JP2545078B2 - Method for producing nucleic acid-related substance - Google Patents

Description

TECHNICAL FIELD The present invention relates to the synthesis of amidophosphoribosyl transferase (EC2.4.2.14, hereinafter also referred to as APTase), which is an enzyme involved in purine nucleotide biosynthesis. Using a recombinant DNA of a DNA fragment containing a gene and a vector DNA, a microorganism belonging to the genus Corynebacterium or Brevibacterium is transformed, and the resulting transformant is cultured in a medium, Inosine 5 '
-A method for producing a nucleic acid-related substance, in which a nucleic acid-related substance such as inosine acid and 5'-xanthylic acid is produced and accumulated, and the nucleic acid-related substance is collected from the culture.

Since the nucleic acid-related substance is useful as a seasoning and a raw material for pharmaceuticals, the present invention belongs to the fields of food and pharmaceutical industry.

2. Description of the Related Art Known methods for producing nucleic acid-related substances include a method of decomposing ribonucleic acid, a method of converting a fermentation-produced precursor substance into a target substance by a synthetic method, and a method of directly fermenting and producing by a microorganism. As a method for producing a nucleic acid-related substance by a fermentation method, a method using a mutant strain derived from a wild strain is known. For example, a method using a 5'-inosinic acid-producing mutant strain (Japanese Patent Publication No. 58-46319), a 5'-xanthylic acid-producing mutant strain (JP-A-60-156399) and the like is known.

Problems to be Solved by the Invention Production of nucleic acid-related substances such as inosine, inosinic acid, and xanthyl acid by conventional methods is not always satisfactory, and higher yields of these nucleic acid-related substances important as seasonings and pharmaceutical raw materials are obtained. It is desired to develop a method of manufacturing at low cost.

Means for Solving the Problems In the production of nucleic acid-related substances using microorganisms, the present inventor is completely different from conventional breeding by imparting mutations,
We have continued research on breeding methods of nucleic acid-related substance-producing strains using recombinant DNA technology. As a result, a strain carrying a recombinant DNA of a DNA fragment containing a gene for APTase, which is an enzyme involved in the biosynthesis of nucleic acid-related substances (hereinafter sometimes abbreviated as purF), and a vector DNA The inventors have found that they have high productivity and completed the present invention.

 Hereinafter, the present invention will be described in detail.

INDUSTRIAL APPLICABILITY The present invention possesses a recombinant DNA of a DNA fragment carrying the genetic information involved in the synthesis of microbial APTase and a vector DNA, belongs to the genus Corynebacterium or the genus Brevibacterium, and produces a nucleic acid-related substance. Provided is a method for producing a nucleic acid-related substance, which comprises culturing a microorganism having an ability in a medium, producing and accumulating the nucleic acid-related substance in the culture, and collecting the nucleic acid-related substance from the culture.

Examples of the nucleic acid-related substance include inosine, inosine acid, xanthyl acid and the like.

As the DNA fragment containing the APTase gene (purF) used in the present invention, those derived from prokaryotes, bacteriophages or plasmids are used. Among them, bacteria belong to the genus Escherichia, Corynebacterium or Brevibacterium. A DNA fragment containing purF derived from a bacterium is preferably used. Specifically, the chromosomal DNA of E. coli K12 strain
An example is a DNA fragment containing the origin purF.

As the vector used in the present invention, any vector can be used as long as it can autonomously grow in Corynebacterium or Brevibacterium. In particular
PCG1, pCG2, pCG4pCG11, pCE52, pCE53, pCE54 and the like are preferably used. Regarding these vectors, Japanese Patent Laid-Open No. 57-
134500, JP-A-57-183799, JP-A-58-35197, JP-A-5
8-105999, JP-A-58-126789, and JP-A-59-156292.

Recombinant DNA of donor DNA containing purF and vector DNA
After cleaving both DNAs with a restriction enzyme in a test tube and religating with a DNA ligase, this ligation mixture was used to transform a PurF-deficient Corynebacterium or Brevibacterium species. It can be obtained by transforming and selecting a transformant in which the defective trait has been complemented. Instead of directly selecting recombinant DNA in Corynebacterium or Brevibacterium species, purF is isolated using a host-vector system with established genetic recombination techniques such as E. coli, The isolated gene can then be expressed in Corynebacterium or Brevibacterium species. That is,
PurF donor DNA is a shuttle vector DN of Escherichia coli and Corynebacterium spp. or Brevibacterium spp.
A is allowed to undergo a binding reaction in vitro and this reaction mixture is used to
A mutant strain of E. coli deficient in urF is transformed. Then, a transformant in which the defective trait has been complemented is selected, and a recombinant plasmid is isolated from this transformant. The target recombinant DNA can also be obtained by transforming a strain of the genus Corynebacterium or Brevibacterium using this recombinant plasmid and selectively isolating the transformant. PCE5 as a shuttle vector between Escherichia coli and Corynebacterium or Brevibacterium
2, pCE53, pCE54, etc. can be used.

The host microorganism of the present invention is a microorganism belonging to the genus Corynebacterium or the genus Brevibacterium and having a DNA uptake ability, in addition to the wild strain, a mutant strain having properties such as drug resistance and auxotrophy, nucleic acid-related Any strain may be used, such as a mutant strain having or lacking substance productivity. Suitable for Brevibacterium ammoniagenes AT
CC6872 or a nucleic acid-related substance-producing strain mutated with this strain as a parent strain, for example, 5'-inosinic acid-producing bacterium Brevibacterium ammoniagenes KY13184 (FERM P-3
790) 5'-xanthylic acid producing bacterium Brevibacterium ammoniagenes ATCC 21075, inosine producing bacterium Brevibacterium ammoniagenes ATCC 21477 and the like.

As a method for transforming a microorganism, JP-A-57-186492 discloses a method for transforming a microorganism belonging to the genus Corynebacterium or the genus Brevibacterium. However, Brevibacterium ammoniagenes that can be preferably used as a host microorganism in the present invention, although belonging to the genus Brevibacterium, is different from other Brevibacterium species such as Brevibacterium flavum. Clearly differ in chromosomal DNA homology [International Journal of Systematic Bacteriology (Int.J.Sys.Bacteriol.) 31 , 131, (198
1)] Therefore, it is not used as a host microorganism in conventional gene recombination techniques. The present invention is further useful in that a genetic recombination technique using Brevibacterium ammoniagenes as a host microorganism has been established by the present invention.

Transformation of host microorganisms with recombinant DNA involves (1) preparation of protoplasts from cultured cells, (2) recombinant DN
It is carried out in a step consisting of a transformation treatment of protoplasts with A, (3) reversion of protoplasts to normal cells and selection of transformants. A specific method is shown below by an example using Brevibacterium ammoniagenes.

(1) Preparation of Protoplasts from Cultured Cells Protoplasts are prepared by growing microorganisms under conditions that inhibit the synthesis of cell walls, and subjecting the cultured cells to lysozyme or achromopeptidase in a hypertonic solution to remove the cell walls. It is carried out by dissolving and removing. The medium used to obtain the vegetative cells may be any medium as long as Brevibacterium ammoniagenes can grow. For example, a complete nutrient medium such as NB medium (Table 1) or a semi-synthetic medium such as G III medium (Table 2) can be used. Table 1 Powdered broth 20 g / Yeast extract 5 g / pH 7.2 Table 2 Glucose 15 g / (NH ₄ ) ₂ SO ₄ 8 g / Urea 1.2 g / Yeast Eski 1.2 g / KH ₂ PO ₄ 0.5 g / K ₂ HPO ₄ 0.5 g / MgSO ₄ / 7H ₂ O 0.1 g / FeSO ₄ / 7H ₂ O 2 mg / ZnSO ₄ / 7H ₂ O 1 mg / MnSO ₄ / 4-6H ₂ O 1 mg / Biotin 0.1 mg / Cyamine hydrochloride 2 mg / Calcium pantothenate 10 mg / adenine 100 mg / guanine 100 mg / pH 7.2 This medium is inoculated with Brevibacterium ammoniagenes and cultured at 20-40 ° C under aeration and stirring conditions. The absorbance (OD) of the medium is measured with a colorimeter over time, and a cell wall synthesis inhibitor is added at the early stage of the logarithmic growth phase of the bacterium (when the OD reaches 0.1 to 0.15). As the drug that inhibits cell wall synthesis, penicillin, glycine and the like can be used. The amount of these agents used is preferably a concentration that suppresses the growth of microorganisms or less, and in the case of penicillin, about 0.1 to 2.0 U / ml in the culture solution, and in the case of glycine, about 10 to 40 mg / ml. To the concentration of After the addition of the drug, cultivation is further continued and propagated for several generations to obtain vegetative cells.

The vegetative cells are collected from the culture solution, washed with a medium and a hypertonic solution, suspended in each hypertonic medium, and subjected to lytic enzyme treatment. As the medium used for washing, the NB medium,
G III medium or the like can be used, and as the hypertonic solution, P3 hypertonic solution (Table 3) can be used. Table 3 NaCl 70 mM MgCl ₂ 5 mM CaCl ₂ 5 mM N-tris (hydroxymethyl) -methyl 2-aminoethane-sulfonic acid 25 mM sorbitol 1.6 M pH 7.6 Also, as hypertonic medium, nutrient medium, semi-synthetic medium, minimal medium 0.25-0.6M sucrose as a hypertonic drug, etc.
It is possible to use a solution to which any one of 3-0.7 M disodium succinate and 0.4-2.0 M sorbitol is added, or a P3 hypertonic solution. Lytic enzyme treatment was performed with egg white lysozyme, achromopeptidase, etc.
Add it to a concentration of about mg / ml, and add 5 at 30-40 ℃.
Hold for -20 hours. The production of protoplasts can be confirmed as spherical cells by observing with a light microscope.

The protoplasts thus prepared grow on hypertonic agar medium to form colonies and regenerate into vegetative cells. For regeneration, it is usually kept at 20-40 ° C for 3 to 20 days. As the hypertonic agar medium, nutrient medium, semi-synthetic medium, minimal medium, etc. to which 0.25-0.6M sucrose or 0.3-0.7M disodium succinate and agar (manufactured by Difco) 14g / are used. To be

(2) Transformation of Protoplasts with Recombinant DNA Transformation of protoplasts with recombinant DNA is carried out by mixing protoplasts and recombinant DNA in a hypertonic solution in which cells can maintain the protoplast state, and incorporating DNA into the mixture. It is carried out by adding a mediating polyethylene glycol (PEG, average molecular weight 1,540-6,000) and a divalent metal cation and treating it. As the stabilizer which gives hypertonic conditions, those which are generally used for retaining protoplasts of microorganisms may be used, and for example, sucrose, disodium succinate: sorbitol and the like can be used. The usable concentration range of PEG is 5-60% at the final concentration. Examples of the divalent metal cation include Ca ²⁺ , Mg ²⁺ , Mn ²⁺ and BA.
²⁺ , Sr ^2+, etc. are effective in the final concentration range of 1-100 mM and can be used alone or in combination. The treatment temperature is preferably 0 to 25 ° C.

(3) Regeneration of protoplasts to normal cells and selection of transformants Regeneration of protoplasts to normal cells and selection of transformants are performed as follows. The regeneration of the protoplasts transformed with the recombinant DNA is carried out on a hypertonic agar medium in the same manner as the regeneration of the protoplasts. The transformant can be obtained by selecting for the trait imparted to the bacterium by the gene derived from the recombinant DNA. Selection based on this characteristic trait acquisition may be performed simultaneously with regeneration on hypertonic agar. Alternatively, non-selective regenerated normal cells may be collected once and then selected on a normal hypotonic agar medium.

By transforming the transformed strain by culturing in a normal nutrient medium,
The trait of the introduced recombinant DNA can be expressed. When properties such as drug resistance are imparted to the transformed strain by the introduction of the recombinant DNA, the medium may be supplemented with the drug depending on the properties.

The nucleic acid-related substance can be produced by culturing the transformant thus obtained by the culture method used in the production of the nucleic acid-related substance by the fermentation method. That is, the transformant in a normal medium containing a carbon source, a nitrogen source, inorganic substances, amino acids, vitamins, etc.
If the culture is carried out under aerobic conditions while adjusting the temperature, pH, etc., the nucleic acid-related substance is produced and accumulated in the culture, so this is collected. The carbon source, glucose, fructose, sucrose, glycerol, starch, starch hydrolyzate, carbohydrates such as molasses, molasses hydrolysate, gluconic acid, pyruvic acid, lactic acid, various organic acids such as acetic acid, glycine, glutamic acid, Any amino acid such as alanine and aspartic acid that can be assimilated by the producing bacterium can be used. As the nitrogen source, ammonia, ammonium chloride, ammonium sulfate, ammonium phosphate,
Various inorganic and organic ammonium salts such as ammonium nitrate, ammonium carbonate and ammonium acetate, urea, peptone, NZ-amine, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, nitrogen-containing organic substances such as fish meal or its digest ,glycine,
Various amino acids such as various amino acids such as glutamic acid can be used. As inorganic substances, potassium dibasic phosphate, dibasic potassium phosphate, magnesium sulfate, magnesium phosphate,
Sodium chloride, ferrous sulfate, manganese sulfate, zinc sulfate, calcium carbonate and the like can be used. Furthermore, when the bacteria to be used require specific nutrients such as amino acids, nucleic acids and vitamins for growth, appropriate amounts of these substances are added to the medium, but they are supplied to the medium along with other medium components as described above. If so, it is not necessary to add it.

The culture is carried out under aerobic conditions such as shaking culture or aeration-agitation culture. Generally, the culture temperature is preferably 20-40 ° C.
The culture period is usually 2-7 days. It is desirable to keep the pH of the medium near neutral with aqueous ammonia, urea solution, sodium hydroxide solution, or the like. In this way, a considerable amount of nucleic acid-related substances accumulate in the culture. After completion of the culture, the nucleic acid-related substance can be recovered from the culture by an ion exchange resin method, an adsorption method, a precipitation method, an extraction method or the like alone or in combination.

 Next, examples will be shown.

Example 1 (1) Cloning of purF into pCE53 The chromosomal DNA of E. coli is E. coli K12 strain (Escherichia coli).
Coli ATCC33525) Bactrypton (made by Difco) 1
0g /, yeast extract (Difco) 5g /, NaCl 5g /
Inoculated into L medium containing pH of 7.2 and adjusted to 1 at 30 ℃
After culturing for 8 hours, Smith's phenol extraction method [Smith.MG .; Methods in Enzymology], 12 , Part A, 545 (196
7)] and isolated. pCE53 was isolated and purified from Escherichia coli 12 strain substrain MM294 (see Japanese Patent Application Laid-Open No. 60-210994) by the method described in this publication.

10 μg of chromosomal DNA of Escherichia coli K12 strain prepared above was added to 10 mM.
A buffer solution containing tris (hydroxymethyl) aminomethane (hereinafter abbreviated as Tris) -hydrochloric acid (pH 7.5), 100 mM NaCl, 7 mM MgCl ₂ and 6 mM _2- mercaptoethanol (hereinafter Y-
It is dissolved in 40 μl, and 24 units of the restriction enzyme Pst I (manufactured by Takara Shuzo, all the restriction enzymes are manufactured by Takara Shuzo unless otherwise specified) and 20 units of the restriction enzyme BamHI are added,
The digestion reaction was performed at 37 ° C for 1 hour. Then, the reaction was stopped by heat treatment at 65 ° C. for 10 minutes. On the other hand, 3 μg of pCE53 plasmid DNA was dissolved in 20 μl of Y-100 buffer, 12 units of Pst I and 10 units of BamHI were added, digestion reaction was carried out at 37 ° C for 1 hour, and the reaction was stopped by heat treatment at 65 ° C for 10 minutes. Let After mixing both digests, 20 mM Tris-HCl (pH 7.
6), 10mM MgCl ₂ , 10mM dithiothreitol and 0.5m
A buffer containing MATP (hereinafter referred to as T4 DNA ligase buffer) 120 μ and 3 units of T4 DNA ligase (manufactured by Takara Shuzo) were added and treated at 4 ° C. for 18 hours. Using the T4 DNA ligase reaction mixture thus obtained, the Escherichia coli FL-46 strain auxotrophic for hypoxanthine due to purF deficiency was coen (Co
hen) et al. [Cohen et al.,: Proceeding of the National Academy of Science (Proc.Natl.Acad.Sci.) USA, 69 , 2110 (1972)], Selective medium (glucose 2g, Na ₂ HP ₄ 6g,
_{KH 2 PO 4 3g, NaCl 0.5g} , NH 4 Cl 1g, MgSO 4 · 7H 2 O 0.5
g, CaCl ₂ · 2H ₂ O 15 mg, thiamine hydrochloride 4 mg, casamino acid 2 g, L-tryptophan 50 mg, kanamycin 50 mg and agar 16 g in water 1 and adjusted to pH 7.2), and then at 37 ° C for 3 By culturing for a day, a transformant strain resistant to kanamycin (50 μg / ml) and complemented with the hypoxanthine auxotrophy exhibited by the host was obtained.

A plasmid was prepared from this transformant by the method of Ann et al. [An.
G. et al., Journal of Bacteriology (J. Bac
teriol.), 140, 400 ( 1979) ] by separating purified Pst I
The structure of the plasmid was analyzed by digestion with restriction enzymes such as. As a result, it was confirmed that this was a recombinant plasmid in which the Pst I-BamH I of pCE53 was inserted with a Pst I-BamH I DNA fragment of about 9 kb derived from the chromosomal DNA of the Escherichia coli K12 strain, and this plasmid was named pEF12. It was using pEF12,
When the E. coli FL-46 strain was retransformed by the method of Cohen et al., All of the transformants selected as kanamycin resistant strains were hypoxanthine non-auxotrophic.
From these, it was confirmed that purF was cloned into pEF12.

(2) Introduction of plasmid pEF12 into Brevibacterium ammoniagenes The plasmid pEF12 separated and purified in (1) was used for transformation of Brevibacterium ammoniagenes ATCC6872.

Transformation was performed using protoplasts of the ATCC 6872 strain prepared as described below. ATCC6872 strain 30 in NB medium
Culture at 16 ℃ for 16 hours with shaking, and add 0.8 ml of the seed culture to G III medium.
An L-shaped test tube containing 8 ml was inoculated and shake-cultured at 30 ° C. using a mono-type shake incubator. The absorbance of the medium was measured with a Tokyo photoelectric colorimeter over time, and penicillin G was adjusted to 0.3 U / ml at the beginning of the logarithmic growth phase (culture time about 3 hours, cell concentration about 10 ⁸ cells / ml). Was added and the culture was continued for another 3 hours. After harvesting the cells from the culture solution and washing with G III medium, 2.0 mg
/ ml egg white lysozyme and 0.6 mg / ml achromopeptidase-containing P3 hypertonic solution (1 ml) were resuspended and allowed to stand at 30 ° C for 16 hours for protoplast formation.

Transfer 0.5 ml of this protoplast suspension to a small test tube.
Centrifuge at 00 × g for 10 minutes and then use TSMC buffer (10 mM MgCl ₂ , 3
0 mM CaCl ₂ , 50 mM Tris-HCl, 0.4 M sucrose, pH
7.5) Resuspended in 1 ml, centrifuged and washed, and then resuspended in 0.1 ml of TSMC buffer. To this suspension, 0.1 ml of TSMC buffer containing 10 μg of pEF12 plasmid DNA was added and mixed, and then 0.8 ml of 20% polyethylene glycol (PEG) 6,000 (manufactured by Hanai Chemical Co., Ltd.) was added to the TSMC buffer. Mixed. Ten
After that, 2 ml of G III medium was added, and the mixture was centrifuged at 2,500 xg for 10 minutes to remove the supernatant. 1 ml of sedimented protoplasts
Suspended in G III medium, and then 0.2 ml of the suspension was added to a hypertonic agar medium containing 200 μg / ml of kanamycin (0.5 M in G III medium.
Disodium succinate, containing 1.4% agar)), 3
After culturing at 0 ° C. for 14 days, a kanamycin-resistant transformant T-51 growing on the medium was obtained. The obtained transformant strain is Brevibacterium ammoniagenes T-51 (FE
RM BP-1332) has been deposited at the Institute of Microbial Science and Technology, Institute of Industrial Science (MIC) on April 3, 1987.

(3) Preparation of plasmid from Brevibacterium ammoniagenes Transformant T-51 (FERM BP-133 obtained in (2)
The pEF12 plasmid from 2) was isolated and purified as follows. Shake culture in NB medium at 30 ℃ for 16 hours, seed culture 4m
l was inoculated into 400 ml of G III medium and cultured at 30 ° C. with shaking. Penicillin G was added to the beginning of the logarithmic growth phase (cell concentration of 10 ⁸ cells / ml) to 0.3 U / ml, and the culture was continued for further 5 hours. The cells were collected from the culture solution and collected in TES buffer [30 mM Tris-
Hydrochloric acid, 5 mM ethylenediaminetetraacetic acid disodium (EDT
A), 50 mM NaCl, pH 8.0], and then lysozyme-acromopeptidase solution (12.5% sucrose, 0.1 M NaC
l, 0.05M Tris-hydrochloric acid, 3mg / ml lysozyme, 1mg / ml achromopeptidase, pH8.0) suspended in 10ml, 4 at 37 ℃
Allowed to react for hours. 2.4 ml of 5M NaCl, 0.5M EDT in the reaction solution
A (pH8.0) 0.6 ml, 4% sodium lauryl sulfate and 0.7M
4.4 ml of a solution consisting of NaCl was sequentially added, gently mixed, and then placed in ice water for 16 hours. The whole lysate was transferred to a centrifuge tube and centrifuged at 69,000 × g at 4 ° C. for 60 minutes to collect a supernatant. PEG 6,000 with a weight percentage of 10%
Was added, and the mixture was gently mixed to dissolve and then placed in ice water. After 10 hours, the pellet was collected by centrifugation at 1,500 × g for 10 minutes. Add 5 ml of TES buffer and gently redissolve the pellet, then add 2.0 ml of 1.5 mg / ml ethidium bromide, add cesium chloride to it and gently dissolve to a density of 1.
I adjusted it to 580. This solution was ultracentrifuged at 105,000 × g for 40 hours at 20 ° C. The circular DNA that was covalently closed by this density gradient centrifugation was found as a high density band in the lower part of the centrifugal chirp under UV irradiation. A liquid containing pEF12 plasmid DNA was separated by extracting this band portion from the side of the centrifuge tube with a syringe. Then, the separated solution was treated five times with an equal volume of isopropanol [volume percentage, isopropanol: TES buffer = 9: 1 (this TES buffer contains a saturated amount of cesium chloride)] to extract and remove ethidium bromide. After that, it was dialyzed against TES buffer.

Structural analysis was performed by digesting the plasmid DNA thus isolated and purified with a restriction enzyme such as Pst I. As a result, it was confirmed that it had the same structure as pEF12 confirmed in (1).

(4) introduction of the plasmid into an inosine-producing strain,
Measurement of APTase activity of the transformant Brevibacterium ammoniagenes ATCC21477, which has the ability to produce inosine, was protoplastized in the same manner as in (2) (3).
Was transformed with the recombinant plasmid pEF12 isolated and purified in (1) to obtain a transformant strain having a kanamycin-resistant trait.

APTase of ATCC21477 and transformant ATCC21477 / pEF12
Of the activity of JM Lewis and SCHartma are known methods.
n, Methods in Enzymology
ology), 51 , 171-178 (1978)] with minor modifications.

The strain to be used for the activity measurement is 50 ml containing 100 ml of G III medium.
Shake culture using a 0 ml Erlenmeyer flask at 30 ° C for 16 hours,
The obtained bacterial cells were disrupted by ultrasonic waves to prepare a lysate. The supernatant obtained by centrifuging this at 6,000 xg for 30 minutes was used as a crude enzyme solution, and 30 mM Tris-hydrochloric acid (pH 8.0), 9 mM MgCl
₂ , 25 mM in 0.3 ml of a solution consisting of 16 mM potassium fluoride (KF), 3 mM phosphoribosyl pyrophosphate, 12 mM glutamine,
The reaction was performed for 5 minutes. A control experiment was conducted without glutamine. After completion of the reaction, 0.1 ml of 1.0 M sodium acetate solution (pH 5.0), 0.05 ml of 3 mM sodium pyrophosphate solution, and 0.1 ml of 0.1 M manganese chloride solution were sequentially added, and then 1,5
The pellet was recovered by centrifugation at 00 × g for 5 minutes. 0.5 ml of 0.01 M manganese chloride solution and 0.1 ml of 10% acetone solution were sequentially added to this, washed, and then centrifuged to collect the pellet. 1.0N sulfuric acid 1.0ml was added to this pellet, 100 ℃,
Heated for 15 minutes. After cooling, phosphorus was quantified using an assay kit for measuring inorganic phosphorus (manufactured by Wako Pure Chemical Industries, Ltd.), and APTase activity was determined by measuring glutamine-dependent activity. Table 4 shows the activity measurement results.

By introducing the recombinant plasmid pEF12 containing purF, the APTase activity was increased by 1.5 times, and the activity increasing effect was clearly observed.

(5) Production of inosine by transformant Brevibacterium ammoniagenes ATCC21477 and its transformant ATCC21477 / pEF12 were examined for inosine productivity.

Seed culture solution obtained by inoculating one strain of both strains into 250 ml Erlenmeyer flasks each containing 20 ml of NB medium and culturing at 30 ° C. for 24 hours was used as a production medium (glucose 130 g, KH ₂ PO ₄ 10 g, K ₂ H
_{PO 4 10g, MgSO 4 · 7H} 2 O 10g, corn steep liquor 20
g, CaCl ₂・ 2H ₂ O 0.1g, FeSO ₄・ 7H ₂ O 10mg, ZnSO ₄・ 7H
₂ O 2 mg, MnCl ₂ -4-6H ₂ O 2 mg, biotin 30 μg, vitamin B ₁₅ 5 mg, calcium pantothenate 10 mg, nicotinic acid 5 mg, adenine 100 mg, guanine 100 mg, urea 4 g are included in pure water 1 and adjusted to pH 7.6. The culture medium) was inoculated into a 250 ml Erlenmeyer flask with a baffle plate containing 20 ml at a ratio of 10% (volume ratio), and cultured at 30 ° C. for 4 days with shaking at 220 rpm.
At the 48th and 72th hours during the culturing, urea that had been separately sterilized was added at a rate of 2 g / to adjust the pH. After the culture was completed, the amount of inosine accumulated in the culture was quantified by paper chromatography. Table 5 shows the results.

By introducing the recombinant plasmid pEF12 containing purF, a remarkable improvement in inosine productivity was observed.

Example 2 Using the recombinant plasmid pEF12 isolated and purified in (3) of Example 1, Brevibacterium ammoniagenes KY13184 (FERM P having the ability to produce 5'-inosinic acid).
-3790) was transformed into protoplasts in the same manner as in (2) of Example 1 and transformed to obtain a transformant having a kanamycin-resistant trait. The transformant KY13184 /
The APTase activity of pEF12 was measured by the same method as in Example 1 (4). The results are shown in Table 6. The transformant KY13184 / pEF12 was subjected to paper chromatography in the same manner as in (5) of Example 1 to quantify 5'-inosinic acid. The amount of 5'-inosinic acid accumulated is shown in Table 7. The same procedure was performed using the parent strain KY13184 as a control strain.

Increased APTase activity due to the introduction of pEF12,
An improvement in 5'-inosinic acid productivity was observed.

Example 3 Using the recombinant plasmid pEF12 isolated and purified in (3) of Example 1, Brevibacterium ammoniagenes ATCC21075 capable of producing 5'-xanthylic acid was transformed. Transformation was performed in the same manner as in (2) of Example 1 except that the concentration of penicillin was adjusted to 0.5 U / ml to adjust protoplasts, and a transformant strain having a kanamycin resistance trait was obtained. APT of the transformant ATCC21075 / pEF12
The ase activity was measured by the same method as in Example 1 (4). As a control, the ATCC21075 strain was used and treated in the same manner.
The results are shown in Table 8.

An increase in APTase activity was observed due to the introduction of the recombinant plasmid pEF12 containing purF.

Next, the transformant FTCC21075 / pEF12 was examined for 5'-xanthylic acid producing ability. Brevibacterium ammoniagenes ATCC21075 and ATCC21075 / pEF12 were inoculated into a 250 ml Erlenmeyer flask containing 20 ml of NB medium, one platinum loop each, 3
The seed culture broth obtained by culturing at 0 ° C. for 24 hours was used in Example 1 (5).
Inoculate a 250 ml Erlenmeyer flask containing 20 ml of production medium of the same composition as the above at a ratio of 10% (volume ratio), and at 220C for 4 days at 30 ° C.
Culture was carried out with shaking at pm. At the 48th and 72th hours during the culturing, urea that had been separately sterilized was added at a rate of 2 g / to adjust the pH. The accumulated 5'-xanthylic acid was quantified by paper chromatography. The results are shown in Table 9.

By introducing the recombinant plasmid pEF12 containing purF, it was confirmed that the productivity of 5'-xanthylic acid was improved.

Effects of the Invention According to the method of the present invention, a recombinant DNA containing a gene for APTase, which is an enzyme involved in purine nucleotide biosynthesis, and a microorganism belonging to the genus Corynebacterium or the genus Brechbacterium, which carries the recombinant DNA. And a method for producing a diffusion-related substance using the microorganism can be provided.

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Claims (2)

(57) [Claims] 1. A recombinant DNA comprising a vector DNA and a DNA fragment responsible for the synthesis of amidophosphoribosyl transferase derived from a microorganism belonging to the genus Escherichia and having a Corynebacterium genus or Brevibacterium genus. A nucleic acid characterized by culturing a microorganism belonging to the genus and having the ability to produce a nucleic acid-related substance in a medium, producing and accumulating the nucleic acid-related substance in the culture, and collecting the nucleic acid-related substance from the culture. Manufacturing method of related substances. 2. The method according to claim 1, wherein the nucleic acid-related substance is inosine, inosine acid or xanthyl acid.