GB2046272A - A novel plasmid and its microbiological preparation - Google Patents

Abstract

The novel plasmid pUC7 can be obtained from the microorganism Streptomyces espinosus subsp. acanthus NRRL 11081. 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 pUC7 and then a suitable host microbe 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 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. 1978. "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. 1 978. "A general method for lysis of Streptomycesspecies". Can J. Microbiol. 24, 631-632.] [Schrempf, H., Bujard, H., Hopwood, D.A. and Goebel, W. 1975. "Isolation of covalently closed circular deoxyribonucleic acid from Streptomyces coelicolorA3(2)". J. Bacteriol. 121, 416-421.] [Umezawa, H. 1977. "Microbial secondary metabolities with potential use in cancer treatment (Plasmid involvement in biosynthesis and compounds)".Biomedicine 26, 236-249.], [Malik, V.S. 1 977. Preparative Method for the isolation of super-coiled DNA from a chloramphenicol producing Streptomycete. J. Antibiotics 30, 897-899.], only one Streptomycete plasmid has 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 chloramphenicol production in Streptomyces venezuelae: Evidence from genetic mapping". J. Gen.

Microbiol. 90, 336-346.

(2) Freeman, R.F. and Hopwood, D.A. 1978. "Unstable naturally occurring resistance to antibiotics in Streptomyces". J. Gen. Microbiol. 106, 377-381.

(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. 1977. "Elimination of the ability of a kanamycinproducing 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 coelicolor". 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. 1969. "Possible control of formation of aerial mycelium and antibiotic production in Streptomyces by episomic factors". J. Antibiotics 23, 45-47.

Plasmid pUC7 is obtainable from the microorganism Streptomyces espinosus subsp. acanthus NRRL 11081. This plasmid can be obtained from NRRL 11081 by growing the culture on a suitable medium, fragmenting the mycelia, incubating the fragmented mycelia, harvesting the culture after a suitable time, and then lysing the mycelia. From this lysate it is possible to isolate essentially pure pUC7. Plasmid pUC7 is advantageously small, and is sensitive to cleavage by a variety of restriction endonucleases. These properties should allow pUC7's ready modification and adaptation to a number of host vector systems.

pUC7 is characterized by standard characterization tests which includes its molecular weight, approximately 10-11 megadaltons, a restriction map as shown in the drawing, and presence at 2-4 copies per S. espinosus subsp. acanthus NRRL 11081 cell.

pUC7 is useful as a cloning vector in DNA work wherein desired genes are incorporated into the plasmid, and the plasmid then transformed into a suitable host.

The drawing depicts the restriction endonuclease cleavage map for pUC7. The map is constructed on ths basis of plasmid pUC7 having a molecular weight of ca. 10.7 megadaltons or a molecular length of ca. 1 6.2 kilobases. The map positions of the various restriction sites are given as kilobase coordinates relative to the Bgl II restriction site at 0.0/16.2 kilobases. The restriction endonuclease abbreviations are as follows: (1) Bgl II is an enzyme from Bacilus globigii; (2) Bam HI is an enzyme from Bacillus amyloliquefaciens HI; (3) Pst I is an enzyme from Providencia stuartii 164; (4) Kpn I is an enzyme from Klebsiella pneumoniae; and (5) Xho I is an enzyme from Xanthomonas holicicola.

In the interpretation of the drawing, it should be noted that two ambiguities exist. One, we cannot determine the orientation of the Bam HI and Pst I restriction sites mapping at ca. 14.5 kilobases. Two, because of symmetry considerations, one of the two Kpn I restriction sites will map at either ca. 0.7 or ca. 3.7 kilobases. The latter ambiguity is represented by dashed lines on the drawing.

The Microorganism pUC7 is obtainable from Streptomyces espinosus subsp. acanthus NRRL 11081. This biologically pure culture is available from the permanent collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Illinois, U.S.A. The culture is taxonomically described in Belgium Patent 864,401.

Characteristics Of pUC7 Molecular Weight: ca. 10-11 megadaltons.

Copies Per Cell: 2-4.

Restriction Endonuclease Sensitivities: pUC7 has the following sensitivities to restriction endonucleases. Please refer to the drawing for the restriction endonuclease cleavage map for pUC7.

Plasmid Sensitivities To Restriction Endonucleases # Cleavage Sites # Cleavage Sites Enzyme pUC7 Enzyme pUC7 Bgl 1 > 7 Bgl II 2 Bam HI 4 Hpa I 0 Hpa II Many Hind lil 2-3 EcoRI 0 Kpn 1 2 Pst I 2 Pvu II > 5 Mbo II > 6 Ava I > 8 Xba 1 3 Xho 1 3 Sal I > 7 Sma I Many Hinc II Many Bcl I Many These results were obtained by digestion of pUC7 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 or 1.0% agarose gels.

pUC7 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 vector plasmid, e.g., pUC7, at a specific site(s) by means of a restriction endonuclease, for example, Bgl II, Pst I, and the like. The plasmid, which is a circular DNA molecule, is thus converted into a linear DNA molecule(s) by the enzyme which cuts the two DNA strands at a specific site. Other non-vector DNA is similarly cleaved with the same enzyme. Upon mixing the linear vector or portions thereof and non-vector DNAs, their single-stranded or blunt ends can pair with each other, and in the presence of a second enzyme known as polynucleotide ligase, can be covalently joined to form a single circle of DNA.

The above procedure also can be used to insert a length of DNA from a higher animal into pUC7. For example, the DNA which codes for ribosomal RNA in the frog can be mixed with pUC7 DNA that has been cleaved. The resulting circular DNA molecules consist of plasmid pUC7 with an inserted length of frog rDNA.

The recombinant plasmids containing a desired genetic element, prepared by using pUC7, can be introduced into a host organism for expression. Examples of valuable genes which can be inserted into host organisms by the above described process are genes coding for somatostatin, rat proinsulin, and proteases.

The usefulness of plasmid pUC7 is derived from its capacity to function as a plasmid vector in industrially important microorganisms, e.g. Streptomyces. Hence, cloning of genetic information from Streptomyces into pUC7 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-1 2 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 pUC7 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. pUC7, 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 pUC7 is a streptomycete plasmid, it is ideally suited for these purposes in the genus Streptomyces.Furthermore, since pUC7 is also a plasmid from a Gram-positive organism, it may serve as a vector in a number of other microoganisms, e.g.

Bacillus, Arthrobacter, etc.

Streptomyces espinosus subsp. acanthus, NRRL 11081, 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 soids, 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 50"C., and preferably between about 20 and 37"C. Ordinarily, optimum growth of the microorganism is obtaned in about 3 to 1 5 days. The medium normally remains acidic during the growth cycle. The final pH is dependent, in part, on the buffers present, if any, and in part on the initial pH of the culture medium.

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 micro-oganism 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 1Isolation Of Plasmid pUC7 From A Biologically Pure Culture Of Streptomyces espinosus subsp. acanthus, NRRL 11081 The spores from a biologically pure culture of Streptomyces espinosus subsp. Acanthus, NRRL 11081 are inoculated into 10 ml of the following Difco Antibiotic Medium No. 3 Broth (Difco Labs., Detroit, Michigan): 0. 15% Beef extract; 0. 15% yeast extract; 0.5% peptone; 0. 1% glucose; 0.35% Nacl; 0.368% K2HPO4; 0.132% KH2PO4.

The medium has previously been sterilized in a 50 ml Erlenmeyer flask. After inoculation, the flask is incubated at 37"C. for about 36 to 48 hours on a Gump or New Brunswick rotary shaker operating at 100-250 rpm. Upon completion of the incubation, the mycelia-broth suspension in the flasks is homogenized under sterile conditions and is then mixed in sterile 1 25 ml Erlenmeyer flask containing 10 ml of the above medium and also, advantageously, 68% (w/v) sucrose and 1 % (w/v) glycine. The addition of sucrose and glycine facilitates the subsequent lysing of the cells. The amounts of sucrose and glycine in the medium can be varied by routine adjustments with the goal being to facilitate the subsequent lysing of the cells.The flask is then incubated further for another 36 to 48 hours at 37"C. on a Gump rotary shaker, as above. After this incubation, the mycelia are separated from the broth by low speed centrifugation, for example, at 6000 x g for 1 5 minutes at 4"C. and decantation of the supernatant from the mycelial pellet.

The supernatant is discarded and the pellet is resuspended in 1.5 ml of an isotonic buffer containing ethylenediaminotetraacetic acid (EDTA) and sucrose, e.g. TES buffer [0.03 M tris(hydroxymethyl)aminomethane (Tris), 0.005 M EDTA and 0.05 M Nacl; pH = 8.0] containing 20% (w/v) sucrose. Next, 1.5 ml of a 5 mg/ml solution of lysozyme in the same buffer is added and the mixture is incubated at 37"C. for 30 minutes with occasional mixing. Then, 1.5 ml of 0.25 M EDTA (pH = 8.0) is added and this mixture is incubated 15 minutes at 37"C.

Subsequently, the cell suspension is lysed by the addition of 2.5 ml of a lytic mixture, e.g.

[1.0% (w/v) Brij-58 (a detergent sold by 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 37"C. for 20 minutes. The lysate is then sheared by passing it 5-10 times through a 10 ml pipet. The sheared lysate is then digested with ribonulease (140 yg/ml) and pronase (300 g/ml) for an additional 20 minutes at 37"C. Alternatively, the cell-lysozyme-EDTA mixture can be digested with ribonuclease and pronase before lysis with a lytic agent such as 2% sodium dodecyl sulfate in water.

This crude lysate material is then mixed with a salt, for example, cesium chloride (preferred) or cesium sulfate, and the intercalating dye ethidium bromide to give a solution of density p = 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 long wave 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 an appropriate buffer, e.g. 0.1 X SSC buffer (0.015 M naCI, 0.0015 M Na3QH5O72H20; pH = 7.4) to yield essentially pure pUC7.

Procedures For Characterizing And Isolating pUC7 The size of pUC7 was determined by sedimentation in neutral and alkaline sucrose gradients using an internal marker plasmid DNA having a molecular weight of approximately 9.0 megadaltons and a corresponding sedimentation value of approximately 34S. From the neutral sucrose gradients the sedimentation value of pUC7 was determined to be 35-37 5. The molecular weight for pUC7 was calculated from the equations by Hudson et al. [Hudson, B., Clayton, DA. and Vinograd, J. 1 968. "Complex mitochondrial DNA". Cold Spring Harbor Symp. Quant. Biol. 33, 435-442]. This molecular weight is in good agreement with that estimated from the alkaline sucrose gradients.

An estimate of pUC7 molecular weight was also obtained by electron microscopy of individual DNA molecules [Kleinschmidt, A.K. (1968). Monolayer techniques in electron microscopy of nucleic acid molecules. In "Method in Enzymology" (S.P. Colowick and N.O. Kaplan, eds.) Vol.

12B, pages 361-377. Academic Press, New York]. Plasmid pUC7 was found to have an average contour length of 5.43 10-8 meters and a corresponding molecular weight of 10.7 megadaltons.

The percent plasmid DNA in Streptomyces espinosussubsp. acanthus, NRRL 11081 was determined by labeling the culture with [methyl-3H]thymidine, preparing crude lysates, and centrifuging samples of the lysates in cesium chloride ethidium bromide density gradients. The gradients are fractionated, the isotopic counting performed, and the percent radioactivity in the plasmid band used to quantitate the plasmid DNA and calculate the plasmid copy number [Radloff, R., Bauer, W. and Vinograd, J. 1967. "A dye-buoyant density method for detection and isolation of closed circular duplex DNA: The closed circular DNA in HeLa cells". Proc. Nat.

Acad. Sci. USA 57, 1514-1520].

Restriction En don uclease Digestion And Agarose Gel Electrophoresis Restriction endonucleases were obtained as commercial preparations from Miles Laboratories and New England Biolabs. Enzyme digestions were prepared in accordance with the conditions specified by the suppliers using at least a two-fold excess of endonuclease.

In some experiments plasmid DNA was digested with more than one endonuclease. Two methods were used in these Experiments. In the first method, the plasmid DNA was digested first with the enzyme having the lower ionic strength requirements, and then digested with the enzyme having higher ionic strength requirements after the addition of an equal volume of 2X buffer of the second enzyme. In the second method, restriction fragments of one enzyme digest were isolated from a preparative agarose gel as described by Tanaka and Weisblum [Tanaka, T., and Weisblum, B. 1975. Construction of a colicin El-R factor composite plasmid in vitro: Means for amplification of deoxyribonucleic acid. J. Bacteriol. 121, 354-362]. The isolated restriction fragments were concentrated by ethanol precipitation and then digested with other restriction enzymes. Double digest experiments were compared with single digest experiments to ensure that no abnormal restriction patterns were obtained, i.e. no non-specific cleavage of DNA by an enzyme occurs after altering the ionic strength of the digestion mixture.

The digested samples were applied to 0.7-1% agarose gels and were electrophoresed for 2 hours at a constant applied voltage of 10-15 v/cm of gel height. [Sharp, P.A., Sugden, J. and Sambrook, J. 1973. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis. Biochemistry 12, 3055-3063]. The molecular weights of restriction fragments were determined relative to the standard migration patterns of bacteriophage lambda DNA digested with enzyme Hind Ill [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] or EcoRI [Helling, R.B., Goodman, H.M. and Boyer, H.W. 1 974. Analysis of endonuclease R. EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. J. Virology 14, 1235-1244].

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

Claims (5)

1. Essentially pure plasmid pUC7 which is characterized by a molecular weight of approximately 10-11 megadaltons, and a restriction endonuclease cleavage map as shown in the drawing.

2. A process for isolating essentially pure pUC7 from Streptomyces espinosus subsp.

acanthus, NRRL 11081, which comprises: (a) growing S. espinosus subsp. acanthus, NRRL 11081 on a suitable S. espinosus growth medium until sufficient mycelial growth is obtained; (b) fragmenting said mycelia; (c) incubating said fragmented mycelia in a suitable growth medium, as above; (d) harvesting the culture after a suitable time; (e) lysing the harvested mycelia; and (f) isolating essentially pure pUC7 from the lysate.

3. A process, according to claim 2, which comprises cultivating Streptomyces espinosus subsp. acanthus, NRRL 11081, in a nutrient medium at a temperature of about 37"C. for about 36 to 48 hours.

4. A process according to claim 2 or claim 3 in which the fragmented mycelia are incubated in a growth medium containing sucrose and glycine.

5. A process according to claim 2 substantially as herein described.