GB2044773A - A plasmid and its microbiological preparation - Google Patents

Abstract

The plasmid pUC3 can be obtained from the microorganism Streptomyces sp. 3022a, NRRL 11441. The plasmid can be used as a cloning vehicle in recombinant DNA work. By way of example, the insulin gene can be inserted into the plasmid and then a suitable host containing the resulting plasmid can be used to produce insulin.

Description

SPECIFICATION A novel plasmid and its microbiological preparation The present invention relates to a novel plasmid which can have utility as a cloning vehicle in recombinant DNA work.

The development of plasmid vectors useful for recombinant DNA genetics among industrially important microorganisms is well known. The editorial in Science, Vol. 196, April, 1977, gives a good summary of DNA research. This editorial is accompanied by a number of supporting papers in the same issue of Science.

Similar DNA work is currently being done on industrially important microorganisms of the genus Streptomyces. [Bibb, M.J., Ward, J.M., and Hopwood, D.A. 1 978. "Transformation of plasmid DNA into Streptomyces at high frequency". Nature 274, 398-400]. Though plasmid DNA's have been detected in several Streptomycetes [Huber, M.L.B. and Godfrey, 0. 1978. "A general method for lysis of Streptomyces species". Can. J. Microbiol. 24, 631-632.] [Schrempf, H., Bujard, H., Hopwood, D.A. and Goebel, W. 1 975. ''Isolation of covalently closed circular deoxyribonucleic acid from Streptomyces coelicolorA3(2)". J.Bacteriol, 121, 416-421.] [Umezawa, H. 1977. "Microbial secondary metabolites with potential use in cancer treatment (Plasmid involvement in biosynthesis and compounds)". Biomedicine26, 236-249.], only three Streptomycete plasmids have been physically isolated and extensively characterized in the literature [Schrempf, supra]. The existence of other plasmids in the genus Streptomyces has been inferred from reported genetic data as follows: (1) Akagawa, H., Okanishi, M. and Umezawa, H. 1975. "A plasmid involved in chloramphen icol production in Streptomyces venezuelae: Evidence from genetic mapping". J. Gen. Micro biol. 90, 336-346.

(2) Freeman, R.F. and Hopwood, D.A. 1978. "Unstable naturally occurring resistance to antibiotics in Streptomyces". J. Gen. Microbiol. 106, 337-381.

(3) Friend, E.J., Warren, M. and Hopwood, D.A. 1 978. "Genetic evidence for a plasmid controlling fertility in an industrial strain of Streptomyces rimosus". J. Gen. Microbiol. 106, 201-206.

(4) Hopwood, D.A. and Wright, H.M. 1973. "A plasmid of Streptomyces coelicolorcarrying a chromosomal locus and its inter-specific transfer". J. Gen. Microbiol. 79, 331-342.

(5) Hotta, K., Okami, Y. and Umezawa, H. 1 977. "Elimination of the ability of a kanamycin producing strain to biosynthesize deoxystreptamine moiety by acriflavine". J. Antibiotics 30, 1146-1149.

(6) Kirby, R., Wright, L.F. and Hopwood, D.A. 1 975. "Plasmid-determined antibiotic synthesis and resistance in Streptomyces coelicolo;'. Nature 254, 265-267.

(7) Kirby, R. and Hopwood, D.A. 1 977. "Genetic determination of methylenomycin synthesis by the SCPI plasmid of Streptomyces coelicolorA3(2)". J. Gen. Microbiol. 98, 239-252.

(8) Okanishi, M., Ohta, T. and Umezawa, H. 1 969. "Possible control of formation of aerial mycelium and antibiotic production in Streptomyces by episomic factors". J. Antibiotics 33, 45-47.

(9) Okanishi, M. 1 977. Involvement of plasmids in the production of secondary metabolites.

Amino Acid-Nucleic Acid 35, 15-30.

(10) Schrempf, H. and Goebel, W. 1 977. Characterization of a plasmid from Streptomyces coelicolorA3. J. Bacteriol. 131, 251-258.

(11) Shaw, P.D. and Piwowarski, J. 1977. Effect of ethidium bromide and acriflavine on streptomycin production by Streptomyces bikiniensis. J. Antibiotics 30, 404-408.

(12) Yagisawa, M., Huang Rossana T-S., and Davies, J.E. 1978. Possible involvement of plasmids in biosynthesis of neumycin. J. Antibiotics, 31, 809-813.

The Journal of Antibiotics, Vol. XXX No. 10, pages 897-899, describes a method for the isolation of plasmid DNA. The source microorganism for the plasmid DNA is not identified sufficiently to enable a person skilled in the art to practice the process. Further, no public repository is disclosed from which the microorganism could be obtained.

Plasmid pUC3 is obtainable from the microorganism Streptomyces sp. 3022a, NRRL 11 441.

This plasmid can be obtained from NRRL 11441 by growing the culture on a suitable medium, harvesting the culture after a suitable time, and then lysing the mycelia from which lysate is obtained essentially pure pUC3. Its sensitivities to a variety of restriction endonucleases should allow its ready modification and adaptation to a number of host vector systems.

pUC3 is characterized by standard characterization tests which includes its molecular weight, approximately 20 X 106 daltons, and a restriction map as shown in the drawing.

pUC3 is useful as a cloning vector in DNA work wherein desired genes are incorporated into the plasm id, and the plasmid then transformed into a suitable host.

The drawing depicts the restriction endonuclease cleavage map for pUC3. The map is constructed on the basis of plasmid pUC3 having a molecular weight of 20 x 106 daltons or a molecular length of 30 kilobase pairs. The map positions of the various restriction sites are given as percentages, the total plasmid length being 100 percent. One of the EcoRI sites is assigned position 0 percent on the circular map.

The restriction endonuclease abbreviations are as follows; (1) EcoRI is an enzyme from Escherichia coli; (2) Hindlll is an enzyme from Hemophilus influenza; (3) Xhol is an enzyme from Xanthomonas holcicola; (4) Pstl is an enzyme from Providencia stuartii; and (5) Bglll is an enzyme from Bacillus globigii.

The Microorganism pUC3 is obtainable from Streptomyces sp. 3002a, NRRL 11441. This biologically pure culture is available from the permanent collection of the Northern Regional Reseach Laboratory, U.S. Department of Agriculture, Peoria, Illinois, U.S.A. This culture is disclosed in several publications, for example: (1) Smith, C.G. 1958. Effect of halogens on the chloramphenicol fermentation. J. of Bacteriol. 75: 577-583; (2) Vining, L.C. and D.W.S. Westlake. 1 964.

Bisynthesis of the phenylpropanoid moiety of chloramphenicol. Can. J. of Microbiol. 10: 705-716.; and (3) Malik, V.S and L.C. Vining. 1970. Metabolism of Chloramphenicol by the producing organism. Can J. of Microbiol. 16: 173-179.

Characteristics Of p UC3 Molecular Weight: ca. 20 x 106 daltons.

Restriction Endonuclease Sensitivities: pUC3 has the following sensitivities to restriction endonucleases. Please refer to the drawing for the restriction endonuclease cleavage map for pUC3.

Plasmid Sensitivities to Restriction Endonucleases # Cleavage Sites # Cleavage Sites Enzyme pUC3 Enzyme pUC3 Bgll Many* Bglll 4 BamHI 4 Hpal O Hindlll 1 EcoRI 3 Kpnl > 5 Pstl 5 Pvull 9 Mboll Many Aval Many Xbal None Xhol 2 Sall Many Hphl Many Haell > 7 Hinfl Many Smal Many Hincll Many *Used to denote more than 10 sites.

These results were obtained by digestion of DNA in the presence of an excess of restriction endonuclease. The number of restriction sites were determined from the number of resolvable fragments in either 0.7 to 1.0% agarose gels.

pUC3 can be used to create recombinant plasmids which can be introduced into host bacteria by transformation. The process of creating recombinant plasmids is well known in the art. Such a process comprises cleaving the isolated plasmid, e.g. pUC3, at one specific site by means of a restriction endonuclease, for example. Hindlll. The plasmid, which is a circular DNA molecule, is thus converted into a linear DNA molecule by the enzyme which cuts the two DNA strands at sites which can be several base pairs apart. Another plasmid is similarly cleaved with the same enzyme. Upon mixing the two linear plasmids, their single-stranded ends can pair with each other to form a single circle of DNA. The two plasmids can be joined covalently by use of a second enzyme known as polynucleotide ligase.

The above procedure also can be used to insert a length of DNA from a higher animal into pUC3. For example, the DNA which codes for ribosomal RNA in the frog can be mixed with pUC3 DNA that has been cleaved. The resulting circular DNA molecules consist of plasmid pUC3 with an inserted length of frog rDNA.

The recombinant plasmids containing a desired genetic element, prepared by using pUC3, can be introduced into a host organism for expression. Examples of valuable genes which can be inserted into host organism by the above described process are genes coding for somatostatin, rat proinsulin, and proteases.

The usefulness of plasmid pUC3 is that it represents a plasmid vector which functions in the industrially important microorganisms of the genus Streptomyces. Hence, cloning of genetic information from Streptomyces onto pUC3 provides a means of increasing the production of commercially important products from these organisms, e.g. antibiotics.

This approach is compared to the concept of cloning genes for antibiotic production into the well characterized Escherichia coli K-12 host-vector system. The E. coli system has the disadvantage that it has been found that genes from some Gram-positive organisms, e.g.

Bacillus, do not express well in the Gram-negative E. coli host. Likewise, plasmids from Gramnegative organisms are not maintained in Gram-positive hosts, and Gram-negative genetic information is either expressed poorly or not at all in Gram-positive hosts. This clearly argues for the advantage of a Gram-positive host-vector system and argues the usefulness of plasmid pUC3 in such a system.

In general, the use of a host-vector system to produce a product foreign to that host requires the introduction of the genes for the entire biosynthetic pathway of the product to the new host.

As discussed above, this may lead to problems of genetic expression, but may also generate new and/or increased problems in the fermentation of the microorganisms and in the extraction and purification of the product. A perhaps more useful approach is to introduce a plasmid vector, e.g. pUC3, into a host which normally produces the product and clone onto that plasmid the genes for biosynthesis of the product. At the very least, problems of fermentation and product extraction and purification should be minimized. Additionally, in this cloning system it may not be necessary to clone and amplify all the genes of the biosynthetic pathway, but rather it may be necessary only to clone regulatory genes or genes coding for the enzymes that are rate limiting in product biosynthesis. Since pUC3 is a streptomycete plasmid, it is ideally suited for these purposes in the genus Streptomyces.Furthermore, since pUC3 is also a plasmid from a Gram-positive organism, it may serve as a vector in a number of other microorganisms, e.g.

Bacillus, Arthrobacter, etc.

Streptomycessp. 3022a, NRRL 11441, can be grown in an aqueous nutrient medium under submerged aerobic conditions. The organism can be grown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate, and a nitrogen source, for example, an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include cornsteep, liquor, yeast, autolyzed brewer's yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, fish meal, distillers' solids, animal peptone liquors, meat and bone scraps, and the like.

Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and the like, need not be added since tap water and unpurified ingredients are used as components of the medium prior to sterilization of the medium.

The inoculated medium can be incubated at any temperature conducive to satisfactory growth of the microorganism, for example, between about 18 and 30 C., and preferably between about 20 and 28 C. Ordinarily, optimum growth of the microorganism is obtained in about 2 to 5 days. At this time pH of the medium becomes alkaline and organism produces chloramphenicol.

When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avoid a pronounced lag in the growth of the microorganism and the attendant inefficient utilization of the equipment.

Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil, liquid N2 agar plug, or a slant culture.

When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the growth of the microorganism so long as a good growth of the microorganism is obtained.

The following examples are illustrative of the process and products of the subject invention but are not to be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example i-Isolation of Plasmid pUC3 From A Biologically Pure Culture of Streptomyces sp.

3022a, NRRL 11441 Streptomyces sp. 3022a, NRRL 11441, spores are inoculated into 100 ml of the following medium: Bacto tryptone 0.05% Brer rabbit molasses 1.0% Glycerol 1.0% Difco yeast extract 0.25% Adjust to pH 7.2 with 1N NaOH Tap water 1000 ml The medium has previously been sterilized in a 500 ml Erlenmeyer flask. After inoculation, the flask is incubated at 25 C. for about 36 to 48 hours on a Gump rotary shaker operating at 250 rpm.

This cell suspension is used to seed 40 flasks of Glycerol-serine-lactate medium at the rate of 1%. Glycerol-serine-lactate medium has the following composition: Glycerol 2% 60% Sodium lactate 2.8% D1 Serine 0.3% Sodium chloride 0.6% Difco yeast extract 0.025% KH2PO4 0.14% K2HPO4 0.2% MgSO4 0.05% MnSO4 H2O 0.0008% CuSO4 5H2O 0.0006% ZnSO4 0.0012% Adjust pH to 7.0 with 1 N NaOH.

After 3 days incubation at 28 C. the cells are separated from the broth by low speed centrifugation, for example, at 10000 x g for 10 minutes at 4" C. and the supernatant is decantated from the mycelial pellet. The supernatant is discarded and the pellet is resuspended in 200 ml of 25% sucrose, 50 mm Tris buffer pH 8.0. Next, 40 ml of a 2 mg/ml solution of lysozyme in TES buffer is added and the mixture is incubated at 25 C. for 30 minutes with occasional mixing. Then, 100 ml of 0.25 M EDTA (pH = 8.0) is added and this mixture is incubated 30 minutes at 25 C. Subsequently, the cell suspension is lysed by the addition of a detergent, for example, 320 ml of 1.0% (w/v) Brij-58 (Pierce Chem.Co., Rockford, illinois), 0.4% (w/v) deoxycholic acid, 0.05 M Tris (pH = 8.0) and 0.06 M EDTA and incubation of this mixture at 25 C. for 30 minutes. The lysate is then incubated at 50* C. for 30 minutes-60 minutes. The viscous lysate is centrifuged (60 min.) at 10,000 rpm in GSA rotor using sorvall centrifuge. The viscous supernatant is then mixed with a salt, for example, cesium chloride (preferred) and cesium sulfate, and the intercalating dye ethidium bromide to give a solution of density - 1.550. This solution is centrifuged to equilibrium at 145,000 x g (isopycnic density gradient centrifugation).The covalently closed circular plasmid DNA is then visible in the centrifuge tube under ultraviolet (320 nm) illumination as a faint fluorescent band below the intensely fluorescent band of linear chromosomal and plasmid DNAs.

Covalently closed circular plasmid DNA is prepared for characterization by removing it from the isopycnic gradients, extracting the ethidium bromide by two treatments with one third volume of isopropyl alcohol and then dialyzing the aqueous phase against 10 mm Tris, 10 mm EDTA pH 8.0 to yield essentially pure pUC3.

Procedures For Characterizing And Isolating pUC3 The size of pUC3 was determined by electron microscopy.

Restriction Endonuclease Digestion and Agarose Gel Electrophoresis All the restriction enzymes used were purchased from New England Biolabs.

Digestion of plasmid DNA was performed in restriction buffer containing 10 mMTris HCI, pH 7.4; 5 mMMgCl2; imM DTT for Ava I, Hph I, Mbo II, Pvu II, Hae II, Kpn I, Hinc II, Hpa land Bgl II. The same buffer containing 50 mM NaCI was used for Bam H-l, Hinf I, Sal I, Xba I, Bgl I EcoRI, Xho I, Hind Ill and Pst I. Sma I Digestion mixtures contained 1 5 mM HCI, pH 9.0; 15 mM KCI; 6 mMMgCl2 [Post, L.E., Arfsten, A.E., Reusser, F. and Nomura, M. 1978. DNA sequences of promoter regions for the strand spc ribosomal protein operons in E. coli. Cell 15, 215-299].

The digestion mixtures were analyzed in 1 % agarose gels prepared as described by Shinnick et al. [Shinnick, T.M., Lund, E., Smithies, 0., and Blattner, F. R. 1 975. Hybridization of labeled RNA to DNA in agarose gels. Nucl. Acids, Res. 2, 1911-1229].

Hind Ill-digested A DNA was used as a molecular weight reference [Murray, K. and Murray, N.E. 1 975. Phage Lambda Receptor Chromosomes for DNA Fragments made with Restriction Endonuclease Ill of Haemophilus influenzae and Restriction Endonuclease I of Escherichia coli.

J. Mol. Biol. 98, 551-564].

The work described herein was all done in conformity with physical and biological containment requirements specified in the NIH Guidelines.

Claims (3)

1. Essentially pure plasmid pUC3 which is characterized by a molecular weight of approxi mately 20 X 106 daltons, and a restriction endonuclease cleavage map as shown in the drawing.

2. A process for isolating essentially pure pUC3 from Streptomyces sp. 3022a NRRL 11441, which comprises: (a) growing Streptomycessp. 3022a NRRL 11441, on a suitable medium; (b) harvesting the culture after a suitable time; (c) lysing the harvested mycelia; and (d) isolating essentially pure pUC3 from the lysate.

3. A process, according to Claim 2, which comprises cultivating Streptomyces sp. 3022a, NRRL 11441, on a nutrient medium at a temperature of about 28 C. for about 36 to 48 hours.