging the solution and washing the obtained DNA with 20ml of 70% ethylalcohol; (f) treating the DNA with restriction enzyme; (g) analyzing DNA fragments by agarose gel electrophoresis.

Description

Method for producing recombinant cloning vector suitable for transformation of actinomycetes host

Figure 1 shows the restriction enzyme cleavage map of prasmid DNA.

2 is a schematic diagram of a method for preparing a recombinant cloning vector.

Figure 3 is a photograph showing the comparison of restriction distribution on the correlation between the plasmid DNA and recombinant cloning vector.

4 is a restriction enzyme cleavage map of the recombinant cloning vector.

The present invention is to isolate the plasmid DNA of Nocardia mediterranei (Nocardia mediterranei) in order to establish the transformation system of actinomycetes to prepare a recombinant cloning vector (recombinant cloning vecoer) suitable for transformation of actinomycetes host The present invention relates to a method for preparing a recombinant cloning vector that autonomously replicates in Streptomyces lividans.

Actinomycetes are more than 70% of the 4,000 antibiotics discovered so far, which have been used industrially. The bacteria that have been industrially used produce artificial mutations in wild strains isolated from the soil. Genetic recombination technology, which has been bred and developed in the 1970s, has been able to transform newly by introducing foreign DNA with properties not originally possessed by living organisms.

In order to improve productivity by breeding certain antibiotic-producing strains using genetic recombination technology, a host vector system must be developed that can deliver desired genes to host cells and keep them stable.

The development of the actinomycet's plasmid vector has been described by Hopwood et al., J. Bacteriology, 121 (2), 416-, which isolated SCP1 and SCP2, the plasmids of Streptomyces coelicoler A3-2. 421 (1975), Thompson et al. [Thompson et al., Nature., 286 (31), 525-527 (1980)] have reported neomycin resistance of Streptomyces fradiae. The genes (neomycin phosphotransferase and neomycin acetyltransferase), as well as [Thompson et al., J. Bacteriology, 151, 668-677 (1982)], represent a thiostrepton resistant gene (23s r) of Streptomyces azureus. -RNA pentose methylase (Thompson et al., Gene, 20, 51-62 (1982)) was cloned into a plasmid vector by the biomycin phosphotransferase of Streptomyces vinaceus. Developed.

Keiser et al., Mol. Gen. Genet., 185,223-238 (1982) developed a plasmid pIJ 101 with a fork-like function, a large number of copies, and a large host area.

Katz et al., J. Gen. Microbiol., 129, 2714 (1983) improved pIJ 101 and developed a highly practical recombinant cloning vector pIJ 702. This pIJ 702 has a melanin pigmentation gene and thiostrapton resistance gene.

Nocardia mediterranei is a actinomycete that produces rifamycin. Rifamycin has a strong bactericidal effect on gland-negative and benign bacteria, and is widely used as an anti-tuberculosis drug. See Sensi et al., Prog Industr. Microbiol., 6,21-60 (1967).

The bacterium has similar morphological and physiological characteristics as it was originally classified as Streptomyces, and according to Schupp et al., J. Bacteriology, 121, 1, 128-136 (1975), It has been reported to be similar to Myces Selicolor (Streptomyces coelicolor) A3-2.

A general review of this has been reported by Moretti et al., Plasmid, 14, 126-133 (1985). It was isolated from mycelium grown in solid medium, but not from mycelium grown in liquid medium, and it was reported that the plasmid had no replication origin and failed to produce recombinant cloning vector. It is.

Of particular interest and value of the present invention is the isolation of the psj 702-derived thiostrepton resistent gene by separating the Psemid of Nocardia mediterranean from the cells grown in a liquid medium and the recombinant cloning made therefrom. A vector is used for transforming an actinomycetes host.

The novel prasmids (or vectors, these terms may be used interchangeably herein) described in the present invention are those present in actinomycetes, in particular Streptomyces lividans, that they amplify the presence of bacterial relicor, Streptomyces And selective genetic markers that detect and act in Seth revidans.

In general, for the purposes of the present invention, the generation of a fusion vector is directed to a hybrid vector, for example a physiologically heterologous microorganism (strains other than Nocardia mediterranean), preferably a vector that acts on Streptomyces lividans. It is based on manufacture.

Plasmid pCK 101 was obtained from the Nocardia mediterranean ATCC 31066 and is about 22 kb in size.

Of the five fragments resulting from partial decomposition of plasmid pCK 202 pCk 101 with BcII, 1kb of thiostraptone was purified from Low melting agarose gel by completely dissolving 7.7kb and pIJ 702 with BcIi. Resistance genes were cloned to prepare.

The prepared recombinant cloning vector pCK 202 transforms Streptomyces lividans JI 1326.

Figure 1 shows the restriction enzyme cleavage map of pCK 101, pCK 101 is one recognition site in the restriction enzymes ClaI, SstI, EcoRI, EcoRV 2 sites, BamHI, BglII, KpnI 4 sites, BclI, PstI, BstE II 5 In some places, Sma I, AccI 6 places, SalI, Pvu II, XhoI 8 or more places, Hind III, HpaI, SphI does not represent a recognition site.

The result confirmed by each restriction enzyme was about 22 kb in size and the restriction enzyme digestion map was prepared by partial digestion and double digesion.

Figure 2 relates to the preparation flow chart of pCK 202, with partial melting of pCK 101 to BclI, partial decomposition of PiJ 702 to BclI, partial decomposition of PIJ 702 to BclI and complete digesion of PIJ 702 to BclI Thiostrepton resistance genes were purified from agarose gels. pCK 101 and thiostrepton resistance genes were bound at a ratio of 7: 1 using T4 DNA ligase.

FIG. 3 shows the restriction digestion of pCK 101 and pCK 202, wherein the agarose gel is BclI treated with X Hind II size standard in the left lane and a) pCK 101 respectively c) pCK 202 d) pCK 202 BclI It contains what was processed.

Figure 4 shows the restriction enzyme cleavage map of pCK 202, pCK 202 has a thiostrepton resistance gene, and in other parts it has one recognition site in Xhol, SlI, two PstI, three Bgl II, EcoRI, BamHI has no recognition site. The size is about 8.7 kb.

Microorganisms used in the present invention are nocardia mediteranai, Streptomyces lividans and nocardia mediteranai ATCC 31066 [Baxter et al., J. Antibiotics. 31, 949-951 (1978), and Streptomyces lividans JI 1326 (KCTC 1159) and Streptomyces lividans JI 1326 (harbor pCK 202) (KFCC-10383).

Streptomyces lividans JI 1326 can be transformed with high frequency and are useful for this purpose. The following microorganisms are used in the present invention.

Nocardia Mediterranei ATCC 31066 (KFCC 11481)

Streptomyces lividans JI 1326 (KCTC 1159)

Streptomyces lividans JI 1326 (harborpCK202) (KFCC 10383)

EXAMPLE

Substances and Methods

Most restriction enzymes, including ClaI, sstI, salI, EcoRI, are purchased from Bethesda Research Laboratories (BRL). T4 DNA ligase and bacterial alkaline phosphatase were purchased from New England Biolab (NEB).

Restriction was analyzed by electrophoresis of 0.8% and 1.2% agarose gelosis using Tris-borate-EDTA supernatant. (Manistis et el., "Molecular cloning", Cold Spring Laboratory, N.Y., 1982)

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The medium of Nocardia mediterranean is YM containing 0.7% malk extract, 0.3% yeast extract and 0.3% glucose.

The medium of Streptomyces lividans is TSB containing 3% of Lipton Soya Broth. To detect pCK202, thiostrepton was aseptically added to TSB medium to a final concentration of 5 ug / ml.

Isolation of pCK101

The cells were recovered by centrifugation of 50 ml of the culture cultured in the YM medium to the resting period, washed with 20 ml of buffer I (25 mM Tris-Cl, 10 mM EDTA-Na2, 50 mM Glucose), and then suspended again in 7 ml of buffer I, followed by lysozyme. (Final concentration 2mg / ml) is added and treated at 37C for 1 hour. Add 1.4 ml of 25% SDS solution and 7 ml of 0.36 N NaOH solution, and mix vigorously.

Add 2.1 ml of 1.5M Tris-Cl solution, stir slowly, add 5M Nacl 4.5, mix strongly, and leave to stand in ice water for 30 minutes.

Transfer the supernatant by centrifugation, add 20 ml of phenol solution and mix strongly.

The supernatant was separated again by centrifugation, and then, 2 ml of 3.3 M sodium acetate solution and 13 ml of isopropyl alcohol were added in this order, and left in ice water for 1 hour.

DNA was recovered by centrifugation, washed with 20 ml of 70% ethyl alcohol, dried on a vacuum evaporator, and dissolved in 7 ml of Buffer II (50 mM Tris-Cl, 10 mM EDTA-Na2).

7.4 g of cesium chloride and 0.5 ml of ethidium bromide (10 mg / ml) solution are added, followed by ultracentrifugation to separate the plasmid.

Transduction of Streptomyces Lividans

The transformation method of Thompson et al. (Thompseon et al., J. Bacteriology 151,668-677 (1982)) was used.

Isolation of Diostreton Resistance Genes

BclI digestion of PIJ 702 was performed by treating 50 μg of PIJ 702 dir with 20 units of restriction enzyme in 200 μl, and the reaction solution treated at night at 37 ° C. was electrophoresed in low melting agarose. After running on the gel for 1 hour, the undesired band was removed with a razor and further proceeded for another 2 hours, and then the desired band (section of 1 kb of thiostrepton resistance gene) was cut and recovered with a razor blade.

The isolation of DNA from agarose gels was based on the method of Perbal ("A practical guide to molecular cloning", A Wiley Interscience Publication.N.Y.1984).

Claims (6)

Characterized by the transfer of DNA containing Nocardia mediterranei and genetic markers to identify it into Streptomyces lividans and Streptomyces lividans Converted resulting Streptomyces lividans JI 1326 with pCK 202 (KFCC 10383). A method for transformation of Streptomyces lividans, characterized by introducing a DNA containing Nocardia mediteranai and a genetic marker capable of identifying the same into Streptomyces lividans. The method of claim 2, wherein the DNA contains a nocardia mediterany DNA and the DNA can be detected in Streptomyces lividans. 4. The method of claim 3, wherein the DNA contains fragments of Nocardia mediterranean. 5. The bacterium according to claim 4, wherein the bacte is a plasmid of Nocardia mediterranean which contains and spontaneously replicates a fragment of the Thiostrepton resistant gene, which fragment is physiologically active in Streptomyces lividans. How to have a gene. Method for isolating plasmid DNA from liquid culture solution of Nocardia mediterany ATCC 31066.

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