JPH07227287A - Gene dna coding for s-adenosylmethionine synthetase - Google Patents

Abstract

PURPOSE:To obtain a new DNA containing a gene coding for an S- adenosylmethionine synthetase derived from a corynebacterium, capable of efficiently producing adenosylmethionine synthetase by transforming the same kind of a coryneform bacterium and then culturing. CONSTITUTION:A coryneform bacterium (e.g. Brevibacterium flavum MJ-233 strain) is inoculated into a medium and cultured up to the latter period of a logarithmic growth phase. A cell is collected and successively treated with a lysozyme and proteinase and subjected to bacteriolysis. The bacteriolytic solution is mixed with a solution of phenol/chloroform and the whole amount is centrifuged to collect a fraction of a supernatant liquid. The fraction is mixed with ethanol, a formed precipitate is separated to obtain the whole DNA. Then the DNA is treated with a restriction enzyme, linked to a vector and inserted into Escherichia coli. The bacterium is cultured and the culture mixture is screened with a labeled probe comprising part of a gene of S-adenosylmethionine synthetase to select a positive clone. Its DNA is recovered, treated with a restriction enzyme to give a DNA fragment containing a gene coding for the objective enzzme derived from the coryneform bacterium.

Description

Detailed Description of the Invention

[0001]

The present invention relates to S derived from coryneform bacteria.
Adenosylmethionine synthetase (EC.2.
5.1.6) gene DNA.

[0002]

BACKGROUND OF THE INVENTION S-adenosylmethionine synthetase (EC 2.5.1.6) is an enzyme that synthesizes S-adenosylmethionine from adenosyltriphosphate and L-methionine. ( Escherichia col
i) well studied [Journal of
Biological Chemistry (J. Biol. Chem.), 255,
9082-9092, 1980], and a gene encoding S-adenosylmethionine synthetase is a gene derived from Escherichia coli [J. Biol. Chem., 259, 14505-14507, 19].
84], and Saccharomyces cerevisiae (Saccharomyc
es cerevisiae) -derived gene [J. Biol. Chem., 262, 167
04-16709, 1987], gene derived from rat kidney [J. Biol. C
hem., 265, 13683-13686, 1990], rat liver-derived gene [European Journal of Biochemistry (Eur. J. Biochem.), 184, 497-501, 1989], human liver-derived gene [biochemistry]・ International (Biochem. Int.), 25, 81-90, 1991] and other primary structures have been determined. However, there is no conventional report on the S-adenosylmethionine synthetase (EC 2.5.1.6) gene derived from coryneform bacterium, which is an industrially important bacterium.

[0003]

The object of the present invention is to isolate a gene encoding S-adenosylmethionine synthetase (EC 2.5.1.6) derived from coryneform bacterium, Is introduced into a coryneform bacterium of the same species, and S-adenosylmethionine is efficiently produced from a new viewpoint using the coryneform bacterium.

[0004]

As a result of intensive studies to solve the above problems, the present inventors succeeded in isolating the S-adenosylmethionine synthetase gene from the coryneform bacterial chromosome. The present invention has been completed.

Thus, the present invention provides a DNA fragment containing a gene encoding S-adenosylmethionine synthetase derived from coryneform bacteria.

The present invention will be described in more detail below. The "gene encoding S-adenosylmethionine synthetase" of the present invention means adenosyl triphosphate and L-.
Gene DNA encoding an enzyme that synthesizes S-adenosylmethionine from methionine, that is, S-adenosylmethionine synthetase (EC 2.5.1.6)
Means

The DNA fragment containing the gene encoding S-adenosylmethionine synthetase of the present invention (hereinafter, may be abbreviated as "A fragment") is synthesized after its nucleotide sequence is determined. However, coryneform bacteria are usually cloned from a microorganism and used as a source of the microorganism.

An example of the basic procedure for preparing A fragments from these feed microorganisms is as follows: A
The fragment is a coryneform bacterium such as Brevibacterium flavum strain MJ-233 (FE
It exists on the chromosome of RM BP-1497) and can be isolated and obtained from this chromosome by the method described below.

First, Brevibacterium flavum MJ
Chromosomal DNA is extracted from a culture of strain -233. The chromosomal DNA is completely digested with a suitable restriction enzyme, eg SalI.

The obtained DNA fragment is inserted into a cloning vector such as pUC118 (Takara Shuzo), and Escherichia coli JM109 (Takara Shuzo) is transformed with this vector to obtain a transformant.

From the obtained transformant, plasmid DNA is obtained.
Of the S. adenosylmethionine synthetase gene from Escherichia coli, Saccharomyces cerevisiae, rat kidney and rat liver as a probe, and the inserted Brevibacterium flavum MJ −
The A fragment derived from the 233 strain chromosome can be confirmed and obtained.

One of the A fragments thus obtained is the Brevibacterium flavum MJ-233.
The size of about 5.5 kb obtained by digesting the chromosomal DNA of the strain by complete digestion with the restriction enzyme SalI
DNA fragments can be mentioned. The number of recognition sites and the size of the cleaved fragment when the DNA fragment containing the approximately 5.5 kb S-adenosylmethionine synthetase gene DNA was cleaved with various restriction enzymes are shown in Table 1 below.

[0013]

[Table 1]

In the present specification, "the number of recognition sites" by a restriction enzyme means that a DNA fragment or a plasmid is completely digested in the presence of a restriction enzyme, and the degradation product thereof is subjected to 1% agarose gel according to a method known per se. Electrophoresis and 5
% Polyacrylamide gel electrophoresis, and the value determined from the number of separable fragments was adopted.

The "size of the cleaved fragment" and the size of the plasmid are the same as those of Escherichia coli lambda phage (λphage) when agarose gel electrophoresis is used.
Based on the standard line drawn by the migration distance on the same agarose gel of the DNA fragment of known molecular weight obtained by cleaving the DNA of E. coli with the restriction enzyme HindIII, and when using polyacrylamide gel electrophoresis, Escherichia coli Phi-X174 Phage (φx174 Phag
Based on the standard line drawn by the migration distance on the same polyacrylamide gel of the DNA fragment of known molecular weight obtained by cleaving the DNA of e) with the restriction enzyme HaeIII, the size of the cleaved DNA fragment or each DNA fragment of the plasmid is determined. calculate. The size of the plasmid is determined by adding the sizes of the cleaved fragments. In addition, in determining the size of each DNA fragment, the size of a fragment of 1 kb or more is 1%.
Using the results obtained by agarose gel electrophoresis, the size of a fragment of about 0.1 kb to 1 kb was 5%.
The results obtained by polyacrylamide gel electrophoresis were adopted.

On the other hand, the size obtained by digesting the chromosomal DNA of the Brevibacterium flavum MJ-233 strain with the restriction enzyme SalI is about 5.5 kb.
For the DNA fragment of p.
The dideoxynucleotide enzyme method [dideoxy chain terminat] using UC118 or pUC119 (Takara Shuzo)
ion method, Sanger, F. et al., Proceedings of National Academy of Science of
United States of America (Proc. Natl.
Acad. Sci. USA), 74, 5463, 1977)]]. The above-mentioned about 5.5 kb determined in this way
The S-adenosylmethionine synthetase gene DNA determined from the presence of the open reading frame of the nucleotide sequence of the DNA fragment of E. coli has the sequence shown in SEQ ID NO: 1 in the late sequence listing and has 412 amino acids (and a stop codon). 1239 base pairs of DNA encoding
Composed of.

The A fragment containing the above-mentioned nucleotide sequence is not limited to that isolated from natural coryneform bacterial chromosomal DNA, but is also a commonly used DNA synthesizer, for example, Applied Biosystems 394.
It may be one synthesized using a DNA / RNA synthesizer.

Further, as described above, S obtained from the chromosomal DNA of Brevibacterium flavum MJ-233 strain
-The gene DNA of the present invention encoding adenosylmethionine synthetase is such that a part of the base sequence is replaced with another base as long as the function of the S-adenosylmethionine synthetase gene product is not substantially impaired. May be added or deleted, or a new base may be inserted, and a part of the base sequence may be rearranged, and any of these derivatives may be used. , Which is included in the gene DNA of the present invention.

[0019]

The present invention has been described above, but the present invention will be described in more detail with reference to the following examples. Example 1 S derived from Brevibacterium flavum MJ-233 strain
Cloning of DNA fragment (A fragment) containing adenosylmethionine synthetase gene DNA (A) Extraction of total DNA of Brevibacterium flavum MJ-233 strain Semisynthetic medium A medium [composition: urea 2 g, (NH ₄ ) ₂ SO ₄
7 g, K ₂ HPO ₄ 0.5 g, KH ₂ PO ₄ 0.5 g,
MgSO ₄ 0.5 g, FeSO ₄ .7H ₂ O ₆ mg, Mn
SO ₄ .4-6H ₂ O 6 mg, yeast extract 2.5 g, casamino acid 5 g, biotin 200 μg, thiamine hydrochloride 20
0 μg, glucose 20 g, distilled water 1 L] 1 L, and Brevibacterium flavum MJ-233 strain (FERM
BP-1497) was cultured until the late logarithmic growth phase, and the bacterial cells were collected. The obtained bacterial cells were treated with 10 mM NaCl-20 mM Tris buffer (pH 8.
It was suspended in 15 ml of 0) -1 mM EDTA.2Na solution. 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 was added so that the final concentration was 0.5%, and the mixture was kept at 50 ° C. for 6 hours for lysis. After adding an equal volume of phenol / chloroform solution to this lysate and gently shaking at room temperature for 10 minutes, the whole volume was centrifuged (5,000 xg, 20 minutes, 10 to 12 ° C), and the supernatant was collected. The fraction was collected, sodium acetate was added to 0.3 M, and then twice the amount of ethanol was slowly added.
The DNA existing between the aqueous layer and the ethanol layer was scattered with a glass rod, washed with 70% ethanol, and then air-dried.
10 mM Tris buffer (pH 7.5) -1 was added to the obtained DNA.
5 ml of mMEDTA · 2Na solution was added and the mixture was allowed to stand at 4 ° C. overnight and used for the subsequent experiments.

(B) Creation of Recombinant Brevibacterium flavum MJ obtained in the above (A)
-90 μl of total DNA solution of 233 strain was added with restriction enzyme Sal
Using I 50 units, the reaction was carried out at 37 ° C. for 1 hour for complete digestion. This SalI digested DNA was cloned into the cloning vector pUC118 (commercially available from Takara Shuzo) with the restriction enzyme Sa.
cleave with lI, mix with dephosphorylated one,
50 mM Tris buffer (pH 7.6), 10 mM dithiothreitol, 1 mM ATP, 10 mM MgCl ₂ and T ₄ DN
Each component of 1 unit of A ligase was added (the concentration of each component is the final concentration), and the mixture was reacted at 4 ° C. for 15 hours for binding.

Using the plasmid mixture obtained above, Escherichia coli JM109 (Takara Shuzo) was transformed by the calcium chloride method (J. Mol. Biol., 53, 159, 1970), and a medium containing 50 mg of ampicillin was used. Tryptone 10
g, yeast extract 5 g, NaCl 5 g and agar 16 g were dissolved in 1 L of distilled water].

The strain grown on this medium was liquid-cultured by a conventional method, plasmid DNA was extracted from the culture, the plasmid was cleaved with a restriction enzyme, and electrophoresed on an agarose gel. DNA was transferred from this agarose gel onto a nylon membrane, and Southern hybridization was carried out using the common region of each S-adenosylmethionine synthetase gene derived from Escherichia coli, Saccharomyces cerevisiae, rat kidney and rat liver as a probe. The probe used was an amino acid sequence deduced from each S-adenosylmethionine synthetase gene derived from Escherichia coli, Saccharomyces cerevisiae, rat kidney and rat liver. More assumed mixed oligonucleotide probes were synthesized using Applied Biosystems' 394 DNA / RNA synthesizer.

The nucleotide sequence of the probe actually used is the following amino acid sequence: Gly Ala Gly Asp Gln Gly and Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr The following nucleotide sequence is assumed: GGY GCI GGY GAY CAG GG and GGY TTI ACY GGY CGY AAG ATY ATY GTI GAY AC (A is adenine, G is guanine, C is cytosine, T is thymine, I is deoxyinosine, and Y is C
Or T. ) 17mer and 32mer.
In the synthesis of the probe, deoxyinosine was used so that the degree of mixing of the nucleotide sequences would not be too great.

The synthesized oligonucleotide probe was radioisotopically 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, 1986
]. Southern hybridization can be carried out by a conventional method ["Molecular Cloning", Cold
Spring Harbor Laboratory Press (1989)]. As a result, a clone producing a positive band could be selected, and in addition to the DNA fragment of plasmid pUC118 having a length of 3.2 kb, an insert fragment having a length of about 5.5 kb was recognized. The number of restriction enzyme recognition sites and the size of the cleaved fragment of a DNA fragment having a length of about 5.5 kb when cleaved with various restriction enzymes were as shown in Table 1 above. A map of restriction enzyme cleavage points of this DNA fragment is shown in FIG. The size of the cleaved fragment was measured by cleaving the plasmid obtained above with various restriction enzymes. The results are shown in Table 2.

[0025]

[Table 2]

The plasmid characterized by the above restriction enzymes was named pUC118-SAL21.

Example 2 D containing the S-adenosylmethionine synthetase gene
Determination of the nucleotide sequence of NA fragment (A fragment) The DN obtained in item (B) of Example 1 was about 5.5 kb in length.
In order to identify the site of the S-adenosylmethionine synthetase gene present on the A fragment, its nucleotide sequence was determined by the dideoxynucleotide enzyme method (Sange) using plasmid pUC118 or pUC119 (Takara Shuzo).
r, F. et al, supra). Determined 5.
There is an open reading frame composed of 1239 base pairs of DNA encoding a protein of 412 amino acid residues having the base sequence shown in SEQ ID NO: 1 in the Sequence Listing, in the base sequence of the 5 kb DNA fragment.
The amino acid sequence of the protein encoded by the open reading frame has high homology with the amino acid sequence deduced from each S-adenosylmethionine synthetase gene derived from Escherichia coli, Saccharomyces cerevisiae, rat kidney and rat liver. The sequence possessed was confirmed.

As a result, the adenosylmethionine synthetase gene derived from coryneform bacterium is composed of 1239 base pair DNA encoding a protein of 412 amino acid residues having the base sequence shown in SEQ ID NO: 1 in the Sequence Listing below. And a DN of about 5.5 kb in length was obtained.
It was found that the full-length gene encoding the S-adenosylmethionine synthetase enzyme was present on A.

[0029]

According to the present invention, S-derived from coryneform bacteria
Adenosylmethionine synthetase (EC 2.5.
A novel gene encoding 1.6) is provided. By using this transformant in which the S-adenosylmethionine synthetase gene has been integrated, S-
It is expected to be possible to produce adenosylmethionine synthetase.

[0030]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1239 Sequence type: Nucleic acid Topology: Linear Sequence type: Genomic DNA Origin organism name: Brevibacterium flavum Strain name: MJ-233 Sequence features Characteristic symbol: CDS present Location: 1-1239 Method of characterizing: E sequence GTG GCT CAG CCA ACC GCC GTC CGT TTG TTC ACC AGT GAA TCT GTA ACT 48 Val Ala Gln Pro Thr Ala Val Arg Leu Phe Thr Ser Glu Ser Val Thr 1 5 10 15 GAG GGA CAT CCA GAC AAA ATA TGT GAT GCT ATT TCC GAT ACC ATT TTG 96 Glu Gly His Pro Asp Lys Ile Cys Asp Ala Ile Ser Asp Thr Ile Leu 20 25 30 GAC GCG CTG CTC GAA AAA GAT CCG CAG TCG CGC GTC GCA GTG GAA ACT 144 Asp Ala Leu Leu Glu Lys Asp Pro Gln Ser Arg Val Ala Val Glu Thr 35 40 45 GTG GTC ACC ACC GGA ATC GTC CAT GTT GTT GGC GAG GTC CGT ACC AGC 192 Val Val Thr Thr Gly Ile Val His Val Val Gly Glu Val Arg Thr Ser 50 55 60 GCT TAC GTA GAG ATC CCT CAA TTA GTC CGC AAC AAG CTC ATC GAA ATC 240 Ala Tyr Val Glu Ile Pro Gln Leu Val Arg Asn Lys Leu Ile Glu Ile 65 70 75 80 GGA TTC AAC TCC TCT GAG GTT GGA TTC GAC GGA CGC ACC TGT GGC GTC 288 Gly Phe Asn Ser Ser Glu Val Gly Phe Asp Gly Arg Thr Cys Gly Val 85 90 95 TCA GTA TCT ATC GGT GAG CAG TCC CAG GAA ATC GCT GAC GGC GTG GAT 336 Ser Val Ser Ile Gly Glu Gln Ser Gln Glu Ile Ala Asp Gly Val Asp 100 105 110 AAC TCC GAC GAA GCC CGC ACC AAC GGC GAC GTT GAA GAA GAC GAC CGC 384 Asn Ser Asp Glu Ala Arg Thr Asn Gly Asp Val Glu Glu Asp Asp Arg 115 120 125 GCA GGT GCT GGC GAC CAG GGC CTG ATT GTT TCG GCT ACG CCA CCA ACG 432 Ala Gly Ala Gly Asp Gln Gly Leu Ile Val Ser Ala Thr Pro Pro Thr 130 135 140 AAA CCG AAG AGT ACA TGC CTC TTC CTA TCG CGT TTG GCG CAC CGA CTG 480 Lys Pro Lys Ser Thr Cys Leu Phe Leu Ser Arg Leu Ala His Arg Leu 145 150 155 160 TCA CGT CGT CTG ACC CAG GTT CGT AAA GAG GGT ATC GTT CCT CAC CTG 528 Ser Arg Arg Leu Thr Gln Val Arg Lys Glu Gly Ile Val Pro His Leu 165 170 175 CGT CCA GAC GGA AAA ACC CAG GTC ACC TTC GCA TAC GAT GCG CAA GAC 576 Arg Pro Asp Gly Lys Thr Gln Val Thr Phe Ala Tyr As p Ala Gln Asp 180 185 190 CGC CCT AGT CAC CTA GAT ACC GTT GTC ATC TCC ACC CAG CAC GAC CCA 624 Arg Pro Ser His Leu Asp Thr Val Val Ile Ser Thr Gln His Asp Pro 195 200 205 GAA GTT GAC CGT GCA TGG TTG GAA ACC CAG CTG CGC GAA CAC GTC ATT 672 Glu Val Asp Arg Ala Trp Leu Glu Thr Gln Leu Arg Glu His Val Ile 210 215 220 GAT TGG GTA ATC AAA GAC GCA GGC ATT GAG GAT CTG GCA ACC GGT GAG 720 Asp Trp Val Ile Lys Asp Ala Gly Ile Glu Asp Leu Ala Thr Gly Glu 225 230 235 240 ATC ACC GTG TTG ATC AAC CCT TCA GGT TCC TTC ATT CTG GGT GGC CCC 768 Ile Thr Val Leu Ile Asn Pro Ser Gly Ser Phe Ile Leu Gly Gly Pro 245 250 255 ATG GGT GAT GCG GGT CTG ACC GGC CGC AAG ATC ATC GTG GAT ACC TAC 816 Met Gly Asp Ala Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr 260 265 270 GGT GGC ATG GCT CGC CAT GGT GGT GGA GCA TTC TCC GGT AAG GAT CCA 864 Gly Gly Met Ala Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro 275 280 285 AGC AAG GTG GAC CGC TCT GCT GCA TAC GCC ATG CGT TGG GTA GCA AAG 912 Ser Lys Val Asp Arg Ser Ala Ala Tla Al a Met Arg Trp Val Ala Lys 290 295 300 AAC ATC GTG GCA GCA GGC CTT GCT GAT CGC GCT GAA GTT CAG GTT GCA 960 Asn Ile Val Ala Ala Gly Leu Ala Asp Arg Ala Glu Val Gln Val Ala 305 310 315 320 TAC GCC ATT GGA CGC GCA AAG CCA GTC GGA CTT TAC GTT GAA ACC TTT 1008 Tyr Ala Ile Gly Arg Ala Lys Pro Val Gly Leu Tyr Val Glu Thr Phe 325 330 335 GAC ACC AAC AAG GAA GGC CTG AGC GAC GAG CAG ATT CAG GCT GCC GTG 1056 Asp Thr Asn Lys Glu Gly Leu Ser Asp Glu Gln Ile Gln Ala Ala Val 340 345 350 TTG GAG GTC TTT GAC CTG CGT CCA GCA GCA ATT ATC CGT GAG CTT GAT 1104 Leu Glu Val Phe Asp Leu Arg Pro Ala Ala Ile Ile Arg Glu Leu Asp 355 360 365 CTG CTT CGT CCG ATC TAC GCT GAC ACT GCT GCC TAC GGC CAC TTT GGT 1152 Leu Leu Arg Pro Ile Tyr Ala Asp Thr Ala Ala Tyr Gly His Phe Gly 370 375 380 CGC ACT GAT TTG GAC CTT CCT TGG GAG GCT ATC GAC CGC GTT GAT GAA 1200 Arg Thr Asp Leu Asp Leu Pro Trp Glu Ala Ile Asp Arg Val Asp Glu 385 390 395 400 CTT CGC GCA GCC TCA AGT TGG CCT AAA AAT CTG ATG TAG 1239 Leu Arg Ala Ala Ser Ser Trp Pro Lys Asn Leu Met Stop 405 410 412

[Brief description of drawings]

FIG. 1 is about 5.5 including a gene DNA (samA) encoding the S-adenosyl synthetase of the present invention.
A restriction map of a kb DNA fragment with a restriction enzyme.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12R 1:13) (C12N 1/21 C12R 1:19) C12R 1:13)

Claims (5)

[Claims] 1. A DNA fragment containing a gene encoding S-adenosylmethionine synthetase derived from coryneform bacteria. 2. The DNA fragment according to claim 1, wherein the coryneform bacterium is Brevibacterium flavum MJ-233 strain. 3. The DNA fragment according to claim 1 or 2, which has restriction enzyme SalI recognition sites at both ends and is cleaved by the following restriction enzymes into the following size-cut fragments having the following restriction enzyme recognition sites. Restriction enzyme Recognition site number Cleavage fragment size (kb) BamHI 2 2.4, 1.7, 1.4 PstI 3 3.3, 1.0, 0.7, 0.5 SalI 0 5.5 4. A gene DNA encoding S-adenosylmethionine synthetase represented by the amino acid sequence of SEQ ID NO: 1 in the sequence listing. 5. A gene DNA encoding S-adenosylmethionine synthetase represented by the nucleotide sequence of SEQ ID NO: 1 in Sequence Listing.