JPS6225038B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a plasmid isolated from a coryneform glutamic acid producing bacterium. Coryneform-glutamate-producing bacteria have a large amount of L
- Known to produce glutamic acid. Moreover, its mutant strains are known to produce amino acids such as lysine and purine nucleotides such as inosinic acid, and are industrially useful microorganisms. On the other hand, recently attempts have been made to breed and improve industrial microorganisms using recombinant DNA technology, such as Escherichia coli, but vectors suitable for using these microorganisms as hosts have not yet been developed for coryneform/glutamate producing bacteria. However, this has hindered the breeding and improvement of coryneform-glutamic acid-producing bacteria through DNA recombination. Against this background, the present inventors conducted intensive research to develop a vector suitable for coryneform bacteria, and finally found a vector suitable for use as a vector or suitable for processing as a vector from coryneform glutamate-producing bacteria. Plasmid pCC1, which has a molecular weight of 4.2 kb and has the restriction enzyme cleavage map shown in Figure 1.
I found out. This plasmid was isolated from Ccrynebaeterium callunae NRRLB-2244, which is a coryneform glutamic acid producing bacterium. Coryneform glutamate-producing bacteria are aerobic, non-sporulating, non-acid-fast, Gram-positive, indeterminate bacilli that produce a large amount of glutamate, and some of them are listed below. Illustrated as an example. Brevibacterium ammoniagenes ATCC13745 Brevibacterium deivaricatam ATCC14020 Brevibacterium flavum ATCC13826 Brevibacterium imariophyllum ATCC14068 Brevibacterium ketoglutamicum ATCC15887 Brevibacterium lactofamentum ATCC13869 Brevibacterium roseus ATCC13825 Brevibacterium satu Corynebacterium glutamicum ATCC14066 Brevibacterium thiogenitalis ATCC19240 Corynebacterium acetophyllum ATCC13870 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium alkanolytecum ATCC21511 Corynebacterium carnae ATCC15991 Corynebacterium glutamicum ATCC13 032 Corynebacterium hydro Carboclusters ATCC15592 Corynebacterium Lilium ATCC15990 Corynebacterium melasecola ATCC17965 Microbacterium ammoniaphyllum ATCC15354 These coryneform/glutamic acid producing bacteria are capable of stably injecting the plasmid of the present invention into cells; It can serve as a host for the plasmid of the present invention. The plasmid of the present invention can proliferate well in the host microorganism as described above, so if a foreign gene is inserted into any position of this plasmid, the genetic information of the foreign gene can be transferred to the host microorganism. - It can be expressed in glutamic acid-producing bacterial cells and can transform the genetic traits of these host microorganisms. To introduce the plasmid of the present invention into host microorganism cells, the bacteria are treated with calcium chloride at low temperatures to increase the permeability of the bacterial membrane, as reported for E. ccli K-12. Methods for transferring plasmids (Mandel, M and, Higa, A., J. Mol.
Biol., 53 , 159 (1970)), a method of transferring plasmids by taking advantage of the fact that it naturally becomes easier to incorporate plasmids at a certain stage of growth (so-called competent state), as reported in Bacillus subtilis. (Duncan, CH, Wilson, GA and Young, F.
E., Gene, 1 , 153 (1977)), and furthermore, as reported for Bacillus subtilis, actinomycetes, and yeast, cells are made into protoplasts or supheplasts and plasmids are transferred (Chang, S and Cohen, SN,
Molec.Gen.Genet., 168 , 111 (1979); Bibb,
MJ, Ward, JMand, Hopwood, D.A.
Nature, 274 , 398 (1978); Hinnen, A.,
Hicks, JBand Fink, GR, Proc. Natl. Acad.
Sci., USA, 75 , 1929 (1978)). In order to insert a foreign gene into the plasmid of the present invention, it is convenient to insert it into a site that can be cut with a restriction enzyme, but it is especially convenient to insert it into a site where only one site is cut with the same restriction enzyme. It is desirable to do so. To insert a foreign gene, the plasmid and the genomic DNA serving as the source of the foreign gene may be partially or completely digested with the same restriction enzyme, and then ligated under appropriate conditions. As a foreign gene, the genomic DMA of a coryneform-glutamate producing bacterium is the most efficient and its genetic information is expressed. The plasmid of the present invention has a large number of copies in cells, and therefore a large amount of gene amplification can be expected when recombining with a foreign gene. Example 1 Isolation of pCC1 DNA Corynebacterium callunae ((Corgnebacterium callunae) NRRL B-
Strain 2244 was cultured in trypticase soy broth (1.5% casein peptone, 0.5% soy peptone, 0.5% Nacl, PH7.0) at 30°C to mid-log phase (OD = 0.6), and penicillin G was added to the final After addition at a concentration of 0.3 u/ml, the culture was further continued for 18 hours. Bacteria were collected by centrifugation, and lysozyme (10
mg/ml) and SDS (10 mg/ml) were added to lyse the bacteria, followed by high-speed centrifugation to obtain superzuke. polyethylene·
The DNA was concentrated by adding glycol, centrifuged to collect the precipitate, and suspended in TEN buffer. This DNA solution was subjected to cesium chloride ethidium bromide density gradient centrifugation, ccc
The DNA portion was separated, ethidium bromide was removed with isoacyl alcohol, and cesium chloride was removed by dialysis. 2 Determination of molecular weight of pCC1 Molecular weight was calculated from agarose gel electrophoresis distance. That is, following the method from Schaap (Biochemistry 12 , 3055 (1973)),
Various plasmid DNAs of E.wli V 517 strain
was used as the standard material for molecular weight measurement (Macrina FL
et, al, Plasurid 1 417 (1978)), the molecular weight of pCC1 was measured. As a result, the molecular weight was calculated to be 4.21±0.23kb. Furthermore, DNA cleaved with restriction enzymes such as Hind, Kpm, Sma and Bal
The molecular weight was also calculated to be 4.27±0.05kb by agarose gel electrophoresis using a Hind-digested molecular weight standard of lambda phage DNA as a control. 3 Cleavage of pCC1 DNA with restriction enzymes The purified DNA of pCC1 was digested with various restriction enzymes, and the results shown in Table 1 were obtained.

【table】

[Table] 4 Restriction enzyme cleavage map of pCC1 From the restriction enzyme sensitivity pattern described in 3.
A restriction enzyme cleavage map as shown in the figure was created. 5 Measuring the copy number of pCC1 Corynebacterium carnae NRRL
B-2244 was cultured in a minimal medium containing 100 μCi of tritium-thymidine, the DNA was labeled with a radioisotope, the bacterial cells were lysed using lysozyme and SDS, and the fraction containing total DNA was subjected to Cscl-EtBr equilibrium density gradient centrifugation. Then, the copy number of the pCC1 plasmid was calculated from the ratio of the radioactivity of the ccc DNA part and the chromosomal DNA part. i.e. chromosome
The copy number was calculated to be 30, assuming that the molecular weight of DNA was 8000 kb, and the molecular weight of pcc1 and DNA was 4.2 kb.

[Brief explanation of the drawing]

Figure 1 shows the pCC1 restriction enzyme cleavage map.

Claims (1)

[Claims] 1. Has a molecular weight of 4.2 Kb, has a restriction enzyme cleavage map shown in the following formula, and has Bam HI, Bc1I,
A plasmid that is not cut by EcoR1, HpaI, PstI, SacII, Sa1I and XhoI restriction enzymes. formula