PLASMID PHM 1519

Abstract: An essentially pure plasmid is provided, which is charac­ terized by a molecular weight of 1.8 megadalton and the re­ striction endonuclease-cleavage chart shown in the Figure, and which is capable of propagating in a Coryneform glutamic acd proaucing bacterium as a host strain. This plasmid may be inserted by a gene which is responsible for the production of a product and the hybrid plasmid may be incorporated in Coryneform glutamic acid producing bacteria as recipient organisms.

Description: PLASMID pHM 1519 The present invention relates to a plasmid, designated as plasmid pHM 1519 which is capable of propagating in Coryneform glutamic acid-producing bacteria. Coryneform glutamic acid producing bacteria, socalled "Coryneform bacteria, are known to produce substantial amounts of L-glutamic acid, and mutants of the Coryneform glutamic acid producing bacteria produce amino acids such as lysine and purine nucleotides such as inosinic acid. Therefore, they are of great importance to the fermentation industry. The recently developed gene splicing techniques can successfully be applied for producing or improving industrial microorganisms, especially in the case of Escherichia coli. It has been difficult, however, to apply the gene splicing techniques for producing or improving industrially useful microorganisms, which are Coryneform glutamic acid-producing bacteria, since suitable plasmids useful for the construction of such industrially useful Coryneform glutamic acid producing bacteria have not yet been developed. Although a plasmid capable of propagating in Coryneform glutamic acid-producing bacteria was reported in Agric. Biol. Chew., 43, 867, (1979), the molecular weight of the known plasmid is 37 megadalton which is too large and inconvenient to use it for the construction of industrial microorganisms of Coryneform bacteria by gene splicing techniques. Therefore a continuous need exists for the development of a novel plasmid useful for producing or improving industrial microorganisms of Coryneform glutamic acidproducing bacteria. It is accordingly one object of the present invention to provide a plasmid useful for producing or improving industrially useful Coryneform glutamic acid-producing bacteria. This and other objects of the present invention, as will hereinafter become readily apparent, can be attained by providing an essentially pure plasmid pHM 1519 which is characterized by a molecular weight of 1.8 megadalton and a restriction endonuclease cleavage chart as shown in the Figure. A more complete appreciation of the invention and of many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawing, wherein the Figure is the restriction endonuclease cleavage chartoftheplasmid pHM 1519. In the following preferred embodiments of the invention are described in more detail. Plasmid pHM 1519 is separated from Corynebacterium glutamicum ATCC 13058. The plasmid has the following characteristics The molecular weights of the plasmid calculated by the migration distance on agarose gel electrophoresis and the length of the DNA-chains under an electron microscope are both 1.8 megadalton. The sensitivity to restriction enzymes and the restriction chart of each plasmid are the same as shown in Table 1 and in the Figure, respectively. The plasmid pHM 1519 can be obtained from the cells of the deposited microorganisms Corynebacterium glutamicum ATCC 13058 by lysing the cells previously harvested at a late exponential growth phase by lysozyme and SDS, adding polyethylene glycol to the supernatant obtained from the lysate by centrifugation at 30,000 xg, and purifying the precipitated DNA by fractionation using cesium chloride-ethidium bromide equilibrium density gradient centrifugation. Table 1 Number of Restriction Restriction Enzyme Sites Ava I Anabena variabilis Bcl I Bacillus caldolyticus 1 BamH I Bacillus amyloliquefaciens H 0 Bgl II Bacillus globigii 1 EcoR I Escherichia coli RI+ 1 Hae II Haemophilus -aegy?tius > 7 HgiA I Herpetosiphon giganteus Hind III Haemophilus influenzae 2 Kpn I Klebsiella pneumoniae 0 Sma I Serratia marcescens 0 Sst I Streptomyces stanford 2 Xba I Xanthomonas badrii 0 Xho I Xanthomonas holicola 0 Xma I Xanthomonas malvacearum 0 Coryneform bacteria are aerobic, gram-positive rods, non-sporulating, and non-acidfast, and are described in Bergy's Manual of Determinative Bacteriology, 8th ed., 599, (1974). Examples of wild strains of Coryneform glutamic acid-producing bacteria useful as hosts in this invention are as follows: Brevibacterium divaricatum ATCC 14020, Rrevibacterium saccharoliticum ATCC 14066, Brevibacterium immariophilum ATCC 14068, Brevibacterium lactofermentum ATCC 13869, Brevibacterium roseum ATCC 13825, Brevibacterium flavum ATCC 13826, Brevibacterium thiogenitalis ATCC 19240, Corynebacterium acetoacidophilum ATCC 13870, Corvnebacterium acetoglutamicum ATCC 15806, Corynebacterium callunae ATCC 15991, Corynebacterium glutamicum ATCC 13032, 13060, Corynebacterium lilium ATCC 15990, Corynebacterium melassecola ATCC 17965, Microbacterium ammoniaphilum ATCC 15354. Coryneform glutamic acid-producing bacteria also include mutants which have lost the ability to produce glutamic acid or are capable of producing other amino acids such as lysine and arginine; purine nucleosides such as inosine; purine nucleotides such as inosine-5'monophosphate; and other fermentation products. In the cells of Corvneform glutamic acid-producing bacteria, the plasmids of the present invention are stably maintained. Since the plasmids of the present invention can propagate in cells of Coryneform glutamic acid-producing bacteria, the information of a foreign gene inserted in the plasmid can be amplified in the hosts. The incorporation -of plasmid DNA into the hosts of Coryneform glutamic acid-producing bacteria can be done by treating the cells of the DNA-recipient with calcium chloride to increase the permeability for DNA, as is reported regarding E. coli K-12 (Mandelt M and High, A., J. Mol. Biol., 53, 159 (1970), or by incorporation into the host at a specific stage of growth when the cells become capable of incorporating DNA therein (competent cells) as is reported for Bacillus subtilis (Duncan, C.H., Wilson, G.A., and Young, F.E.1 Gene 1, 153 (1977)). The plasmid can also be incorporated into the DNArecipient by forming protoplasts or spheroplasts of the DNA-recipient which easily incorporate plasmid DNA therein, as is known for Bacillus subtilis, actinomycetes and yeast (Chang, S. and Choen, S.N., Molec. Gen. Genet., 168, 111 (1979); Bibb, M.J., Ward, J.M. and Hopwood, O.A., Nature, 274, 398 (1978); Hinnen, A., Hicks, J.B. and Fink, G.R., Proc. Nat'l Acad. Sci., USA 75, 1929 (1978)). A foreign gene can be inserted into the plasmid of the present invention at the positions cleaved by the restriction enzymes shown in Table 1 and more preferably at the cleavage position whose number is one. Genom DNA of Coryneform glutamic acid-producing bacteria is the most preferred of the foreign genes. A preferred vector can be obtained from the present plasmid by removing all or a part of the DsA regions ot'ner than the drive-unit (vehicle unit), since the molecular weight becomes small. The replication number of the present plasmid is sufficiently large and therefore is suitable for amplifying foreign genes. Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified. Example 1 Pretaration of PHM 1519 DNA Corynebacterium glutamicum ATCC 13058 which possessed gIM 1519 was cultured at 30"C in CMG medium at pH 7.2 containing 1 g/dl peptone, l g/dl yeast extract, 0.5 g/dl glucose and 0.5 g/dl NaCl until the late exponential growth phase was reached. Cells were harvested and lysed by treatment with lysozyme and SDS. The lysate was centrifuged to obtain a supernatant, which was added to polyethylene glycol, and the resulting mixture was evaporated to precipitate DNA. The precipitate was dissolved in Tris-EDTA-NaCl buffer (containing 20mM Tris, 20mM NaCl and lmM EDTA) at pH 8.0, and the solution was subjected to cesium chloride-ethidium bromide equilibrium density gradient centrifugation,whereby a circular plasmid was obtained. Thus, 51 pg of pHM 1519 plasmid DNA was obtained. This method is disclosed in Tamio Yamakawa (ed.), SEIRAGAKU JIKREN KOZA, vol 2, 73, Tokyo-Ragaku-Donin Co. (1975). As such pHM 1519 can be obtained easily from ATCC 13058. Agarose-gel electrophoresis of digested plasmid Commercially available restriction enzymes sold by Bethesda Research Laboratory Co., Boehringer Mannheim Co., New England Biolab Co., Worthington Biochemical Co. were used. The digestion by restriction enzyme was carried out using an excessive amount of restriction enzyme, e.g. more than 3 times the amount required. The digestion reaction was carried out according to the directions by the distributors. The DNA fragments were thereafter subjected to agarose-gel electrophoresis by the method shown in P.A Sharp et al., Biochemistry 12, 3055 (1973). The agarose concentration was 0.7 to 1%, the voltage was 5 to 20 v and electrophoresis was continued for 1 to 3 hours. Molecular weight of pHM 1519 The molecular weight of pHM 1519 was calculated by its migration distance inthe electrophoresis on agarose gel and by the length of its DNA-chain as determined under an electron microscope, pHM 1519 was cleaved with Bcl I which cut the plasmid at one point, and subjected to agarose-gel electrophoresis shown above. The molecular weight was calculated based on the mobility differences of the l DNA-Hind III fragment produced by Bethesda Research Laboratory. Determination of the replication number of DHM 1519 Corynebacterium glutamicum ATCC 13058 containing pHM 1519 was cultured at 300C in 4 ml of CiMG medium at pH 7.0 to obtain cells at the exponential growth phase. The cells harvested were lysed by lysozyme-SDS, and the DNAs in the lysate were separated by precipitation. The DNAs were thereafter subjected to 0.7% agarose-gel electrophoresis, and from the gel obtained a photograph under ultra-violet light was taken. The negative-film thus obtained was put on a densitometer, and the density ratio of plasmid DNA fraction to total DNA was calculated. The replication number was determined as 100 when chromosomal DNA of ATCC 13058 is supposed as 3 x lO9 dalton.

Claims: CLAIMS 1. A substantially pure plasmid which is characterized by a molecular weight of 1.8 megadalton and the restriction endonuclease-cleavage chart shown in the Figure, and which is capable of propagating in a Coryneform glutamic acid producing bacterium as a host strain. 2. The substantially pure plasmid of Claim 1, which is pHM 1519. 3. A substantially pure plasmid, which is obtained by insertion of a foreign gene into a plasmid according to Claim 1 or Claim 2. 4. A process for producing a microorganism from Corvneform glutamic acid producing bacteria, which comprises incorporating into a Coryneform glutamic acid producing bacterium as a recipient a plasmid into which a foreign gene is inserted, which is responsible for the genetic information for the production of a product, characterized in that the plasmid according to claim 3 is incorporated into the recipient.

Inventors: MATSUI, HIROSHI, MIWA, KIYOSHI, NAKAMORI, SHIGERU, SANO, KONOSUKE, TERABE, MAHITO
Applicants: AJINOMOTO CO., INC.
Application year: 1982
Main classification: C12N15
IPC codes: C12R1, C12N15, C12P19, C07H21