JPS63248394A - Production of nucleic acid-relating substance - Google Patents

Abstract

PURPOSE:To obtain a nucleic acid-relating substance in high efficiency at a low cost, by culturing a microbial strain belonging to Corynebacterium genus or Brevibacterium genus and containing a recombinant DNA of a vector DNA and a DNA fragment containing gene of APTase. CONSTITUTION:A microbial strain belonging to Corynebacterium genus or Brevibacterium genus is transformed with a recombinant DNA derived from (A) a DNA fragment containing gene (hereinafter called as purF) of amidophosphoribosyl transferase (APTase) which is an enzyme participating in biosynthesis of purine nucleotide, preferably a DNA fragment containing purF originated from bacterium belonging to Escherichia genus, Corynebacterium genus or Brevibacterium genus and (B) a vector DNA which can be proliferated in bacterial cell of Corynebacterium genus or Brevibacterium genus by autonomous replication. The obtained transformant is cultured in a medium to obtain nucleic acid-relating substances such as inosine, 5'-inosinic acid and 5'-xanthylic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to amidophosphoribosyl transferase (E), an enzyme involved in purine nucleotide biosynthesis.
Using a recombinant DNA of a DNA fragment containing a gene involved in the synthesis of C2,4,2,14 (hereinafter sometimes referred to as APTase) and vector DNA, a gene of the genus Corynebacterium or Brevibacterium spp. The resulting transformed strain is cultured in a medium, and nucleic acid-related substances such as inosine, 5'-inosinic acid, and 5'-xanthylic acid are produced and accumulated in the culture, and the resulting transformed strain is cultured in a culture medium. The present invention relates to a method for producing a nucleic acid-related substance by collecting the nucleic acid-related substance.

Since nucleic acid-related substances are useful as seasonings and pharmaceutical raw materials, the present invention belongs to the field of food and pharmaceutical industries.

Conventional techniques Known methods for producing nucleic acid-related substances include a method of decomposing ribonucleic acid, a method of converting a fermented precursor into a target substance by a synthetic method, and a method of direct fermentation production using microorganisms. As a method for producing nucleic acid-related substances by fermentation, a method using a mutant strain derived from a wild strain is known. For example, methods using 5'-inosinic acid-producing mutant strains (Japanese Patent Publication No. 58-46319), 5'-xanthylic acid-producing mutant strains (Japanese Patent Application Laid-Open No. 60156399), etc. are known.

Problems to be Solved by the Invention The production of nucleic acid-related substances such as inosine, inosinic acid, and xanthylic acid by conventional methods is not necessarily satisfactory. It is desired to develop a method for manufacturing it at low cost.

Means for Solving the Problems In the production of nucleic acid-related substances using microorganisms, the present inventor conducted research on a method of breeding nucleic acid-related substance-producing strains using recombinant DNA techniques, which is completely different from the conventional breeding by imparting mutations. layered. As a result, the gene for APTa se, an enzyme involved in the biosynthesis of nucleic acid-related substances (hereinafter p
The present inventors have discovered that a strain containing a recombinant DNA of a DNA fragment containing vector DNA (sometimes abbreviated as urF) has high productivity of nucleic acid-related substances, and has completed the present invention.

The present invention will be explained in detail below.

The present invention possesses a recombinant DNA of a DNA fragment carrying genetic information involved in the synthesis of APTase of a microorganism and a vector DNA, belongs to the genus Corynebacterium or the genus Brevibacterium, and is associated with nucleic acids. Provided is a method for producing a nucleic acid-related substance, which comprises culturing a microorganism capable of producing the substance in a medium, producing and accumulating a nucleic acid-related substance in the culture, and collecting the nucleic acid-related substance from the culture. do.

Examples of the nucleic acid-related substance include inosine, inosinic acid, and xanthylic acid.

APTase gene used in the present invention (pur F
) are derived from prokaryotes, bacteriophages, or plasmids, but
Among them, pu derived from bacteria, especially those belonging to the genus Escherichia, Corynebacterium or Brevibacterium.
A DNA fragment containing rF is preferably used. Specifically, D containing purF derived from the chromosomal DNA of E. coli K12 strain
NA fragments are listed.

Any vector that can be used in the present invention can be used as long as it can autonomously proliferate within a species of the genus Corynebacterium or Brevibacterium. Specifically, p
CGI, pCG2, pcc, 1pcG11, pCE52
, pCE53, pCE54, etc. are preferably used. Regarding these vectors, see JP-A-5'1-134500;
Japanese Patent Application Publication No. 183799, Japanese Patent Application Publication No. 58-35197
, JP-A-58-105999, JP-A-58-12678
9 and JP-A No. 59-156292, respectively.

The recombinant DNA of the donor DNA containing purF and the vector DNA was obtained by cutting both DNAs with restriction enzymes in a test tube, religating them with DNA IJ gauze, and then using this ligation reaction mixture. It can be obtained by transforming a Corynebacterium genus or Brevibacterium genus mutant lacking purF, and selecting a transformed strain in which the defective trait is complemented. Directly recombinant DN with Corynebacterium or Brevibacterium species
Instead of selecting A, purF can be isolated using a host-vector system for which recombinant techniques are already established, such as E. coli, and then purified in Corynebacterium or Brevibacterium spp. Separate genes can also be expressed. That is, the donor DNA of purF is subjected to a binding reaction in a test tube with the shuttle vector DNA of Escherichia coli and Corynebacterium or Brevibacterium genus, and this reaction mixture is used to generate purF
Transform a mutant strain of E. coli that is defective. Then, a transformant strain in which the defective trait has been complemented is selected, and a recombinant plasmid is isolated from this transformant strain.

The desired recombinant DNA can also be obtained by transforming a Corynebacterium or Brevibacterium species using this recombinant plasmid and selectively separating the transformed strain. pCE52, pCE53, pCE54, etc. can be used as shuttle vectors between E. coli and Corynebacterium or Brevibacterium species.

The host microorganisms of the present invention include wild strains, mutant strains having properties such as drug resistance and auxotrophy, and nucleic acid-related microorganisms that belong to the genus Corynebacterium or Brevibacterium and have DNA uptake ability. Any strain may be used, including mutant strains that have or have lost substance productivity. Preferably Brevibacterium ammoniagenes Δ
TCC6872 and nucleic acid-related substance producing strains mutated using this strain as a parent strain, such as 5'-inosinic acid producing bacterium Brevibacterium ammoniagenes KY1318
4 (FERM P-3790), 5'-xanthylic acid producing bacterium Brevibacterium ammoniagenes ΔT
CC21075, inosine-producing bacterium Brevibacterium
Examples include ammoniagenes^TCC21477.

As a method for transforming microorganisms, JP-A-57-186492
A method for transforming microorganisms belonging to the genus Corynebacterium or Brevibacterium has been reported. However, although Brevibacterium ammoniagenes, which can be suitably used as a host microorganism in the present invention, belongs to the genus Brevibacterium, it is different from other species of Brevibacterium, such as Brevibacterium flavum. have clearly different chromosomal DNA homology [International Journal of Systematics, l.
Bacterium di(Int, J, Sys, B
acteriol, )■, 131, (198
1)], therefore, they are not used as host microorganisms in conventional genetic recombination techniques. The present invention is further useful in that it establishes a genetic recombination technique using Brevibacterium ammoniagenes as a host microorganism.

Transformation of host microorganisms with recombinant DNA involves (]) preparation of protoplasts from cultured cells, (2) recombinant D
The process consists of transformation of protoplasts with NA, (3) regeneration of protoplasts into normal cells, and selection of transformed strains. A specific method will be shown below using an example using Brevibacterium ammoniagenes.

(1) Preparation of protoplasts from cultured cells To prepare protoplasts, microorganisms are grown under conditions that inhibit cell wall synthesis, and the cultured cells are treated with lysozyme or achromopeptidase in a hypertonic solution to form cell walls. This is done by dissolving and removing. Any medium that can grow Brevibacterium ammoniagenes can be used to obtain vegetative cells. For example, a complete nutrient medium such as NB medium (Table 1) or a semi-synthetic medium such as GI medium (Table 2) can be used.

Table 1 Powder bouillon 20g/j! yeast extract
5 g/f! pH7.2 Table 2 Glucose 15g/j! (NH4)2
SO, 8g/n Urea 1.2g/β
Yeast eski 1.2g/βKH2P0
．． 0.5g/IK28P0.
0.5g/βMg5O, 7820
0.1g/, i! Fe50.・7H202mg/j
! Z n S 04 ・7 HzO1mg/j! Mn
S○-'46H201mg/j Biotin 0.1mg/β Thiamine hydrochloride 2mg/β Calcium pantothenate IO+mg/j! adenine 10
0mg/R Guanine 100mg/j+ pH 7,2 Brevibacterium ammoniagenes is inoculated into this medium and cultured at 20-40°C under aeration and stirring conditions. The optical density (OD) of the medium is measured over time using a colorimeter, and a cell wall synthesis inhibitor is added at the beginning of the logarithmic growth phase of the bacteria (when the OD reaches 0.1 to 0.15). Penicillin, glycine, etc. can be used as drugs that inhibit cell wall synthesis. The amount of these drugs to be used is preferably a concentration that partially suppresses the growth of microorganisms or less, and in the case of penicillin, 0.1 to 2. It is added to a concentration of about OU/ml, and in the case of glycine, it is added to a concentration of about 10 to 49 mg/ml. After adding the drug, the culture is continued and the cells are grown 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 the respective hypertonic medium, and treated with a lytic enzyme. The medium used for washing is the above-mentioned NB medium,
GI medium etc. can be used, and the hypertonic solution is P3 hypertonic solution (
Table 3) can be used.

Table 3 NaC 170mM MgCI2 5mM CaCL 5nnM Sorbitol sulfonate 1.6M pH 7,6 In addition, as a hypertonic medium, nutrient medium, semi-synthetic medium, minimal medium, etc. may contain 0.25-0.6 M Nucrose, 0.3-M as a hypertonic agent. 0.7M disodium succinate, 0.4
- supplemented with either 2.0 M sorbitol,
Alternatively, P3 hypertonic solution or the like can be used. In the lytic enzyme treatment, egg white lysozyme or achromopeptidase is added to a concentration of about 0.1-5.0 mg/ml, and the mixture is kept at 30-40°C for 5-20 hours. The production of protoplasts can be confirmed as spherical cells by observing with an optical microscope.

The protoplasts thus prepared grow on a hypertonic agar medium to form colonies and regenerate into vegetative cells. In order to perform regeneration, it is usually kept at 20-40°C for 3 to 20 days. As a hypertonic agar medium, 0.25-0.6M can be used as a nutrient medium, semi-synthetic medium, minimal medium, etc.
of sucrose or 0.3-0.7 M succinic acid 2
Sodium and agar (manufactured by Difco) 14g/j! Added products are used.

(2) Transformation of protoplasts with recombinant DNA Transformation of protoplasts with recombinant DNA involves mixing protoplasts and recombinant DNA in a hypertonic solution that allows cells to maintain their protoplast state, and then incorporating the DNA into the mixture. Polyethylene glycol (PEG,
average molecular weight 1,540-6.000) and divalent metal cations. Stabilizers that provide hypertonic conditions may be those commonly used to maintain protoplasts of microorganisms, such as sucrose, disodium succinate: sorbitol, and the like. The usable concentration range for PEG is 5-60% final concentration. Examples of divalent metal cations include Ca'', y, g2-1Mn'', Ba2-1Sr''-
etc. are effective at a final concentration of 1-100 mM and can be used alone or in combination.

The temperature of the treatment is preferably 0-25°C.

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

The transformed strain can be cultured in an ordinary nutrient medium to express the characteristics of the introduced recombinant DNA. If the transformed strain is endowed with properties such as drug resistance due to the introduction of the recombinant DNA, the culture medium may be supplemented with drugs depending on the properties.

Nucleic acid-related substances can be produced by culturing the thus obtained transformed strain using a culture method that is used for producing nucleic acid-related substances by fermentation. That is, the transformed strain is treated with carbon sources, nitrogen sources, inorganic substances,
If the culture is carried out under aerobic conditions in a normal medium containing amino acids, vitamins, etc. while adjusting the temperature, pH, etc., nucleic acid-related substances will be generated and accumulated in the culture, which will be collected. Carbon sources include carbohydrates such as glucose, fructose, sucrose, glyceronove starch, starch hydrolyzate, molasses, and molasses hydrolysates, various organic acids such as gluconic acid, pyruvic acid, lactic acid, and acetic acid, glycerol, and glutamic acid. Any amino acid that can be assimilated by the producing bacteria can be used, such as amino acids such as , alanine, and aspartic acid. Nitrogen sources include ammonia, various inorganic and organic ammonium salts such as ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium nitrate, ammonium carbonate, ammonium acetate, urea, peptone, NZ-amine, meat extract, yeast extract, corn steep liquor, casein. Nitrogen-containing organic matter such as hydrolyzate, fikunyumir or its digested product, glycine,
Various amino acids such as glutamic acid can be used. As the material, potassium phosphate, dipotassium phosphate, magnesium sulfate, magnesium phosphate, sodium chloride, potassium sulfate, manganese sulfate, zinc sulfate, calcium carbonate, etc. can be used. Furthermore, if the bacteria used require specific nutrients such as amino acids, nucleic acids, and vitamins for growth, appropriate amounts of these substances are added to the culture medium, but they are not supplied to the culture medium along with the pond culture medium components mentioned above. If so, there is no need to add anything.

Cultivation is performed under aerobic conditions such as shaking culture or aerated 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 culture medium near neutral using aqueous ammonia, urea solution, sodium hydroxide solution, etc. In this way, significant amounts of nucleic acid-related substances are present in the culture. After completion of the culture, nucleic acid-related substances can be recovered from the culture using ion exchange resin method, adsorption method, precipitation method, extraction method, etc. alone or in combination.

Next, examples will be shown.

Example 1 (1) Cloning of purF into pCE53 The chromosomal DNA of Escherichia coli was obtained from Escherichia coli K1.2 strain (E.
Coli ATCC 33525), Bactodributon (manufactured by Difco)], [) g/L yeast extract (manufactured by Difco) 5
g/C NaC15g/j! After adjusting the pH to 742, the cells were inoculated into a medium, and after culturing at 30°C for 18 hours, Smith's phenol extraction method [Sm+th, M
, G, , ;Methods in Enzymology (M
methods in Enzy+nology).

! 2, Parj A, 545 (1967)).

pCE53 is a 12-strain substrain of E. coli MM294 that carries this plasmid (see Japanese Patent Application Laid-Open No. 60-210994).
It was separated and purified by the method described in the publication.

Chromosomal DNA 10 of the K12 strain of F% bacteria prepared above
in 10mM tris(hydroxymethyl)aminomethane (
(hereinafter abbreviated as Tris)-hydrochloric acid (pH 7.5), 100m
M NaC,i! .

Dissolved in 40I of a buffer (hereinafter referred to as Y-100 buffer) containing 7mM MgCl2 and 6mM2-mercaptoethanol, and added 24 units of restriction enzyme pst.
1 (manufactured by Takara Shuzo Co., Ltd.; unless otherwise specified, all restriction enzymes are manufactured by Takara Shuzo Co., Ltd.) and 20 units of restriction enzyme BamHI,
Digestion reaction was carried out at 37°C for 1 hour. After that, 65℃, 1
The reaction was stopped by heat treatment for 0 minutes. On the other hand, pCE5
3 Plasmid DNA 3■ was dissolved in 20ml of Y-100 buffer, 12 units of PStl and 10 units of BamHI were added, the digestion reaction was performed at 37°C for 1 hour, and the reaction was stopped by heat treatment at 65°C for 10 minutes. .

After mixing both digests, 20mM Tris-HCl (pH 7)
, 6), a buffer containing 10mM MgCL, 1QmM dithiothreitol and 0.5mM ATP (hereinafter referred to as T
Add 120ρ (referred to as 4DNA ligase buffer) and 3 units of T4DNA'J gauze (manufactured by Takara Shuzo Co., Ltd.) at 4°C.
Treated for 18 hours. T4DNΔ obtained in this way
Cohen ((:
Cohen et al.'s method [Cohen et al.:
Proceedings of the National Academy of Sciences
, Sci, ) U, S, A, , Dan.

2110 (1972)) and transformed with selective medium (glucose 2 g, Na2HPO + 6 g-KH
2P 04 3 gSN a C10,5g-NH<C
R1g, Mg Sc4.7 H2O0.5g, Ca
Cl2・2H2015mg, thiamine hydrochloride 4mg,
2 g of casamino acid, 50 mg of L-tryptophan, 50 mg of kanamycin, and 16 g of agar in 11 parts of water, pH
After coating on a medium adjusted to 7.2, and culturing at 37°C for 3 days, a transformed strain was obtained which was resistant to kanamycin (>50Ag/ml) and complemented the hypoxanthine auxotrophy exhibited by the host.

From this transformed strain, a plasmid was extracted using the method of Ahn et al.
G. et al., Journal of Bacteriology, 140.
400 (1979)) and was digested with restriction enzymes such as Pstl to analyze the structure of the plasmid. As a result, Psll-BamHI of pCE53
It was determined that this was a recombinant plasmid in which an approximately 9 kb PstI-BamHI DNA fragment derived from the chromosomal DNA of E. coli strain 12 was inserted [812], and this plasmid was named pEF12. When E. coli FL-46 strain was re-transformed using pEF 12 by the method of Cohen et al., all of the transformed strains selected as kanamycin-resistant strains were non-auxotrophic for hyboxanthin. From these results, it was confirmed that purF was cloned into pEF12.

(2) Introduction of plasmid pEF 12 into Brevibacterium ammoniagenes Plasmid pEF12 isolated and purified in (1) was used to transform Brevibacterium ammoniagenes ΔTCC68j2.

Transformation was prepared as follows. This was carried out using protoplasts of ATCC6872 strain.

ATCC6872 strain was cultured with shaking in NB medium at 30°C for 16 hours, and 8 ml of the seed culture solution Q was mixed with GII [medium 8 ml].
The cells were inoculated into an L-shaped test tube containing a 100-mL tube, and cultured with shaking at 30°C using a mono-type shaking culture machine. The absorbance of the medium was measured over time using a Tokyo Photoden colorimeter, and at the beginning of the logarithmic growth phase, the culture time was about 3 hours, and the bacterial cell concentration was about 108 cells/mt).
ml of penicillin G was added, and the culture was continued for an additional 3 hours. Collect bacterial cells from the culture solution and add GII [after washing with medium, 2.0 mg/ml egg white lysozyme, 0.6 mg
The cells were resuspended in 1 ml of P3 hypertonic solution containing /ml achromopeptidase and allowed to stand at 30°C for 16 hours to form protoplasts.

Transfer 5 ml of this protoplast suspension Q to a small test tube, centrifuge at 2,500 xg for 10 minutes, add TSMC buffer (I OmM Mg (1, 30mM CaC
C, 50mM Tris-HCl, 0.4M'/:L
After centrifugal washing, the suspension was resuspended in 0.1 ml of TSMC buffer. TSMC containing 10Ag of pEF12 plasmid DNA in this suspension.
Add 6.0 l in 3MC buffer and mix, then add 20% polyethylene glycol (PEG) 6.0 in TSMC buffer.
3 ml of Solution Q containing 00 (manufactured by Hanui Chemical Co., Ltd.) was added and mixed. After 10 minutes, add 2 liters of GIrlIrl medium,
The mixture was centrifuged at 2,500×g for 10 minutes, and the supernatant was removed. After suspending the precipitated protoplasts in 1 ml of G medium, 2 ml of Q of the suspension was added to Kanamai Schiff 20.
Hypertonic agar medium containing 0 g/ml (0.5
The medium was coated on a medium containing 1.4% agar) and cultured at 30°C for 14 days to obtain a kanamycin-resistant transformant strain T-51 that grew on the medium. The obtained transformed strain was deposited as Brevibacterium f' Jum ammoniagenes T-51 (FERIJBP-1332) at the Institute of Microbial Technology, Agency of Industrial Science and Technology on August 3, 1986. ing.

(3) Preparation of plasmid from Brevibacterium ammoniagenes (2) Transformant strain T-51 (FERI, I
BP-7332), the pEF12 plasmid was isolated and purified as follows. Cultured with shaking in NB medium at 30°C for 16 hours, and 4 ml of the seed culture was added to G [[400 ml of medium
and cultured with shaking at 30°C. Early logarithmic growth phase (
Penicillin G was added to give a bacterial cell concentration of 108 cells/ml and 0.3 U/ml, and the culture was continued for an additional 5 hours. Bacterial cells were collected from the culture solution and added to TES buffer (30mM) Lis-HCl, 5mM disodium ethylenediaminetetraacetic acid (EDTA), 50mM NaCf1SpH8,0).
After washing with lysozyme-achromopeptidase solution (12
,5%nxCrose,O,],M NaCj! ,04
05M Tris-HCl, 3mg/ml lysozyme, 1mg
/m17 chromobeptidase, pH 8,0) at 10m
1 and reacted at 37°C for 4 hours. 5M to the reaction solution
NaCj! 2.4ml, 0.5M EDT
0.6 ml of A (pH 8.0) and 4.4 ml of a solution consisting of 4% sodium lauryl sulfate and 0.7 M NaCf were sequentially added, mixed gently, and then placed in ice water for 16 hours. The entire lysate was transferred to a centrifuge tube and centrifuged at 69,000 x g for 60 minutes at 4°C to collect the supernatant. PEG 6,000 equivalent to 10% by weight was added thereto, mixed gently to dissolve, and then placed in ice water. After 10 hours, the pellet was collected by centrifugation at 1.500 x g for 10 minutes. After adding 5 ml of T8S buffer and gently redissolving the pellet, 20 ml of 1.5 mg/ml ethidium bromide was added, and cesium chloride was added thereto and gently dissolved to adjust the density to 1.580.

This solution was subjected to ultracentrifugation at 20° C. and 105.000×g for 40 hours. The circular DNA covalently closed by this density gradient centrifugation was found as a high-density band in the lower part of the centrifuged tube under ultraviolet irradiation. A liquid containing pEF 12 plasmid DNA was separated by extracting this band portion from the side of the centrifuge tube with a syringe. Next, the separated solution was added to an equal volume of Impropatool [volume percentage, Impropatool: TBS buffer = 9 + 1 (
Note that this TBS buffer solution contains cesium chloride in a saturating amount)] to extract and remove ethidium bromide five times, followed by dialysis against a TES buffer solution.

Structural analysis was performed by digesting the thus isolated and purified plasmid DNA with a restriction enzyme such as Pstl. As a result, it was confirmed that it had the same structure as pEF 12 confirmed in (1).

(4) Introduction of plasmid into inosine-producing strain and measurement of APTa se activity of the transformed strain Brevibacterium ammoniagenes AT capable of producing inosine
CC21477 was made into a protoplast in the same manner as in (2), and then transformed using the recombinant plasmid pEF 12 isolated and purified in (3) to obtain a transformant having kanamycin resistance.

The PTase activity of ATCC21477 and the transformed strain ATCC21477/pEF 12 was measured using a known method CJ,! J, Lewis a
ndS, C, tlartman, Methods in Enzymology
ymology), 51. 171-178 (1
978) :] was slightly modified and carried out as follows.

The strain to be subjected to activity measurement was incubated at 30°C for 1 hour using a 50 Qml Erlenmeyer flask containing 10 Qml of GITI medium.
After culturing with shaking for 6 hours, the resulting bacterial cells were disrupted using ultrasound to prepare a lysate. This was centrifuged at 6,000xg for 30 minutes, and the supernatant obtained was used as the crude enzyme solution.
J-Su-hydrochloric acid (pH 8,0), 9mM MgCL,
The reaction was carried out at 25° C. for 5 minutes in 0.31 Tll of a solution consisting of 16 mM potassium fluoride (KF), 3111 M phosphoribosylbirophosphate, and 12 mM glutamine. Note that a control experiment was one that did not contain glutamine. After the reaction, add 0.1 ml of 1.0M sodium acetate solution (pH 5,
0), 0.05 ml of 3mM sodium picrate solution, O,! After sequentially adding 0.1 ml of 0.1 M manganese chloride solution, centrifugation was performed at 1.500 x g for 5 minutes.
l- was recovered. After washing by sequentially adding Q, 5 ml of 0.01M manganese chloride solution, and Q, 1 ml of 10% acetone solution, the pellet was centrifuged to collect the pellet. This pellet contains 1. ON sulfuric acid 1. Add Qml, 100℃, 1
Heated for 5 minutes. After cooling, phosphorus was quantified using an assay kit for measuring inorganic phosphorus (manufactured by Wako Hyakuyaku Co., Ltd.), and APTase activity was determined by measuring glutamine-dependent activity. Table 4 shows the activity measurement results.

Table 4 ATCC214,778,2 ATCC21477/pEF12 1.2.7purF
By introducing the recombinant plasmid pEF12 containing , APTase activity was increased by 1.5 times, and an activity-increasing effect was clearly observed.

(5) Inosine production by transformed strain Inosine productivity of Brevibacterium ammoniagenes ATCC21477 and its transformed strain ATCC21477/pEF 12 was investigated.

A loopful of both strains was inoculated into a 250 ml Erlenmeyer flask containing 20 ml of NB medium, and cultured at 30°C for 24 hours.
KH2PO410gXK2HPOn 10g, Mg5
O<・7H20Log, corn steep liquor - 20g,
CaCj! z'2Hzo O, 1g.

FeS○+・7H2010mg, ZnSO4・71h
0 2mg, MnCA'z・4-68202wg, biotin 30■, vitamin Bl 5mL calcium pantothenate 10. Nicotinic acid 5 mg 5 adenine i o O
``L-guanine 100mg.

Culture medium containing 4g of urea in 1β of pure water and adjusting the pH to 7.6
i) Inoculate a 250 ml Erlenmeyer flask with a baffle plate at a ratio of 10% (by volume) and incubate at 30°C.
The cells were cultured with shaking at 220 rpm for 4 days. Cultivating 48
And at 72 hours, separately sterilized urea was added at a rate of 2 g/β to adjust the pH. After completion of the culture, the amount of inosine accumulated in the culture was quantified using E-Park D-7 Togelafy. The results are shown in Table 5.

Table 5 Bacterial strain Inosine (g/β) ATCC2
14776,2 ^TCC21477/ρEF12 9.4pu
By introducing the recombinant plasmid pEF12 containing rF, a remarkable improvement in inosine productivity was observed.

Example 2 Using the recombinant plasmid pEF 12 isolated and purified in Example 1, column 1 (3), Brevibacterium ammoniagenes KY1318 having 5'-inosinic acid productivity was produced.
4 (FERM P 3790) was transformed into a protoplast in the same manner as in (2) of Can Example 1, and then transformed, and a transformed strain (power strain) having kanamainone resistance was obtained. The transformed strain KY13184 The APTa se activity of /pEF12 was measured in the same manner as in Example 1 r.1). The results are shown in Table 6. Furthermore, 5'-ynonic acid was determined from the transformed strain KY1.3184/pEF12 by paper chromatography in the same manner as in Example 1 (5). The amount of 5'-inosinic acid accumulated is shown in Table 7. KY13 as a control strain
The same procedure was carried out using 184.

Table 6 KY13184 7. I KYi3184/pEFI2 1.0.4 No. 7
Omotekan strain 5'-inosinic acid <g/l> KYI3
1f14 20. IKY131.
84/pEF12 24.9Introduction of pEF 12 increased APTase activity, 5'
- Improvement in inosinic acid productivity was observed.

Example 3 Using the recombinant plasmid pEF 12 isolated and purified in (3) of Example 1, Brevibacterium ammoniagenes ATCC21 having the ability to produce 5'-xanthylic acid was produced.
075 was transformed. Transformation was carried out in the same manner as in Example 1 (2) except that protoplasts were prepared at a penicillin concentration of 0.5 U/ml, and a transformed strain that acquired kanamycin resistance was obtained. PTa of the transformed strain ATCC21075/IFεF12
Measurement of se activity was carried out in the same manner as in Example 1 (4). As a control, eight TCC21075 strains were used and treated in the same manner. The results are shown in Table 8.

Table 8 ATC ro 21075
8.9ATCro21θ75/pEF12
1 By introducing the recombinant plasmid pEF 12 containing 4.7pN1rF, APTase
An increase in activity was observed.

Next, the transformed strain, TCC2+, (175/pEFI
The 5'-xanthylic acid producing ability of No. 2 was investigated. Brevibacterium ammoniagenes ATCC21075 and
TCC21, O75/pEF 12 to NB medium 20m
A loopful of 25 Qml containing 1 was inoculated into each flask and cultured at 30°C for 24 hours.
The cells were inoculated into a 5Qml flask at a ratio of 10% (volume f ratio), and cultured at 30°C for 4 days with shaking at 22Orpm. Separately sterilized urea was added at 48 and 72 hours during incubation.
g/f! , to adjust the DH. Accumulated 5'-xanthylic acid was quantified by paper chromatography. The results are shown in Table 9.

Table 9 Strain 5'-xanthylic acid (g/l2) AT
C(:21075 14.8^TCC2
1075/i'1EF12 19.5purF
By introducing the recombinant plasmid pEF 12 containing pEF 12, an improvement in the productivity of 5'-xanthylic acid was observed.

Effects of the Invention By the method of the present invention, a recombinant DNA containing the gene of APTase, which is an enzyme involved in the main synthesis of purine nucleotides, and a Corynebacterium genus or Prehybacterium harboring the recombinant DNA can be obtained. It is possible to provide a microorganism belonging to the genus P. genus and a method for producing a nucleic acid-related substance using the microorganism.

Claims (5)

[Claims] (1) Contains recombinant DNA of a DNA fragment carrying genetic information involved in the synthesis of microbial amidophosphoribosyl transferase and vector DNA, belongs to the genus Corynebacterium or Brevibacterium, and is related to nucleic acids. 1. A method for producing a nucleic acid-related substance, which comprises culturing a microorganism capable of producing a substance in a medium, producing and accumulating a nucleic acid-related substance in the culture, and collecting the nucleic acid-related substance from the culture. (2) The production method according to claim 1, wherein the DNA fragment is a DNA fragment obtained from a microorganism belonging to the genus Escherichia, Corynebacterium, or Brevibacterium. (3) Claim 1, wherein the nucleic acid-related substance is inosine, inosinic acid, or xanthylic acid.
Manufacturing method described in section. (4) Recombinant DNA of vector DNA and a DNA fragment carrying genetic information involved in the synthesis of amidophosphoribosyl transferase of a microorganism belonging to the genus Escherichia, Corynebacterium, or Brevibacterium
A. (5) Recombinant DNA of a DNA fragment carrying genetic information involved in the synthesis of amidophosphoribosyl and transferase of a microorganism belonging to the genus Escherichia, Corynebacterium, or Brevibacterium and vector DNA
A microorganism that possesses A, belongs to the genus Corynebacterium or the genus Brevibacterium, and has the ability to produce nucleic acid-related substances.