KR19990074792A - Method for preparing 5'-guanylic acid by recombinant microorganism - Google Patents

Abstract

The present invention provides the production of 5'-guanylic acid using a recombinant plasmid constructed by genetic engineering of a gene derived from glutamate producing bacteria to activate intracellular ATP-regeneration system in a microorganism of the genus Brevibacterium. A method for increasing yield and a host-vector system used therein.

Description

Method for producing 5'-guanylic acid by recombinant microorganism

The present invention relates to coryneform glutamate producing bacteria (eg, Brevibacterium flavum, Brevibacterium lactofermetum, Brevibacterium ammoniagenes, or Corynebacterium) Novel recombinant plasmid vectors capable of replicating in Corynebacterium glutamicum, coryneform glutamate producing strains transformed with these vectors, and methods for preparing 5'-guanylic acid (hereinafter referred to as "GMP") using them will be.

GMP is one of the important items in the food industry because it is directly used as a seasoning with monosodium glutamate (MSG) and 5'-inosinic acid (IMP) and is required as a food additive in seasoning foods.

Known methods for producing GMP include (1) enzymatically degrading ribonucleic acid extracted from yeast cells, (2) chemically phosphorylating guanosine produced by microbial fermentation, and (3) produced by microbial fermentation. Method of converting 5'-xanthyl acid (XMP) to GMP using microorganisms (J. Ferment. Technol. Vol. 62, No. 2, pp. 131-137), (4) Direct GMP by microbial fermentation There are production methods, among which the method of (4) has a very low membrane membrane permeability of GMP and cannot be used industrially. The method of (3) is the most widely used industrially because of the low production cost. .

As in the method of (3), a method of preparing GMP from XMP using enzyme cells involves an enzyme called XMP aminase, and its enzymatic reaction mechanism can be expressed by the following reaction formula.

Methods for enhancing the activity of XMP aminase enzymes can be conferred to microorganisms by the resistance to the enzyme, the specific inhibitor of tecoinin (Biotechnol. Bioengineer. Vol. 13, 229-240, 1971, Vol. 16, 795). -803, 1973, Korean Patent Application No. 87-5453), Method of using a transformant transformed with recombinant DNA consisting of a vector fragment and a DNA fragment containing a gene (guaA) encoding an XMP aminase (published in Japan) Patent Publication No. 85-224498) and the like are known.

In addition, since ATP, which is another substrate used in the method of (3), is expensive, there has been a demand for the development of an economical process capable of producing GMP by enzymatic reaction in a method involving the addition of ATP. The GMP synthesis reaction system and the reaction system regenerating 5'-adenylic acid (AMP) in parallel produce ATP repeatedly, eliminating the need to add ATP to the reaction process. In other words, when adenine (Adenine), a substrate for ATP regeneration, is supplied into the medium, adenine enters the cells and receives ribosyl-phosphate group from PRPP (5'-phosphoribosyl-1'-pyrophosphate), which is one of the metabolites in vivo. Is converted to AMP, followed by a reaction system for regenerating ATP from the resulting AMP (hereinafter referred to as "ATP-regeneration system") and a reaction system for generating GMP from XMP, ammonia and / or glutamine, and ATP (hereinafter " The reaction of the enzymatic production of GMP from XMP can be carried out using a conjugated reaction in combination with a "conversion reaction system". As the ATP-regeneration system used in the conjugated reaction, a reaction system capable of regenerating ATP using a low-cost substrate is suitable. Among them, systems using saccharides or other carbohydrates such as glucose in cells may be economical in producing GMP.

In view of this, methods using coryneform microorganisms or E. coli have been developed (Japanese Patent Laid-Open No. 85-224498). In this method, a method using two microorganisms having a single activity and the activity required in two reaction systems, that is, the activity of converting XMP to GMP and the activity of regenerating ATP are dependent on the same one microorganism It can be roughly divided into. The method using one microorganism having two activities simultaneously as an enzyme source for two activities has the advantage that the reaction system is simpler than the method using two microorganisms having a single activity. However, in the case where the two activities constituting the reaction system cannot be controlled separately from each other, there is a problem that it is difficult to maximize the productivity of adjusting the balance between the two activities to an optimal level.

In addition to the problems mentioned above, the methods described in the prior art still have problems which must be further improved. That is, in the conventional method, ATP-regeneration activity is a limiting step when reducing the amount of microorganisms with increasing XMP aminase activity. Therefore, in order to enhance the productivity of GMP by reinforcing the deficiency of ATP-regeneration activity, there is a problem that a relatively large amount of microorganisms involved in ATP regeneration should be used.

When E. coli is used, strains with markedly increased XMP aminase activity can be obtained and utilized by genetic recombination techniques. As a result, GMP productivity was markedly increased (Japanese Laid-Open Patent Publication No. 85-224498). However, this method still entails the problem of requiring a relatively large amount of microorganisms since ATP-regeneration activity also acts as a limiting step.

In addition, industrial XMP producing bacteria have the properties of guanine-urea and lack XMP aminase, an XMP metabolic enzyme involved in purine nucleotide biosynthesis reactions, but their ATP-regenerating activity is generally strong. Efforts have been made to utilize the potent ATP-regeneration activity of XMP producing microorganisms as an ATP-regeneration system in converting the reaction system from XMP to GMP. That is, a method of producing GMP by separating the two activities of the ATP regeneration system and the conversion reaction system by using the waste cells of the XMP producing bacteria as an enzyme source having ATP-regeneration activity is known (Biosci. Biotech. Biochem. Vol. 61 (5), 840-845, 1997). However, there is still a problem that it is difficult to maximize the productivity by adjusting the balance between the two activities (ATP-regeneration system, conversion reaction system) to the optimum level as described above.

The present invention improves APT regeneration ability by introducing genes encoding enzymes that catalyze each step of the ATP production reaction into a specific vector system developed by the present inventors in view of the problems of the prior art as described above. To provide a host-vector system and to provide a method for producing 5'-guanylic acid using such a microbial system.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the construction of the recombinant plasmid pMPSE82 and its structure according to the present invention.

Important factors in GMP production include: ① First, XMP-producing strains capable of producing high concentrations of XMP must be developed, and ② The XMP fermentation process must be industrialized. As a next step, the development of microorganisms possessing a powerful enzyme that converts from XMP to GMP and the industrialization of the conversion process are the most important in the manufacture of GMP. Especially in the latter stage, the potent converting enzyme strain from XMP to GMP and APT (adenosine 5'-phosphate) -regenerating ability of the converting enzyme strain are particularly important for GMP production.

The present inventors use a microorganism transformed with a DNA fragment containing a guaA gene encoding XMP aminase as an enzyme source and an ATP source, that is, one microorganism having two activities simultaneously as an enzyme source for two activities. In order to solve the problem of the ATP-regeneration step, which can be called a limiting step of the method using the method has been studied. In particular, the inventors of the present invention, among the genes encoding the enzyme catalyzing each step of the ATP production reaction, E. coli or Corynebacterium glutamicum strain of prs (PPRP synthase), apt (adenine phosphoribosyl transferase) The base sequences of, adk (adenylate kinase) and pyk (pyruvate kinase) were found by searching the Genenbank Database (NCBI; National Center for Biotechnology Information), and these E. coli or Corynebacterium glutamicum (Corynebacterium) After obtaining DNA fragments by PCR method using chromosomal DNA of glutamicum) strain, these DNA fragments were respectively prepared by the inventors of the guaA gene-containing plasmid pGX1-2 (Korean Patent Application Publication No. 97-43051). ). As a result of performing a GMP conversion reaction using the transformants transformed into the recombinant plasmids thus prepared, when the transformants transformed with the recombinant plasmid containing the prs gene were used, ATP-regeneration of the cells was performed. Enhancement of the ability to confirm that the GMP conversion reaction is significantly improved and came to complete the present invention.

For ATP-regeneration, the stepwise progression from the externally supplied adenine to the generation of ATP can be defined as in the following scheme.

That is, adenine supplied from the outside as a medium component receives ribose-phosphate groups from PRPP to form AMPs, and the AMPs thus generated are converted to ADP by receiving phosphate groups from ATP by adenylate kinase. ADP receives phosphate groups from high-energy phosphate compounds (acetyl-phosphate, phosphoeno pyruvate, etc.) present in vivo, and finally ATP is produced. At this time, PRPP, which delivers a ribose-phosphate group to adenosine, is produced by an enzyme called PRPP syntase and is a very important intermediate product in vivo that is involved in amino acid biosynthesis, pyrimidine metabolism, etc. in addition to the purine nucleotide metabolism process. .

E. coli-derived prs gene used in the present invention is registered in the Genebank database with the registration number of M13714, its nucleotide sequence is as shown in SEQ ID NO: 1, the PRPP synthase enzyme encoded by the gene is ribose Catalyzes the reaction to generate PRPP, a precursor of purine nucleotide biosynthesis from -1'-phosphate and ATP (adenosine 5'-triphosphate).

To clone the prs gene from the prs-containing gene sequence, the following two primers (SEQ ID NO: 1 and SEQ ID NO: 2) were prepared, and these primer sequences and the chromosomal DNA of E. coli W3110 were used as templates. PCR reaction was performed.

The obtained DNA fragment is introduced into the recombinant plasmid pGX1-2 containing two copies of the guaA gene developed by the present inventors (Korean Patent Application Publication No. 97-43051). By using the transformant transformed with pMPSE82, the recombinant plasmid thus prepared, the conversion efficiency of GMP is increased by performing the GMP conversion reaction.

The present inventors isolated the cryptic plasmid pCG1 from the Corynebacterium glutamicum strain ATCC13058 strain for the development of a host-vector system of Corynebacterium glutamate producing bacteria, and then the pACYC177 cloning vector of Escherichia coli. In conjunction with the Corynebacterium and Escherichia coli, pECCG1, a shuttle vector that can be transformed and expressed, was removed from the vector, and the ampicillin resistance gene was removed from the vector and the multiple cloning site of the plasmid pBluescript II KS +. Was introduced to develop a shuttle vector pECCG117 that is stably expressed in strains of the genus Corynebacterium (see Korean Patent Publication No. 92-7401). The shuttle vector pECCG117 is capable of expressing the gene of interest in the microorganisms of Brevibacterium because the expression and stability are maintained not only in strains of Corynebacterium but also strains of Brevibacterium, a strain of coryneform glutamate. May be advantageously used to prepare vectors

Examples of XMP producing bacteria used in the present invention are Brevibacterium ammoniagenes D1550-40 (Korean Patent Publication No. 78-344) and its mutants, and a considerable amount of these microorganisms are cultured by culturing in a conventional culture method. XMP can be accumulated in the medium.

That is, the above-mentioned microorganisms are cultured while controlling temperature and pH increase under aerobic conditions in a conventional medium containing a suitable carbon source, nitrogen source, inorganic substance, amino acid, vitamin, and the like.

As the carbon source, various organic acids such as carbohydrates such as glucose, fructose, sucrose and maltose, mannitol sorbitol, glycerol and the like and various amino acids such as glutamic acid, methionine, lysine and the like can be used. Furthermore, natural organic nutrients such as starch hydrolyzate, molasses, corn steep liquor and the like may be used. Nitrogen sources that can be used include various inorganic and organic ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium acetate and the like, amino acids such as glutamic acid, glutamine, methionine, and the like, and peptone, N2-amine, corn steep liquor, meat extract, casein Nitrogen-containing organic substances such as hydrolyzates and the like. In addition, as an inorganic substance, potassium dihydrogen phosphate, sodium monohydrogen phosphate, magnesium sulfate, sodium chloride, calcium chloride, iron chloride, copper sulfate, manganese chloride, ammonium molybdate, zinc sulfate and the like may be added to the medium as necessary. Vitamins, amino acids, nucleic acids, etc. necessary for microbial growth can be added to the medium. Of course, if they are supplied as the other medium components described above, there is no need to add these specific nutrients separately.

In general, the culturing is carried out under aerobic conditions, for example, by stirring under shaking or aeration conditions. Preferred incubation temperatures are generally 25-35 ° C. and the pH of the medium is preferably maintained near neutral during incubation. Incubation time is usually 10 to 120 hours is preferred.

The XMP fermentation broth obtained as a result of the culture is slightly sterilized (eg, 80 ° C., 5 minutes) to inactivate the XMP cells and then culture the microorganisms having the XMP aminase activity.

The XMP-> GMP converting strains usable in the present invention are any microorganisms with XMP aminase activity that produce GMP from XMP, ammonia and / or glutamine and ATP. Suitable examples of these strains include E. coli and Coryneform microorganisms that can be transformed by the recombinant plasmids prepared above. Specific examples include Brebibacterium ATCC6872, Corynebacterium glutamicum (Corynebacterium). glutamicum) ATCC13059, E. coli. In addition to these strains, strains of these strains that have increased XMP aminase activity using conventional artificial variations, such as drug-resistant variations, can also be used.

The above-mentioned microorganisms are cultured by adjusting the temperature, pH and the like under aerobic conditions in a conventional medium containing a suitable carbon source, a suitable nitrogen source, an inorganic substance, amino acids, vitamins and the like.

As the carbon source used in the medium, carbohydrates such as glucose, fructose, sucrose, maltose and the like, various organic acids such as mannitol sorbitol and glycerol and various amino acids such as glutamic acid, methionine, lysine and the like can be used. Furthermore, natural organic nutrients such as starch hydrolyzate, molasses, corn steep liquor and the like may be used. Nitrogen sources that can be used include various inorganic and organic ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium acetate and the like, amino acids such as glutamic acid, glutamine, methionine, and the like, and peptone, N2-amine, corn steep liquor, meat extract, casein Nitrogen-containing organic substances such as hydrolyzates and the like. In addition, as an inorganic substance, potassium dihydrogen phosphate, sodium monohydrogen phosphate, magnesium sulfate, sodium chloride, calcium chloride, iron chloride, copper sulfate, manganese chloride, ammonium molybdate, zinc sulfate and the like may be added to the medium as necessary. Vitamins, amino acids, nucleic acids, etc. necessary for microbial growth can be added to the medium. Of course, if they are supplied as the other medium components described above, there is no need to add these specific nutrients separately.

In general, the culture is carried out under aerobic conditions, for example, by stirring under shaking or aeration conditions. Preferred incubation temperatures are generally 25-35 ° C. and the pH of the medium is preferably maintained near neutral during incubation. Incubation time is typically 10-120 hours.

Ammonia and / or glutamine and phosphate ions, magnesium ions and surfactants, and / or organic solvents were adjusted to pH 7-8 in an XMP fermentation broth containing the obtained conversion cells, and the temperature was maintained at 30 to 50 ° C. By addition as needed for 48 hours.

As the ATP-regenerating substrate, carbohydrates such as glucose, arabinose, lactose, maltose, sucrose, mannitol, sorbitol, trehalose, starch hydrolyzate, pyruvic acid, lactic acid, acetic acid, 2-ketoglu as long as XMP producing bacteria are available Organic acids such as taric acid and the like, amino acids such as glycine, alanine, aspartic acid, glutamic acid and the like can be used. It is also possible to use phosphorylated compounds such as sodium or potassium salts such as acetyl phosphate, carbamyl phosphate and creatine phosphate.

As the surfactant, cationic surfactants, anionic surfactants, amphoteric surfactants and the like can be used, and these are usually used at a concentration of 1-20 mg / ml.

During the conversion of XMP to GMP, it is appropriate to keep the concentration of phosphate ions and magnesium ions within the range of 4-400 mM. If the content of these ions provided in the conversion system by the medium or cells is within the above-mentioned concentration, it is not necessary to add these ions to the conversion system, while if the content of these ions is insufficient, it is not within the above-mentioned range of serf These ions should be added as much as possible. As the phosphate ion, any salt such as sodium salt, potassium salt, magnesium salt and the like of phosphoric acid can be used, and any inorganic and organic salts can be used as the magnesium salt. As ammonia and / or glutamine sources, any ammonia gas, various inorganic and organic ammonium salts, glutamine and yeast extracts, glutamine containing natural products such as casamino acid, corn steep liquor and the like can be used.

Recovery of GMP from the culture can be carried out using activated carbon, ion exchange resins and the like according to conventional methods.

<Example>

Although an Example and a comparative example demonstrate this invention further in detail below, the scope of the present invention is not limited to these.

<Example 1>

Selection and cloning of enzymes involved in ATP-regeneration system

(1) Selection of enzymes involved in ATP-regeneration system

In order to make ATP from the adenine supplied in the medium, as shown in Reaction 2 in the above-mentioned cells, the ribosil-phosphate group is delivered from PRPP and the AMP is converted to ADP after receiving the phosphate group from ATP. In addition, high energy phosphate groups are transferred from other high energy compounds in vivo (eg, acetyl phosphate, phosphophenol pyruvate) and converted to ATP. Genes encoding enzymes that catalyze each step of this series of ATP production pathways: the prs (PRPP) gene, which catalyzes the synthesis of PRPP, and the apt (adenosine phosphoribosyl transferase) gene, which transfers ribosyl-phosphate groups from PRPP to adenine , Genebank databse, such as adk (adenosyl kinase) gene to form ADP from AMP and pyk (pyruvate kinase) gene to produce ATP in the most important glycolysis pathway of carbon metabolism for substrate-level phosphorylation As a result, the prs, adk, and apt genes have been reported in Escherichia coli, and there are no reports from coryneform microorganisms. Therefore, the E. coli gene was cloned, and the pyk gene was cloned from the E. coli and Corynebacterium glutamicum strains and cloned from the Corynebacterium glutamicum strain. Cloning was performed as follows.

Corynebacterium glutamicum strain ATCC13032 was incubated for 16 hours in LB medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride) at a temperature of 32 ° C., followed by centrifugation. Was recovered. It was washed with 10 ml of 1 mM Tris buffer (pH 8.0), followed by 40 ml of lysozyme reaction buffer (10 mM Tris, 5 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.5 M sucrose, 10 mg / ml lysozyme , pH 8.0), and then treated at 37 ° C. for 90 minutes. The cells were collected by centrifugation, resuspended in the same solution without 10 ml of sucrose and lysozyme, and 0.6 ml of 0.5 M EDTA and 4.4 ml of 4% SDS were added to digest the cells. 1 g / ml of cesium chloride was added thereto, and 2 ml of ethidium bromide concentrate (10 mg / ml) was mixed. The mixture was centrifuged at 50,000 rpm for 48 hours at 16 ° C. using an ultra-centrifuge. The chromosomal DNA was taken using a syringe. This was treated with isopropyl alcohol 3-4 times to remove ethidium bromide, and then dialyzed in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) to separate and purify chromosomal DNA.

Chromosome DNA of Escherichia coli K-12 was also isolated and purified according to the process diagram as shown in FIG. 1 using the same method as above.

(2) Cloning of ATP-regenerating related genes

In order to clone the genes of enzymes involved in the ATP-regeneration pathway, the genebank database was first searched to obtain the nucleotide sequences of the genes. As a result, the base sequence was registered with the accession number of Escherichia coli x03038, Escherichia coli prs gene M13174, Escherichia coli apt gene M38777, Corynebacterium glutamicum strain pyk gene L27126. It could be confirmed. Primer DNAs (SEQ ID NOs: 1 and 2) were prepared to include all of the structural genes and the promoter sequences of the structural genes upstream in the respective nucleotide sequences, and then the primers prepared in this manner and E. coli or Coryne PCR reactions were performed using chromosomal DNA of the bacterium glutamicum strain as template DNA. At this time, the thermophilic DNA polymerase used was Vent DNA polymerase (commercially available from New England Biolab, NEB, USA) or Pfu DNA polymerase (obtained from Boehringer Mannheim, Germany). Molecular Cloning; A laboratory manual, 2nd ed, Sambrrok, J., EF Frisch, and T. Maniatis.

The DNA fragments thus obtained were mixed with the SmaI cleavage DNA of pUC18, the cloning vector of each E. coli, treated with T4 DNA ligase, and transformed into E. coli. The restriction map of each of the resulting DNAs (insert DNA) of the resulting recombinant plasmids confirmed that the DNA fragments were correct and also confirmed the orientation of the DNA fragments. In this way, each of the introduced DNA fragments were cut and isolated by restriction enzymes BamHI and KpnI, and then the isolated DNA fragments were each synthesized by the inventors of two copies of the recombinant plasma pGX1-2 containing guaA gene. Korean Patent Application Publication No. 97-43051) was mixed with a mixture digested with restriction enzymes BamHI and KpnI, treated with T4 DNA ligase, and transformed into E. coli. The resulting recombinant plasmid was named pMADE8 (containing the adk gene of E. coli), pMAPE18 (containing the apt gene of E. coli), pMPKC38 (containing the pyk gene of Corynebacterium), and pMPSE82 (containing the prs gene of E. coli), respectively. It was.

<Example 2>

Conversion of XMP to GMP by Brevibacterium Transgenic Strains

Plasmid DNA was isolated from the transformants containing the recombinant plasmids obtained in Example 1 by a conventional method, and the Brevibacterium ammonia genes strain BA 17-2 (XMP aminase-enhanced mutants, Korean Patent Publication No. 78 The transformation of XMP to GMP was performed using the transformants obtained by the transformation of the present invention. 30 ml each of 500 ml shaking Erlenmeyer flasks using C medium as a seed culture medium and autoclaving at 115 ° C. for 15 minutes were used to transform strains BA17-2 / pMADE8, BA17-2 / pMAPE18, BA17-2 / pMPKC38, And BA17-2 / pMPSE82 bacteria were inoculated by one platinum and then incubated at 30 ° C. for 24 hours to use as a seed solution. Fermentation medium 1.5 L (glucose 100 g / L, potassium phosphate 1,2,10 g / L, magnesium sulfate 10 g / L, calcium chloride 0.1 g / L, iron sulfate 10 mg / L, manganese sulfate 2 mg / L) , 10 g / l yeast extract, 10 g / l casein hydrolyzate, 30 μg / l d-biotin, 10 mg / l adenine) into a 5 L fermenter and autoclaved at 120 ° C. for 30 minutes, followed by seed culture. The cells were inoculated at% dose and incubated at 30 ° C. for pH 24 for 24 hours. At this time, the rotation speed was 750 rpm and the air addition speed was 1 vvm.

The sterile XMP fermentation broth was added to the cultured enzyme cell-containing solution so that the final concentration was 40 mg / ml, and the reaction was carried out for 24 hours while maintaining the pH at 7.0 to 8.0 at 35 to 50 ° C. BA17-2 / pGX1-2 strain was used as a control. Substances such as magnesium salt, phosphate, ammonium sulfate or glutamine required for the conversion reaction were added in an appropriate amount, and adenine and glucose were added together for ATP regeneration by enzyme cells. The conversion reaction was initiated by adding an appropriate amount of surfactant (SDS, hyamine, etc.). Table 1 shows the conversion of GMP over time during the reaction.

TABLE 1

Conversion of GMP Over Time by Transformants

Strain Conversion rate over response time 1 hours 2 hours 3 hours 4 hours 6 hours BA17-2 / pGX1-2 16 29 47 62 91 BA17-2 / pMPSE 25 42 73 92 -

As can be seen from the results of the above table, in the case of a host transformed with a vector containing a gene encoding an enzyme that catalyzes each step of the ATP production pathway according to the present invention, the rate of XMP-> GMP conversion reaction in the reaction is a control group. It can be seen that the increase significantly (about 50%) compared to the case.

The vector-host system developed according to the present invention can increase the rate of XMP-> GMP conversion reaction significantly compared to the conventional one can provide a cost-effective method for producing 5'-guanylic acid.

<Sequence list>

SEQ ID NO: 1

Length of sequence: 1741

Type of sequence: nucleic acid

Number of chains: 2 printing

Topology: Straight

Type of sequence: chromosomal DNA

Origin: E. coli

SEQ ID NO: 2

Sequence length: 24

Type of sequence: nucleic acid

Number of chains: two prints

Topology: Straight

Type of sequence: other synthetic DNA

Characteristic of the sequence

Characteristic symbol: primer

Presence Location: 1..24

CGCGCAATAC GCCAAAAAGG TCAA 24

SEQ ID NO: 3

Sequence length: 24

Type of sequence: nucleic acid

Number of chains: two prints

Topology: Straight

Type of sequence: other synthetic DNA

Characteristic of the sequence

Characteristic symbol: primer

Presence Location: 1..24

GGGTA AGCAA CTAAT CCGAC GGTT 24

Claims (5)

Group consisting of Brevibacterium flavum, Brevibacterium lactofermetum, Brevibacterium ammoniagenes, and Corynebacterium glutamicum A recombinant plasmid vector containing a gene for enhancing ATP-regeneration in a genus of microorganisms of the genus Brevibacterium, which is a coryneform glutamate producing bacterium selected from among. The vector of claim 1, wherein the gene capable of enhancing ATP-renewal ability is prs (PRPP synthase). The vector of claim 1, wherein the vector contains two guaA (XMP aminase) genes. Preparation of 5'-guanylic acid characterized by reacting XMP with a culture of microorganisms of Brevibacterium, a coryneform glutamate producing strain transformed with a recombinant plasmid vector containing a gene that enhances ATP-regeneration ability Way. The microorganism of claim 5, wherein the microorganism of the genus Brevibacterium is Brevibacterium flavum, Brevibacterium lactofermetum, Brevibacterium ammonia genes ), And Corynebacterium glutamicum.