FR2581653A1 - DNA fragment containing a gene encoding phosphoenolpyruvate carboxylase, recombinant DNA containing it and microorganism containing this recombinant DNA. - Google Patents

Abstract

The invention relates to a DNA fragment containing a gene encoding phosphoenolpyruvate carboxylase. According to the invention, the gene is derived from a glutamic acid-producing bacterium belonging to the genus Corynebacterium. It also relates to a recombinant DNA containing this fragment and to a microorganism containing the recombinant DNA. The accompanying drawing shows the restriction endonuclease cleavage map for a plasmid pAG203. The invention applies especially to the production of amino acids or nucleic acids with a high yield.

Description

 The present invention relates to a DNA fragment containing a gene encoding phosphoenolpyruvate carboxylase (often hereinafter referred to as "PEPC").

More particularly, the present invention relates to a DNA fragment which contains a gene coding for PEPC and which is derived from a bacterium producing glutamic acid belonging to the genus Corynebacterium. The present invention also relates to a recombinant DNA containing the DNA fragment and a microorganism containing the recombinant DNA.

 In the present description, the restriction endonucleases (often referred to hereinafter simply as "restriction enzyme") represented by the following abbreviations are those obtained from the following microorganisms, respectively.

Abbreviation Microorganism
EcoRI: Escherichia coli RY13
Hindili: Haemophilus influenza Rd
PstI: Providencia stuartii 164
BamHI: Bacillus amyloliquefaciens H
XbaI: Xanthomonas badrii
BglII: Bacillus globigii
SalI: Streptomyces albus G
HaeIII: Haemophilus aegyptius
SacI: Streptomyces achromogenes
XhoI: Xanthomones holcicola
It is known that a bacterium producing glutamic acid belonging to the genus Corynebacterium produces L-glutamic acid in a high yield and that some mutant or variant of the bacterium producing glutamic acid produces other amino acids such as
L-lysine and purine nucleotides such as inosinic acid. Such a bacterium producing glutamic acid belonging to the genus Corynebacterium and its mutant or variant have been industrially used for the production of amino acids and purine nucleotides as mentioned above.

To obtain a bacterium belonging to the genus
Corynebacterium in a form capable of producing the desired products efficiently and in a high yield, is usually carried out a reproduction of the bacterium. Particularly in recent years attempts have been made to reproduce a bacterium producing glutamic acid belonging to the genus Corynebacterium by a recombinant DNA technique.Until now, in the practice of reproduction of an acid-producing bacterium Glutamic acid belonging to the genus Corynebacterium by recombinant DNA technique, the following genes were cloned (1) a gene for resistance to S- (2-aminoethyl) cysteine derived from Corynebacterium glutamicum (ATCC 21543) and a gene coding for anthranilate synthetase derived from Brevibacterium flavum (ATCC 14067) atsumata and others, Japanese Patent Application Publication No. 58-126789 / 19833 (2) a gene encoding ATP-phosphoribosyltransferase derived from Corynebacterium glutamicum UMizugami and others, Abridged lectures given at the annual meeting of the Agricultural Chemical Society of Japan P. 249 (1984) 0 etc.

 Most amino acids, nucleic acids, etc., are directly or indirectly produced from intermediates formed in the tricarboxylic acid cycle (hereinafter referred to as "TCA cycle"). The TCA cycle is a sequence enzymatic reactions occurring in the cells.

Oxalacetate, one of the TCA cycle intermediates, is given by the PEPC reaction. Therefore, if the PEPC activity can be increased in the cells of the aforementioned glutamic acid-producing bacterium belonging to the genus Corynebacterium, amino acids, nucleic acids, etc. high yield by fermentation using the bacteria.

In general, in order to increase the activity of a desired enzyme in the cells of a microorganism, cloning of the gene encoding the desired enzyme and transformation of the microorganism cells by the gene has been proposed. cloned. For the gene coding for PEPC, a gene coding for PEPC derived from Brevibacterium lactofermentum lItoh and others, abbreviated from the lectures given at the annual meeting of Agricultural Chemical, was cloned.
Society of Japan, page 431 (1984) 3. Furthermore, it has been proposed that the cloned gene coding for PEPC derived from the bacterium Brevibacterium lactofermentum be transformed into the cells of the bacterium Brevibacterium lactofermentum to increase the activity of PEPC of the bacterium (description of the patent application in Japan).
No. 60-87788). In order to obtain a bacterium belonging to the genus Corynebacterium capable of producing amino acids, nucleic acids, etc., at a high yield, an attempt has been made to transform the PEPC gene cloned below. above mentioned derivative of Brevibacterium lactofermentum in cells of a bacterium producing glutamic acid belonging to the genus
Corynebacterium, which bacterium is industrially used for the production of amino acids, nucleic acids, etc. However, satisfactory results have not been obtained with respect to the production of amino acids. Indeed, it has not obtained a bacterium producing glutamic acid belonging to the genus Corynebacterium capable of producing amino acids, nucleic acids, etc ... at a high yield.

In order to overcome the above-mentioned problem, the present inventors have carried out extensive and intensive studies. As a result, they succeeded in isolating a fragment of DNA, derived from a bacterium producing glutamic acid, belonging to the genus
Corynebacterium, containing a gene encoding phosphoenolpyruvate carboxylase and cloning the DNA fragment. They also found that when such a DNA fragment is inserted into a vector to obtain a
Recombinant DNA and a cell of a microorganism belonging to the genus Corynebacterium is transformed by the obtained recombinant, the activity of PEPC in the cell of the transformed microorganism can be increased. Thus, the present invention has been accomplished.

 Therefore, the object of the present invention is to produce a DNA fragment containing a gene encoding phosphoenolpyruvate carboxylase, useful for the production of amino acids, nucleic acids, etc., at a high yield by technique. recombinant DNA.

 Another object of the present invention is a recombinant DNA containing the above-mentioned DNA fragment.

 Another object of the present invention is the production of a microorganism containing the recombinant DNA mentioned above.

The invention will be better understood and other objects, features, details and advantages thereof will appear more clearly in the following explanatory description made with reference to the appended schematic drawings given solely by way of example illustrating several embodiments and in which
FIG. 1 illustrates restriction endonuclease cleavage map of a pAG203 plasmid of the present invention.
FIG. 2 illustrates the restriction endonuclease cleavage map of a plasmid pAG211 of the present invention.
FIG. 3 illustrates the restriction endonuclease cleavage map of a pAG103 plasmid used in the present invention.
FIG. 4 illustrates the restriction endonuclease cleavage map of a pAG204 plasmid of the present invention.
FIG. 5 illustrates the restriction endonuclease cleavage map of a plasmid pAG221 of the present invention.
FIG. 6 illustrates the restriction endonuclease cleavage map of a pAG1 plasmid used in the present invention.
FIG. 7 illustrates the restriction endonuclease cleavage map of a pAG3 plasmid used in the present invention.
FIG. 8 illustrates the restriction endonuclease cleavage map of a plasmid pAG14 used in the present invention.
FIG. 9 illustrates the restriction endonuclease cleavage map of a pAG50 plasmid used in the present invention.
FIG. 10 illustrates the restriction endonuclease cleavage map of plasmid pAG2001 of the present invention; and
FIG. 11 illustrates the restriction eridonuclease cleavage map of a plasmid pAG2002 of the present invention.

In FIGS. 1 to 11, the positions on the map of the various restriction sites are given in kilobase coordinates (hereinafter referred to as "kb") relative to the PstI restriction site at 0.0 / 11. 5 kb in Figure 1, at the restriction site of
SalI at 0.0 / 9.3 kb in FIG. 2, at the restriction site of EcoRI at 0.0 / 11.4 kb in FIG. 3, at the XbaIII restriction site at 00 / 22.9 kb on the FIG. 4, at the XbaI restriction site at 0.0 / 17.5 kb in FIG. 5> XbaI restriction site at 0.0120.4 kb in FIG. 6, at the Hind III restriction site at 0, 0 / 4.5 kb in Fig. 7, at the 0.01 / 10.6 kb XbaI restriction site in Fig. 8, at the 0.07 → 6 kb BarsHI restriction site in Fig. 9 at the 0.01 / 10.9 kb SalII restriction site in Figure 10- and at the 0.01 / 19.1 kb xbai restriction site in Figure 11. In Figure 1, DNA-A is a DNA fragment of the present invention having a molecular length of about 5.5 kb and pBR 325- (A) is a DNA fragment obtained by PstI cleavage of plasmid pBR325. In Figure 2, DNA- (B) is a DNA fragment of the present invention having a molecular length of about 3.3 kb and pBR325- (B) is a fragment of DNA obtained by SalI cleavage of the plasmid pBR325.In FIG. 3, DNA- (C) is a DNA fragment containing a gene coding for glutamate dehydrogenase and having a molecular length of about 5.4 kb and pBR325- (C) is a DNA fragment obtained by EcoRI cleavage of plasmid pBR325. in Figure 4, DNA- (D) is a DNA fragment of the present invention having a molecular length of about 11.5 kb and pAG103- (A) is a fragment of DNA obtained by XbaI cleavage of the plasmid pAG103. In Figure 5, pBR325- (D) is a DNA fragment obtained by XbaI cleavage of the plasmid derived from plasmid pBR325.

In Figure 9, DNA- (E) is a DNA fragment obtained by BamHI cleavage and BglII cleavage of plasmid pAG14. In FIG. 10, pAG50- (A) is a DNA fragment obtained by SalI cleavage of plasmid pAG50 and DNA- (B) is identical to that of FIG. 2. In FIG.
DNA- (D) is identical to that of FIG. 4 and pAG50- (B) is a fragment of DNA obtained by Xbai cleavage of plasmid pAG50.

Essentially, according to the present invention, there is provided a DNA fragment derived from a bacterium producing glutamic acid belonging to the genus
Corynebacterium, containing a gene encoding phosphoenolpyruvate carboxylase.

 Furthermore, according to the present invention, there is provided a recombinant DNA comprising a first DNA fragment, derived from a bacterium producing glutamic acid belonging to the genus Corynebacterium, containing a gene coding for phosphoenolpyruvate carboxylase and a second fragment of DNA containing a gene for replication in a cell of a bacterium, said first DNA fragment being directly or indirectly linked to said second DNA fragment.

 Furthermore, according to the present invention, there is provided a microorganism which comprises a bacterium and the recombinant DNA mentioned above, said recombinant DNA being contained in said bacterium.

 The bacterium producing glutamic acid belonging to the genus Corynebacterium is a gram-positive, non-motile, non-mobile bacterium that does not form a spore and requires biotin for its growth, and that produces L-glutamic acid at a high level. yield in a medium containing biotin in a limited amount or in a medium supplemented with a surfactant containing biotin in a large amount. The DNA fragment of the present invention is derived from the above-mentioned bacterium producing glutamic acid which belongs to the genus Corynebacterium.

 The DNA fragment of the present invention contains a gene encoding phosphoenolpyruvate carboxylase. When the gene is expressed in a cell of a microorganism, phosphoenolpyruvate carboxylase (PEPC) is produced in the cell. Phosphoenolpyruvate carboxylase is an enzyme and catalyzes the conversion between phosphoenolpyruvic acid and oxaloacetic acid.

The DNA fragment containing a gene coding for
PEPC can be isolated from the coryneform bacteria producing glutamic acid using a usual host-vector system. For the host, for example, Escherichia coli (often referred to as "E.

For E. coli, strain K12 can be used.
E. coli and its variants. As a vector used in a host-vector system, where E. coli or its variant is used as a host, mention may be made of the plasmids usually employed to transform E. coli and its variants. It is also possible to employ a host-vector system in which a coryneform bacterium producing glutamic acid is used as the host and a usual plasmid compatible with the coryneform bacterium producing glutamic acid is used as vector.

As a coryneform bacterium producing glutamic acid, there may be mentioned bacteria belonging to
Corynebacterium, Brevibacterium or Microbacterium.

The DNA fragment containing a gene coding for
PEPC can be isolated from the bacterium producing glutamic acid belonging to the genus Corynebacterium by the following method.

 (1) All of the DNA is extracted from a cell of a bacterium producing glutamic acid belonging to the genus Corynebacterium and is partially cleaved by various restriction enzymes to obtain DNA fragments. Extraction of the DNA from the bacterium can be accomplished by a usual method in which lysozyme-SDS treatment and phenol-chloroform treatment are carried out. As restriction enzymes to be used to split all of the above-mentioned bacterium DNA, any restriction enzyme can be employed as long as it can split not only the above-mentioned bacterium DNA but also 1 DNA of the below-mentioned vector of a host-vector system to be used for cloning the DNA fragment of the present invention. The DNA fragments thus obtained contain a DNA fragment containing a gene coding for PEPC.

 (2) a vector DNA is completely cleaved by at least one of the appropriate restriction enzymes mentioned above to obtain a split vector DNA.

 For the vector, one must use those that are compatible with a host to transform as well. It is necessary to confirm, in advance, that the vector is not digested by the bacterium that is to be used as a host.

(3) The respective DNA fragments obtained in step (1) above are separately inserted into the cleavage site of the cleaved vector DNA obtained in step (2) above according to any usual method. , for example the method of Maniatis and others (T. Maniatis, EF Fritsch, J Sambrook: molecular Cloning A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, New York 1982). Thus, circular recombinant DNAs are obtained which contain a recombinant DNA having a DNA fragment containing a gene encoding PEPC.

 (4) The respective recombinant DNAs are separately transferred into an E. coli cell to form transformants.

The conversion can be carried out by a usual method, for example the method of Nandel and others Vandel, M., Higa, A., J. Nol. Biol. t 53, 159-162 (1970g
As E. coli to be used for transformation, a mutant strain deficient in PEPC of E. coli is used. The reason is the following. In order for the mutant strain to grow, glutamic acid or an organic acid to be formed as an intermediate in the TAC cycle must be present in the medium, and therefore the mutant strain can not be killed in a medium which does not contain the However, when the mutant strain is transformed by a recombinant DNA containing a gene encoding PEPC, the mutant strain no longer requires glutamic acid or acid. organic for its growth and can grow even in the medium containing neither glutamic acid nor this organic acid. Moreover, even if the mutant strain is transformed by a recombinant DNA, if the recombinant DNA does not contain a gene coding for PEPC, the strain still requires glutamic acid or organic acid for its growth and can not fester in the medium containing neither glutamic acid nor this organic acid.Therefore, when the above transformants obtained are cultured in a medium containing neither glutamic acid nor this organic acid, only the transformant which retains a recombinant DNA containing a gene coding for PEPC grows in the medium.

Accordingly, the desired transformant can be efficiently and easily selected and isolated using a mutant strain of E. coli deficient in PEPC.

 Such a mutant strain of E. coli can be obtained by the usual methods. For example, such a mutant strain can be obtained as follows.

First, E. coli cells are mutated using N-methyl-N'-nitro-N-nitrosoguanidine to obtain mutant cells. Mutant cells, auxotrophic mutant cells that require glutamic acid are selected and isolated. Mutant cells thus isolated are subject to PEPC activity determinations, and a mutant cell lacking PEPC activity is selected. The cell thus selected is cultured to obtain a mutant strain of E. coli deficient in PEPC. The mutation of
E. coli can also be carried out by usual techniques, for example ultraviolet irradiation, X-ray irradiation, treatment with various mutagens, treatment with transposons, etc.

Alternatively, the known strain deficient in PEPC can be obtained from public deposits having such a strain.

 (5) transformants obtained in step (4) above, the transformant which retains a recombinant DNA containing a gene coding for PEPC is selected and isolated by the criterion of auxotrophy of glutamic acid. By way of example, the transformants are cultured in a synthesis medium containing no glutamic acid at 20 to 370 ° C. according to a usual method. The cells growing in the synthesis medium not containing glutamic acid are isolated. The cells thus isolated are cultured separately in the medium to obtain strains. The cells of the respective strains are collected, followed by extraction to obtain cell extracts. The respective cell extracts are subjected to a measurement of PEPC activity according to a customary method for selecting a strain exhibiting PEPC activity. The strain exhibiting high activity of PEPC has a DNA fragment containing a gene encoding PEPC. . The strain is cultured to clone the DNA fragment.

The DNA fragment is isolated from the cells of the strain according to a customary method.

One of the specific examples of the DNA fragment of the present invention is obtained from the microorganism
Corynebacterium melassecola 801 deposited in Fermentation
Research Institute (hereinafter referred to as "FR1") under accession number FERM BP-558, and has the following characteristics
(1) The DNA fragment has a molecular length of about 11.5 kb.

 (2) The DNA fragment has its two sticky ends formed by XbaI splitting.

(3) the DNA fragment has the following restriction endonuclease cleavage sites
a first PstI site, a second PstI site, a first EcoRi site, a third PstI site, a first HindIII site, a first BamHI site, a fourth site
PstI, a fifth PstI site, a second HindIII site, a third HindIII site, a first SalI site, a second EcoRi site, a fourth HindIII site, a third EcoRi site, a sixth PstI site, a seventh PstI site, a fifth site HindIII, an eighth PstI site, a second BamHI site, a second SalI site, a ninth PstI site and a third BamHI site,
said second PstI site, said first EcoRi site, said third PstI site, said first Hindili site, said first BamHI site, said fourth site
PstI, said fifth PstI site, said second site
HindIII, said third HindIII site, said first site
SalI, said second EeoRI site, said fourth Hindili site, said third EcoRI site, said sixth site
PstI, said seventh PstI site, said fifth Hindili site, said eighth PstI site, said second site
BamHI, said second SalI site, said ninth PstI site and said third BamHI site are respectively located at about 1.0 kb, about 1.9 kb, about 2.5 kb, about 2.6 kb, about 3.5 kb, about 3.7 kby about 4.0 kb, about 4.3 kb, about 4.4 kb, about 5.0 kb, about 6.3 kb, about 6.5 kb, about 6.7 kb, about 6 kb, 9 kb, about 7.0 kb, about 7.0 kb, about 7.4 kb, about 7.4 kb, about 8.3 kb, about 9.5 kb and 10.3 kb of said first PstI site.

 Restriction endonuclease cleavage sites (often referred to simply as below).

"restriction") of the DNA fragment are determined as follows: First, the recombinant DNA containing the DNA fragment is obtained from cells of the aforementioned strain by a usual method. DNA is obtained from the recombinant DNA using the restriction enzymes, the DNA fragment thus obtained is completely digested in the presence of an excess of a restriction enzyme, and the resulting digested products are electrophoresed on a gel. 1% (w / v) agarose followed by a 4% (w / v) polyacrylamide gel The number of restriction sites is determined from the number of resolvable fragments in the agarose gel and in the polyacrylamide gel. The molecular length of each of the cleaved fragments is determined as follows. In the case of agarose gel electrophoresis, fragments which are obtained by digestion of the phage DNA of E. coli by HindIII and whose molecular lengths are respectively 23130 bp, 9419 bp, 6557 bp, 4371 bp, 2322 bp, 2028 bp, 564 bp and 125 bp (base pair) in the order of long molecular length undergo electrophoresis on the same agarose gel to form patterns standards for fragment migration. The molecular length of each of the fragments obtained by digestion of the DNA fragment is determined relative to the standard migration patterns. In the case of polyacrylamide gel electrophoresis, the fragments which are obtained by digestion of the Fox174 phage DNA of E. coli by HaeIII and whose molecular lengths are respectively 1353 bp, 1078 bp, 872 bp, 603 bp, 310 bp, 281 bp, 271 bp, 234 bp, 194 bp, 118 bp and 72 bp> in the order of great molecular length, undergo electrophoresis on the same polyacrylamide gel, thus forming standard patterns of migration of fragments. The molecular length of each fragment obtained by digestion of the DNA fragment is determined relative to the standard patterns of migration. The molecular length of the DNA fragment is obtained by adding the molecular lengths of the fragments obtained by restriction enzyme cleavage.

 The restriction sites of the DNA fragment can be determined by completely digesting the DNA fragment with various kinds of restriction enzymes, electrophoresing the resulting fragments on an agarose gel and then on a polyacrylamide gel and analyzing the results obtained. .

 The DNA fragment can be modified as long as it does not harm the gene coding for PEPC. For example, a portion of the DNA fragment may be left while retaining the PEPC-encoding gel in the DNA fragment to obtain a deletion derivative of the DNA fragment. Partial deletion of the DNA fragment can be accomplished by digesting the DNA fragment with one or more restriction enzymes.

As specific examples of such a deletion derivative, there may be mentioned the following DNA fragments: (I) a DNA fragment having
(1) a molecular length of about 8.3 kb
(2) a sticky tip formed by SalI and the other sticky tip formed by XbaI; and
(3) the following restriction sites
a first PstI site, a second PstI site, a first EcoRi site, a third PstI site, a first Hindili site, a first BamHI site, a fourth site
PstI, a fifth PstI site, a second HindIII site, a third HindIII site, a SalI site, a second EcoRi site, a fourth HindIII site, a third EcoRi site, a sixth PstI site, a seventh PstI site, a fifth HindIII site , an eighth PstI site, and a second BamHI site,
said second PstI site, said first EcoRi site, said third PstI site, said first site
HindIII, said first BamHI site, said fourth site
PstI, said fifth PstI site, said second site
HindIII, said third HindIII site, said SalI site, said second EcoRi site, said fourth HindIII site, said third EcoRi site, said sixth PstI site, said seventh PstI site, said fifth HindIII site, said eighth PstI site and said second BamHI site. being respectively placed at about 1.0 kb, about 1.9 kb, about 2.5 kb, about 2.6 kb, about 3.5 kb, about 3.7 kb, about 4.0 kb, about 4.3 kb kb, about 4.4 kb, about 5.0 kb, about 6.3 kb, about 6.5 kb, about 6.7 kb, about 6.9 kb, about 7.0 kb, about 7.0 kb, about 7.4 kb, and about 7.4 kb from said first site
Pst.

(II) a DNA fragment having
(1) a molecular weight of about 6.4 kb
(2) a sticky tip formed by splitting with
EcoRI and the other sticky tip formed by split with
SalI; and
(3) restriction sites as follows
a first PstI site, a first HindIII site, a first BamHI site, a second PstI site, a third PstI site, a second Hindili site, a third Hindili site, a SalI site, a first EcoRi site, a fourth Hindili site, a second EcoRi site, a fourth PstI site, a fifth PstI site, a fifth Hindili site, a sixth PstI site and a second site
Bam,
said first HindIII site, said first site
BamHI, said second PstI site, said third PstI site, said second HindIII site, said third HindIII site, said SalI site, said first EcoRI site, said fourth HindIII site, said second EcoRi site, said fourth PstI site, said fifth PstI site said fifth HindIII site, said sixth PstI site and said second BamHI site being respectively located at about 0.1 kb, about 1.0 kb, about 1.2 kb, about 1.5 kb, about 1.8 kb, about 1.9 kb, about 2.5 kb, about 3.8 kb, about 4.0 kb, about 4.2 kb, about 4.4 kb, about 4.5 kb, about 4.5 kb, about 4 kb 9 kb and about 4.9 kb from said first PstI site.

(III) a DNA fragment having
(1) a molecular weight of about 5.5 kb
(2) the two sticky ends formed by splitting with PstI; and
(3) restriction sites as follows
a first HindIII site, a second HindIII site, a first SalI site, a first EcoRi site, a third HindIII site, a second EcoRI site, a first PstI site, a second PstI site, a fourth Hindili site, a third PstI site, a BamHI site and a second SalI site,
said second site Hindili, said first site
SalI, said first EcoRi site, said third site
HindIII, said second EcoRi site, said first site
PstI, said second PstI site, said fourth site
HindIII, said third PstI site, said BamHI site and said second SalI site being respectively located at about 0.1 kb, about 0.7 kb, about 2.0 kb, about 2.2 kb, about 2.4 kb, about 2.6 kb, about 2.7 kb, about 2.7 kb, about 3.1 kb, about 3.1 kb and about 4.0 kb of said first HindIII site.

(IV) a DNA fragment having
(1) a molecular weight of about 4.8 kb;
(2) a sticky tip formed by splitting with
BamHI and the other sticky tip formed by splitting with SalI; and
(3) restriction sites as follows
a first PstI site, a second PstI site, a first HindIII site, a second HindIII site, a site
SalI, a first EcoRi site, a third HindIII site, a second EcoRi site, a third PstI site, a fourth PstI site, a fourth HindIII site, a fifth PstI site and a BamHI site,
said second PstI site, said first site
HindIII, said second HindIII site, said SalI site, said first EcoRi site, said third HindIII site, said second EcoRi site, said third PstI site, said fourth PstI site, said fourth Hindili site, said fifth PstI site and said BamHI site being placed at about 0.3 kb, about 0.6 kb, about 0.7 kb, about 1.3 kb, about 2.6 kb, about 2.8 kb, about 3.0 kb, about 3.2 kb , about 3.3 kb, about 3.3 kb, about 3.7 kb, and about 3.7 kb of said first PstI site.

(V) a DNA fragment having
(1) a molecular weight of about 3.9 kb
(2) a sticky tip formed by splitting with HindIII and the other sticky tip formed by splitting with
SalI; and
(3) restriction sites as follows
a SalI site, a first EcoRi site> a first HindIII site, a second EcoRi site> a first PstI site, a second PstI site, a second HindIII site, a third PstI site, and a BamHI site,
said first EcoRi site, said first HindIII site, said second EcoRi site, said first site
PstI, said second PstI site, said second HindIII site, said third PstI site and said BamHI site are respectively located at about 1.3 kb, about 1.5 kb, about 1.7 kb, about 1.9 kb, about 2 kb , 0 kb, about 2.0 kb, about 2.4 kb, and about 2.4 kb from said site
Sall.

(VI) a DNA fragment having
(1) a molecular weight of about 3.3 kb
(2) the two sticky ends formed by splitting with SalI; and
(3) restriction sites as follows
a first EcoRi site, a first HindIII site, a second EcoRi site, a first PstI site, a second PstI site, a second HindIII site, a third PstI site and a BamHl site,
said first HindIII site, said second EcoRi site, said first PstI site, said second PstI site, a second HindIII site, a third PstI site and a site
Wherein BamHI are placed at about 0.2 kb, about 0.4 kb, about 0.6 kb, about 0.7 kb, about 0.7 kb, about 1.1 kb, and about 1.1 kb, respectively, of said first EcoRi site. .

The DNA fragment containing a gene coding for
PEPC mentioned above can also be easily modified by changing the base sequence of at least one restriction site of the DNA fragment such that the restriction enzyme effective to split the restriction site is changed into a different kind. restriction enzyme, while keeping the gene intact for
PEPC. The DNA fragment can also be changed at least at its first end relative to the base sequence so that the end of the DNA fragment can be adapted to the base sequence of a split portion of a vector which cleaved portion must be linked to the end of the DNA fragment.

 As described above, the aforementioned PEPC-deficient strain of E. coli does not grow without glutamic acid or an organic acid to form in the TAC cycle and does not produce PEPC.

However, when a recombinant DNA which comprises a DNA fragment containing a gene encoding PEPC is incorporated into the aforementioned strain, the aforementioned strain is forced to produce PEPC without requiring glutamic acid or organic acid to form in the TAC cycle. Therefore, the presence of a gene encoding PEPC in the DNA fragment of the present invention can be confirmed by the fact that when the DNA fragment of the present invention is inserted into a vector to form recombinant DNA then that the recombinant DNA is transferred to cells of the aforementioned strain, the cells grow in a medium containing no glutamic acid or organic acid as mentioned above and produce PEPC in the cells.

 The recombinant DNA of the present invention comprises a first DNA fragment containing a gene encoding PEPC and a second DNA fragment containing a gene for replication in a cell of a bacterium.

The first DNA fragment is linked directly or indirectly to the second DNA fragment.

 For the first DNA fragment, a DNA fragment of the present invention is used.

For the second DNA fragment, there may be mentioned any DNA fragment derived from a vector of a host-vector system which is usually employed in the recombinant DNA technique. For this DNA fragment, there may be mentioned, for example, a DNA fragment containing a vector for a host-vector system where a coryneform bacterium producing glutamic acid is used as a host, a DNA fragment containing a vector for a host-vector system where Escherichia coli is used as a host, a vector-containing DNA fragment for a host-vector system where Bacillus subtilis is used as a host, and the like.For the coryneform bacterium producing glutamic acid above mentioned, mention may be made of a bacterium belonging to the genus
Corynebacterium, a bacterium belonging to the genus
Brevibacterium and a bacterium belonging to the genus
Microbacterium. For the vector-containing DNA fragment for a host-vector system where a coryneform bacterium producing glutamic acid is used as a host, there may be mentioned, for example, DNA fragments derived from pAG1, pAG3, pAG14, pAG50. and pAG103. The plasmid pAG1 can be obtained from the microorganism Corynebacterium melassecola 22243 deposited at the FRI under accession number FERM BP-560. The plasmid pAG3 can be obtained from Corynebacterium melassecola 22220 deposited with FRI under accession number FERM BP-559. Plasmid pAG14 is a deletion derivative of plasmid pAG1. Plasmid pAG50 comprises part of plasmid pAG14 and part of plasmid pAG3. The plasmid pAG103 comprises a plasmid pBR325 cleaved with EcoRI and a DNA fragment derived from
Corynebacterium melassecola 801 (FERM BP-558), containing a gene coding for glutamate dehydrogenase and having a molecular length of about 5.4 kb. The methods for obtaining these plasmids will be described in the examples below. Restriction endonuclease cleavage maps of plasmids pAG103, pAG1, pAG3, pAG14, pAG50 are shown in FIGS. 3, 6, 7, 8 and 9, respectively. As a vector-containing DNA fragment for a host-vector system or Escherichia coli is used as a host, for example, fragments of DNA derived from plasmid pBR325 may be mentioned. As a vector-containing DNA fragment for a host-vector system where Bacillus subtilis is used as a host, there may be mentioned, for example, DNA fragments derived from plasmid pUB110.

 The recombinant DNA of the present invention can be prepared by ligation of the first DNA fragment directly to the second DNA fragment. Alternatively, the recombinant DNA of the present invention may be prepared by ligation of the first DNA fragment indirectly to the second DNA fragment by another type of DNA fragment. To prepare the recombinant DNA, a standard hSte-vector system may be employed. As a host-vector system, there may be mentioned, for example, a host-vector system using E. coli as host, a host-vector system using Bacillus subtilis as host, a host-vector system using a coryneform bacterium producing glutamic acid as a host, etc.

 For recombinant DNA comprising the first DNA fragment and the second DNA fragment, there may be mentioned, for example, plasmids pAG203, pAG211, pAG204, pAG221, pAG2001, pAG2002, etc. Plasmid pAG203 comprises, as the first DNA fragment, the above-mentioned DNA (III) fragment having a molecular length of about 5.5 kb and for the second DNA fragment, a PstI-cleaved plasmid pBR325 . Plasmid pAG211 comprises, for the first DNA fragment, the above-mentioned DNA fragment (IV) having a molecular length of about 3.3 kb and for the second DNA fragment, a SalI-cleaved plasmid. Plasmid pAG204 comprises, for the first DNA fragment, the above-mentioned DNA fragment having a molecular length of about 11.5 kb and for the second DNA fragment, a plasmid pAG103 cleaved by XbaI. Plasmid pAG221 comprises, for the first DNA fragment, the above-mentioned DNA fragment having a molecular length of about 11.5 kb and for the second DNA fragment a plasmid-derived plasmid-cleaved plasmid. pBR325. The plasmid pAG2001 comprises, for the first DNA fragment, the DNA fragment (VI) having a molecular length of approximately 3.3 kb and for the second DNA fragment, a plasmid pAG50 cleaved with SalI. The plasmid pAG2002 comprises, for the first DNA fragment 1 the above-mentioned DNA fragment having a molecular length of about 11.5 kb and for the second DNA fragment, a plasmid pAG50 split by XbaI.

 The above-mentioned recombinant DNA of the present invention can be modified by changing the base sequence of at least one restriction site of the recombinant DNA so that the restriction enzyme is effective in splitting the restriction site. be changed to a different type of restriction enzyme, while retaining the intact gene encoding PEPC and the gene for replication.

 The microorganism of the present invention comprises a bacterium and a recombinant DNA which comprises a DNA fragment containing a gene encoding PEPC and a DNA fragment containing a gene for replication.

The recombinant DNA is contained in the bacterium.

 As recombinant DNA, the above-mentioned recombinant DNA of the present invention is used.

 For the bacterium, there may be mentioned, for example, the known strains of Escherichia coli and the coryneform bacteria producing glutamic acid mentioned above.

 The microorganism of the present invention is prepared by transforming the bacterium with recombinant DNA using a standard recombinant DNA technique.

As the microorganism of the present invention, there may be mentioned, for example, strain 342-167 of
E. coli K12 (pAG203), E. coli K12 strain 342-167 (pAG211), E. coli K12 strain 342-167 (pAG204), E. coli K12 strain 342-167 (pAG221), Corynebacterium melassecola 801 (pAG2001), Corynebacterium melassecola 801 (pAG2002), etc. E. coli K12 strain 342-167 (pAG203), E. coli K12 strain 342-167 (pAG211), E. coli K12 strain 342-167 (pAG204) and E. strain 342-167. coli K12 (pAG221) are prepared by transforming cells of E. coli strain 342-167
K12 by the above-mentioned plasmids pAG203, pAG211, pAG204 and pAG221, respectively. E. coli K12 strain 342-167 may be publicly available at the E. coli Genetic Stock Center, Department of Human
Genetics, Yale University, School of Medicine, USA

Corynebacterium melassecola 801 (pAG2001) and
Corynebacterium melassecola 801 (pAG2002) are prepared by transformation of Corynebacterium melassecola 801 cells by the above-mentioned plasmids pAG2001 and pAG2002, respectively. Corynebacterium melassecola 801 is, as mentioned above, deposited at the Fermentation Research Institute under accession number FERM BP-558 and is available for Fermentation
Research Institute.

 The DNA fragment of the present invention has the following advantages.

When the DNA fragment of the present invention is inserted into a vector for a host-vector system where a bacterium belonging to the genus
Corynebacterium is used as a host to form a
Recombinant DNA, and that the recombinant DNA thus formed is transferred into a bacterium belonging to the genus
Corynebacterium, the PEPC production capacity of the bacterium can be improved and the bacterium can produce PEPC at a high yield. As previously mentioned, reactions in the TAC cycle are promoted by the action of PEPC to form organic acids as intermediates. From the intermediates thus formed, various amino acids, desired nucleic acids and the like are produced. Therefore, by culturing the bacterium with improved ability to produce PEPC, such amino acids, nucleic acids and the like can be high yield products. Indeed, the DNA fragment of the present invention is useful for obtaining a bacterium producing glutamic acid belonging to the genus
Corynebacterium, which bacterium is capable of producing amino acids, nucleic acids, etc., at a high yield.

 The recombinant DNA of the present invention has the following advantages.

When the second DNA fragment of the recombinant DNA is a DNA fragment containing a vector for a host-vector system where a bacterium belonging to the genus Corynebacterium is used as a host, the recombinant DNA of the present invention can be advantageously used to transform a bacterium belonging to the genus Corynebacterium to obtain the above-mentioned bacterium belonging to the genus
Corynebacterium having excellent ability to produce amino placids, nucleic acids, etc.

On the other hand, when the second DNA fragment of the recombinant DNA is a vector-containing DNA for a host-vector system where a bacterium other than that belonging to the genus is used as the host.
Corynebacterium, as a bacterium belonging to the genera Escherichia, Bacillus, Brevibacterium,
Microbacterium, etc., the recombinant DNA of the present invention may be advantageously employed for the multiplication of the DNA fragment of the present invention.

 The microorganism of the present invention has the following advantages.

 In the case where the microorganism containing the recombinant DNA of the present invention is a bacterium belonging to the genus Corynebacterium, the microorganism of the present invention is useful for the production of various amino acids, various nucleic acids, etc. at a high efficiency. In the case where the microorganism is a bacterium other than that mentioned above, the microorganism of the present invention can be directly used for the multiplication of the DNA fragment of the present invention.

 The present invention will be further illustrated by reference to the following examples, which should not be construed as limiting the scope of the present invention.

 Example 1

In this example, Corynebacterium melassecola 801 (FERM BP-558) was used as a DNA donor to clone a gene encoding PEPC (Corynebacterium melassecola 801 is deposited at
Fermentation Research Institute under accession number FERM BP-558). The gene encoding PEPC was cloned using the host-vector system where Escherichia coli K12 strain 342-167 was used as a host and plasmid pBR325 was used as the vector.

Escherichia coli K12 strain 342-167 was obtained from Dr. Barbara J. Bachmann of E. coli Genetic Stock
Center, Department of Human Genetics, University of
Yale, School of Medicine, 333 Ceder Street PO Box 3333
New Haven, Connecticut 06510 EUA. Strain 342-167 of Escherichia coli K12 is publicly available in E.

coli Genetic Stock Center. Plasmid pBR325 was purchased from Bethesda Research Laboratories, E.U.A.

Step 1. Extraction of the DNA from
Corynebacterium melassecola 801 (FERM BP-558) and its split3.

 The Corynebacterium melassecola 801 microorganism (FERM BP-558) was inoculated into 100 ml of a molasses medium (prepared by dissolving 80 g black beetle ligule molasses, 0.5 g MgSO 4 .7H 2 O, 8 g of urea and 1.5 g of phosphoric acid in water so that the total volume is 1 liter and adjusting the pH to 6.2), followed by sterilization at 1200C for 15 minutes and culturing at 320C overnight while stirring, to thereby obtain a culture of the microorganism.

From the culture, cells of the microorganism were collected, washed and then suspended in 8 ml of a buffer containing 10 mM tris-HCl (pH 8.0) and 1 mM EDTA. To this suspension, lysozyme was added in such an amount that the resulting mixture had a lysozyme concentration of 5 mg / ml. The resulting mixture was allowed to stand at 370 ° C. for 4 hours. Then, to the mixture was added Pronase E (manufactured and sold by
Sigma Chemical Company, USA) in an amount such that the resulting mixture has a Pronase E concentration of 200 μg / ml. The resulting mixture was allowed to stand at room temperature for 15 minutes. Then, to the mixture, sodium dodecyl sulfate was added in such an amount that the resulting mixture had the concentration of sodium dodecyl sulfate 1% (w / v). The mixture was allowed to stand at 370 ° C for 1 hour. Then, an equal volume of phenol saturated with a TNE buffer solution (containing 50 mM tris-HCl, 5 mM EDTA, 100 mM NaCl and pH 8.0) was added to the above mixture. Then, the resultant mixture was subjected to centrifugation at 10,000 rpm (11000 g) for 10 minutes and an aqueous phase was collected.

To the aqueous phase, an equal volume of phenol-chloroform (1: 1, v / v) was added, followed by mixing. Then, the resulting mixture was centrifuged at 10,000 rpm (11000 g) for 10 minutes and an aqueous phase was recovered. To the aqueous phase, an equal volume of chloroform was further added, followed by mixing. Then, the resulting mixture was centrifuged at 10,000 rpm (11000 g) for 10 minutes and an aqueous phase was recovered. To the aqueous phase, ribonuclease A (manufactured and sold by Sigma Chemical Company, U.S.A.) was added in an amount such that the resulting mixture had a ribonuclease A concentration of 40 ug / ml. The mixture was allowed to stand at 370 ° C for one hour. Then, 1/5 volume of an aqueous solution containing 5M NaCl and 1/4 volume of an aqueous solution containing 50% (w / v) was added to the mixture. ) of polyethylene glycol 6000, followed by mixing. The resulting mixture was allowed to stand at 40C for 4 hours. Then, the mixture was centrifuged at 5000 rpm (2700 g) for 20 minutes to obtain precipitates and a supernatant. The precipitates were collected and dissolved in 4 ml of TE buffer containing 10 mM tris-HCl and 1 mM EDTA, pH 7.5. To the solution thus obtained, sodium acetate was added in such an amount that the resulting mixture had a sodium acetate concentration of 300 millimoles. Subsequently, twice the volume of ethanol was added to the mixture. The mixture thus obtained was stirred, allowed to stand at -300 ° C. for 3 hours and centrifuged at 10,000 ° C. / min (11000g) for 20 minutes to obtain a supernatant and precipitates. The precipitates were collected, dried under reduced pressure and dissolved in 2 ml of TE buffer, thereby obtaining a DNA solution containing 0.85 mg / ml of DNA.

The DNA in the DNA solution was split as follows. 16 units of the PstI restriction enzyme (manufactured and sold by Bethesda Research Laboratories,
EUA) were added to 145 μg of the DNA, and the resulting mixture was mixed with 200 μl of a buffer containing 10 mM Tris-HCl (pH 7.4), 50 mM NaCl, and 1 mM dithiothreitol and left stand at 300C for 2 hours so that the reaction takes place. Then, the reaction mixture was heated at 700C for 10 minutes to stop the reaction.

Step 2. (Preparation of plasmid pBR325 and its split)
Escherichia coli K12 strain 342-167 was inoculated into 50 ml of L broth (containing 10 g / l of polypepone, 5 g / l of yeast extract, 5 g / l of NaCl and pH 7.2). and cultured at 370C until the cell concentration reaches 5x108 cells / ml. Cells were harvested from the culture by centrifugation at 20C. The cells were suspended in 50 ml of 100 mM MgCl 2 while cooling with ice. From the suspension, the cells were collected and suspended in 25 ml of CaCl 2 in 100 mM while cooling with ice. The suspension thus obtained was allowed to stand ice for 30 minutes. From the suspension, the cells were collected and suspended in 5 ml of 100 mM CaCl 2 while cooling with ice. The suspension thus obtained was allowed to stand in ice for 1 hour to obtain a suspension of competent cells. To 200 μl of the suspension was added 0.1 μg of pBR325 DNA (manufactured and sold by Bethesda Research Laboratories, USA) and the resulting mixture was allowed to sit in ice for 1 hour.

Subsequently, the mixture was allowed to stand at 420 ° C. for 2 minutes and then 5 ml of L broth was added to the mixture. The resulting mixture was incubated stationary at 370 ° C. for 900 minutes. Then, the mixture was diluted with sterile water and spread on agar medium L (medium obtained by adding 15 g of agar to L broth so that the total volume was 1 liter) containing 20 μg. ml of chloramphenicol, followed by overnight incubation at 370C to obtain
Escherichia coli transformed with the plasmid pBR325.

 Escherichia coli K12 strain 342-167 transformed with plasmid pBR325 was inoculated into 100 ml L-broth containing 20 μg / ml chloramphenicol and cultured overnight at 370 ° C.

From the mixture, cells were harvested by centrifugation and washed with TE buffer. The cells were suspended in 2 ml of an aqueous solution consisting of 15% (w / v) sucrose, 50 mM Tris-HCl (pH 8.5), 50 mM EDTA and 2 mg / ml lysozyme (manufactured and sold by Sigma Chemical
Company, USA) and allowed to stand at room temperature for 30 minutes. To the resulting suspension was added 2 ml Triton solution (w / v) of Triton X-100 (manufactured by Rohm & Haas
Co., USA and sold by Wako Pure Chemical Industries
Ltd., Japan), 50 mM Tris-HCl (pH 8.5) and 50 mM EDTA was allowed to stand at 370 ° C. for 30 minutes. The resulting solution was centrifuged at 30,000 rpm (64,000 g) at 50 ° C for 1 hour to obtain a supernatant. To the supernatant, a TE buffer was added in such a quantity that the resulting mixture had a volume. of 18 ml. 1.2 ml of a 10 mg / ml aqueous solution of ethidium bromide and 18.64 g of cesium chloride were gently dissolved in the above mixture, followed by centrifugation at 40000 rpm ( 100000 g) at 150C for 48 hours. After completion of the centrifugation, two bands were visualized under ultraviolet light. The lower band was taken from the centrifuge tube through the side of the tube using a syringe to obtain a fraction of plasmid pBR325. The fraction thus obtained was extracted four times with an equal volume of isopropyl alcohol to obtain an extract. From the extract, the ethidium bromide was removed and the resulting solution was dialyzed with TE buffer for obtain 1 ml of a dialysate containing plasmid pBR325 which had a DNA concentration of 150 wug / ml.

 To an aliquot of the dialysate containing 15 μg of the resulting pBR325 plasmid DNA, 45 units of the PstI restriction enzyme were added. The resulting mixture was added to 150nul of a buffer solution containing 10mM Tris-HCl (pH 7.4), 10mM MgSO4, 50mM NaCl and 1mM dithiothreitol, followed by incubation at 300C for 2 hours to advance a reaction. .

Then, the mixture was heated at 700C for 10 minutes to stop the reaction. To the reaction mixture was added sodium acetate in such an amount that the resulting mixture contained sodium acetate at a concentration of 300 millimoles. To the mixture was added twice its volume of ethanol and the resulting mixture was allowed to stand at -300C for 3 hours. Then, the mixture was centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes to obtain a supernatant and precipitates. The precipitates were collected and dried under reduced pressure. The precipitates thus obtained were dissolved in 200 μl of a BAPT buffer (50 mM Tris-HCl, pH8.4). To the solution thus obtained was added a unit of bacterial alkaline phosphatase (manufactured and sold by Takara
Shuzo Co., Ltd., Japan) followed by incubation at 650C for 30 minutes. Then, one unit of bacterial alkaline phosphatase was added to the solution, followed by incubation at 650C for 30 minutes. Then, an equal volume of saturated phenol of the TNE buffer was added to the solution with mixing. The resulting mixture was centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes and an aqueous phase was recovered. To the aqueous phase thus obtained, an equal volume of phenol saturated with TNE buffer was added, followed by mixing. The resulting mixture was centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes and an aqueous phase was recovered. An equal volume of a mixture of phenol-chloroform ( 1: 1> volume / volume), to the aqueous phase, followed by mixing. The resulting mixture was centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes and an aqueous phase was recovered. Then, an equal volume of chloroform was added to the aqueous phase with stirring, and centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes to recover an aqueous phase. To the aqueous phase thus obtained was added sodium acetate in such an amount that the resulting mixture contained sodium acetate at a concentration of 300 millimoles and twice the volume of ethanol with stirring. The resulting mixture was allowed to stand at -300C for 3 hours.

Subsequently, the mixture was centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes to obtain DNA precipitates. The DNA precipitates were dried under reduced pressure and dissolved in 30 μl of a TE buffer solution.

Step 3. (DNA Recombination) 2.9 μg of the DNA obtained in Step 1 of Example 1 was added, and 1.5 μg of the DNA obtained in Step 2 of Example 1 and 3 units of phage T4 DNA ligase (manufactured and sold by Nippon Gene Co., Ltd., Japan) at 100, or buffer containing 50 mM Tris-HCl (pH 7.4), MgCl 2 mM, 10mM dithiothreitol, 1mM spermidine, 1mM ATP and 0.1mg / ml BSA (bovine serum albumin) (manufactured and sold by Bethesda Research).
Laboratories, USA). The resulting mixture was allowed to stand to advance a reaction at 150C overnight. The thus obtained reaction mixture was heated at 700C for 10 minutes to stop the reaction.

Step 4. (Transfer of the recombinant plasmid into Escherichia coli)
A suspension of competent cells from
Escherichia coli K12 strain 342-167 was prepared in substantially the same manner as in step 2 of Example 1. 400 μL of the competent cell suspension and 40 μL of the reaction mixture obtained in step 3 were mixed. of Example 1, and allowed to stand in ice for 1 hour.

Then, the mixture was heated at 420C for 2 minutes and then 5ml of the L-broth was added to the mixture. The resulting mixture was incubated stationarily at 370 ° C. for 90 minutes. Then, from the mixture, the cells were collected and suspended in sterile water to obtain a suspension of cells. The cell suspension thus obtained was applied to a synthetic agar medium (containing 6 g / l of Na2HPO4, 3 g / l of KH2PO4, 0.5 g / l of
NaCl, 1 g / l NH4Cl, 1 mM MgSO4, 0.1 mM CaCl2, 2 gil glucose, 15 g / l agar, 0.3 mM L-threonine,
0.3 mM L-leucine, 0.1 mM L-histine,
0.6mM L-arginine and 0.05mM thiamine) and incubated.

Step 5. t (Selection and isolation of E. coli having a gene coding for PEPC derived from a Corynebacterium melassecola 801 microorganism (FERM BP-558
The cells obtained in step 4 of Example 1 were separately cultured on the above-mentioned synthetic agar medium containing ampicillin (30 μg / ml) and tetracycline (10 μg / ml). ml) and the above-mentioned synthetic agar medium containing tetracycline (10 μg / ml) alone. Cells that did not grow on the synthetic agar medium containing ampicillin and tetracycline but grew on the synthetic agar medium containing tetracycline were selected and isolated.

The cells thus isolated were sensitive to ampicillin but resistant to tetracycline, not requiring glutamic acid in the medium. Cells thus isolated were designated Escherichia coli strain K12 (pAG203) 342-167 which had the desired gene encoding PEPC. Isolated E. coli was cultured to clone the gene encoding PEPC.

The PEPC activity of isolated E. coli was determined by the following method to confirm that the cloned gene was a gene encoding PEPC. Escherichia coli K12 strain 342-167 (pAG203) was inoculated into 100 ml of a synthetic liquid medium (containing 13.6 gXl of KH2PO4, 2.61 g / l of K2SO4, -0.2 g / l of MsSO4 7H20, 10 mg / l CaCl2, 0.5 mg / l FeSO4.7H2O, 4 gjl glucose, 3 gXl NH4Cl, 0.3 mx L-threonine, 0.3 mx L-leucine 0.1 mM L-histidine, 0.6 mM
L-arginine and 0.05 mx thiamine, pH7.2) and cultured at 37 ° C. for one day. From the culture thus obtained, the E. coli cells were collected and suspended in 2 ml of MES buffer 2- (N-morpholino) ethanesulfonic acid (MES) 50 mM, 10 mM MgSO4, 10 mM EDTA, pH7.02 to obtain a suspension. The suspension was sonicated and centrifuged at 14000 rpm (20000 g) for 20 minutes to obtain a cell extract (crude enzyme solution). On the other hand, Escherichia coli K12 strain 342-167 and Escherichia coli K12 strain 342-167 (pBR325) were cultured in the above-mentioned liquid synthesis medium containing 10 mM sodium glutamate. PEPC activity was determined by measuring an increase in absorbance at 412 nm of 2.5 ml of a reaction solution of the enzyme containing 10 mM MES, 2 mM phosphoenolpyruvate, 10 mM NaHCO 3, CoA acetal 0.1 mM (manufactured and sold by
Sigma Chemical Company, USA) 3.3 mM MnSO4, 0.05 mM 5,5-dithiobis (2-nitrobenzoic acid) (hereinafter referred to as DTNB), 5 units / ml of pig heart citrate synthase ( manufactured and sold by Sigma Chemical
Company, USA) and 10 to 100 μl of the cell extract, pH 7.4 using a model 228 spectrophotometer (manufactured and sold by Hitachi, Ltd., Japan). Moreover, the concentration of the protein in the extract of cells was measured by the method of Lowry et al. Lowry, NJ Rowebrough, RJ Randall, J.

Biol. Chem. 193, 265 (1988) j. To measure protein concentration, bovine serum albumin was used (manufactured and sold by Wako Pure
Chemical Industries Ltd., Japan) as a standard protein for the determination mentioned above.

 The results are shown in Table 1 given in Step 7 which will be mentioned below.

 As is apparent from the results shown in Table 1, strain 342-167 of Escherichia coli K12 (pAG203) had an extremely high specific activity of PEPC.

Step 6. (Isolation of a pAG203 hybrid plasmid and its analysis)
a plasmid pAG203 DNA was isolated from E. coli K12 strain 342-167 (pAG203) and purified substantially in the same manner as in step 2 of Example 1, except that used E. coli K12 strain 342-167 (pAG203) instead of pBR325 transformed E. coli K12 strain 342-167. Thus, 170 μg of plasmid pAG203 DNA was obtained. 0.3 μg of the DNA thus isolated was digested with an excess of each of the various restriction enzymes, i.e. EcoRi,
BamHI, HindIII, SalI and PstI (each manufactured and sold by Nippon Gene Co., Ltd., Japan) under conditions appropriate to each of the restriction enzymes. The digested products thus obtained were subjected to electrophoresis on 1% (weight / volume) of agarose gel and electrophoresis on 4% (weight / volume) of polyacrylamide gel according to a usual method.

Electrophoresis was carried out constantly at 5 V per cm of gel length for 4 hours. After completion of the electrophoresis, the gel was immersed in a solution of ethidium bromide containing 1 μg / ml of ethidium bromide for 30 minutes to tint the gel. Then, the gel was irradiated with ultraviolet light to determine the number of bands corresponding to the number of DNA fragments obtained. The respective molecular lengths of the DNA fragments were determined by comparing the respective migration distances of the fragments in the electrophoresis with those of the DNA fragments respectively having known molecular lengths.For such a DNA fragment having a length known molecule, DNA fragments obtained by HindIII digestion of phasic X-DNA (manufactured and sold by Nippon Gene
Co., Ltd., Japan) were used in the case of agarose gel electrophoresis and DNA fragments obtained by HaeIII digestion of the phalDNA174 DNA (manufactured and sold by Bethesda
Research Laboratories, USA) were used in the case of polyacrylamide gel electrophoresis. Moreover, by analyzing the DNA fragments obtained by digestion of the plasmid with various restriction enzymes, the restriction sites of the plasmid were determined.

As a result, plasmid pAG203 was found to have the structure shown in the restriction endonuclease cleavage or cleavage map shown in Figure 1 and consisted of the pBR325 vector and inserted at the site cut or cleaved by the PstI restriction, a foreign PstI fragment having a molecular length of about 5.5 kb. The PstI fragment is a DNA fragment containing a gene coding for PEPC derived from
Corynebacterium melassecola 801 (deposited with FRI accession number FERM BP-558).

Cells of E. coli strain 342-167
K12 were transformed with the DNA of the above plasmid pAG2O3 obtained in substantially the same manner as in step 2 of Example 1, except that the plasmid pAG203 DNA was used in the first place. pBR325, to obtain transformants. The resulting transformants were examined for drug resistance and auxotrophy. All transformants examined were found to be tetracycline-resistant but ampicillin-sensitive, not requiring glutamic acid. On the other hand, the restriction endonuclease cleavage map of the plasmid retained in the transformant was found to be the same as that of the plasmid incorporated in the above transformation.

Step 7. (Partial deletion of a DNA fragment containing a gene coding for PEPC and having a molecular length of about 5.5 kb)
To 3 μM DNA of plasmid pBR325 obtained in step 2 of Example 1, 20 units of SalI restriction enzyme were added. The resulting mixture was allowed to react in 50 μl of a buffer containing
50 mM Tris-HCl (pH 7.4), 10 mM MgSO 4, and 100 mM NaCl at 370 ° C. for 2 hours. Then, an equal volume of a 1: 1 (volume / volume) mixture of phenol and chloroform was added to the mixture and stirred with subsequent recovery of an aqueous phase. To the aqueous phase an equal volume of chloroform was added and stirred, followed by recovery of an aqueous phase. To the aqueous phase, sodium acetate was added to a final concentration of 300 ml. millimoles and then a double volume of ethanol was added, and stirred. Then, the mixture was kept at -300C for 3 hours, and centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes to obtain a DNA pellet. The thus-obtained DNA pellet was dried under vacuum to obtain DNA sample I.

To 3.4 μg of the plasmid pAG203 DNA obtained in step 6 of Example 1, 20 units of SalI restriction enzyme were added. The resulting mixture was allowed to react in 50 μl of a buffer containing
50 mM Tris-HCl (pH 7.4), 10 mM MgSO 4 and 100 mM NaCl at 37 ° C. for 2 hours. To the resulting reaction mixture was added an equal volume of a 1: 1 (v / v) mixture of phenol and chloroform. The mixture was stirred, followed by recovery of an aqueous phase. To the aqueous phase, an equal volume of chloroform was added and stirred, followed by recovery of an aqueous phase. To the aqueous phase, sodium acetate was added to a final concentration of 300 millimoles and then a double volume of ethanol was added. Then, the mixture was kept at -300C for 3 hours, and centrifuged at 12000 rpm (8900g) at room temperature for 10 minutes to obtain a DNA pellet. The thus-obtained DNA pellet was dried under vacuum to obtain a DNA II sample.

 All of the above DNA I and II samples obtained were reacted with 3 units of phage T4 DNA ligase in 50 μl of a buffer containing 50 mM Tris-HCl (pH 7.4), 10 mM MgCl 2, 10mM dithiothreitol, 1mM spermidine, 1mM ATP and 0.1mg / ml BSA at 150C overnight. Then, the resulting reaction mixture was heated at 700C for 10 minutes to complete the reaction.

 Using the above obtained ligase reaction mixture, the transformation of E. coli K12 strain 342-167 cells was carried out in substantially the same manner as in step 4 of Example 1. Transformants that were resistant to ampicillin and chloramphenicol but sensitive to tetracycline, did not require glutamic acid.

 The plasmid retained in each strain was separated in substantially the same manner as in step 6 of Example 1. The plasmid thus obtained was designated pAG211. For strains retaining plasmid pAG211, the activity of PEPC was determined in substantially the same manner as in step 5 of Example 1 except that the synthetic liquid medium contained 20 μg / ml of chloramphenicol. . As a result, it was found that strains retaining plasmid pAG211 had an extremely high specific activity of PEPC as shown in Table 1.

Table 1.

<Tb>

<SEP> Strain <SEP> PEPC <SEP> Specific <SEP> Activity <SEP> of
<tb><SEP> PEPC <SEP> * 1)
<tb> 342-167 <SEP> * 2) <SEP> - <SEP><SEP> 0.003
<tb> 342-167 (pBR325) <SEP> - <SEP><SEP> 0.002
<tb> 342-167 (pAG203) <SEP> + <SEP> 0.081
<tb> 342-167 (pAG211) <SEP> + <SEP> 0.26
<Tb>
Notes: * 1) expressed in terms of the amount (micromoles) of oxaloacetic acid forced to be formed by 1 mg of the protein in the enzyme reaction solution for 1 minute. The amount of oxaloacetic acid formed is determined as follows. The formed oxaloacetic acid is reacted with excesses of citrate synthase and acetyl CoA to form citric acid and coenzyme A. The coenzyme A thus formed is measured using a reagent for the quantitative analysis of the SH groups for determine the amount of coenzyme A formed. The amount of oxaloacetic acid is calculated based on the amount of coenzyme A formed.

* 2) a variant of the K12 strain of Escherichia coli obtained from Dr. Barbara J. Bachmann of E. coli
Genetic Stock Center, Department of Human Genetics,
Yale University, School of Medicine, 333 Ceder Street
PO Box 3333 New Haven, Connecticut 06510 USA. This strain is a strain deficient in PEPC.

The plasmid pAG211 had the structure shown in FIG. 2 and was a recombinant DNA composed of a SalI fragment of plasmid pBR325 and a fragment of
SalI foreign having a molecular length of about 3.3 kb. The foreign SalI fragment was a DNA fragment derived from Corynebacterium melassecola 801 deposited at
FRI under accession number FERM BP-558, containing a gene coding for PEPC.

The cells of E. coli strain 342-167
K12 were transformed with the plasmid pAG211 DNA, obtained above, in substantially the same manner as in step 2 of Example 1 except that the plasmid pAG211 DNA was used. instead of pBR325, to obtain transformants. The resulting transformants were examined for drug resistance and auxotrophy. All transformants examined were found to be resistant to chloramphenicol and ampicillin but sensitive to tetracycline, not requiring glutamic acid. On the other hand, the restriction endonuclease cleavage map of the plasmid retained in the transformant was found to be the same as that of the plasmid incorporated in the above transformation.

 Step 8. (Isolation of a DNA fragment containing a gene encoding PEPC and having a molecular length of about 3.3 kb).

To 30 μg of the plasmid pAG203 DNA obtained in step 7 of Example 1, 100 units of each SalI restriction enzyme were added. The resulting mixture was allowed to react in 100 μl of a buffer containing
50 mM Tris-HCl (pH 7.4), 10 mM MgSO 4 and 100 mM NaCl at 370 ° C. for 2 hours. The plasmid thus digested was electrophoresed using a 1% agarose gel in substantially the same manner as in step 6 of Example 1 except that a gel was used. 1% agarose prepared by LMP Agarose (trade name for a product manufactured and sold by Bethesda
Research Laboratories, USA), and that the electrophoresis was carried out at 40 ° C. The agarose gel was dyed with ethidium bromide and exposed to ultraviolet irradiation, so as to visually observe the DNA fragments containing a protein. gene encoding PEPC and having a molecular length of about 3.3kb. Part of the gel where such DNA fragments were found was cut. To the cut gel, TE buffer was added in an amount of three times the weight of the gel, and heated at 650C for 10 minutes to completely dissolve the agarose gel in the buffer. To the resulting solution, an equal volume of phenol was added and stirred, followed by recovery of an aqueous phase. To the aqueous phase, an equal volume of a 1: 1 (volume / volume) mixture of phenol and chloroform was added and stirred with subsequent recovery of an aqueous phase. To the aqueous phase an equal volume of chloroform was added and stirred with subsequent recovery of an aqueous phase. To the aqueous phase, sodium acetate was added to a final concentration of 300 millimoles, then a double volume of ethanol was added, and stirred. Then, the mixture was kept at -300C for 3 hours and centrifuged at 10,000 rpm (9000g) at room temperature for 10 minutes to obtain a DNA pellet. The thus-obtained pellet of DNA was dried under vacuum and then dissolved in 20 μl of TE buffer. Thus, a solution containing about 3 μg of a DNA fragment containing a gene coding for PEPC and having a length was obtained. molecular weight of about 3.3 kb.

 Step 9. (Isolation of a DNA fragment containing a gene coding for PEPC and having a molecular length of about 5.5 kb).

To 50 μg of the plasmid pAG203 DNA obtained in step 6 of Example 1, 150 units of each of BamHI and SalI restriction enzymes were added. The resulting mixture was allowed to react in 100 μl of a buffer containing 50 mM Tris-HCl (pH 7.4), 10 mM MgSO 4 and
100 mM NaCl at 37 ° C. for 3 hours to digest the plasmid. The digested plasmid was subjected to agarose gel electrophoresis in substantially the same manner as in step 8 of Example 1. As a result, a DNA fragment having a molecular length of about 3 was observed. 8 kb and a DNA fragment having about 4.1 kb. A portion of the gel where such DNA fragments were found was cut out. On the cut gel, TE buffer was added in an amount of three times the weight of the gel and heated at 650C for 10 minutes to completely dissolve. the agarose gel in the buffer. To the resulting solution, an equal volume of phenol was added and stirred, followed by recovery of aqueous phase. To the aqueous phase an equal volume of a 1: 1 (volume / volume) mixture of phenol and chloroform was added and stirred, followed by recovery of an aqueous phase. To the aqueous phase an equal volume of chloroform was added and stirred, followed by recovery of an aqueous phase. To the aqueous phase, sodium acetate was added to a final concentration of 300 millimoles, then a double volume of ethanol was added, and stirred.

Then, the mixture was kept at -300C for 3 hours and centrifuged at 10000 rpm (9000g) at room temperature for 10 minutes to obtain a DNA pellet. The thus-obtained DNA pellet was dried under vacuum and then dissolved in 50 μl of a buffer containing 10 mM Tris-HCl (pH 7.4), 10 mM MgSO 4, 50 mM NaCl and 1 mM dithiothreitol. To the resulting solution, 50 units of PstI were added, followed by incubation at 300C for 3 hours to digest the DNA pellet. The digested pellet was subjected to agarose gel electrophoresis in substantially the same manner as in step 8 of Example 1. As a result, a DNA fragment having a molecular length of about 1 was observed. 0 kb and a DNA fragment having about 1.2 kb. Part of the gel where such DNA fragments were found was cut.

To the cut gel, TE buffer was added in an amount of three times the weight of the gel, and heated at 650C for 10 minutes to completely dissolve the agarose gel in the buffer. To the resulting solution, an equal volume of phenol was added and stirred, followed by recovery of an aqueous phase.

To the aqueous phase, an equal volume of a 1: 1 (volume / volume) mixture of phenol and chloroform was added and stirred with subsequent recovery of an aqueous phase. To the aqueous phase an equal volume of chloroform was added and stirred with subsequent recovery of an aqueous phase. To the aqueous phase, sodium acetate was added to a final concentration of 300 millimoles, then a double volume of ethanol was added, and stirred. Then, the mixture was kept at -300C for 3 hours and centrifuged at 10,000 rpm (9000g) at room temperature for 10 minutes to obtain a pellet of DNA. was dried under vacuum to obtain about 2 μg of the DNA pellet containing a PstI-SalI fragment having a molecular length of about 1.2 kb and a PstI-SalI fragment having a molecular length of about 1.0 kb which are parts of the DNA fragment containing the PEPC gene and having a molecular length of about 5.5 kb. The DNA pellet was dissolved in 20> 11 of TE buffer.

 There were thus obtained three DNA fragments having molecular lengths of about 1.0 kb, about 1.2 kb and about 3.3 kb, respectively, which were the constituents of the DNA fragment containing the PEPC gene and having a molecular length of about 5.5kb. The DNA fragment having a molecular length of about 3.3 kb was the same as that obtained in step 8 of Example 1.

 Example 2

In this example, Corynebacterium melassecola 801 (FERM BP-558) was used as a DNA donor to clone a gene coding for PEPC (Corynebacterium melassecola 801 is deposited at
Fermentation Research Institute under accession number FERM BP-558). The gene encoding PEPC was cloned using a host-vector system where Escherichia coli K12 strain 342-167 was used as a host and a hybrid plasmid pAG103 was used as the vector. Strain 342-167 of Escherichia coli K12 was obtained from Dr. Barbara J. Bachmann of E. coli Genetic
Stock Center, Department of Human Genetics, University of
Yale, School of Medicine, 333 Ceder Street PO Box 3333
New Haven, Conneeticut 06510 EUA.The strain 342-167 of
Escherichia coli K12 is publicly available in E.

coli Genetic Stock Center. Plasmid pAG103 has the structure shown on the restriction endonuclease cleavage map shown in Figure 3.

As is apparent in FIG. 3, the plasmid pAG103 comprises the plasmid pBR325 and, inserted at its EcoRI site, a DNA fragment containing a gene coding for glutamate dehydrogenase (which will be often called hereinafter "GDH ") and having a molecular length of about 5.4 kb, which DNA fragment is derived from
Corynebacterium melassecola 801 (FERM BP-558). The DNA fragment containing a gene encoding GDH contained an XbaI site. Plasmid pAG103 was prepared as follows.

Step 1. (Preparation of plasmid pAG103)
(1) Extraction of Corynebacterium melassecola 801 DNA (FERM BP-558) and its split3
The same processes as in step 1 of Example 1 were repeated substantially to obtain a DNA solution containing the Corynebacterium melassecola 801 DNA.

The DNA in the DNA solution was split as follows. 160 units of the restriction enzyme were added
EcoRI (manufactured and sold by Nippon Gene Co., Ltd.,
Japan) at 34 g of the DNA, and the resulting mixture was mixed with 67 l of a buffer containing 50 mM Tris-HCl (pH 7.4), 10 mM MgSO 4 and 100 mM NaCl and allowed to stand at 370C for 30 minutes so that the reaction proceeds. Then, the reaction mixture was heated at 700C for 10 minutes to stop the reaction.

 (2) (Preparation of plasmid pBR325 and its cleavage).

The PA340 strain of Escherichia coli K12 which is a strain deficient in GDH and glutamate synthase and requires glutamic acid for its growth was inoculated into 50 ml of L broth (containing 10 g / l of polypetone, 5 g / l). 1 yeast extract, 5 g / l NaCl and having a pH of 7.2) and cultured at 370C until the cell concentration reaches 5 x 108 cells / ml. Cells were harvested from the culture by centrifugation at 20C. The cells were suspended in 50 ml of 100 mM MgCl 2 while cooling with ice. From the suspension, the cells were collected and suspended in 25 ml of 100 mM CaCl 2 while cooling with ice. The suspension thus obtained was allowed to stand in ice for 30 minutes. From the suspension, the cells were collected and suspended in 5 ml of 100 mM CaCl 2 while cooling with ice. The suspension thus obtained was allowed to stand in ice for 1 hour. To 200 μl of the suspension was added 0.1 g of pBR325 DNA (manufactured and sold by
Bethesda Research Laboratories, USA) and the resulting mixture was allowed to stand in ice for 1 hour.

Subsequently, the mixture was allowed to stand at 420 ° C for 2 minutes and then 5 ml broth was added.
L. The resulting mixture was incubated stationarily at 370C for 90 minutes. Then, the mixture was diluted with sterile water and spread on agar medium L (medium obtained by adding 15 g of agar to L broth so that the total volume was 1 liter) containing 10 μg. / ml tetracycline, followed by incubation overnight at 370C to obtain Escherichia coli transformed with plasmid pBR325.

 Escherichia coli K12 PA340 transformed with plasmid pBR325 was inoculated into 100 ml of L-broth containing 10 μg / ml of tetracycline and cultured overnight at 37 ° C.

From the mixture, cells were harvested by centrifugation and washed with TE buffer. The cells were suspended in 2 ml of an aqueous solution consisting of 15% (w / v) sucrose, 50 mM Tris-HCl (pH 8.5) 50 mM EDTA and 2 mg / ml lysozyme (manufactured and sold by Sigma Chemical
Company, USA) and allowed to stand at room temperature for 30 minutes. To the resulting suspension, 2 ml Triton solution 0.1% (w / v) of Triton X-100 manufactured by Rohm & Haas was added.
Co., USA and sold by Wako Pure Chemical Industries
Ltd., Japan), 50 mM Tris-HCl (pH 8.5) and 50 mM EDTA was allowed to stand at 370 ° C. for 30 minutes. The solution thus obtained was centrifuged at 30000 rpm (64000 g) at 50 ° C. for 1 hour to obtain a supernatant. To the supernatant was added a buffer.
TE in an amount such that the resulting mixture has a volume of 18 ml. 1.2 ml of a 10 mg / ml aqueous solution of ethidium bromide and 18.64 g of cesium chloride were gently dissolved in the mixture obtained above, followed by centrifugation at 40000 rpm ( 100000 g) at 150C for 48 hours. After completion of the centrifugation, two bands were visualized under ultraviolet light. The lower band was taken from the centrifuge tube through the side of the tube using a syringe to obtain a fraction of plasmid pBR325. The fraction thus obtained was extracted four times with an equal volume of isopropyl alcohol to obtain an extract. From the extract, the ethidium bromide was removed and the resulting solution was dialyzed with TE buffer for obtain 1 ml of a dialysate containing plasmid pBR325 which had a DNA concentration of 130 μg / ml.

 To an aliquot of dialysate containing 17 μg of the resulting plasmid pBR325 DNA was added 40 units of the restriction enzyme EcoRI. The result was added to 150 μl of a buffer solution containing 50 mM Tris-HCl (pH 7.4), 10 mM MgSO 4 and 100 mM NaCl, followed by incubation at 370 ° C. for 2 hours to advance a reaction.

Then, the mixture was heated at 700C for 10 minutes to stop the reaction. To the reaction mixture was added sodium acetate in such an amount that the resulting mixture contained sodium acetate at a concentration of 300 millimoles. To the mixture was added a double volume of ethanol and the resulting mixture was allowed to stand at -300C for 3 hours. Then, the mixture was centrifuged at 12,000 rpm (8900 g) at room temperature for 10 minutes to obtain a supernatant and precipitates. The precipitates were collected and dried under reduced pressure. The precipitates thus obtained were dissolved in 200 μl of a BAPT buffer (50 mM Tris-HCl, pH8.4). To the solution thus obtained was added a unit of bacterial alkaline phosphatase (manufactured and sold by Takara Shuzo Co.,
Ltd., Japan) followed by incubation at 650C for 30 minutes. Then, one unit of bacterial alkaline phosphatase was added to the solution, followed by incubation at 650C for 30 minutes. Then, an equal volume of phenol saturated with TNE buffer was added to the solution, followed by mixing. The resulting mixture was centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes and an aqueous phase was recovered. To the aqueous phase thus obtained, an equal volume of phenol saturated with TNE buffer was added, followed by mixing. The resulting mixture was centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes. an aqueous phase was recovered. Then, an equal volume of a phenol-chloroform mixture (1: 1, v / v) was added to the aqueous phase, followed by mixing. The resulting mixture was centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes and an aqueous phase was recovered. Then, an equal volume of chloroform was added to the aqueous phase with stirring, and centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes to recover an aqueous phase. obtained, sodium acetate was added in such an amount that the resulting mixture contained 300 millimoles sodium acetate and twice the volume of ethanol with stirring. The resulting mixture was allowed to stand at -300C for 3 hours. Subsequently, the mixture was centrifuged at 12,000 rpm (8900 g) at room temperature for 10 minutes to obtain DNA precipitates. The DNA precipitates were dried under reduced pressure and dissolved in 23 μl of a buffer solution
YOU.

(3) (Recombination of DNA)
2.4 μg of the DNA obtained in step 1 (1) of Example 2 and 1.4 μg of the DNA obtained in step 1 (2) of Example 2 and 3 phage T4 DNA ligase units (manufactured and sold by Nippon Gene Co., Ltd., Japan) were added to 100 μl of a buffer containing 50 μl of Tris-HCl (pH 7.4), 10 μl of MgCl 2, 10 mM of dithiothreitol, 1 mM spermidine, 1 mM ATP and 0.1 mg / ml BSA (bovine serum albumin) (manufactured and sold by Bethesda Research
Laboratories, USA). The resulting mixture was allowed to stand for a reaction at 150C overnight. The reaction mixture thus obtained was heated at 700 ° C. for 10 minutes to stop the reaction.

(4) (Transfer of the recombinant plasmid into
Escherichia coli)
A suspension of competent cells of Escherichia coli K12 strain PA340 was prepared in substantially the same manner as in step 1- (2) of Example 2. 400 μl of the competent cell suspension was mixed with 40 μl of the reaction mixture obtained in step 1 (3) of Example 2 and allowed to stand in ice for 1 hour.

Then, the mixture was heated at 420 ° C for 2 minutes and then 5 ml of the L broth was added to the mixture. The resulting mixture was incubated stationary at 370 ° C. for 90 minutes. Then, from the mixture, the cells were collected and suspended in sterile water to obtain a cell suspension. The suspension thus obtained from cells was applied to a synthetic agar medium (containing 6 g / l of Na2HPO4, 3 g / l of KH2PO4, 0.5 g / l of NaCl, 1 g / l of NH4Cl, 1 mM MgSO4, 0.1 mM CaCl2, 2 g / l glucose, 15 g / l agar, 0.3 mM L-threonine, 0.3 mM
L-leucine, 0.1 mM L-histidine, 0.6 mM L-arginine and 0.05 mM thiamine, and incubated.

(5) [Selection and isolation of E. coli having a gene encoding GDH derived from the microorganism
Corynebacterium melassecola 801 (FERM BP-558)
The cells obtained in step 1 (4) of Example 2 were cultured separately on the above-mentioned synthetic agar medium containing chloramphenicol (20 μg / ml) and tetracycline (10 μg / ml). pg / ml) and the above-mentioned synthetic agar medium containing tetracycline alone (10 μg / ml). Cells that did not grow on the synthetic agar medium containing chloramphenicol and tetracycline but grew on the synthetic agar medium containing tetracycline were selected and isolated.

The cells thus isolated were sensitive to chloramphenicol but resistant to tetracycline, not requiring glutamic acid in the medium. The cells thus isolated were designated by the strain
PA340 Escherichia coli K12 (pAG103), which had the desired gene encoding GDH. Isolated E. coli was cultured to clone the gene encoding GDH.

Isolated E. coli GDH activity was determined by the following method to confirm that the cloned gene was the gene encoding GDH. The PA340 strain of Escherichia coli K12 (pAG103) was inoculated into 100 ml of a synthetic liquid medium (containing 13.6 g / l of KH2PO4, 2.61 g / l of K2SO4, 0.2 g / l of MgSO 4 .7H 2 O, 10 mg / l CaCl 2, 0.5 mg / l FeSO 4 .7H 2 O, 4 g / l glucose, 3 g / l NH 4 Cl, 0.3 mM L-threonine, 0.3 mM L-leucine, 0.1 mM L-histidine, 0.6 mM
L-arginine and 0.05 mM thiamine pH7.2) and cultured at 370C for 1 day. From the culture thus obtained, E. coli cells were collected and suspended in 2 ml of TM buffer (50 mM
Tris-HCl, 10 mM 2-mercaptoethanol, pH 7.6) to obtain a suspension. The suspension was sonicated and then centrifuged at 14000 rpm (20000 g) for 20 minutes to obtain an extract. cells (crude enzyme solution). Furthermore, the PA340 strain of Escherichia coli and the PA340 strain of
Escherichia coli K12 (pBR325) were cultured in the above-mentioned liquid synthesis medium containing 10 millimoles of sodium glutamate.

The activity of GDH was determined by measuring a decrease in absorbance at 340 nm of 2.5 ml of an enzyme reaction solution (containing 50 mM
Tris-HCl, 40 mM NH4Cl, 0.25 Mm NADPH, 5 mx 3-ketoglutaric acid and 10-100 μl of the cell extract, pH 7.6 using a Model 228 spectrophotometer (manufactured and sold by Hitachi, Ltd., Japan). Moreover, the concentration of the protein in cell extract was measured by the method of Lowry and other WO.H. Lowry, NJ Rowebrough, RJ Randall, J. Biol. Chem. 193, 265 (1951) 2. To measure protein concentration, bovine serum albumin (manufactured and sold by Wako Pure Chemical) was used.
Industries Ltd., Japan) as the standard protein for the determination mentioned above. The results are shown in Table 2. e
TABLE 2.

<Tb>

<SEP> Strain <SEP> GDH <SEP><SEP> Specific <SEP> Activity of <SEP> GDH
<tb><SEP> * 1) <SEP>
<tb> PA340 <SEP> - <SEP> 0.009
<tb> PA340 (pBR325) <SEP> i <SEP> 0.003
<tb> PA340 (pAGlQ3) <SEP> + <SEP> 20.8
<tb> PA340 (pAGl2) <SEP> + <SEP> 28.9
<Tb>
Note: * 1) expressed in terms of the amount (micromoles) of NADPH (-nicotinamide adenine dinucleotide phosphate, reduced form) forced to be oxidized with 1 mg of protein-in the reaction solution for 1 minute.

As is apparent from the results shown in Table 2, Escherichia coli strain PA340
K12 (pAG103) had a very high specific GDH activity.

 (6) (Isolation of a hybrid plasmid pAG103 and its analysis).

Plasmid pAG103 DNA was isolated from E. coli K12 strain PA340 (pAG103) and purified in substantially the same manner as in step 1 (2) of Example 2 except that we used the strain
E. coli K12 PA340 (pAG103) instead of the E. coli K12 strain PA340 transformed with pBR325. Thus, 150 μg of the plasmid pAG103 DNA was obtained. 0.3 μg of the DNA thus isolated was digested with an excess of each of the various restriction enzymes, i.e. EcoRI, BamHI, BglII, HindIII, SalI and XbaI (each manufactured and sold by
Nippon Gene Co., Ltd., Japan), PstI (manufactured and sold by Bethesda Research Laboratories, USA), and SacI and
XhoI (each manufactured and sold by Takara Shuzo Co.,
Ltd., Japan) under the conditions appropriate for each of the restriction enzymes. The digested products thus obtained were subjected to electrophoresis on 1% of agarose gel (weight / volume) and electrophoresis on 4% of polyacrylamide gel (weight / volume) according to a usual method. The electrophoresis was carried out constantly at 5 V per cm of gel length for 4 hours. After completion of the electrophoresis, the gel was immersed in a solution of ethidium bromide containing 1 μg / ml of ethidium bromide for 30 minutes to tint the gel. Then, the gel was irradiated with ultraviolet light to determine the number of bands corresponding to the number of DNA fragments obtained. The respective molecular lengths of the DNA fragments were determined by comparing the respective migration distances of the fragments in the electrophoresis with those of the DNA fragments respectively having known molecular lengths. For this DNA fragment having a known molecular length, DNA fragments obtained by digestion with HindIII of a phage DNA A (manufactured and sold by
Nippon Gene Co., Ltd., Japan) in the case of agarose gel electrophoresis, and DNA fragments obtained by HaeIII digestion of Fox174 phage DNA (manufactured and sold by Bethesda Research Laboratories,
EUA) in the case of polyacrylamide gel electrophoresis. Moreover, by analyzing the DNA fragments obtained by digestion of the plasmid with various restriction enzymes, the restriction sites of the plasmid were determined.

As a result, plasmid pAG103 was found to have a structure exemplified by the restriction endonuclease cleavage map shown in Figure 3 and consisted of the pBR325 vector and inserted at the site cleaved or cleaved with the restriction enzyme EcoRi. a foreign EcoRI fragment having a molecular length of about 5.4 kb. The EcoRI fragment is a DNA fragment containing a gene encoding GDH derived from
Corynebacterium melassecola 801 (deposited with FRI accession number FERM BP-558).

 The E. coli K12 PA340 strain cells were transformed with the DNA of the above-mentioned plasmid pAG103 in substantially the same manner as in step 1 (2) of Example 2, to except that plasmid pAG103 DNA was used instead of pBR325 to obtain transformants. The resulting transformants were examined for drug resistance and auxotrophy. All transformants examined were found to be resistant to tetracycline and ampicillin but to be sensitive to chloramphenicol, not requiring glutamic acid. On the other hand, the restriction endonuclease cleavage map of the plasmid retained in the transformant was found to be the same as that of the plasmid incorporated in the above transformation.

 To 15 μg of the plasmid pAG103 DNA obtained above, 100 units of restriction enzyme XbaI were added. The resulting mixture was allowed to react in 150 μl of a buffer containing 50 mM Tris-HCl (pH7.4), 10 mM MgSO4 and 100 mM NaCl at 3700C for 2 hours. Then, the mixture was heated at 700C for 10 minutes to stop the reaction. Thus the DNA sample III was obtained.

Step 2. (Scission of the DNA obtained from
Corynebacterium melassecola 801)
The same processes as in step 1 of Example 1 were repeated substantially to obtain the DNA of
Corynebacterium melasssecola 801.

 50 μg of the thus-obtained DNA were added to 150 μl of a buffer containing 50 mM Tris-HCl (pH7.4), 10 mM MgSO4 and 100 mM NaCl. To the mixture was added 50 units of XbaI, followed by incubation at 370C for 2 hours to advance a reaction. Then, the mixture was heated at 700C for 10 minutes to stop the reaction. The DNA sample IV was thus obtained.

 Step 3. (Recombination of AD.

1.8 μg of the DNA sample III obtained in step 1 of example 2 and 1.5 μg of the DNA obtained in step 2 of example 2 and 3 units of DNA ligase T4 phage (manufactured and sold by Nippon Gene Co., Ltd.,
Japan) was added to 100 μl of a buffer containing 50 mM Tris-HCl (pH 7.4), 10 mM MgCl 2, 10 mM dithiothreitol, 1 mM spermidine, 1 mM ATP and 0.1 mg / ml. ml of BSA (bovine serum albumin) (manufactured and sold by Bethesda Research Laboratories, USA). The resulting mixture was allowed to stand for a reaction at 150C overnight. The reaction mixture thus obtained was heated at 70 ° C for 10 minutes to stop the reaction.

 Step 4. (Transfer of the recombinant plasmid into Escherichia coli).

 A suspension of competent cells of Escherichia coli K12 strain 342-167 was prepared in substantially the same manner as in Step 2 of Example 1. The same procedures as in Step 4 were repeated substantially of Example 1 except that a culture agar medium containing 10 μg / ml of tetracycline was used as the culture medium.

Step 5. Selection and isolation of E. coli having a gene coding for PEPC derived from a Corynebacterium melassecola 801 microorganism (FERM BP-558,1
The cells obtained in step 4 of Example 2 were cultured separately on the above-mentioned synthetic agar medium containing ampicillin (30 μg / ml) and tetracycline (10 μg / ml). ml) and the above-mentioned synthetic agar medium containing tetracycline (10 μg / ml) alone. Cells that did not grow on the synthetic agar medium containing ampicillin and tetracycline but grew on the synthetic agar medium containing tetracycline were selected and isolated. The cells thus isolated were ampicillin sensitive. but resistant to tetracycline, not requiring glutamic acid in the medium. The cells thus isolated were designated by the strain 342-167 of Escherichia coli
K12 (pAG204) which had the desired gene encoding PEPC.

Isolated E. coli was cultured to clone the gene encoding PEPC.

 The PEPC activity of E. coli isolated was determined in the same manner as in step 5 of Example 1 to confirm that the cloned gene was a gene encoding PEPC.

 The results are shown in Table 3 given below.

 As is apparent from the results shown in Table 3, Escherichia coli K12 strain 342-167 (pAG204) had an extremely high specific activity of PEPC.

TABLE 3.

<Tb>

<SEP> Strain <SEP> PEPC <SEP><SEP> Specific <SEP> Activity of <SEP> PEPC
<tb><SEP> * 1) <SEP>
<tb> 342-167 <SEP> * 2) <SEP> - <SEP><SEP> 0.003
<tb> 342-167 (pBR325) <SEP> - <SEP><SEP> 0.002
<tb> 342-167 (pAG204) <SEP> + <SEP> 0.036
<Tb>
Notes: * 1) and * 2) are identical to the notes of the
table 1.

 Step 6. (Isolation of a hybrid plasmid pAG204 and its analysis).

 Plasmid pAG204 DNA was isolated from E. coli K12 strain 342-167 (pAG204) and purified in substantially the same manner as in step 1 (2) of Example 2, except that was used at strain 342-167 of E. coli K12 (pAG204) instead of the strain 342-167 of E. coli K12 transformed with pBR325. Thus, 165 μg of the plasmid pAG204 DNA was obtained. The DNA was analyzed in substantially the same manner as in step 6 of Example 1 to prepare the restriction endonuclease cleavage map which is shown in Figure 4. Plasmid pAG204 comprises plasmid pAG103 and inserted at its XbaI site, an XbaI fragment having a molecular length of about 11.5 kb, which fragment is a DNA fragment containing a gene coding for PEPC derived from Corynebacterium melassecola 801.

The cells of E. coli strain 342-167
K12 were transformed with the DNA of the above-mentioned plasmid pAG204 in substantially the same manner as in step 1 (2) of Example 2 except that the DNA of plasmid pAG204 instead of pBR325, to obtain transformants. The resulting transformants were examined for drug resistance and auxotrophy. All transformants examined were found to be resistant to tetracycline and ampicillin but to be sensitive to chloramphenicol, not requiring glutamic acid. On the other hand, the restriction endonuclease cleavage map of the plasmid retained in the transformant was found to be the same as that of the plasmid incorporated in the above transformation.

 Step 7. (Isolation of a DNA fragment containing a gene encoding PEPC and having a molecular length of about 11.5 kb from plasmid pAG204).

It was impossible to isolate a DNA fragment containing a gene encoding PEPC and having a molecular length of about 11.5 kb from plasmid pAG204 by agarose gel electrophoresis because the remaining DNA fragment of plasmid pAG204 which remaining DNA fragment was an XbaI-split plasmid pAG103, had almost the same molecular length as that of the DNA fragment containing a gene encoding PEPC. Therefore, the remaining DNA fragment, i.e., an XbaI-cleaved pAG103 plasmid was changed to the other vector-containing DNA fragment, followed by isolation of the DNA fragment containing a coding gene. for PFPC using agarose gel electrophoresis. For the other DNA fragment, a split plasmid obtained by cleaving plasmid pBR325 by EcoRI and ligating XbaI linkers (manufactured and sold by
Takara Shuzo Co., Ltd., Japan) at both ends of the split pBR325. Below, the method will be explained in detail.

 (1) Preparation of a split plasmid which comprises an EcoRI-cleaved plasmid and XbaI linkers bound at both ends thereof.

 To 5 μg of the plasmid pBR325 DNA obtained in step 2 of Example 1, 20 units of restriction enzyme EcoRI were added. The resulting mixture was allowed to react in 100 μl of a buffer containing 50 mM Tris-HCl (pH 7.4), 10 mM MgSO4, and 100 mM NaCl at 370 ° C for 2 hours. Then, the reaction mixture was heated at 70 ° C. for 10 minutes to stop the reaction. To the resulting mixture was added sodium acetate to a final concentration of 300 millimoles, then a double volume of ethanol was added, and stirred. The mixture was then kept at -300 ° C. for 3 hours and centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes to obtain a DNA pellet. The thus-obtained pellet of DNA was dried under vacuum to obtain DNA sample V.

To DNA sample V thus obtained, 3 units of T4 DNA polymerase (manufactured and sold by Takara Shuzo Co., Ltd., Japan) were added. The resulting mixture was allowed to react in 44 μl of a solution containing 33 mM Tris-CH3COOH (pH7.9), 66 ml
CH3COOK, 10mM (CH3C00) 2Mg, 0.5mM dithiothreitol, 0.1mg / ml BSA, 0.1mM 2'-deoxyadenosine 5'-triphosphate (manufactured and sold by Sigma Chemical
Company, USA), 0.1 mM 2'-deoxycytidine 5'-triphosphate (manufactured and sold by Sigma Chemical
Company, USA), 0.1 mM 2'-deoxyguanosine 5'-triphosphate (manufactured and sold by Sigma Chemical
Company, USA) and 0.1 mM thymidine 5'-triphosphate (manufactured and sold by Sigma Chemical Company, USA).

The reaction was advanced at 300C for 20 minutes.

Then, to the reaction mixture, sodium acetate was added to a final concentration of 300 millimoles and then a double volume of ethanol was added and stirred. Then, the mixture was kept at -300C for 3 hours and centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes to obtain a DNA pellet. The thus-obtained DNA pellet was dried under vacuum to obtain the DNA sample VI.

To 1.5 μg of an XbaI linker (manufactured and sold by Takara Shuzo Co., Ltd., Japan) 2.5 units of T4 polynucleotide kinase (manufactured and sold by
Takara Shuzo Co., Ltd., Japan). The resulting mixture was allowed to react at 370 ° C. for 1 hour in 100 μl of a solution containing 66 mM Tris-HCl (pH 7.6), 1 mM
ATP, 10 mM MgCl 2, 1 mM spermidine, 15 mM dithiothreitol, 0.2 mg / ml BSA. The sample VII of DNA was thus obtained.

One quarter of the amount of the DNA sample VI, the entire amount of the DNA sample VII and 6 units of the phage T4 DNA ligase were added to 22 μl of a solution containing 66 mM of
Tris-HCl (pH7.6), 1mM ATP, 10mM MgCl2, 1mM spermidine, 15mM dithiothreitol and 0.2mg / ml BSA.

The resulting mixture was incubated at 220C for 4 hours. Then, to the reaction mixture, an equal volume of a 1: 1 (volume / volume) mixture of phenol and chloroform was added and stirred, followed by recovery of an aqueous phase. To the aqueous phase, an equal volume of chloroform was added and stirred, followed by recovery of an aqueous phase. To the aqueous phase, sodium acetate was added to a final concentration of 300 millimoles and then a double volume of ethanol was added and stirred. Then, the mixture was kept at -300C for 3 hours and centrifuged at 10,000 rpm (9000g) at room temperature for 10 minutes to obtain a DNA pellet. The resulting pellet of DNA was dried under vacuum to obtain DNA sample VIII.

 To the full amount of the DNA sample VIII was added 15 units of XbaI. The resulting mixture was allowed to react at 370 ° C. for 2 hours in 50 μl of a buffer containing 50 mM Tris-HCl (pH 7.4), 10 mM MgSO 4 and 100 mM NaCl, thereby obtaining digested DNA. The digested DNA was electrophoresed on 1% (w / v) agarose gel in substantially the same manner as in step 6 of Example 1 except that LMP agarose (manufactured and sold by Bethesda Research Laboratories, USA) as agarose and electrophoresis was performed at 40C.

The agarose gel was tinted with ethidium bromide and exposed to ultraviolet radiation so as to visually observe DNA fragments containing a gene encoding PEPC and having a molecular length of about 6.0 kb. Part of the gel where such DNA fragments were found was cut out. To the cut gel was added TE buffer in an amount of three times the weight of the gel, and heated at 650C for 10 minutes to completely dissolve the agarose gel in the buffer. To the resulting solution, an equal volume of phenol was added and stirred, followed by recovery of an aqueous phase. To the aqueous phase, an equal volume of a 1: 1 (volume / volume) mixture of phenol and chloroform was added and stirred, followed by the recovery of an aqueous phase. an equal volume of chloroform and stirred, followed by recovery of an aqueous phase.

To the aqueous phase was added sodium acetate to a final concentration of 300 millimoles and then a double volume of ethanol was added, and stirred. Then, the mixture was kept at -300C for 3 hours and centrifuged at 10,000 rpm (9000g) at room temperature for 10 minutes to obtain a DNA pellet. The thus-obtained DNA pellet was dried under vacuum to obtain a DNA sample IX.

 (2) Scission of plasmid pAG204 by the restriction enzyme XbaI.

 To 5 μg of the plasmid pAG2G3 DNA obtained in step 6 of Example 2, 15 units of an XbaI restriction enzyme were added. The resulting mixture was allowed to react in 100 μl of a buffer containing 50 mM Tris-HCl (pH 7.4), 10 mM MgSO4 and 100 mM NaCl at 370 ° C for 3 hours. Then, the mixture was heated at 700C for 10 minutes to stop the reaction. To the resulting reaction mixture was added sodium acetate to a final concentration of 300 millimoles and then a double volume of ethanol was added and stirred. Then, the mixture was kept at -300C for 3 hours and centrifuged at 12000 rpm (8900g) at room temperature for 10 minutes to obtain a DNA pellet. DNA was dried under vacuum to obtain DNA sample X.

 (3) Recombination of DNA.

The entire amount of the DNA sample IX, the entire amount of the DNA sample X and three units of phage T4 DNA ligase were added to 100 μl of a solution containing 50 mM of
Tris-HCl (pH 7.4), 10mM MgCl 2, 10mM dithiothreitol, 1mM spermidine, 1mM ATP and 0.1mg / ml BSA. The mixture was incubated at 150C overnight. Then, the mixture was heated at 700C for 10 minutes to stop the reaction.

 (4) Transformation of E. coli by recombinant DNA.

 The same processes as in step 4 of Example 2 were repeated substantially except that 40 μl of the reaction mixture obtained in Step 7- (4) of Example 2 was used. instead of 40 μl of the reaction mixture obtained in step 3 of Example 2 to obtain transformants.

 (5) Selection of E. coli having a gene encoding PEPC derived from a Corynebacterium melassecola 801 microorganism (FERM BP-558) and isolating a hybrid plasmid from the microorganism.

 The same processes were repeated substantially as in step V of Example 1 to isolate transformants obtained in step 7- (4) of Example 2, a transformant that is ampicillin-resistant and with tetracycline but sensitive to chloramphenicol. From the transformant thus isolated, a plasmid contained in the transformant was isolated and purified substantially in the same manner as in step 1 (2) of Example 2. The plasmid thus obtained was analyzed for the same way as in step 1 (6) of Example 2. The plasmid was designated plasmid pAG221. pAG221 has a structure shown by the restriction endonuclease cleavage map shown in Figure 5, and composed of plasmid pBR325 having an XbaI linker at its EcoRI site and, inserted at its XbaI split site, a DNA fragment containing a gene encoding PEPC and having a molecular length of about 11.5 kb. The DNA fragment having a molecular length of about 11.5 kb was the same as that containing a gene coding for PEPC and having a molecular length of about 11.5 kb.

 (6) Isolation of a DNA fragment containing a gene encoding PEPC and having a molecular length of about 11.5 kb of plasmid pAG221.

To 20 μg of plasmid pAG221 obtained in step 7- (5) of Example 2, 60 units of XbaI were added. The resulting mixture was allowed to react at 370 ° C. for 2 hours in 100 μl of a buffer containing 50 mM Tris-HCl (pH 7.4), 10 mM MgSO 4 and 100 mM NaCl, thereby obtaining a digested plasmid. The digested plasmid was subjected to 1% (w / v) agarose gel electrophoresis in substantially the same manner as in Step 6 of Example 1 except that agarose
LMP (manufactured and sold by Bethesda Research
Laboratories, USA) as agarose and electrophoresis was performed at 40C. The agarose gel was tinted with ethidium bromide and exposed to ultraviolet radiation, so as to visually observe DNA fragments containing a coding gene. for PEPC and having a molecular length of about 11.5 kb. Part of the gel where such DNA fragments were found was cut out. To the cut gel was added TE buffer in an amount of three times the weight of the gel, and heated at 650C for 10 minutes to completely dissolve the agarose gel in the buffer. To the resulting solution, an equal volume of phenol was added and stirred, followed by recovery of an aqueous phase. To the aqueous phase, an equal volume of a 1: 1 (v / v) mixture of phenol and chloroform was added and stirred, followed by the recovery of an aqueous phase. added an equal volume of chloroform and stirred, followed by recovery of an aqueous phase.

To the aqueous phase was added sodium acetate to a final concentration of 300 millimoles and then a double volume of ethanol was added and stirred. Then, the mixture was kept at -300C for 3 hours and centrifuged at 10,000 rpm (9000g) at room temperature for 10 minutes to obtain a DNA pellet. The DNA pellet thus obtained was dried under vacuum and then dissolved in 20 μl of a TE buffer. There was thus obtained a solution containing about 5 μg of a DNA fragment containing a gene coding for PEPC and having a molecular length of about 11.5 kb.

 Step 8. (Isolation of a DNA fragment containing a gene coding for PEPC and having a molecular length of about 8.3 kb).

 At 5 μg of the XbaI-XbaI DNA fragment obtained in substantially the same manner as in step 7- (5) of Example 2, 20 units of SalI restriction enzyme were added. The resulting mixture was allowed to react in 50 μl of a buffer containing 50 mM Tris-HCl (pH 7.4), 10 μM MgSO 4 and 100 mM NaCl at 370 ° C for 3 hours to digest the DNA fragment. The digested DNA fragment was subjected to agarose gel electrophoresis in substantially the same manner as in step 7- (5) of Example 2. As a result, an XbaI-DNA fragment was observed. SalI having a molecular length of about 5.0 kb and a SalI-SalI DNA fragment having about 3.3 kb. A gel portion where such DNA fragments were found was cut out.

From the cut gel, about 1 μg of a XbaI-SalI DNA fragment having a molecular length of about 5.0 kb and about 0.5 μg of a SalI-SalI DNA fragment having a length molecular weight of about 3.3 kb, in substantially the same manner as in step 7- (5) of Example 2. The DNA fragments were analyzed for their restriction pattern. As a result, it was found that the DNA fragment having a molecular length of about 3.3 kb was the same as that having a molecular length of about 3.3 kb obtained in step 8 of Example 1 Thus, a DNA fragment containing a gene coding for PEPC and having a molecular length of about 8.3 kb was obtained in the form of a DNA fragment having a molecular length of about 5.0 kb. and a DNA fragment having a molecular length of about 3.3 kb.

 Step 9. (Isolation of a DNA fragment containing a gene coding for PEPC and having a molecular length of about 6.4 kb).

To 2 μg of the XbaI-SalI DNA fragment having a molecular length of about 5.0 kb obtained in substantially the same manner as in Step 8 of Example 2, 10 units of an enzyme were added. EcoRI restriction. The resulting mixture was allowed to react in 50 μl of a buffer containing 50 mM Tris-HCl (pH 7.4), 10 mM MgSO 4 and 100 mM NaCl at 370 ° C for 2 hours to digest the DNA fragment. The digested fragment of DNA was subjected to agarose gel electrophoresis in substantially the same manner as in step 7- (5) of Example 2. As a result, an EcoRI-DNA fragment was observed. SalI having a molecular length of about 3.1 kb. Part of the gel where such a DNA fragment was found was cut out. From the cut gel, about 0.5 μg of a DNA fragment was obtained.
EcoRI-SalI having a molecular length of about 3.1 kb, substantially in the same manner as in step 7- (5) of Example 2. Thus, a DNA fragment containing a gene encoding PEPC and having a molecular length of about 6.4 kb was obtained as two DNA component fragments, i.e., a DNA fragment having a molecular length of about 3.1 kb and a DNA fragment having a molecular length of about 3.3 kb obtained in step 8 of Example 2.

 Step 10. (Isolation of a DNA fragment containing a gene coding for PEPC and having a molecular length of about 4.8 kb).

To 4 μg of the XbaI-SalI DNA fragment having a molecular length of about 5.0 kb obtained in substantially the same manner as in step 8 of Example 2, 20 units of an enzyme were added. BamHI restriction. The resulting mixture was allowed to react in 50 μl of a buffer containing 10 mM Tris-HCl (pH 7.4), 10 mM MgSO 4, 50 mM NaCl and 1 mM dithiothreitol at 370 ° C. for 2 hours to digest the DNA fragment. . The digested DNA fragment was subjected to agarose gel electrophoresis in substantially the same manner as in step 7- (5) of Example 2. As a result, a BamHI-DNA fragment was observed. SalI having a molecular length of about 1.5 kb. Part of the gel was cut off where this fragment was found. From the cut gel, about 0.5 μl of a DNA fragment was obtained.
BamHI-SalI having a molecular length of about 1.5 kb, substantially in the same manner as in step 7- (5) of Example 2. Thus, a DNA fragment containing a gene was obtained. coding for PEPC and having a molecular length of about 4.8 kb, in the form of two constituent DNA fragments, i.e., a DNA fragment having a molecular length of about 1.5 kb and a DNA fragment having a molecular length of about 3.3 kb obtained in step 8 of Example 2.

 Step 11. (Isolation of a DNA fragment containing a gene encoding PEPC and having a molecular length of about 3.9 kb).

 To 4 μg of the XbaI-SalI DNA fragment having a molecular length of about 5.0 kb obtained in substantially the same manner as in step 8 of Example 2, 20 units of the enzyme were added. HindIII restriction.

The resulting mixture was allowed to react in 50 μl of buffer containing 10 mM Tris-HCl (pu7.4), 10 mM MgSO4, 50 mM NaCl and 1 mM dithiothreitol at 370 ° C for 2 hours to digest the DNA fragment. The digested DNA fragment was subjected to agarose gel electrophoresis in substantially the same manner as in step 7- (5) of Example 2. As a result, a DNA fragment was observed
HindIII-SalI having a molecular length of about 0.6 kb. A portion of the gel where such a DNA fragment was found was cut. From the cut gel, about 0.2 μg of a HindIII-SalI DNA fragment having a molecular length of about 0.6 kb was obtained, in substantially the same manner as in step 7- (5) of FIG. Example 2. Thus, a DNA fragment containing a gene coding for PEPC and having a molecular length of about 3.9 kb was obtained in the form of two constituent DNA fragments, i.e. a DNA fragment having a molecular length of about 0.6 kb and a DNA fragment having a molecular length of about 3.3 kb obtained in step 8 of Example 2.

 Example 3

In this example, a DNA fragment, derived from a bacterium producing glutamic acid belonging to the genus Corynebacterium, containing a gene coding for
PEPC was inserted into a vector derived from a glutamic acid-producing coryneform bacterium to prepare a recombinant DNA for the production of PEPC.

A transformant capable of forming PEPC at a high yield was then prepared using the recombinant DNA. The SalI fragment obtained in step 8 of Example 1, that is to say a SalI fragment containing a gene coding for PEPC and having a molecular length of about 3.3 kb and the XbaI fragment obtained at the step 7- (6) of Example 2, that is, an XbaI fragment containing a gene encoding PEPC and having a molecular length of about 11.1 kb, was used as a DNA fragment , derived from a bacterium producing glutamic acid belonging to the genus Corynebacterium, containing a gene encoding PEPC. A plasmid pAG50 was used as a vector derived from a coryneform bacteria producing glutamic acid. Plasmid pAG50 is a plasmid which was prepared from pAG1 and pAG3 plasmids derived from a coryneform bacterium producing glutamic acid by the method that will be mentioned below. The plasmid pAG1 is a tetracycline-resistant plasmid that has been isolated from
Corynebacterium melassecola 22243 deposited with the FRI under accession number FERM BP-560. The plasmid pAG3 is a cryptic plasmid isolated from Corynebacterium melassecola 22220 deposited at the FRI under accession number FERM
BP-559.

Step 1. CPreparation of plasmid pAG50 and its isolation of Corynebacterium melassecola 801 (pAG5O) 3
(1) Isolation of a plasmid pAGi from
Corynebacterium melassecola 22243 (FERM BP-560).

 The microorganism Corynebacterium melassecola 22243 was cultured in a semi-synthetic medium Medium prepared by dissolving 10 g of (NH4) 2SO4, 3 g of urea, 1 g of K2HPO4, 50 mg of Nazi, 400 mg of MgSO4 .7H2, 2 mg of MnSO4.4-6H2O, 2 mg of FeSO4.4-6H2O, 20 g of glucose, 50 of biotin, 200 of thiamine hydrochloride and 1 g of yeast extract in exchanged water. ion and distilled so that the total volume is 1 liter and adjusting to pH 7 at 320C overnight, while stirring.Inoculated 8 ml of the culture thus obtained in 200 ml of the same semi-synthetic medium, with then culturing at 320C for 5 hours while stirring to obtain a culture of the microorganism.

From the culture, cells of the microorganism were collected and suspended in 10 ml of a solution of lysozyme (pH8.0) containing 50 mM glucose, 10 mM EDTA, 25 mM Tris (hydroxylmethyl) - aminomethane (often referred to hereinafter as "Tris") and 10 mg / ml lysozyme (manufactured and sold by Sigma Chemical
Company, USA) followed by incubation at 420C for 1 hour. To the suspension was added 20 ml of an alkali-SDS solution containing 0.2 N NaOH and 1% (w / v) sodium dodecyl sulphate (often referred to hereinafter as "SDS"). The mixture was stirred and then allowed to stand in ice for 5 minutes. To the mixture was then added 15 ml of ice-cold potassium acetate solution (a mixture of 60 ml. 5 M potassium acetate, 11.5 ml of acetic acid and 28.5 ml of distilled water and exchanged for ions). The mixture was stirred and allowed to stand in ice for 10 minutes to obtain a lysate. The entire lysate was transferred to a centrifuge tube and centrifuged at 12,000 rpm (13,000 g) at 40 ° C for 5 minutes to thereby obtain a supernatant. The supernatant was extracted with an equal volume of phenol-chloroform (1: 1) and an aqueous phase was collected. To the aqueous phase, a double volume of ethanol was added and the resulting mixture was stirred and allowed to stand at room temperature for 5 minutes. Then, the mixture was centrifuged at 10,000 rpm (11000 g) at 200 ° C. for 10 minutes to thereby obtain the precipitates. The precipitates were washed with 70% (v / v) ethanol, dried under reduced pressure and dissolved in 20 ml of TE buffer (pH 7.5) containing 10 mM Tris and 1 mM EDTA in a tube. centrifuge. To the resulting solution, 1.2 ml of a 10 mg / ml aqueous solution of ethidium bromide and 23.6 g of cesium chloride were gently added and dissolved with centrifugation at 40000 rpm ( 100000 g) at 150C for 48 hours. After completion of the centrifugation, two bands were visualized with ultraviolet light. The lower band was removed from the centrifuge tube through the side wall of the tube using a syringe to obtain a pAG1 plasmid. The plasmid fraction thus obtained was extracted with an equal volume of isopropyl alcohol four times to obtain an extract. From the extract, the ethidium bromide was removed and the resulting solution dialyzed with TE buffer to obtain 1 ml of a dialysate containing a pAG1 plasmid at a concentration of 50 μg / ml.

 (2) In vitro recombination of plasmid pAGI.

To 0.5 μg of the plasmid pAGI DNA prepared in step 1- (1) of Example 3 above, 10 units of the restriction enzyme EcoRI was added. The resulting mixture was allowed to react in 40 μl of a buffer containing
50 mM Tris-HCl (pH 7.4), 10 mM MgSO 4 and 100 mM Nac at 370 ° C. for 2 hours. Then, the temperature was raised to 700 ° C. and held at 700 ° C. for 10 minutes to terminate the reaction. 20 μl of the reaction mixture thus obtained were reacted with 3 units of phage T4 DNA ligase (manufactured and sold by Nippon Gene Co. , Ltd.,
Japan) in 50-11 of a buffer containing 50 mM Tris-HCl (pH 7.4), 10 mM MgCl 2, 10 mM dithiothreitol, 1 mM spermidine, 1 mM ATP and 0.1 mg / ml BSA ( bovine serum albumin) (manufactured and sold by Bethesda
Research Laboratories, USA) at 150C overnight.

 (3) Isolation of plasmid pAG14.

 (Curage of the plasmid).

 A loop of Corynebacterium melassecola 22243 (FERM BP-560) was inoculated into 5 ml of LG medium (medium prepared by dissolving 10 g of Trypton, 5 g of yeast extract, 5 g of NaCl and 2 g of glucose in ion-exchanged water and distilled so that the total volume is 1 liter and adjusting to pH7.2) and grown at 370C overnight while stirring. The culture thus obtained was diluted with sterilized water. The diluted culture was then extended on LG agar medium (medium prepared by adding agar to LG medium so that the agar concentration of the resulting medium was 1.5% by volume), followed by culturing at 320C for 2 days to form colonies. 100 colonies thus formed were replated-plating onto an LG agar plate containing 10 μg / ml tetracycline and cultured at 320 ° C for 2 days. Then, two cells sensitive to tetracycline were selected. For the two cells thus selected, the existence of the pAG1 plasmid in the cells was determined by the plasmid isolation method described above. As a result, it was found that both cells did not contain a plasmid, i.e., the cells were a strain of the plasmid. One of the cells was used as a host in the next step.

 (Transformation).

 The pretreated cell of the plasmid prepared in the above step was cultured in the semi-synthetic medium mentioned above at 320C for 12 hours while stirring. 0.5 ml of the culture thus obtained was inoculated into 50 ml of the same semi-synthetic medium and cultured at 320 ° C. with stirring. The optical density (OD) of the 660 nm culture was monitored using a Model 228 spectrophotometer manufactured and sold by Hitachi, Ltd., Japan. At the point in time when the OD value became 0.2, penicillin G was added to the culture so that the penicillin concentration was 0.3 units / ml. The resulting culture was incubated at 320C for 1.5 hours.

 From the culture thus obtained, the cells were collected and suspended in 5 ml of medium R (medium prepared by dissolving 5 g of glucose, 10 g of Casamino acid, 10 g of yeast extract, 0, 35 g of K 2 HPO 4, 0.15 g of KH 2 PO 4, 1.37 g of sucrose, 5.73 g of N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (hereinafter referred to as TES), 0.95 g of MgCl 2 and 1.11 g of CaCl 2 in distilled water and ion exchanged so that the total volume is 1 liter, and adjusting to pH7.2) to obtain a cell suspension. To 4.5 ml of the cell suspension was added 0.5 ml of R medium containing 3 mg / ml lysozyme (sterilized using a Millipore filter) followed by incubation at 350C for 5 hours to thereby form protoplasts. The resulting culture was centrifuged at 7000 rpm (4500 g) at 50 ° C. for 7 minutes to obtain the protoplasts. The protoplasts thus obtained were suspended in 5 ml of medium R. The suspension of the protoplasts was subjected to centrifugation under the same conditions as those mentioned above to obtain the protoplasts. The protoplasts were suspended in 5 ml of R medium to obtain a suspension of the protoplasts.

To 0.5 ml of the protoplast suspension was added a mixture of 50 μl of the ligase reaction mixture obtained in step 1 (2) of Example 3 and 50 μl of 2 x TSMC solution (solution Aqueous solution containing 50 mM TES, 0.8 M sucrose, 20 mM MgCl 2 and 60 mM CaCl 2 and adjusted to pH 7.2 using NaOH). Then, to the resulting mixture was gently added a solution of PEG solution prepared by dissolving a polyethylene glycol. having a molecular weight of 6,000 in a solution of
TSMC containing 25mM TES, 0.4M sucrose, 10mM MEC12 and 30mM CaCl2, which solution was adjusted to pH 7.2 using NaOH, to a final concentration of 40% (w / v) 3 and allowed to stand at room temperature for 2 minutes. Then, to the mixture, 5 millimoles of an R-PVP solution solution prepared by dissolving polyvinyl pyrrolidone (hereinafter referred to as "PVP") in medium R at a gel concentration was added, followed by centrifugation at 4000. rpm (1800 g) at room temperature for 10 minutes to form a supernatant and precipitates. The supernatant was removed. To the precipitates, 5 ml of the R-PVP solution was added, followed by centrifugation under the same conditions as mentioned above to obtain protoplasts as precipitates. The protoplasts thus obtained were gently suspended in 0.5 ml of an R-PVP solution. The suspension was incubated at 300C for 3 hours and diluted with R-PVP solution to obtain a dilute suspension. The diluted suspension was inoculated into a regenerating medium containing 10 μg / ml tetracycline. The regenerating medium was a two-layer agar medium comprising a lower layer of agar medium obtained by adding PVP and agar to the concentrations. 40 g / l and 15 g / l respectively in the middle
R and, on the lower layer, an upper layer of agar medium obtained by adding PVP and agar at concentrations of 40 g / l and 6 g / l respectively, in the middle
R; and inoculation of the above-mentioned diluted suspension was carried out by mixing with 3 ml of the upper layer of the molten agar medium before forming the double layer. The resulting regenerating medium was incubated at 320C for 4 days to form tetracycline resistant strains.

 Of the tetracycline-resistant strains so formed, 10 strains were randomly selected and grown in biologically pure form on LG agar medium containing 10 μg / ml tetracycline. From each of the strains, a plasmid was isolated in substantially the same manner as in step 1 (1) of Example 3. The restriction sites of the plasmid thus isolated were determined using 0.5 μg of plasmid in substantially the same manner as in step 6 of Example 1, thereby to obtain a plasmid pAG14.

Using the plasmid DNA thus obtained, cells of the strain with Corynebacterium melassecola 22243 plasmid (Fermentation deposited
Research Institute under accession number FERM
BP-560) were transformed in much the same manner as mentioned above to obtain a tetracycline-resistant transformant. From the transformant thus obtained, a plasmid was separated, followed by analysis in substantially the same manner as mentioned above. As a result, it was found that the separated plasmid had the same restriction endonuclease cleavage map as plasmid pAG14.

 (4) Isolation of a DNA fragment containing a gene for tetracycline resistance of plasmid pAG14.

To 20 μg of pAG14 plasmid DNA obtained in step 1 (3) of Example 3, 100 units of each of BamHI and BglII restriction enzymes were added. The resulting mixture was allowed to react in 100 μl of a buffer containing 10 mM Tris-HCl (pH 7.4), 10 mM MgSO 4, 50 mM NaCl and 1 mM dithiothreitol at a temperature of 370 ° C for 2 hours. The digested plasmid was electrophoresed at 40C using a 1% agarose gel prepared from LMP Agarose (a trade name for a product manufactured and sold by Bethesda Research.
Laboratories, USA) in substantially the same manner as in step 6 of Example 1. The agarose gel was tinted with ethidium bromide and exposed to irradiation of ultraviolet light so as to visually observe fragments of DNA containing a gene for tetracycline resistance and having a molecular length of about 3.1 kb. Part of the gel where such DNA fragments were found was cut. To the cut gel, TE buffer was added in an amount of three times the weight of the gel and heated at 650C for 10 minutes to complete dissolution of the agarose gel. To the resulting solution, an equal volume of phenol was added and stirred, followed by recovery of an aqueous phase. To the aqueous phase, an equal volume of a 1: 1 (v / v) mixture of phenol and chloroform was added and stirred with subsequent recovery of an aqueous phase. To the aqueous phase, an equal volume of chloroform was added and stirred, followed by recovery of an aqueous phase. To the aqueous phase, sodium acetate was added to a final concentration of 300 millimoles and then a double volume of ethanol was added and stirred.

Then, the mixture was kept at -300C for 3 hours and centrifuged at 10,000 rpm (9000g) at room temperature for 10 minutes to obtain a DNA pellet. The thus obtained DNA pellet was dried under vacuum.

 (5) Preparation of plasmid pAG3 and its treatment with BamHI restriction enzyme.

20 units of the BamHI restriction enzyme were added to 4 μg of plasmid pAG3 DNA obtained from Corynebacterium melassecola 22220 (deposited at
Fermentation Research Institute under accession number FERM BP-559) and was purified substantially in the same manner as in step 1 of Example 3. The resulting mixture was allowed to react in 100 μl of a buffer. containing 10 mM Tris-HCl (pH 7.4), 10 mM MgSO 4, 50 mM NaCl and 1 mM dithiothreitol at a temperature of 370 ° C for 2 hours. Then, an equal volume of a 1: 1 (volume / volume) mixture of phenol and chloroform was added to the mixture and stirred, followed by the recovery of an aqueous phase. added an equal volume of chloroform and stirred, followed by recovery of an aqueous phase.

To the aqueous phase, sodium acetate was added to a final concentration of 300 millimoles and then a double volume of ethanol was added and stirred. Then, the mixture was kept at -300C for 3 hours and centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes to obtain a DNA pellet. The thus obtained DNA pellet was dried under vacuum.

 (6) Isolation of plasmid pAG5O.

 The totality of each of the DNAs obtained in substeps (4) and (5) of step 1 of Example 3 were reacted with three units of phage T4 DNA ligase in a buffer containing Tris. 50 mM HCl (pH 7.4), 10 mM MgCl 2 10 mM dithiothreitol, 1 mM spermidine, 1 mM ATP and 0.1 mg / ml BSA at a temperature of 150C overnight. The temperature was raised to 700C and held at 700C for 10 minutes to complete the reaction.

 Using 50 μl of the ligase reaction mixture, a Corynebacterium melassecola 801 tetracycline-resistant transformant (Fermentation Research Institute accession number FERM BP-558) was obtained according to the same processing technique as employed. in step 1- (3) of Example 3, except that the incubation in the regenerating medium was undertaken for 7 days. There was thus obtained a tetracycline resistant transformant. A plasmid retained in the above tetracycline-resistant transformant was separated in substantially the same manner as used in step 1 (1) of Example 3. Then, the plasmid thus obtained was subjected to an analysis in substantially the same manner as in step 6 of Example 1. The plasmid thus obtained was designated pAG5O.

In much the same way as mentioned above, cells of the
Corynebacterium melassecola 801 (Fermentation deposit
Research Institute under accession number FERM
BP-558) were transformed using the above plasmid to obtain a tetracycline-resistant transformant. The restriction endonuclease cleavage map of the plasmid retained in the transformant was found to be the same as that of the plasmid incorporated in the above transformation.

 Step 2. (Insertion of a DNA fragment containing a gene coding for PEPC and having a molecular length of about 3.3 kb in the plasmid pAG5O).

To 5 μg of the plasmid pAG50 DNA prepared in step 1- (6) of Example 3, 15 units of the restriction enzyme EcoRI were added. The resulting mixture was allowed to react in 60 μl of a buffer containing
50 mM Tris-HCl (pH 7.4), 10 mM MgSO 4 and 100 mM NaCl at 370 ° C. for 2 hours. Then, the temperature was raised to 700 ° C. and maintained at this value for 10 minutes to terminate the reaction. To the mixture, sodium acetate was added to a final concentration of 300 millimoles and then the mixture was added twice. volume of ethanol. The resulting mixture was allowed to stand at -300 ° C. for 3 hours and then centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes to obtain a DNA pellet. The resulting pellet was dried under reduced pressure. Then, the pellet was dissolved in 200 μl of BAPT buffer containing 50 mM Tris-HCl (pH8.4).

To the resulting solution was added a unit of bacterial alkaline phosphatase (manufactured and sold by
Takara Shuzo Co., Ltd., Japan).

 The resulting mixture was allowed to react at 650C for 30 minutes. Then, a bacterial alkaline phosphatase unit was added to the reaction mixture. The resulting mixture was allowed to react at 650C for 3 hours. To the reaction mixture thus obtained, an equal volume of phenol saturated with TNE buffer was added and stirred. Then the mixture was centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes followed by recovery of an aqueous phase. To the aqueous phase an equal volume of phenol saturated with TNE buffer was added and stirred. The mixture was subjected to centrifugation under the same conditions as mentioned above to obtain an aqueous phase. To the aqueous phase, an equal volume of a 1: 1 mixture (volume / volume) was added. of phenol and chloroform, was stirred and centrifuged at 12,000 rpm (8900 g) for 10 minutes, followed by recovery of an aqueous phase. To the aqueous phase, an equal volume of chloroform was added, stirred and centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes followed by recovery of an aqueous phase. To the aqueous phase was added sodium acetate to a final concentration of 300 millimoles and then a double volume of ethanol was added and stirred. Then, the mixture was allowed to stand at -300C for 3 hours and centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes to obtain a DNA pellet. The DNA pellet thus obtained was dried under vacuum.

The entire amount of the thus obtained DNA was reacted with 1 μg of the DNA prepared in step 8 of Example 1 with 3 units of phage T4 DNA ligase (manufactured and sold by Nippon Gene Co. ., Ltd., Japan) in 50 μl of a buffer containing 50 mM Tris-HCl (pH 7.4) 10 mM MgCl 2, 10 mM dithiothreitol, 1 mM spermidine, 1 mM ATP and 0.1 mg / ml BSA ( manufactured and sold by Bethesda
Research Laboratories, USA) at a temperature of 150C overnight. The temperature was raised to 700C and held at this value for 10 minutes to complete the reaction.

Step 3. (Isolation of a hybrid plasmid pAG2001 containing a gene coding for PEPC)
Corynebacterium melassecola 801 (Fermentation Research Institute accession number FERM accession number BP-558) was cultured in the semi-synthetic medium mentioned above at 320C for 12 hours while shaking. 0.5 ml of the culture thus obtained was inoculated in 50 ml of the same semi-synthetic medium and cultured at 320C while shaking. The optical density (OD) of the 660 nm culture was monitored using a Model 228 spectrophotometer (manufactured and sold by Hitachi, Ltd., Japan). At the point where the OD value became 0.2, penicillin G was added to the culture so that the penicillin concentration was 0.3 units / ml. The resulting culture was incubated at 320C for 1 hour. ,5 hours.

Cells were collected from the culture thus obtained. The collected cells were suspended in 5 ml of R medium to obtain a cell suspension. To 4.5 ml of the cell suspension was added 0.5 ml of R medium containing 3 mg / ml of lysozyme (sterilized using a filter
Millipore) followed by incubation at 350C for 5 hours to thereby form protoplasts. The resulting culture was centrifuged at 7000 rpm (4500 g) at 50 ° C for 7 minutes to obtain the protoplasts. The protoplasts thus obtained were suspended in 5 ml of medium R. The protoplast suspension was centrifuged under the same conditions as mentioned above to obtain protoplasts. The protoplasts were suspended in 5 ml. medium R to obtain a suspension of protoplasts.

 To 0.5 ml of the protoplast suspension was added a mixture of 50 μl of the mixture obtained in step 2 of Example 3 and 50 μl of a solution of 2 × TSMC (aqueous solution containing 50 mM TES). 0.8 M sucrose, 20 mM MgCl 2 and 60 mM CaCl 2 and adjusted to pH 7.2 using NaOH). Then, to the resulting mixture was added 1.5 ml PEG solution, gently stirred and allowed to stand at room temperature for 2 minutes. Then, to the mixture was added 5 ml of the R-PVP solution, followed by centrifugation at 4000 rpm (1800 g) at room temperature for 10 minutes to form a supernatant and precipitates. The supernatant was removed. To the precipitates, 5 ml of the R-PVP solution was added followed by centrifugation under the same conditions as mentioned above to obtain protoplasts as precipitates. The protoplasts thus obtained were gently suspended in 0.5 ml of the R-PVP solution. The suspension was incubated at 300C for 3 hours and diluted with R-PVP solution to obtain a dilute suspension. The diluted suspension was mixed with 3 ml of the molten agar medium having the same composition as that of the top layer of the regenerating medium agar containing 10 μg / ml of tetracycline in step 1- (3) of Example 3 and the resulting mixture was applied to the lower layer of agar medium. The resulting regenerating medium was incubated at 320C for 7 days to form tetracycline resistant cells.

 The thus formed tetracycline-resistant cells were grown in biologically pure form on LG agar medium containing 10 μg / ml tetracycline. From each of the cells, a plasmid was isolated in substantially the same manner as in step 1 of Example 3, followed by analysis in substantially the same manner as in step 6 of Example 1. The plasmid thus obtained has been designated by pAG2001. The restriction endonuclease cleavage map of plasmid pAG2001 is shown in FIG. 10. As is apparent from FIG. 10, plasmid pAG2001 is a hybrid plasmid containing plasmid pAG50 and inserted at the site cleaved by the restriction enzyme. SalI, a DNA fragment derived from a bacterium producing glutamic acid, belonging to the genus Corynebacterium, containing a gene coding for PEPC and having a molecular length of about 3.3 kb.

 Step 4. (Measurement of the activity of PEPC of a transformant retaining the plasmid pAG2001).

 The Corynebacterium melassecola 801 cell (pAG2001) retaining the plasmid pAG2001 was cultured at 320C overnight in 50 ml of a molasses medium of the type employed in step 1 of Example 1 containing 10 μg / ml. of tetracycline. From the culture thus obtained, the cells were collected, washed twice with 20 ml of 0.8% aqueous NaCl solution and suspended in 10 ml of a MES buffer (pH7.0) containing 50 mM 2- (N-morpholino) ethanesulfonic acid (hereinafter referred to as MES), 10 mM MnSO4 and 10 mM EDTA.

The suspension was homogenized using a cell homogenizer (Model 85321 MSK Cell Homogenizer manufactured and sold by Braun Inc., West Germany) followed by centrifugation at 14000 rpm (20000 g) at room temperature for 20 minutes. to obtain a cell extract (crude enzyme solution).

The PEPC activity and the cell extract protein concentration were determined in substantially the same manner as in step 5 of Example 1. The results are shown in Table 4 given in Step 5 which will be mentioned below.

 Step 5. (Insertion of a DNA fragment containing a gene encoding PEPC and having a molecular length of about 11.5 kb in plasmid pAG50).

 5 μg of the pAG50 plasmid DNA prepared in step 1 (6) of Example 3 were reacted with 15 units of an XbaI restriction enzyme in 60 μl of a buffer containing Tris-HCl 50. mM (pH 7.4), 10 mM MgSO 4 and 100 mM NaCl at a temperature of 370C for 2 hours.

Then, the temperature was raised to 700C and held at this value for 10 minutes to complete the reaction. To the reaction mixture was added sodium acetate to a final concentration of 300 millimoles and a double volume of ethanol and allowed to stand at -300C for 3 hours. Then, the mixture was centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes to obtain a DNA pellet. The resulting pellet of DNA was dried under vacuum.

The dried DNA was dissolved in 200 μl of BAPT buffer containing 50 mM Tris-HCl (pH8.4). To the resulting solution was added a unit of bacterial alkaline phosphatase (manufactured and sold by Takara
Shuzo Co., Ltd., Japan) followed by incubation at 650C for 30 minutes. Then, to the resulting mixture was further added a unit of bacterial alkaline phosphatase followed by incubation at 650C for 30 minutes. To the reaction mixture thus obtained an equal volume of saturated phenol of the TNE buffer was added and stirred. Then the mixture was centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes followed by recovery of an aqueous phase. To the aqueous phase, an equal volume of phenol saturated with TNE buffer was added and stirred. The mixture was subjected to centrifugation under the same conditions as those mentioned above to obtain an aqueous phase. To the aqueous phase, an equal volume of a 1: 1 (volume / volume) mixture of phenol and chloroform was added, stirred and centrifuged at 12,000 rpm (8900 g) for 10 minutes. then recovering an aqueous phase.

To the aqueous phase, an equal volume of chloroform was added, stirred and centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes followed by recovery of an aqueous phase. To the aqueous phase was added sodium acetate to a final concentration of 300 millimoles and then a double volume of ethanol was added, and stirred. Then, the mixture was allowed to stand at -300 ° C for 3 hours and centrifuged at 12000 rpm (8900 g) at room temperature for 10 minutes to obtain a DNA pellet. The thus obtained DNA pellet was dried under vacuum.

The entire amount of the thus obtained DNA was reacted with 1 μg of the DNA prepared in step 7 of Example 2 with 3 units of phage T4 DNA ligase (manufactured and sold by Nippon Gened Co., Ltd., Japan) in 50nul of a buffer containing 50mM Tris-HCl (pH 7.4) 10mM MgCl2, 10mM dithiothreitol, 1mM spermidine, 1mM ATP and 0.1mg / ml BSA (manufactured and sold by
Bethesda Research Laboratories, USA) at a temperature of 150C overnight. The temperature was raised to 700C and held at this value for 10 minutes to complete the reaction. There was thus obtained a ligase reaction mixture.

 Step 6. (Isolation of a hybrid plasmid pAG2002 containing a gene coding for PEPC).

Using the ligase reaction mixture obtained in step 5 of Example 3, cells of
Corynebacterium melassecola 801 (Fermentation deposit
Resarch Institute accession number FERM BP-558) were transformed in substantially the same manner as in step 3 of Example 3 to obtain tetracycline resistant transformants. The tetracycline resistant transformants thus obtained were grown in a biologically pure form on the agar medium.
LG containing 10 μg / ml of tetracycline.

 A plasmid was isolated from each of the transformants in substantially the same manner as in Step 1 of Example 2, followed by analysis in substantially the same manner as in Step 6 of Example 1. obtained a plasmid. The resulting plasmid was designated pAG2002. The restriction endonuclease cleavage map of the plasmid pAG2002 is shown in FIG. 11. As is apparent in FIG. 11, the plasmid pAG2Q02 is a hybrid plasmid containing the plasmid pAG5O, and inserted at the site cleaved by the XbaI restriction, a DNA fragment derived from a bacterium producing glutamic acid belonging to the genus Corynebacterium, containing a gene encoding PEPC and having a molecular length of about 11.5 kb.

 Step 7. (Measurement of the PEPC activity of a transformant retaining pAG2002).

The cells of Corynebacterium melassecola 801 (pAG2002) retaining the plasmid pAG2002 were cultured and an extract of cells (crude enzyme solution) of the cultured cells was obtained in substantially the same manner as Step 4 of Example 3. Using the cell extract thus obtained, the PEPC activity of
Corynebacterium melassecola 801 (pAG2002) in substantially the same manner as in step 5 of Example 1. The result is also shown in Table 4 given below as well as the data obtained in step 4 of the example 3, in comparison with data relating to cells retaining plasmid pAG50 and cells not retaining plasmid.

 As is apparent from Table 4, cells retaining plasmid pAG2001 and plasmid-retaining cells pAG2002 had high specific activity of PEPC compared to cells retaining plasmid pAG50 and non-plasmid-retaining cells.

In addition, the incubation of the
Corynebacterium melassecola 801, not retaining plasmid, was carried out in the absence of tetracycline.

TABLE 4.

<Tb>

<SEP> Strain <SEP> Specific <SEP> activity <SEP> of <SEP> PEPC <SEP> * 1)
<tb><SEP> 801 <SEP> * 2) <SEP> 0.06
<tb><SEP> 801 <SEP> (pAG50) <SEP> 0.06
<tb> 1 <SEP> 801 <SEP> (pAG2001) <SEP> 0.22
<tb><SEP> 801 <SEP> (pAG2002 <SEP> 0.79
<Tb>
Notes: * 1) as in Table 1
* 2) Corynebacterium melassecola 801 deposited at
Fermentation Research Institute under the number
FERM BP-558.

Claims (33)

 1. A DNA fragment characterized in that it is derived from a bacterium producing glutamic acid belonging to the genus Corynebacterium, containing a gene encoding phosphoenolpyruvate carboxylase.  2. DNA fragment according to claim 1, characterized in that the aforementioned bacterium producing 1 t glutamic acid is the microorganism Corynebacterium melassecola 801 deposited at the Fermentation Research Institute under accession number FERM BP-558.  3. DNA fragment according to claim 2, characterized in that it has the following restriction sites  a first PstI site, a second PstI site, a first EcoRI site, a third PstI site, a first HindIII site, a first BamHI site, a fourth site PstI, a fifth PstI site, a second HindIII site, a third HindIII site, a first SalI site, a second EcoRI site, a fourth HindIII site, a third site EcoRI, a sixth PstI site, a seventh PstI site, a fifth HindIII site, an eighth PstI site, a second BamHI site, a second SalI site, a ninth PstI site and a third BamHI site,  said second PstI site, said first site EcoRI, said third PstI site, said first HindIII site, said first BamHI site, said fourth site PstI, said fifth PstI site, said second HindIII site, said third HindIII site, said first site SalI, said second EcoRI site, said fourth HindIII site, said third EcoRI site, said sixth site PstI, said seventh PstI site, said fifth HindIII site, said eighth PstI site, said second site BamHI, said second SalI site, said ninth PstI site and said third BamHI site are respectively located at about 1.0 kb, about 1.9 kb, about 2.5 kb, about 2.6 kb, about 3.5 kb, about 3.7 kb, about 4.0 kb, about 4.3 kb, about 4.4 kb, about 5.0 kb, about 6.3 kb, about 6.5 kb, about 6.7 kb, about 6 kb , 9 kb, about 7.0 kb, about 7.0 kb, about 7.4 kb, about 7.4 kb, about 8.3 kb, about 9.5 kb and 10.3 kb of said first PstI site.  4. DNA fragment according to claim 2, characterized in that it has the following restriction sites.  a first PstI site, a second PstI site, a first EcoRI site, a third PstI site, a first HindIII site, a first BamHI site, a fourth site PstI, a fifth PstI site, a second HindIII site, a third HindIII site, a SalI site, a second EcoRi site, a fourth HindIII site, a third site EcoRI, a sixth PstI site, a seventh PstI site, a fifth HindILI site, an eighth PstI site, and a second BamHI site,  said second PstI site, said first site EcoRI, said third PstI site, said first HindIII site, said first BamHI site, said fourth site PstI, said fifth PstI site, said second HindIII site, said third HindIII site, said SalI site, said second EcoRi site, said fourth HindIII site, said third EcoRi site, said sixth PstI site, said seventh PstI site, said fifth HindIII site. , said eighth PstI site and said second BamHI site being respectively located at about 1.0 kb, about 1.9 kb, about 2.5 kb, about 2.6 kb, about 3.5 kb, about 3.7 kb, about 4.0 kb, about 4.3 kb, about 4.4 kb, about 5.0-kb, about 6.3 kb, about 6.5 kb, about 6.7 kb, about 6.9 kb, about 7.0 kb, about 7.0 kb, about 7.4 kb and about 7.4 kb of said first PstI site.  5. DNA fragment according to claim 2, characterized in that it has the following restriction sites  a first PstI site, a first HindIII site, a first BamHI site, a second PstI site, a third PstI site, a second HindIII site, a third site HindIII, a SalI site, a first EcoRI site, a fourth HindIII site, a second EcoRI site, a fourth PstI site, a fifth PstI site, a fifth HindIII site, a sixth PstI site, and a second site. Bam,  said first HindIII site, said first site BamHI, said second PstI site, said third PstI site, said second HindIII site, said third HindIII site, said SalI site, said first EcoRI site, said fourth HindIII site, said second EcoRi site, said fourth PstI site, said fifth PstI site said fifth HindIII site, said sixth PstI site and said second BamHI site being respectively located at about 0.1 kb, about 1.0 kb, about 1.2 kb, about 1.5 kb, about 1.8 kb, about 1.9 kb, about 2.5 kb, about 3.8 kb, about 4.0 kb, about 4.2 kb, about 4.4 kb, about 4.5 kb, about 4.5 kb, about 4 kb 9 kb and about 4.9 kb from said first PstI site.  6. DNA fragment according to claim 2, characterized in that it has the following restriction sites.  a first site HindîlI, a second site HindIII, a first SalI site, a first EcoRI site, a third HindIII site, a second EcoRI site, a first PstI site, a second PstI site, a fourth HindIII site, a third PstI site, a BamHI site and a second SalI site. ,  said second HindIII site, said first site SalI, said first EcoRI site, said third HindIII site, a second EcoRI site, said first PstI site, said second PstI site, said fourth HindIII site, said third PstI site, said BamHI site and said second SalI site being placed at approximately 0.1 kb, about 0.7 kb, about 2.0 kb, about 2.2 kb, about 2.4 kb, about 2.6 kb, about 2.7 kb, about 2.7 kb, about 3 kb 1 kb, about 3.1 kb and about 4.0 kb of said first HindIII site.  DNA fragment according to claim 2, characterized in that it has the following restriction sites.  a first PstI site, a second PstI site, a first HindIII site, a second HinaIII site, a site SalI, a first EcoRI site, a third HindIII site, a second EcoRI site, a third PstI site, a fourth PstI site, a fourth HindIII site, a fifth PstI site, and a BamHI site,  said second PstI site, said first HindIII site, said second HindIII site, said SalI site, said first EcoRI site, said third HindIII site, said second EcoRI site, said third PstI site, said fourth PstI site, said fourth HindIII site, said fifth PstI site, and said BamHI site being respectively placed at about 0.3 kb, about 0.6 kb, about 0.7 kb, about 1.3 kb, about 2.6 kb, about 2.8 kb, about 3 kb , 0 kb, about 3.2 kb, about 3.3 kb, about 3.3 kb, about 3.7 kb, and about 3.7 kb of said first PstI site.  8. DNA fragment according to claim 2, characterized in that it has the following restriction sites  a SalI site, a first EcoRi site, a first HindIII site, a second EcoRI site, a first PstI site, a second PstI site, a second HindIII site, a third PstI site and a BamHI site,  said first EcoRI site, said first HindIII site, said second EcoRI site, said first site PstI, said second PstI site, said second HindIII site, said third PstI site, and said BamHI site are respectively located at about 1.3 kb, about 1.5 kb, about 1.7 kb, about 1.9 kb, about 2.0 kb, about 2.0 kb, about 2.4 kb and about 2.4 kb of said SalI site.  9. DNA fragment according to claim 2, characterized in that it has the following restriction sites.  a first EcoRi site, a first HindIII site, a second EcoRI site, a first PstI site, a second PstI site, a second HindIII site, a third site PstI, and a BamHI site,  said first HindIII site, said second site EcoRI, said first PstI site, said second PstI site, a second HindIII site, a third PstI site and a site Wherein BamHI are placed at about 0.2 kb, about 0.4 kb, about 0.6 kb, about 0.7 kb, about 0.7 kb, about 1.1 kb, and about 1.1 kb, respectively, of said first EcoRI site. .  10. Recombinant DNA characterized in that it comprises a first DNA fragment according to any one of claims 1 or 2 and a second DNA fragment containing a gene for replication in a cell of a bacterium, said first DNA fragment being directly or indirectly linked to said second DNA fragment.  Recombinant DNA according to claim 10, characterized in that the first DNA fragment mentioned above is the DNA fragment according to any one of claims 3 to 9.  The recombinant DNA according to any one of claims 10 or 11, characterized in that said second DNA fragment is a DNA fragment containing a vector for a host-vector system where Escherichia coli is used as a host.  13. Recombinant DNA according to claim 12, characterized in that the above-mentioned vector is a member selected from the group consisting of a plasmid pBR325 and its derivatives.  The recombinant DNA according to any one of claims 10 or 11, characterized in that said second DNA fragment is a DNA fragment containing a vector for a host-vector system where a coryneform bacterium producing the acid Glutamic is used as a host.  15. Recombinant DNA according to claim 14, characterized in that the aforementioned vector is a member selected from the group consisting of plasmid pAGi, plasmid pAG3, plasmid pAG50 and their derivatives.  The recombinant DNA of claim 10, characterized by a molecular length of about 11.5 kb and a restriction endonuclease cleavage map shown in Figure 1 and is designated pAG203.  The recombinant DNA of claim 10, characterized by a molecular length of about 9.3 kb and a restriction endonuclease cleavage map shown in Figure 2 and is designated pAG211.  The recombinant DNA of claim 10, characterized by a molecular length of about 22.9 kb and a restriction endonuclease cleavage map shown in Figure 4 and is designated pAG204.  The recombinant DNA of claim 10, characterized by a molecular length of about 17.5 kb and a restriction endonuclease cleavage map shown in Figure 5 and is designated pAG221.  The recombinant DNA of claim 10, characterized by a molecular length of about 10.9 kb and a restriction endonuclease cleavage map shown in Figure 10 and is designated pAG2001.  21. Recombinant DNA according to claim 10, characterized by a molecular length of about 19.1 kb and a restriction endonuclease cleavage map shown in Figure 11 and is designated pAG2Q02.  22. Microorganism characterized in that it contains a bacterium and recombinant DNA according to any one of claims 10 to 21, which is contained in said bacterium.  23. Microorganism according to claim 22, characterized in that the bacterium mentioned above is a bacterium belonging to the genus Escherichia.  24. Microorganism according to claim 23, characterized in that the aforementioned bacterium belonging to the genus Escherichia is Escherichia coli.  25. Microorganism according to claim 22, characterized in that the aforementioned bacterium is a coryneform bacterium producing glutamic acid.  26. Microorganism according to claim 25, characterized in that the above-mentioned coryneform bacterium is a bacterium belonging to the genus Corynebacterium.  27. Microorganism according to claim 26, characterized in that the aforementioned bacterium belonging to the genus Corynebacterium is Corynebacterium melassecola.  28. Microorganism according to claim 22, characterized in that the aforementioned bacterium is Escherichia coli K12 strain 342-167 and the recombinant recombinant DNA is the plasmid pAG203 according to claim 16 and which is called strain 342-167 of Escherichia coli K12 (pAG203).  29. Microorganism according to claim 22, characterized in that the aforementioned bacterium is Escherichia coli K12 strain 342-167 and the aforementioned recombinant DNA is the plasmid pAG211 of claim 17, which is called Escherichia coli K12 strain 342-167 (pAG211).  30. Microorganism according to claim 22, characterized in that the aforementioned bacterium is Escherichia coli strain K12 342-167 and the aforementioned recombinant DNA is the plasmid pAG204 of claim 18 which will be called Escherichia coli K12 strain 342-167 (pAG204).  31. Microorganism according to claim 22, characterized in that the aforementioned bacterium is Escherichia coli K12 strain 342-167 and the aforementioned recombinant DNA is the plasmid pAG221 of claim 19, which will be called the strain 342-167 of Escherichia coli K12 (pAG221).  32. Microorganism according to claim 22, characterized in that the aforementioned bacterium is Corynebacterium melassecola 801 and the aforementioned recombinant DNA is the plasmid pAG2001 of claim 20, which will be called Corynebacterium melassecola 801 (pAG2001).  33. Microorganism according to claim 22, characterized in that the aforementioned bacterium is Corynebacterium melassecola 801 and the aforementioned recombinant DNA is the plasmid pAG2002 according to claim 21, which will be called Corynebacterium melassecola 801 (pAG2002).