JPH0866189A - New dna fragment - Google Patents

Abstract

PURPOSE: To obtain a new DNA fragment containing a gene coding for phosphoenolpyruvic acid carboxylase derived from Brevibacterium flavum MJ-233, and capable of giving the enzyme enabling amino acids, etc., to be efficiently produced through genetic engineering technique. CONSTITUTION: This new DNA fragment is derived from Brevibacterium flavum MJ-223 (FERM BP-1497), having an amino acid sequence containing an amino acid sequence of the formula, containing a gene coding for phosphoenolpyruvic acid carboxylase (PEPC) involving the production of useful substances such as amino acids and carbon dioxide fixation, and being capable of improving the production efficiency for useful substances such as amino acid, etc., by its transfection into a manifestation vector to transform a host cell to effect manifestation. This DNA fragment is obtained by extracting the whole DNA of Brevibacterium flavum MJ-223 followed by treating the whole DNA with a restriction enzyme, linking to a cloning vector, and then cloning by a conventional method.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to Brevibacterium
Brevibacterium flavum MJ-233 (F
ERM BP-1497) -derived phosphoenolpyruvate carboxylase (Phosphoenolpyruvate carboxyl)
ase) (EC 4.1.1.31) (hereinafter this may be abbreviated as "ppc gene")
To a DNA fragment containing

[0002]

2. Description of the Related Art Phosphoenolpyruvate carboxylase (hereinafter referred to as "PEPC") which is a ppc gene product.
May be abbreviated as), carbon dioxide (bicarbonate ion) is fixed to phosphoenolpyruvate, which is a metabolic intermediate compound of glycolysis, to produce oxaloacetate, and 4 carbons (tricarbonic acid (TCA) cycle) are generated. C4) It is said to play a physiological role of supplementing the compound. The TCA cycle is an important metabolic pathway in the biosynthesis system of various useful substances such as amino acids. By utilizing the gene, it is expected that the substance supply to the TCA cycle will be strengthened, the productivity of useful substances such as amino acids will be enhanced, and the yield of bacterial cells will be improved. Further, since it has a catalytic function of fixing carbon dioxide to an organic compound, it can be expected to contribute to global environment conservation.

Phosphoenolpyruvate carboxylase has been confirmed to exist in most bacteria, protozoa and all plants. Regarding the gene encoding this enzyme, although many studies have been conducted on higher plants, regarding bacteria, Escherichia coli
Genes [see Journal of Bichemistry (J. Biochem.), 95 , 909-916 (1984)] and Corynebacterium glutamicum (Corynebacterium gl)
utamicum) -derived gene (Gene, 77 , 237-25
1 (1989)] has been isolated. Regarding Escherichia coli, although basic research on the reaction mechanism and the like of the enzyme has been vigorously carried out, its use from an industrial viewpoint has hardly been examined.
Furthermore, basic research and industrial application studies on the ppc gene derived from coryneform bacteria including the bacterium belonging to the genus Brevibacterium, which is an industrially important bacterium, are still insufficient, and various problems to be solved are piled up.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have found that p of a Brevibacterium bacterium, which is an industrially important bacterium, has.
As a result of intensive studies aimed at isolation of the pc gene and industrial application, it was found that the pcc gene can be isolated from a bacterium belonging to a certain genus Brevibacterium, and the present invention was completed. It was

[0005]

The present invention thus provides a DNA fragment containing a gene encoding phosphoenolpyruvate carboxylase derived from Brevibacterium flavum MJ-233.

The present invention will be described in more detail below. DNA containing a gene (ppc gene) encoding “phosphoenolpyruvate carboxylase” of the present invention
The term “fragment” means a DNA fragment containing a gene encoding an enzyme that catalyzes a reaction of fixing carbon dioxide to phosphoenolpyruvate to produce oxaloacetate.

The DNA fragment containing the ppc gene of the present invention can be synthesized after the nucleotide sequence thereof has been determined, but it is usually cloned from a bacterium of the genus Brevibacterium and used as a source thereof. Brevibacterium flavum MJ as a bacterium of the genus
Those derived from -233 are preferable. An example of the basic procedure for preparing the ppc gene from this source bacterium is as follows.

The ppc gene is the Brevibacterium flavum MJ-233 described above.
Exists on the chromosome of the (FERM BP-1497) strain,
From this chromosome, it can be separated and obtained by the method described below. First, chromosomal DNA is extracted from a culture of Brevibacterium flavum MJ-233 strain.
The chromosomal DNA is completely digested with an appropriate restriction enzyme such as SalI.

The obtained DNA fragment is inserted into a cloning vector, for example pUC118 (Takara Shuzo), and Escherichia coli JM109 (Takara Shuzo) is transformed with this vector to obtain a transformant. A plasmid DNA was extracted from the obtained transformant, and the ppc gene derived from Escherichia coli [see Journal of Bichemistry (J. Biochem.), 95 , 909-916 (1984)] and the ppc gene derived from corn [ plant·
Molecular Biology (Plant Mol. Biol.), 1
2 , 579-589 (1989)], the inserted ppc gene derived from Brevibacterium flavum MJ-233 strain can be confirmed by Southern hybridization using the common region sequence as a probe.

One of the DNA fragments containing the ppc gene is the Brevibacterium flavum MJ-2.
A DN having a size of about 3.3 kb obtained by completely decomposing the chromosomal DNA of 33 strains with a restriction enzyme SalI.
A fragment is mentioned. The number of recognition sites and the size of the cleaved fragment when this approximately 3.3 kb DNA fragment was cleaved with various restriction enzymes are shown in Table 1 below.

[0011]

Table 1 size of Table 1 restriction enzyme recognition sites number cleavage fragment (kb) AatII 1 1.0,2.3 AccI 1 0.5,2.8 ClaI 2 0.9,1.0,1. 4 NcoI 2 0.7, 1.2, 1.4

In the present specification, "the number of recognition sites" by a restriction enzyme means that a DNA fragment or a plasmid is completely decomposed in the presence of a restriction enzyme, and those decomposition products are subjected to 1% agarose gel according to a method known per se. Electrophoresis and 4%
It was subjected to polyacrylamide gel electrophoresis and the value determined from the number of separable fragments was adopted.

Further, the "size of the cleaved fragment" and the size of the plasmid are such that, when agarose gel electrophoresis is used, Escherichia coli lambda phage (λ ph
based on the standard line drawn by the migration distance on the same agarose gel of a DNA fragment of known molecular weight obtained by cleaving the DNA of age) with the restriction enzyme HindIII, and in the case of using polyacrylamide gel electrophoresis, Escherichia・ Coli's Phi-X174 phage (φX
174 page) DNA to the restriction enzyme HaeIII
The size of the cleaved DNA fragment or each DNA fragment of the plasmid is calculated based on the standard line drawn by the migration distance on the same polyacrylamide gel of the DNA fragment with a known molecular weight obtained by cleaving with. In the determination of the size of each DNA fragment, the size of a fragment of 1 kb or more is 1
The results obtained by% agarose gel electrophoresis were adopted, and the results obtained by 4% polyacrylamide gel electrophoresis were adopted for fragment sizes of about 0.1 kb to less than 1 kb.

On the other hand, the chromosomal DNA of the Brevibacterium flavum MJ-233 strain described above is digested with a restriction enzyme SalI to obtain a D of about 3.3 kb in size.
Regarding the NA fragment, its base sequence is the plasmid pUC
Dideoxy nucleotide termination method [118] (manufactured by Takara Shuzo)
F. et al., Proceedings of National
Academy of Science of United
States of America (Proc. Natl. Acad. Sci. U
SA), 74 , 5463, (1977)]. The above-mentioned DNA of about 3.3 kb determined in this way
The ppc gene determined from the presence of the open reading frame of the base sequence in the fragment has the sequence shown in SEQ ID NO: 1 in the Sequence Listing, and is composed of 2760 base pairs encoding 920 amino acids.

The ppc of the present invention containing the above-mentioned nucleotide sequence
The DNA fragment containing the gene is not only isolated from the natural chromosomal DNA of the genus Corynebacterium of the genus Brevibacterium, but is also a commonly used DNA synthesizer, for example, 394 manufactured by Applied Biosystems.
It may be one synthesized using a DNA / RNA synthesizer.

As described above, the DNA fragment containing the ppc gene of the present invention obtained from the chromosomal DNA of Brevibacterium flavum MJ-233 has one of the nucleotide sequences unless it substantially impairs the function of PEPC. Part of the base may be replaced with another base, or may be deleted, or a new base may be inserted,
Furthermore, a part of the base sequence may be transposed, and any of these derivatives is included in the DNA fragment of the present invention.

DNA containing the ppc gene DNA of the present invention
By introducing the fragment into an appropriate plasmid, for example, a plasmid vector containing at least a gene that controls the replication / proliferation function of the plasmid in coryneform bacteria, a recombinant plasmid capable of highly expressing PEPC in coryneform bacteria can be obtained. it can. Here, in the above recombinant plasmid, the promoter for expressing the ppc gene may be a promoter possessed by a bacterium of the genus Brevibacterium, but is not limited thereto, and
Any promoter may be used as long as it is a nucleotide sequence derived from a prokaryote for initiating gene transcription.

The plasmid vector into which the ppc gene of the present invention can be introduced is not particularly limited as long as it contains at least a gene that controls the replication / proliferation function in coryneform bacteria. Specific examples thereof include, for example, the plasmid pCR described in JP-A-3-210184.
Y30: plasmids pCRY21, pCRY2KE, pCRY2KX described in JP-A-2-276575.
pCRY31, pCRY3KE and pCRY3KX; plasmid pCR described in JP-A-1-191686.
Y2 and pCRY3; pAM330 described in JP-A-58-67679; pHM1519 described in JP-A-58-77895; pAJ655, pAJ611 and pAJ1 described in JP-A-58-192900.
844; pC described in JP-A-57-134500
G1; pCG described in JP-A-58-35197
2; pCG4 described in JP-A-57-183799
And pCG11 and the like.

Among them, the plasmid vector used in the coryneform bacterium host-vector system includes a gene that controls the replication / proliferation function of the plasmid in the coryneform bacterium and a gene that controls the plasmid stabilizing function in the coryneform bacterium. Are preferred, for example plasmid pCR
Y30, pCRY21, pCRY2KE, pCRY2K
X, pCRY31, pCRY3KE and pCRY3K
X and the like are preferably used.

As a method of preparing the above-mentioned plasmid vector pCRY30, Brevibacterium stationis IFO12144 (F
ERM BP-2515) to the plasmid pBY503.
(For details of this plasmid, see Japanese Patent Laid-Open No. 1-9578
(See Japanese Patent Laid-Open No. 5) DNA is extracted, a DNA fragment (replication region) containing a gene that controls the replication / proliferation function of a plasmid having a size of about 4.0 kb is cut out with a restriction enzyme XhoI, and the size thereof is cut with restriction enzymes EcoRI and KpnI. About 2.1 kb
A DNA fragment (stabilizing region) containing a gene that controls the stabilizing function of the plasmid is cut out. Both of these DNA fragments were EcoRI-containing plasmid pHSG298 (Takara Shuzo).
The plasmid vector pCRY30 can be prepared by incorporating it into the KpnI site and the SalI site, respectively.

Pp of the invention into the plasmid pCRY30
The introduction of the DNA fragment containing the c gene is carried out using the plasmid pCR
Y30 is cleaved with a restriction enzyme XhoI, and the pp
It can be carried out by ligating a DNA fragment containing the c gene with a DNA ligase. The plasmid pCRY30 thus constructed has a size of about 3.
The recombinant plasmid into which the DNA fragment containing the 3 kb ppc gene was introduced was designated as pCRY30-ppc. Details of the method for constructing the plasmid pCRY30-ppc will be described in Examples below.

Host microorganisms that can be transformed with the recombinant plasmid of the present invention include coryneform bacteria such as Brevibacterium flavum MJ-233 (FERM BP-).
1497), Brevibacterium flavum MJ-23.
3-AB-41 (FERMBP-1498), Brevibacterium flavum MJ-233-ABT-11 (F
ERM BP-1500), Brevibacterium flavum MJ-233-ABD-21 (FERM BP-1)
499) and the like.

In addition to these microorganisms, Brevibacterium ammoniagenes
) ATCC 6871, ATCC 13745, AT
CC13746; Brevibacterium divaricatum ATCC14020;
Brevibacterum lactofermentum (Brevibac
terium lactofermentum) ATCC13869; Corynebacterium glutam
icum) ATCC31831 etc. can also be used as a host microorganism.

When a strain derived from the Brevibacterium flavum MJ-233 strain is used as a host, the plasmid pBY502 possessed by this strain (Japanese Patent Laid-Open No. 63-367) is used.
Since it may be difficult to transform because of this, it is desirable to remove the plasmid pBY502 from this strain in such a case. As a method for removing such a plasmid pBY502, for example, it is possible to spontaneously delete it by repeating subculture, or it is possible to remove it artificially [Bacteriological Review (Bact . Rev.), 36 ,
361-40 (1972)].

As a method for transforming the thus obtained plasmid into the strain derived from Brevibacterium flavum MJ-233, as known for Escherichia coli and Erwinia carotovora [Journal of Bacterio Rosie (J. Bacteriol.), 17
0 , 2796 (1988); Agric. Biol. Chem., 52 , 293 (19).
88)], and it is possible to introduce a plasmid by pulse wave energization [see Journal of Industrial Microbiology (J. Indust. Microbiol.), 5 , 159 (1990)] to DNA recipient bacteria. .

The coryneform bacterium obtained by transformation by the above-mentioned method has PEPC-producing ability. By culturing the coryneform bacterium in a commonly used medium known per se, it is possible to enhance PEPC. The containing bacterial cells can be obtained.

[0027]

The present invention has been described above, but the present invention will be described in more detail with reference to the following examples. However, it should be understood that the examples are to be considered as an aid to gain a specific appreciation of the invention and are not intended to limit the scope of the invention.

Example 1 Brevibacterium flavum
Of a DNA fragment containing the ppc gene derived from the MJ-233 strain
Extraction of total DNA of cloned (A) Brevibacterium flavum MJ-233 Semi-synthetic medium A medium 1 l [composition: urea 2 g, (NH ₄ ).
₂ SO ₄ 7 g, K ₂ HPO ₄ 0.5 g, KH ₂ PO <sub>4
0. 5g, MgSO 4 0. 5g,</sub> FeSO 4 · 7H
₂ O 6 mg, MnSO ₄ .4-6H ₂ O 6 mg, yeast extract 2.5 g, casamino acid 5 g, biotin 2
00 μg, thiamine hydrochloride 200 μg, glucose 2
Brevibacterium flavum MJ-233 (FERM BP-1497) was inoculated into 0 g of distilled water 1 l] using a platinum loop, and the cells were collected by shaking culture at 33 ° C. until the late logarithmic growth phase.

The cells obtained were adjusted to a concentration of 10 mg / ml, 10 mg / ml lysozyme, 10 mM Na.
Cl, 20 mM Tris buffer (pH 8.0) and 1 mM
15ml of solution containing each component of EDTA / 2Na
(The concentration of each component is the final concentration). Next, proteinase K was added so that the final concentration was 100 μg / ml, and the mixture was incubated at 37 ° C. for 1 hour. Further, sodium dodecyl sulfate (SDS) was added so that the final concentration was 0.5%, and the mixture was kept at 50 ° C. for 6 hours for lysis. To this lysate, an equal amount of phenol / chloroform solution was added and gently shaken at room temperature for 10 minutes, and then the whole was centrifuged (5,000 xg, 20 minutes, 10 to 12 ° C),
The supernatant fraction was collected. Sodium acetate was added to this supernatant in an amount of 0.
After adding 3 M, double volume of ethanol was slowly added. DN existing between water layer and ethanol layer
A was scattered with a glass rod, washed with 70% ethanol, and then air dried. 10mM Tris buffer (pH7.5) and 1mM EDTA / 2Na solution 5m to the obtained DNA
1 (concentration of each component is the final concentration) was added, and the mixture was allowed to stand at 4 ° C. overnight and used in the subsequent experiments.

(B) Creation of Recombinant Brevibacterium flavum MJ obtained in the above (A)
90 μl of the total DNA solution of S-233 with the restriction enzyme SalI
Using 50 U (units), the reaction was carried out at 37 ° C. for 1 hour for complete decomposition. The cloning vector pUC118 (manufactured by Takara Shuzo) was added to the SalI-degraded DNA with the restriction enzyme Sa.
cleaved with Il and then dephosphorylated, mix and mix
0 mM Tris buffer (pH 7.6), 10 mM dithiothreitol, 1 mM ATP, 10 mM MgCl ₂ ,
And each component of T4 DNA ligase 1U were added (concentration of each component is final concentration) and reacted at 4 ° C. for 15 hours,
Combined.

Using the resulting plasmid mixture, Escherichia coli JM109 (Takara Shuzo) was transformed by the calcium chloride method [J. Molecul. Biol., 53 , 159 (1970)]. It was converted and smeared on a medium containing 50 mg of ampicillin [trypton 10 g, yeast extract 5 g, NaCl 5 g and agar 16 g dissolved in distilled water 11].

A strain grown on this medium was subjected to a conventional method [Molecular cloning, Cold Spring Ha
rbor Laboratory Press (1989)]
Plasmid DNA was extracted from the culture medium, the plasmid was cleaved with the restriction enzyme SalI, and electrophoresed using 0.7% agarose gel. DNA was transferred from this agarose gel onto a nylon membrane, and Escherichia.
Southern hybridization was carried out using the common region of the ppc gene derived from E. coli and maize as a probe.
As the probe used, attention was focused on a region of high homology in the amino acid sequence deduced from the Escherichia coli and maize-derived ppc genes, and a mixed oligonucleotide probe assumed from the amino acid sequence was applied to Applied Biosystems (Applied Biosystems). Biosystems) 3
It was synthesized using a 94 type DNA / RNA synthesizer.

The base sequence of the probe actually used is the following two amino acid sequences: (1) Val Leu Thr Ala His Pro Thr Glu, (2) Ser
The following nucleotide sequences predicted from Trp Net Gly Gly Asp: (1) GTI CTI ACI GCI CAY CCI ACI GAR, (2) TUI
TGG ATG GGI GGI GAY (in the sequence, R represents A or G, Y represents C or T, where A represents adenine, G represents guanine, C represents cytosine, T represents thymine, and I represents deoxyinosine). 24 mer and 18 mer (base pair). In the synthesis of the probe, deoxyinosine was used so that the degree of mixing would not become too great.

The synthesized oligonucleotide probe was radioisotope-labeled with [γ- 32 P] ATP at the 5′-terminal phosphate group by a method using T4 polynucleotide kinase (Takara Shuzo) [Analytical Biochemistry (Anal Biochem.), 158 , 307-315 (198
6)]. Southern hybridization can be carried out by a conventional method [Molecular Cloning, Cold
Spring Harbor Laboratory Press (1989)].

As a result, it was found that only a DNA fragment having a size of about 3.3 kb of the digested product of restriction enzyme SalI hybridizes with the above probe, and ppc is contained in the DNA fragment.
It became clear that the gene was included. This plasmid carrying a DNA fragment of this size of about 3.3 kb was
It was named UC118-ppc. The size obtained is 3.
When a 3 kb DNA fragment was cleaved with various restriction enzymes,
The number of restriction enzyme recognition sites and the size of the cleaved fragment were as shown in Table 1 above. A map of restriction enzyme cleavage points of this DNA fragment is shown in FIG. Also, the plasmid pUC obtained above
118-ppc was cleaved with various restriction enzymes, and the size of the cleaved fragment was measured. The results are shown in Table 2 below.

[0036]

TABLE 2 The size of the second table restriction enzyme recognition sites number cleavage fragment (kb) EcoRI 3 0.5,1.4,4.6 HindIII 2 1.6,4.9 KpnI 1 6.5

As described above, a DNA fragment (SalI fragment) containing the ppc gene and having a size of about 3.3 kb could be obtained.

Example 2 Nucleotide sequence of ppc gene DNA
Of the DNA fragment having a size of about 3.3 kb containing the ppc gene obtained in the item (B) of Example 1, its nucleotide sequence was determined by the dideoxynucleotide enzymatic method.
nation method) [Sanger, F. et al., Proceeding of National Academy of Science
Of United States of America (Pro
Nat. Acad. Sci. USA), 74 , 5463 (1977)]. From the presence of the open reading frame in the base sequence, it was revealed that the ppc gene was composed of 2760 base pairs encoding 920 amino acids having the base sequence shown in SEQ ID NO: 1 in the sequence listing below.

Example 3 PPC residue in coryneform bacteria
Expression of gene product (A) Preparation of plasmid pBY503 The plasmid pBY503 was prepared from Brevibacterium statinis IFO12144 (FERM BP-251).
The plasmid was isolated from 5) and had a molecular weight of about 10 megadalton, and was prepared as follows based on the method described in JP-A-3-210184.

Brevibacterium statinis IFO12144 was cultured in the semi-synthetic medium A medium until the logarithmic growth phase, which was described later, to collect the cells. 10 mg / ml of the obtained bacterial cells
Buffer containing lysozyme at a concentration of [25 mM tris (hydroxymethyl) aminomethane, 10 mM EDTA,
50 mM glucose] was suspended in 20 ml and reacted at 37 ° C. for 1 hour. Alkali-SDS solution [0.2N]
40 ml of NaOH, 1% (W / V) SDS] was added, mixed gently and allowed to stand at room temperature for 15 minutes. Next, a potassium acetate solution [60 ml of 5M potassium acetate solution, 11.5 ml of acetic acid, 28.5 m of distilled water] was added to the reaction solution.
l] Add 30 ml, mix well, and then add 15 ml in ice water.
Let stand for minutes.

The total amount of the lysate was transferred to a centrifuge tube and centrifuged at 4 ° C. for 10 minutes at 15,000 × g to obtain a supernatant. After adding an equal volume of phenol / chloroform solution (phenol: chloroform = 1: 1 mixture) to the suspension, suspend it, transfer to a centrifuge tube, and 15,000 xg for 5 minutes at room temperature.
Was centrifuged and the aqueous layer was recovered. Add twice the amount of ethanol to the aqueous layer, leave at -20 ° C for 1 hour, and then at 4 ° C for 10 hours.
The precipitate was recovered by centrifugation at 15,000 × g for 1 minute.

After drying the precipitate under reduced pressure, TE buffer [Tris 1
0 mM, EDTA 1 mM; pH adjusted to 8 with HCl]
Dissolved in 2 ml. To the solution, 15 ml of a cesium chloride solution [a solution of 170 g of cesium chloride dissolved in 100 ml of a 5 times concentration of TE buffer] and 10 ml of 10 mg / ml ethidium bromide solution were added to give a density of 1.392 g / ml.
Was adapted to. This solution was treated at 12 ° C for 42 hours at 116,0
Centrifugation at 00 × g was performed.

The plasmid pBY503 is found as a lower band in a centrifuge tube by ultraviolet irradiation. This band was extracted from the side of the centrifuge tube with a syringe to obtain a fraction containing the plasmid pBY503. Next, this fraction was treated with an equal amount of isoamyl alcohol four times to extract and remove ethidium bromide, and then dialyzed against TE buffer. A 3 M sodium acetate solution was added to the final concentration of 30 mM to the dialysate containing the plasmid pBY503 thus obtained, and then double the amount of ethanol was added, and the mixture was allowed to stand at -20 ° C for 1 hour. 15, this solution
The DNA was precipitated by centrifugation at 000 × g to obtain 50 μg of plasmid pBY503.

Plasmid pHSG298 (Takara Shuzo)
The restriction enzyme SalI (5 U) was reacted with 0.5 μg at 37 ° C. for 1 hour to completely decompose the plasmid DNA. 2 μg of the plasmid pBY503 prepared above was added to the restriction enzyme Xh.
oI (1 U) was reacted at 37 ° C. for 30 minutes to partially decompose the plasmid DNA. Both plasmid DNA degradation products were mixed and heat-treated at 65 ° C. for 10 minutes to inactivate the restriction enzyme, and then the components in the inactivation solution were each brought to a final concentration of 50 mM Tris buffer pH 7.6, 10 mM M
gCl ₂ , 10 mM dithiothreitol, 1 mM AT
Each component was strengthened so that P and T4 DNA ligase was 1 U, and incubated at 16 ° C. for 15 hours. This solution was used to transform Escherichia coli JM109 competent cells (Takara Shuzo).

Transformant is 30 μg / ml (final concentration)
X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) containing L medium (tryptone 10 g, yeast extract 5 g, NaCl 5 g,
And cultured in distilled water (1 liter, pH 7.2) at 37 ° C. for 24 hours to obtain a growing strain. Of these growing strains,
Those that grew in white colonies were selected, and each plasmid was treated with the alkaline-SDS method [T. Maniatis, EF Frit.
Sch, J. Sambrook, Molecular Cloning (Mo
lecular Cloning), 90-91, (1982)]. Cut this plasmid with the restriction enzyme EcoRI,
The size of the cut fragment was measured.

As a result, S of plasmid pHSG298
A plasmid pHS in which an approximately 4 kb DNA fragment (replication region) derived from the plasmid pBY503 is inserted into the alI site.
G298-ori was obtained. Then use the same method,
The plasmid pBY503DNA was digested with the restriction enzyme KpnI.
And a DNA fragment of about 2.1 kb (stabilization region) obtained by treating with EcoRI and the above plasmid pHSG2.
The plasmid vector pCRY30 was prepared by cloning at the KpnI and EcoRI sites of 98-ori.

(B) Plasmid pCRY30-ppc
Preparation and introduction into coryneform bacterium Plasmid pUC118 obtained in section (B) of Example 1
-Ppc 5 μg with the restriction enzyme SalI5U
What was decomposed by reacting at 1 ° C for 1 hour and 1 µg of the plasmid pCRY30 obtained in the above (A) were digested with the restriction enzyme Xh.
Using 1 U of oI, the mixture was decomposed by reacting at 37 ° C. for 1 hour, mixed, and mixed with 50 mM Tris buffer (pH 7.6), 1
0 mM dithiothreitol, 1 mM ATP, 10 mM
Each component of MgCl ₂ and 1 U of T4 DNA ligase was added (concentration of each component is final concentration), and 1 at 12 ° C.
The reaction was allowed to proceed for 5 hours for binding. With this plasmid,
The Escherichia coli JM109 strain was transformed according to the above method and smeared on a medium containing 50 μg / ml of kanamycin [trypton 10 g, yeast extract 5 g, NaCl 5 g and agar 16 g dissolved in distilled water 1 l].

The strain grown on this medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture solution, the plasmid was cleaved with a restriction enzyme and examined by agarose gel electrophoresis to find that the length of plasmid pCRY30 was long. In addition to the DNA fragment of 8.7 kb, an inserted DNA fragment of 3.3 kb was observed. The plasmid DNA prepared as described above was transformed into a coryneform bacterium. Transformation was performed as follows using the electric pulse method.

Brevibacterium flavum MJ-23
3 (FERM BP-1497) plasmid pBY50
The 2 removed strains were cultivated in 100 ml of the medium A until the initial logarithmic growth, penicillin G was added at 1 U / ml, and the mixture was further cultivated with shaking for 2 hours, and the cells were collected by centrifugation to collect the cells. 20 ml pulse solution (272 mM sucrose, 7 mM KH ₂ PO ₄ , 1 mM MgCl ₂ ; pH
It was washed in 7.4). Collect the cells by centrifugation.
The cells were suspended in 5 ml of the pulse solution, 0.75 ml of the cells were mixed with 50 μl of the plasmid DNA solution obtained above, and the mixture was allowed to stand in water for 20 minutes. The whole amount was transferred to 3 ml of the A medium and cultured at 30 ° C. for 1 hour, then inoculated into the A medium containing 15 μg / ml (final concentration) of kanamycin, and cultured at 30 ° C. for 2 to 3 days. The emerged kanamycin-resistant strain was used as a buffer containing lysozyme at a concentration of 10 mg / ml [25 mM tris (hydroxymethyl)
Aminomethane, 10 mM EDTA, 50 mM glucose] was suspended in 20 ml and reacted at 37 ° C. for 1 hour. Alkali-SDS solution [0.2N NaOH, 1%
(W / V) SDS] 40 ml was added, mixed gently and allowed to stand at room temperature for 15 minutes. Next, potassium acetate solution [60 ml of 5M potassium acetate solution, 1 ml of acetic acid was added to this reaction solution.
1.5 ml, distilled water 28.5 ml] 30 ml,
After mixing well, the mixture was left to stand in ice water for 15 minutes.

The total amount of the lysate was transferred to a centrifuge tube and centrifuged at 4 ° C. for 10 minutes at 15,000 × g to obtain a supernatant. After adding an equal volume of phenol / chloroform solution (phenol: chloroform = 1: 1 mixture) to the suspension, suspend it, transfer to a centrifuge tube, and 15,000 xg for 5 minutes at room temperature.
Was centrifuged and the aqueous layer was recovered. Add twice the amount of ethanol to the aqueous layer, leave at -20 ° C for 1 hour, and then at 4 ° C for 10 hours.
After centrifugation for 1 minute at 15,000 × g, the precipitate was collected to obtain a plasmid. This plasmid was cleaved with various restriction enzymes and the size of the cleaved fragment was measured. The results are shown in Table 3 below.

[0051]

Table 3 Table 3 restriction enzyme recognition sites number cutting size of fragments (kb) EcoRI 3 0.4,3.2,8.4 KpnI 1 12.0 BamHI 3 0.2,1.2,10. 6 The plasmid characterized by the above restriction enzymes was designated as pCR
It was named Y30-ppc. This plasmid pCRY3
A map of 0-ppc restriction enzyme cleavage points is shown in FIG.

(C) With the plasmid pCRY30-ppc
Ppc gene product by transformed coryneform bacterium
Expression of (PEPC) Plasmid pCRY30-pp obtained in the above (B)
Brevibacterium flavum MJ transformed with c
-233 strain was shake-cultured in 100 ml of the A medium at 30 ° C. for 24 hours to obtain cells of the transformed strain. Bacterial cells after culture were collected by centrifugation (5,000 × g), tris buffer (tris -HCl 100mM, MgSO ₄ ·
7H ₂ O 10 mM, dithiothreitol 1 mM, p
After washing with H8.5), the cells were disrupted with an ultrasonic disruptor. The disrupted cell suspension was centrifuged at 15,000 × g for 15 minutes, and the resulting supernatant was further centrifuged at 100,000 × g for 1 hour to obtain a crude enzyme solution.

The PEPC activity of the obtained crude enzyme solution was measured. For activity measurement, coexist with malate dehydrogenase, N
By the method of spectroscopically measuring the rate of decrease of ADH [J. Bioche (J. Bioche
m.), 68 , 747-750 (1970)]. As a control, the host Brevibacterium flavum MJ-2
A crude enzyme solution was similarly prepared using 33 wild strains. The measurement result is a relative value with the activity of the control bacterium (wild strain) as 1,
The results are shown in Table 4 below.

[0054]

From Table 4 Table 4 Strain PEPC activity transformed strain 6 wild strain 1 or more, the strain transformed with the plasmid pCRY30-ppc, PEPC that are considerable amounts generated was confirmed.

[0055]

[Sequence list]

SEQ ID NO: 1 Sequence length: 2760 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Chromosomal DNA Origin organism name: Brevibacterium flavum
um flavum) Strain name: MJ-233 Sequence features Characteristic symbols: CDS Location: 1-2760 Method of determining features: E sequence ATG ACT GAT TTT TTA CGC GAT GAC ATC AGG TTC CTC GGT CGA ATC CTC 48 Met Thr Asp Phe Leu Arg Asp Asp Ile Arg Phe Leu Gly Arg Ile Leu 1 5 10 15 GGT GAG GTA ATT GCG GAA CAA GAA GGC CAG GAG GTT TAT GAA CTG GTC 96 Gly Glu Val Ile Ala Glu Gln Glu Gly Gln Glu Val Tyr Glu Leu Val 20 25 30 GAA CAA GCG CGC CTG ACT TCT TTT GAT ATC GCC AAG GGC AAC GCC GAA 144 Glu Gln Ala Arg Leu Thr Ser Phe Asp Ile Ala Lys Gly Asn Ala Glu 35 40 45 ATG GAT AGC CTG GTT CAG GTT TTC GAC GGC ATT ACT CCA GCC AAG GCA 192 Met Asp Ser Leu Val Gln Val Phe Asp Gly Ile Thr Pro Ala Lys Ala 50 55 60 ACA CCG ATT GCT CGC GCA TTT TCC CAC TTC GCT CTG CTG GCT AAC CTG 240 Thr Pro Ile Ala Arg Ala Phe Ser His Phe Ala Leu Leu Ala Asn Leu 65 70 75 80 GCG GAA GAC CTC CAC GAT GAA GAG CTT CGT GAA CAG GCT CTC GAT GCA 288 Ala Glu Asp Leu His Asp Glu Glu Leu Arg Glu Gln Ala Leu As p Ala 85 90 95 GGC GAC ACC CCT CCG GAC AGC ACT CTT GAT GCC ACC TGG CTG AAA CTC 336 Gly Asp Thr Pro Pro Asp Ser Thr Leu Asp Ala Thr Trp Leu Lys Leu 100 105 110 AAT GAG GGC AAT GTT GGC GCA GAA GCT GTG GCG GAT GTG TTG CGT AAT 384 Asn Glu Gly Asn Val Gly Ala Glu Ala Val Ala Asp Val Leu Arg Asn 115 120 125 GCT GAG GTG GCG CCA GTT CTG ACT GCG CAC CCA ACT GAG ACT CGC CGC 432 Ala Glu Val Ala Pro Val Leu Thr Ala His Pro Thr Glu Thr Arg Arg 130 135 140 CGC ACT GTT TTT GAT GCG CAA AAG TGG ATC ACC ACC CAC ATG CGT GAA 480 Arg Thr Val Phe Asp Ala Gln Lys Trp Ile Thr Thr His Met Arg Glu 145 150 155 160 CGC CAC GCT TTG CAG TCT GCG GAG CCA ACC GCT CGT ACG CAA AGC AAG 528 Arg His Ala Leu Gln Ser Ala Glu Pro Thr Ala Arg Thr Gln Ser Lys 165 170 175 TTG GAT GAG ATC GAA AAG AAC ATC CGC CGT CGC ATC ACC ATT TTG TGG 576 Leu Asp Glu Ile Glu Lys Asn Ile Arg Arg Arg Ile Thr Ile Leu Trp 180 185 190 CAG ACC GCG TTG ATT CGT GTG GCC CGC CCA CGT ATC GAG GAC GAG ATC 624 Gln Thr Ala Leu Ile Arg Val Ala Arg Pro Arg Ile G lu Asp Glu Ile 195 200 205 GAA GTA GGG CTG CGC TAC TAC AAG CTG AGC CTT TTG GAA GAG ATT CCA 672 Glu Val Gly Leu Arg Tyr Tyr Lys Leu Ser Leu Leu Glu Glu Ile Pro 210 215 220 CGT ATC AAC CGT GAT GTG GCT GTT GAG CTT CGT GAG CGT TTC GGC GAG 720 Arg Ile Asn Arg Asp Val Ala Val Glu Leu Arg Glu Arg Phe Gly Glu 225 230 235 240 GAT GTT CCT TTG AAG CCC GTG GTC AAG CCA GGT TCC TGG ATT GGT GGA 768 Asp Val Pro Leu Lys Pro Val Val Lys Pro Gly Ser Trp Ile Gly Gly 245 250 255 GAC CAC GAC GGT AAC CCT TAT GTC ACC GCG GAA ACA GTT GAG TAT TCC 816 Asp His Asp Gly Asn Pro Tyr Val Thr Ala Glu Thr Val Glu Tyr Ser 260 265 270 ACT CCA CGC GCT GCG GAA ACC GTG CTC AAG TAC TAT GCA CGC CAG CTG 864 Thr Pro Arg Ala Ala Glu Thr Val Leu Lys Tyr Tyr Ala Arg Gln Leu 275 280 285 CAT TCC CTC GAG CAT GAG CTC AGC CTG TCG GAC CGC ATG AAT AAG GTC 912 His Ser Leu Glu His Glu Leu Ser Leu Ser Asp Arg Met Asn Lys Val 290 295 300 ACC CCG CAG CTG CTT GCG CTG GCA GAT GCC GGG CAC AAC GAC GTG CCA 960 Thr Pro Gln Leu Leu Ala Leu Ala Asp A la Gly His Asn Asp Val Pro 305 310 315 320 AGC CGC GTG GAT GAG CCT TAT CGA CGC GCC GTC CAT GGC GTT CGC GGA 1008 Ser Arg Val Asp Glu Pro Tyr Arg Arg Ala Val His Gly Val Arg Gly 325 330 335 CGT ATC CTC GCG ACG ACG GCT GAG CTG ATC GGC GAG GAC GCC GTT GAG 1056 Arg Ile Leu Ala Thr Thr Ala Glu Leu Ile Gly Glu Asp Ala Val Glu 340 345 350 GGC GTG TGG TTC AAG GTC TTT ACT CCA TAC GCA TCC CCG GAA GAA TTC 1104 Gly Val Trp Phe Lys Val Phe Thr Pro Tyr Ala Ser Pro Glu Glu Phe 355 360 365 TTA AAC GAT GCG TTA ACC ATC GAT CAT TCT CTG CGT GAA TCC AAT GAC 1152 Leu Asn Asp Ala Leu Thr Ile Asp His Ser Leu Arg Glu Ser Asn Asp 370 375 380 ACT CTC ATC GCC GAT GAT CGT TTG TCT GTG CTG ATT TCT GCC ATC GAG 1200 Thr Leu Ile Ala Asp Asp Arg Leu Ser Val Leu Ile Ser Ala Ile Glu 385 390 395 400 AGC TTC GGA TTC AAC CTC TAC TCA CTG GAT CTG CGC CAG AAC TCT GAG 1248 Ser Phe Gly Phe Asn Leu Tyr Ser Leu Asp Leu Arg Gln Asn Ser Glu 405 410 415 AGC TAC GAA GAC GTA CTC ACA GAG CTT TTC GAG CGT GCC CAA GTC ACC 1296 Ser Tyr Glu As p Val Leu Thr Glu Leu Phe Glu Arg Ala Gln Val Thr 420 425 430 GCA AAC TAC CGC GAG CTG TCT GAA GAG GAG AAG CTT GAG GTG CTG CTG 1344 Ala Asn Tyr Arg Glu Leu Ser Glu Glu Glu Lys Leu Glu Val Leu Leu 435 440 445 AAG GAA CTG CGC AGC CCT CGT CCG TTG ATC CCG CAC GGT TCA GAT GAA 1392 Lys Glu Leu Arg Ser Pro Arg Pro Leu Ile Pro His Gly Ser Asp Glu 450 455 460 TAC AGC GAG GTC ACC GAC CGC GAG CTC GGT ATC TTC CGC ACC GCG TCG 1440 Tyr Ser Glu Val Thr Asp Arg Glu Leu Gly Ile Phe Arg Thr Ala Ser 465 470 475 480 GAA GCT GTC AAG AAA TTC GGC CCA CGC ATG GTG CCT CAC TGC ATC ATT 1488 Glu Ala Val Lys Lys Phe Gly Pro Arg Met Val Pro His Cys Ile Ile 485 490 495 TCC ATG ACA TCA TCG GTC ACC GAT GTG CTC GAG CCG ATG GTG TTG CTC 1536 Ser Met Thr Ser Ser Val Thr Asp Val Leu Glu Pro Met Val Leu Leu 500 505 510 AAA GAA TTC GGC CTC ATC GCG GCC AAC GGC GAC AAT CCA CGC GGC ACC 1584 Lys Glu Phe Gly Leu Ile Ala Ala Asn Gly Asp Asn Pro Arg Gly Thr 515 520 525 GTC GAT GTC ATC CCA CTG TTC GAA ACC ATC GAA GAC CTT CGA GCC GGC 1632 Val Asp Val Ile Pro Leu Phe Glu Thr Ile Glu Asp Leu Arg Ala Gly 530 535 540 GCC GGA ATC CTC GGC GAA CTG TGG AAA ATT GAT CTC TAC CGC AAC TAC 1680 Ala Gly Ile Leu Gly Glu Leu Trp Lys Ile Asp Leu Tyr Arg Asn Tyr 545 550 555 560 CTC CTG CAG CGC GAC AAC GTC CAG GAA GTC ATG CTC GGC TAC TCC GAT 1728 Leu Leu Gln Arg Asp Asn Val Gln Glu Val Met Leu Gly Tyr Ser Asp 565 570 575 TCC AAC AAG GAT GGC GGA TAT TTC TCC GCA AAC TGG GCG CTT TAC GAC 1776 Ser Asn Lys Asp Gly Gly Tyr Phe Ser Ala Asn Trp Ala Leu Tyr Asp 580 585 590 GCG GAA CTG CAG CTT GTC GAA CTA TGC CGA TCA GCC GGG GTC AAC GTT 1824 Ala Glu Leu Gln Leu Val Glu Leu Cys Arg Ser Ala Gly Val Asn Val 595 600 605 CGC CTG TTC CAC GGC CGC GGT GGC ACC GTT GGA CGT GGT GGC GGA CCT 1872 Arg Leu Phe His Gly Arg Gly Gly Thr Val Gly Arg Gly Gly Gly Pro 610 615 620 TCC TAC GAT GCG ATT CTT GCC CAG CCC AAG GGG GCT GTC CAA GGT TCC 1920 Ser Tyr Asp Ala Ile Leu Ala Gln Pro Lys Gly Ala Val Gln Gly Ser 625 630 635 640 GTG CGC ATC ACC GAG CAG GGC GAA ATC ATC T CA GCT AAG TAC GGC AAC 1968 Val Arg Ile Thr Glu Gln Gly Glu Ile Ile Ser Ala Lys Tyr Gly Asn 645 650 655 CCT GAA ACT GCG CGC CGA AAC CTC GAG GCA CTG GTC TCA GCC ACG CTT 2016 Pro Glu Thr Ala Arg Arg Asn Leu Glu Ala Leu Val Ser Ala Thr Leu 660 665 670 GAG GCA TCG CTT CTC GAC GTC TCT GAA CTC ACC GAT CAC CAA CGC GCG 2064 Glu Ala Ser Leu Leu Asp Val Ser Glu Leu Thr Asp His Gln Arg Ala 675 680 685 TAT GAC ATC ATG AGT GAG ATC TCT GAG CTC AGC CTG AAG AAG TAC ACC 2112 Tyr Asp Ile Met Ser Glu Ile Ser Glu Leu Ser Leu Lys Lys Tyr Thr 690 695 700 TCC TTG GTG CAC GAG GAT CAA GGC TTC ATC GAT TAC TTC ACC CAA TCC 2160 Ser Leu Val His Glu Asp Gln Gly Phe Ile Asp Tyr Phe Thr Gln Ser 705 710 715 720 720 ACA ACA CTG CAG GAG ATC GGA TCC CTC AAC ATC GGA TCC AGG CCT TCC 2208 Thr Thr Leu Gln Glu Ile Gly Ser Leu Asn Ile Gly Ser Arg Pro Ser 725 730 735 TCA CGC AAG CAA ACT TCC TCT GTG GAA GAT TTG CGA GCC ATC CCA TGG 2256 Ser Arg Lys Gln Thr Ser Ser Val Glu Asp Leu Arg Ala Ile Pro Trp 740 745 750 GTT CTT AGC TGG TCA CA G TCT CGT GTG ATG CTG CCA GGA TGG TTT GGT 2304 Val Leu Ser Trp Ser Gln Ser Arg Val Met Leu Pro Gly Trp Phe Gly 755 760 765 GTC GGA ACG GCA CTT GAA CAG TGG ATT GGA GAA GGG GAG CAA GCT ACC 2352 Val Gly Thr Ala Leu Glu Gln Trp Ile Gly Glu Gly Glu Gln Ala Thr 770 775 780 CAG CGC ATC GCC GAG CTG CAA ACA CTC AAC GAG TCC TGG CCA TTT TTC 2400 Gln Arg Ile Ala Glu Leu Gln Thr Leu Asn Glu Ser Trp Pro Phe Phe 785 790 795 800 ACC TCA GTG TTG GAC AAC ATG GCT CAG GTG ATG TCC AAG GCA GAG CTG 2448 Thr Ser Val Leu Asp Asn Met Ala Gln Val Met Ser Lys Ala Glu Leu 805 810 815 CGT TTG GCA AAG CTC TAT GCA GAC CTC ATC CCA GAT AGG GAA GTC GCC 2496 Arg Leu Ala Lys Leu Tyr Ala Asp Leu Ile Pro Asp Arg Glu Val Ala 820 825 830 GAG CGC GTC TAT TCC GTC ATC CAC GAG GAA TAT TTC CTG ACT AAA AAG 2544 Glu Arg Val Tyr Ser Val Ile His Glu Glu Tyr Phe Leu Thr Lys Lys 835 840 845 ATG TTC TGC GTG ATC ACC GGC TCC GAT GAT CTC CTT GAT GAC AAC CCA 2592 Met Phe Cys Val Ile Thr Gly Ser Asp Asp Leu Leu Asp Asp Asn Pro 850 855 860 CTT CTG GCA CGC TCT GTC CAG CGT CGT TAC CCC TAC CTG CTT CCA CTC 2640 Leu Leu Ala Arg Ser Val Gln Arg Arg Tyr Pro Tyr Leu Leu Pro Leu 865 870 875 880 AAT GTG ATC CAG GTA GAG ATG ATG CGA CGC TAC CGA AAA GGC GAC CAA 2688 Asn Val Ile Gln Val Glu Met Met Arg Arg Tyr Arg Lys Gly Asp Gln 885 890 895 AGC GAG CAA GTG TCC CGC AAT ATT CAG CTG ACA ATG AAC GGT CTT TCC 2736 Ser Glu Gln Val Ser Arg Asn Ile Gln Leu Thru Met Asn Gly Leu Ser 900 905 910 ACT GCA CTG CGC AAC TCC GGC TAG 2760 Thr Ala Leu Arg Asn Ser Gly *** 915 920

[0056]

INDUSTRIAL APPLICABILITY According to the present invention, a DNA fragment containing a gene (ppc gene) encoding a phosphoenolpyruvate carboxylase involved in production of a useful substance derived from Brevibacterium flavum MJ-233 and carbon dioxide fixation. Will be provided.

The transformant in which the DNA fragment of the present invention is incorporated is phosphoenolpyruvate carboxylase (P
EPC) activity is improved, and it is expected that the efficiency of production of useful substances such as amino acids will be improved by using the bacterium or PEPC prepared from the bacterium.

[0058]

[Brief description of drawings]

[0059]

FIG. 1 is a restriction enzyme cleavage point map of a DNA fragment containing the ppc gene having a size of about 3.3 kb according to the present invention.

[0060]

FIG. 2 is a restriction enzyme map of the plasmid pCRY30-ppc of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12R 1:13) (C12N 9/88 C12R 1:13) C12R 1:13) (72) Inventor Makoto Kobayashi 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Mitsubishi Petrochemical Co., Ltd. Tsukuba Research Institute (72) Inventor Hideaki Yukawa 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Mitsubishi Petrochemical Co., Ltd. Inside the research institute

Claims (4)

[Claims] 1. Brevibacterium flavum
acterium flavum) A DNA fragment containing a gene encoding phosphoenolpyruvate carboxylase derived from MJ-233. 2. The size is about 3.3 kb, has SalI sites at both ends, and when it is cleaved with the restriction enzymes shown in the table below, the number of recognition sites and the size of the cleaved fragment shown in the table below. The DNA fragment according to claim 1, wherein Restriction enzyme number of recognition sites Size of cleavage fragment (kb) AatII 1 1.0, 2.3 AccI 1 0.5, 2.8 ClaI 2 0.9, 1.0, 1.4 NcoI 2 0.7, 1.2, 1.4 3. A DNA fragment containing a gene encoding phosphoenolpyruvate carboxylase represented by the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing. 4. A DNA fragment containing a gene encoding phosphoenolpyruvate carboxylase represented by the nucleotide sequence set forth in SEQ ID NO: 1 in Sequence Listing.