KR20100044302A - Microorganisms of corynebacterium having enhanced nucleotide or nucleoside productivity and method of producing nucleotide or nucleoside using the same - Google Patents

Abstract

PURPOSE: A Corynebacterium sp. microorganism having improved nucleic acid material is provided to strengthen enzyme activity and reduce production cost. CONSTITUTION: A Corynebacterium sp. microorganism has improved nucleic acid material by strengthening enzyme activity of purine biosynthesis pathway. The enzyme is phosphoribosyl pyrophosphate amidotransferase, phosphoribosylamine glycine ligase, phosphorybosylglycine amide formyltransferase, phosphoribosylformylglycine amidine synthetase, or adenylosuccinate lyase. A gene encoding the enzymes is purF, purD, purN, purS, purL, purM, purKE, purC, purB or purH.

Description

MICROORGANISMS OF CORYNEBACTERIUM HAVING ENHANCED NUCLEOTIDE OR NUCLEOSIDE PRODUCTIVITY AND METHOD OF PRODUCING NUCLEOTIDE OR NUCLEOSIDE USING THE SAME}

The present invention relates to an important enzyme in the purine biosynthesis pathway, i) phosphoribosylpyrophosphate amidotransferase, ii) phosphoribosylamine glycine ligase, iii) phospholibosylglycinamide Phosphoribosylglycinamide formyltransferase, ⅳ) phosphoribosylformylglycinamidine synthetase, ⅴ) phosphoribosylformylglycine amidine synthetase Ⅱ (phosphoribosylformyl phosphobosylformyl lysyl phosphinyl phosphinbo) Phosphoribosylaminoimidazole synthetase (ⅶ) phosphoribosylaminoimidazole carboxylase (ⅷ) phosphoribosylaminoimidazole carboxylase (ⅷ) phosphoribosylaminoimidazole carboxylase (phosph) Adenillo Cinnamic acid microorganisms with enhanced activity of enzymes selected from the group of adenylosuccinate lyase and iii) inosinic acid cyclohydrolase and 5'-xanthyl acid or 5'-inosinic acid or To a method of producing inosine.

One of the nucleic acid-based materials, 5'-guanylic acid (hereinafter referred to as GMP) is widely used as a food seasoning additive, and is known to taste mushrooms by itself, but mainly monosodium glutamic acid It is known to enhance the flavor of (MSG). This property is particularly strong when used with 5'-inosinic acid (hereinafter referred to as IMP).

Known methods for manufacturing GMP include (1) a method of digesting ribonucleic acid (RNA) extracted from yeast cells with enzymes, (2) a direct fermentation of GMP by microbial fermentation, and (3) a guanosine produced by microbial fermentation. Phosphorylation by chemical method, (4) enzymatic phosphorylation of guanosine produced by microbial fermentation method, (5) 5'-xanthylic acid (XMP) produced by microbial fermentation method The method of converting into GMP using Nebacterium microorganism, (6) The method of converting XMP produced by microbial fermentation into GMP using E. coli. The method of (1) has a problem in the supply and demand of raw materials, and the method of (2) has a disadvantage of low yield due to the problem of GMP cell membrane permeability. Therefore, other methods are mainly used industrially.

If GMP is produced by producing XMP and converting it to GMP, the strategy for enhancing the productivity of XMP is needed. For this reason, conventional Corynebacterium strains with enhanced genes related to XMP biosynthesis and XMP production methods using the same have been known. For example, Korean Patent No. 0117443 discloses a highly sensitive susceptible strain of lysozyme, which is resistant to guanosine analogues and is a guanosine analogue inactive strain of adenine and guanine antireflective forms to increase XMP yield. Was prepared and started. Korean Patent No.0402320 discloses a mutation causing agent such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) based on corynebacterium ammoniagenes, which produces XMP, as a parent. By processing according to conventional methods, the strains of the parent strain are modified to select norvaline resistant strains, which are analogues to valine, which affect the biosynthesis of XMP, resulting in high yield and high concentration of XMP. An XMP production method using microorganisms that accumulate directly in culture is disclosed.

IMP, another nucleic acid-based substance, is an intermediate in nucleic acid biosynthetic metabolism. It plays a physiologically important role in the body of animals and plants, and is used in various fields such as food, medicine, and various medical uses. When used together with sodium glutamic acid, it is one of nucleic acid-based seasonings that has been spotlighted as a seasoning seasoning because of its synergistic effect.

Methods for producing IMP include a method of enzymatically decomposing ribonucleic acid extracted from yeast cells (Japanese Patent Publication No. 1614/1957, etc.), and a method of phosphorylating inosine produced by fermentation by chemical method (Agri. Biol. Chem , 36, 1511 (1972), and the like, and methods of culturing microorganisms capable of producing IMP and recovering IMP accumulated in the medium. The most widely used method among these methods is a method of producing IMP using microorganisms. Among the microorganisms used to produce IMP, a method of preparing by fermenting a strain of the genus Corynebacterium, for example, Corynebacterium ammonia genes (Korean Patent No. 0446113) is known.

Inosine, another nucleic acid-based substance, is an important substrate for the chemical synthesis of IMP, which has been spotlighted as a seasoning seasoning, and the synthesis by enzyme transfer reaction. Inosine is an intermediate of the nucleic acid biosynthesis metabolic system and has a physiological significance in the body of animals and plants, and is used in various fields such as food, medicine, and various medical uses. In order to produce such inosine, Bacillus (Agric. Biol. Chem., 46, 2347 (1982); Korean Patent No. 27280) or Corynebacterium ammonia genes (Agric. Biol. Chem., 42, 399 (1978)) and direct fermentation using microorganisms or pyrolysis of IMP (Japanese Patent Application No. 43-3320). However, in the case of pyrolysis, a large amount of heat is required to decompose IMP, and practicality is a problem, and the method of direct fermentation produces and studies various genotype strains using Escherichia coli (Biosci Biotechnol Biochem. 2001 Mar; 65 ( 3): 570-8), but the production cost is high due to the low titer of the inosine production strain. In addition, existing inosine studies are concentrated on Escherichia coli and Bacillus bacteria, and development of inosine-producing strains using Corynebacterium ammonia gene strains for higher yield and concentration has been reported (Korea Patent No. 0330705, No. 0671080 and 0671081).

Conventional Corynebacterium strains with enhanced genes related to purine biosynthesis and IMP or XMP production methods using the same have been known. For example, Korean Patent No. 085248 discloses a microorganism overexpressing the gene purC encoding phosphoribosylaminoimidazole leucinecarboxamide synthetase and a method of producing IMP using the same. In addition, the Republic of Korea Patent No. 0578379, a microorganism Corynebacterium ammonia gene strain overexpressing the phosphoribosylaminoimidazole carboxylase gene encoded by purKE and breeding and cultivating it to produce IMP in high concentration and high yield A method is disclosed. In addition, Korean Patent Laid-Open No. 10-2007-0056491 discloses a Corynebacterium ammonia genes strain enhanced with phosphoribosylpyrophosphate amidotransferase enzyme encoded by purF and a method for preparing XMP using the same It is. In addition, in Korean Patent Application No. 10-2008-006537 (unpublished), the activity of phosphoibosylglycine amide formyl transferase encoded by purN and inosine acid cyclohydrolase enzyme encoded by purH is enhanced than intrinsic activity. And, preferably, further relates to a microorganism of the genus Corynebacterium in which 5'-nucleotidase is inactivated, and a method for producing a high concentration of XMP using the same.

An object of the present invention is to provide a microorganism of the genus Corynebacterium that can produce a high concentration of nucleic acid-based material XMP or IMP or inosine.

Still another object of the present invention is to provide a method of culturing the microorganisms of Corynebacterium and producing XMP or IMP or inosine from the culture solution.

In order to achieve the above object, the present invention is a phosphoribosyl pyrophosphate amidotransferase, phosphoribosylamine glycine ligase, phosphoribosyl glycine amide formyl transferase, phosphoribosyl formyl glycine amidine syntheta Azedes, phosphoribosylformylglycineamidine synthetase II, phosphoribosylaminoimidazole synthetase, phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole leucinocarboxamide synthetase, adenyl Enhancing the enzymatic activity of rotoxylate lyase and inosine acid cyclohydrolase to enhance the activity of the purine biosynthetic pathway, thereby providing a microorganism of the genus Corynebacterium with improved production of XMP or IMP or inosine.

The present invention also provides a method of culturing the microorganism of the genus Corynebacterium, to produce XMP or IMP or inosine from the culture.

In accordance with the present invention, by enhancing the activity of enzymes in the purine biosynthetic pathway of Corynebacterium sp. Microorganisms, production of XMP or IMP or inosine at high concentrations can be achieved to reduce production costs by improving yield.

The present invention is phosphoribosylpyrophosphate amidotransferase, phosphoribosylamine glycine ligase, phosphoribosylglycinamide formyltransferase, phosphoribosylformylglycineamidine synthetase, phosphoribosylformylglycineamidine Synthetase II, phosphoribosylaminoimidazole synthetase, phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole rosinocarboxamide synthetase, adenylolycinate lyase and inosinoic acid cyclase Enhancing the enzymatic activity of the rollases enhances the activity of the purine biosynthetic pathway, thereby providing microorganisms of the genus Corynebacterium with enhanced production of XMP or IMP or inosine.

More preferably, the present invention relates to phosphoribosylamine glycine ligase, phosphoribosylglycine amide formyltransferase, phosphoribosylformylglycine amidine synthetase, phosphoribosylformylglycine amidine synthetase II, ade Microorganisms of the genus Corynebacterium in which activity of genes encoding nilorotinate lyase and inosinic acid cyclohydrolase has been enhanced, in addition to phosphogibosylpyrophosphate amidotransferase and phosphoribosyl Genes encoding aminoimidazole synthetase or genes encoding phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazole leucinocarboxamide synthetase have been introduced to enhance activity. Provide microorganisms. In addition, all of the genes encoding the above-mentioned enzymes are introduced to provide a microorganism of the genus Corynebacterium with enhanced activity.

Nucleotide sequences of the genes purF, purD, purN, purS, purL, purM, purKE, purC, purB and purH are already known, and SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, respectively. And 44 nucleotide sequences. Preferably, the genes may be derived from Corynebacterium ammonia genes CJIP2401 (KCCM-10610).

In the present invention, the endogenous gene refers to a gene that the microorganism of Corynebacterium has in its natural state, and the method of enhancing the activity of the enzymes increases enzyme activity by increasing gene copy number and mutation or both. All things are included. The increase in the number of gene copies includes not only an increase in the number due to introduction of a foreign gene but also an amplification of an endogenous gene. Amplification of endogenous genes can be readily accomplished by methods known in the art, such as culturing under appropriate selection pressure and the like.

In the present invention, the activity of the enzyme is preferably achieved by introducing one or more new genes in addition to the intrinsic genes of the native microorganisms of Corynebacterium, preferably two copies of the genes for homologous recombination. One copy of the gene is inserted next to the endogenous gene, and finally two copies of the gene are amplified and mutated to enhance the activity of the enzyme.

The present invention provides a recombinant vector comprising each gene encoding the enzyme.

The vector usable at the time of introduction of a foreign gene in the present invention is preferably a chromosome insertion vector pDZ, more preferably a chromosome insertion vector pDZ- having a cleavage map of Figs. 2, 3, 4, 5, 6 and 7, respectively. 2purFM, pDZ-2purDB, pDZ-2purNH, pDZ-2purSL, pDZ-2purKE, pDZ-2purC. These vectors may be introduced into the Corynebacterium microorganisms in sequence or in combination, and the insertion of the gene into the chromosome may be by any method known in the art, for example homologous recombination.

In a preferred embodiment of the present invention, the present invention produces Corynebacterium ammonia genes CJXFT0301 (KCCM-10530) for producing XMP and Corynebacterium ammonia genes CJIP2401 (KCCM-10610) for producing IMP and inosine The vector pDZ-2purFM, pDZ-2purDB, pDZ-2purNH, pDZ-2purSL, pDZ-2purKE, and pDZ-2purC were introduced into Corynebacterium ammonia gene CJIS-H273 (KCCM-10905), respectively. To provide a strain cultured in a selective medium.

In the present invention, the transformed microorganisms are each transformed to produce high concentrations of Corynebacterium ammonia genes CN02-0058, CN02-0078, CN02-0120 strains, IMP transformed to produce high concentrations of XMP Corynebacterium ammonia genes CN01-0118, CN01-0300 strains, and Corynebacterium ammonia genes CN04-0049, CN04-0050 strains transformed to produce high concentrations of inosine. The Corynebacterium ammonia genes CN01-0118 strain was deposited on September 23, 2008 at the Korean Culture Center of Microorganisms (KCCM), Hongje 1-dong, Seodaemun-gu, Seoul under the Budapest Treaty (Accession No. KCCM). 10965P). The strains have higher XMP or IMP or inosine production efficiency than their respective parent strains, respectively.

In addition, the present invention relates to a method for producing XMP or IMP or inosine from the culture medium by culturing each of the transformed Corynebacterium genus microorganisms.

Each of the transformed Corynebacterium genus microorganisms can be cultured under aerobic conditions in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins and the like while controlling the temperature, pH and the like.

As a carbon source, glucose and fructose may be used, and as a nitrogen source, various inorganic nitrogen sources such as ammonia, ammonium chloride, and ammonium sulfate and peptone, NZ-amine, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, and fish Or organic nitrogen sources such as degradation products thereof, degreasing soy cakes, or degradation products thereof. As the inorganic compound, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate and the like may be used. In addition, vitamins and nutrient-containing bases may be added as necessary.

The culturing may be carried out under aerobic conditions, for example by shaking culture or aeration stirred culture, preferably at a temperature of 20 to 40 ° C. The pH of the medium is preferably maintained near neutral during the culture. Cultivation can be performed for 4 to 5 days. In a specific example, each Corynebacterium microorganism transformed by the present invention produced XMP or IMP or Inosine in higher yield compared to the Corynebacterium microorganism not transformed.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrating the present invention, and the present invention is not limited by these examples.

Example

Reference Example 1 Gene Insertion Method Using a Chromosome Insertion Vector (pDZ)

The chromosome insertion of a gene is performed using the vector pDZ for chromosome insertion. In order to insert an extra gene into the chromosome of Corynebacterium ammonia genes, a pDZ vector containing two consecutive genes of interest is prepared. 1 shows the vector pDZ for chromosome insertion.

In the present invention, Corynebacterium ammonia genes CJXFT0301 (KCCM-10530) strain XMP producing strain, Corynebacterium ammonia genes CJIP2401 (KCCM-10610) strain IMP production strain, Corynebacter strains of inosine production After transforming the ammonia genes CJIS-H273 (KCCM-10905) strain by electroporation, it was inserted by homology with homologous genes on a chromosome in a selection medium containing 25 mg / l of kanamycin. Strains were selected.

Successful chromosomal insertion of the vector is possible by identifying whether blue color appears in solid media, including X-gal (5-bromo-4-chloro-3-indolyl-B-D-galactosid).

The primary chromosome-inserted strain is shaken in nutrient medium (30 ° C., 4 hours), and then plated on solid medium containing X-gal from 10 ⁻⁴ to 10 ⁻¹⁰ , respectively. While most colonies are blue, by selecting white colonies that appear at a low rate, strains from which the vector sequence on the chromosome inserted by the crossover have been removed are selected.

The strains selected as described above are finally selected through the process of gene structure confirmation through PCR and confirmation of susceptibility to the antibiotic kanamycin.

Example 1 PurFM Gene Cloning and Recombinant Vector (pDZ-2purFM) from IMP Production Strain Corynebacterium Ammonia Genes CJIP2401 (KCCM-10610)

In the present embodiment, since the purF gene and the purM gene are located in close proximity, a purFM vector including a promoter region was prepared to simultaneously express the two genes.

The chromosome of the Corynebacterium ammonia genes CJIP2401 strain was isolated, and as a template, the polymerase was used as PfuUltra ^™ high-trust DNA polymerase (Stratagene), and PCR conditions were denaturation 96 ℃, 30 seconds and annealing 53 ℃. , 30 seconds and the polymerization reaction 72 ℃, 2 minutes was repeated 30 times. As a result, purFM containing a promoter site Two pairs of genes (purFM-A, purFM-B) were obtained. purFM-A was amplified using SEQ ID NOs: 1 and 2 as primers, and purFM-B was amplified using SEQ ID NOs: 3 and 4 as primers.

The pDZ-2purFM vector was transformed into Xine-producing strain Corynebacterium ammonia genes CJXFT0301 (KCCM-10530) strain, IMP-producing strain Corynebacterium ammonia genes CJIP2401 (KCCM-10610) strain, inosine production strain The strain of Corynebacterium ammonia genes CJIS-H273 (KCCM-10905) was transformed by electroporation, and a second crossover process was performed to insert one additional purFM gene right next to the intrinsic purFM gene on the chromosome. To obtain a strain in which the total number was increased to two. Subsequently inserted purFM genes are two purFMs It was finally confirmed by PCR using primers of SEQ ID NOs: 5 and 6, which can amplify the linking sites.

The sequences of the primers used for the PCR reaction for constructing the pDZ-2purFM vector and the primers used for identifying the corresponding amplification strains were as follows.

SEQ ID NO: 1 cgacgagaat tccccgaccc gcatgagatg

SEQ ID NO: 2 gtatcgtcta gagcggtagc ggtggcttcg

SEQ ID NO: 3 cgacgatcta gacccgaccc gcatgagatg

SEQ ID NO: 4 gtatcgaagc ttgcggtagc ggtggcttcg

SEQ ID NO: gctatcgttt cccctgaa

SEQ ID NO: 6 tgattctact aagtttgc

Example 2: PurDB gene cloning and recombinant vector (pDZ-2purDB) derived from IMP producing strain Corynebacterium ammonia gene CJIP2401 (KCCM-10610) strain, purDB insertion strain development

In this embodiment, since the purD gene and the purB gene are located in close proximity, a purDB vector including a promoter region was prepared to simultaneously express the two genes.

The chromosome of the Corynebacterium ammonia genes CJIP2401 strain was isolated and the polymerase was used as a template, and PfuUltra ^™ high-reliability DNA polymerase was used, and PCR conditions were denatured 96 ° C, 30 seconds and annealing 53 ° C, 30 seconds. And 30 degreeC of the polymerization reaction 72 degreeC and 2 minutes was repeated. As a result, purDB including the promoter site Two pairs of genes (purDB-A, purDB-B) were obtained. purDB-A was amplified using SEQ ID NOs: 7 and 8 as primers, and purDB-B was amplified using SEQ ID NOs: 9 and 10 as primers.

The amplification product was cloned into E. coli vector pCR2.1 using the TOPO cloning kit to obtain pCR-purDB-A and pCR-purDB-B vectors. The pCR vector was treated with restriction enzymes (purDB-A: HindIII + SpeI, purDB-B: SpeI + XbaI) contained at each end of purDB-A and purDB-B, and each purDB was extracted from the pCR vector. Gene was isolated. Next, the pDZ vector treated with the restriction enzymes HindIII and XbaI was cloned through three-piece conjugation to prepare a pDZ-2purDB recombinant vector in which two purDB genes were cloned. 3 is a diagram showing a vector pDZ-2purDB for inserting Corynebacterium chromosome.

The pDZ-2purDB vector was used in the Corynebacterium ammonia genes CJXFT0301 strain, an XMP producing strain, and the Corynebacterium ammonia genes CJIP2401, an IMP producing strain, and the corynebacterium ammonia genes, which is an inosine producing strain, CJIS-H273. The strain was transformed through the electroporation method, and a second crossover process was performed to insert one additional purDB gene immediately next to the intrinsic purDB gene on the chromosome to obtain a total strain increased to two. Subsequently inserted purDB genes are two purDBs Final confirmation was carried out by PCR using primers of SEQ ID NOs: 11 and 12 capable of amplifying the linking sites.

The sequences of the primers used for the PCR reaction for constructing the pDZ-2purDB vector and the primers used for identifying the corresponding strains are as follows.

SEQ ID NO: 7 ggcacaagct tccgcgtagt atcataagca gtc

SEQ ID NO: 8 cagcactagt gctggctcta gtgcgaagtc at

SEQ ID NO: 9 cagcactagt ccgcgtagta tcataagcag tc

SEQ ID NO: 10 gccctctaga gctggctcta gtgcgaagtc at

SEQ ID NO: 11 tcgctgacaa gcacgcgttt atcggc

SEQ ID NO: 12 ggtagcgggg tcggccgcaa ggcc

Example 3: PurNH gene cloning and recombinant vector (pDZ-2purNH) derived from IMP production strain Corynebacterium ammonia gene CJIP2401 (KCCM-10610) strain, purNH insertion strain development

In this embodiment, since the purN gene and the purH gene are located in close proximity, a purNH vector including a promoter region is prepared to simultaneously express the two genes.

The chromosome of the Corynebacterium ammonia genes CJIP2401 strain was isolated and the polymerase was used as a template, and PfuUltra ^™ high-reliability DNA polymerase was used, and PCR conditions were denatured 96 ° C, 30 seconds and annealing 53 ° C, 30 seconds. And 30 degreeC of the polymerization reaction 72 degreeC and 2 minutes was repeated. As a result, purNH including the promoter site Two pairs of genes (purNH-A, purNH-B) were obtained. purNH-A was amplified using SEQ ID NOs: 13 and 14 as primers, and purNH-B was amplified using SEQ ID NOs: 14 and 15 as primers. The amplified product was cloned into the E. coli vector pCR2.1 using the TOPO cloning kit to obtain pCR-purNH-A and pCR-purNH-B vectors. The pCR vector was treated with restriction enzymes (purNH-A: BamHI + SalI, purNH-B: SalI) contained at each end of purNH-A and purNH-B, and each purNH from the pCR vector. Gene was isolated.

Next, the pDZ vector treated with the restriction enzymes BamHI and SalI was cloned through three-piece conjugation, to finally prepare a pDZ-2purNH recombinant vector in which two purNH genes were continuously cloned. Figure 4 is a diagram showing the vector pDZ-2purNH for Corynebacterium chromosome insertion.

The pDZ-2purNH vector to Corynebacterium ammonia genes CJXFT0301 strain, which is an XMP producing strain, and Corynebacterium ammonia genes, CJIP2401 strain, which is an IMP producing strain, and Corynebacterium ammonia genes, which is an inosine producing strain, CJIS-H273 The strain was transformed through an electroporation method, and a second crossover process was performed to further insert one purNH gene immediately next to the intrinsic purNH gene on the chromosome to obtain a strain of which the total number was increased to two. Subsequently inserted purNH genes are two purNH genes. Final confirmation was carried out by PCR using primers SEQ ID NOs: 16 and 17 capable of amplifying the linking sites.

The sequences of the primers used for the PCR reaction for constructing the pDZ-2purNH vector and the primers used for identifying the corresponding amplification strains were as follows.

SEQ ID NO: 13 cgggatcccg aggcgaagac gatattgagg acag

SEQ ID NO: 14: acgcgtcgac gtgggaaacg cagacgagaa ca

SEQ ID NO: 15 acgcgtcgac gaggcgaaga cgatattgag gacag

SEQ ID NO: 16: tcgatgcctg catcttgg

SEQ ID NO: 17 ggcgataagg cttcgagt

Example 4: IMP production strain Corynebacterium ammonia genes CJIP2401 strain-derived purSL gene cloning and recombinant vector (pDZ-2purSL) production, purSL insertion strain development

In the present embodiment, since the purS gene and the purL gene are located in close proximity, a purSL vector including a promoter region was prepared to simultaneously express the two genes.

The chromosome of the Corynebacterium ammonia genes CJIP2401 strain was isolated and the polymerase was used as a template, and PfuUltra ^™ high-reliability DNA polymerase was used, and PCR conditions were denatured 96 ° C, 30 seconds and annealing 53 ° C, 30 seconds. And 30 degreeC of the polymerization reaction 72 degreeC and 2 minutes was repeated. As a result, purSL containing the promoter site Two pairs of genes (purSL-A, purSL-B) were obtained. purSL-A was amplified using SEQ ID NOs: 18 and 19 as primers, and purSL-B was amplified using SEQ ID NOs: 20 and 21 as primers.

The amplified product was cloned into E. coli vector pCR2.1 using the TOPO cloning kit to obtain pCR-purSL-A and pCR-purSL-B vectors. The pCR vector was treated with restriction enzymes (purSL-A: BamHI + SalI, purSL-B: SalI + BamHl) contained at each end of purSL-A and purSL-B, and each purSL from the pCR vector. Gene was isolated. Next, the pDZ vector treated with the restriction enzyme BamHI was cloned through three-piece conjugation to prepare a pDZ-2purSL recombinant vector in which two purSL genes were cloned. 5 is a diagram showing a vector pDZ-2purSL for corynebacterium chromosome insertion.

The pDZ-2purSL vector is used in the Corynebacterium ammonia genes CJXFT0301 strain, which is an XMP producing strain, and the Corynebacterium ammonia genes, CJIP2401, which is an IMP producing strain, and the corynebacterium ammonia genes, which is an inosine producing strain, CJIS-H273. The strain was transformed through an electroporation method, and a second crossover process was performed to further insert one purSL gene right next to the intrinsic purSL gene on the chromosome to obtain a strain of which the total number was increased to two. Subsequently inserted purSL genes are two purSL Final confirmation was carried out by PCR using primers of SEQ ID NOs: 22 and 23 which can amplify the linking sites.

The sequences of the primers used for the PCR reaction for constructing the pDZ-2purSL vector and the primers used for identifying the corresponding amplification strains were as follows.

SEQ ID NO: 18 gctcggatcc gcgatactca gccccagcaa cagcagaaaa tgaagc

SEQ ID NO: 19: cagcgtcgac gcagccgtcg caggcaccat cgcagcagt

SEQ ID NO: 20 cagcgtcgac gcgatactca gccccagcaa cagcagaaaa tgaagc

SEQ ID NO: 21 gctcggatcc gcagccgtcg caggcaccat cgcagcagt

SEQ ID NO: 22 acttgacctc cagcccta

SEQ ID NO: 23 aagaacaacg tcggcgtc

Example 5: IMP production strain Corynebacterium ammonia genes CJIP2401 strain derived purKE gene cloning and recombinant vector (pDZ-2purKE) production, purKE insert strain development

In the present embodiment, since the purK gene and the purE gene are located in close proximity, a purKE vector including a promoter region is prepared to simultaneously express the two genes.

The chromosome of the Corynebacterium ammonia genes CJIP2401 strain was isolated and the polymerase was used as a template, and PfuUltra ^™ high-reliability DNA polymerase was used, and PCR conditions were denatured 96 ° C, 30 seconds and annealing 53 ° C, 30 seconds. And 30 degreeC of the polymerization reaction 72 degreeC and 2 minutes was repeated. As a result, purKE including the promoter site Two pairs of genes (purKE-A, purKE-B) were obtained. purKE-A was amplified by using SEQ ID NOs: 24 and 25 as primers, and purKE-B was amplified using SEQ ID NOs: 26 and 27 as primers.

The amplification products were cloned into E. coli vector pCR2.1 using the TOPO cloning kit to obtain pCR-purKE-A and pCR-purKE-B vectors. The pCR vector was treated with restriction enzymes (purKE-A: BamHI + KpnI, purKE-B: KpnI + XbaI) contained at each end of purKE-A and purKE-B, and each purKE from the pCR vector. Gene was isolated. Next, the pDZ vector treated with the restriction enzymes BamHI and XbaI was cloned through three-piece conjugation to prepare a pDZ-2purKE recombinant vector in which two purKE genes were continuously cloned. Fig. 6 shows the vector pDZ-2purKE for corynebacterium chromosome insertion.

The pDZ-2purKE vector was used in the Corynebacterium ammonia genes CJXFT0301 strain, which is an XMP producing strain, and the Corynebacterium ammonia genes, CJIP2401, which is an IMP producing strain, and the corynebacterium ammonia genes, which is an inosine producing strain, CJIS-H273. The strain was transformed through the electroporation method, and a second crossover process was performed to further insert one purKE gene immediately next to the intrinsic purKE gene on the chromosome to obtain a strain of which the total number was increased to two. Subsequently inserted purKE genes are two purDBs It was finally confirmed by PCR using the primers of SEQ ID NOs: 28 and 29 that can amplify the linking sites.

The primers used in the PCR reaction for constructing the pDZ-2purKE vector and the primers used for identifying the corresponding strains were as follows.

SEQ ID NO: 24 acgtcaggat cccctatcgt gctttgctgt

SEQ ID NO: 25 ctctaaggta ccattggtac tagtagccgc

SEQ ID NO: 26 acgtcaggta cccctatcgt gctttgctgt

SEQ ID NO: 27: ctctaatcta gaattggtac tagtagccgc

SEQ ID NO: 28: ccagctgggg ttccggtt

SEQ ID NO: 29 tttcgatgcg cttcgttt

Example 6: PurC gene cloning and recombinant vector (pDZ-2purC) derived from IMP producing strain Corynebacterium ammonia gene CJIP2401 strain, purC insertion strain development

The chromosome of the Corynebacterium ammonia genes CJIP2401 strain was isolated and the polymerase was used as a template, and PfuUltra ^™ high-reliability DNA polymerase was used, and PCR conditions were denatured 96 ° C, 30 seconds and annealing 53 ° C, 30 seconds. And 30 degreeC of the polymerization reaction 72 degreeC and 2 minutes was repeated. As a result, purC containing the promoter site Two pairs of genes (purC-A, purC-B) were obtained. purC-A was amplified using SEQ ID NOs: 30 and 31 as primers, and purC-B was amplified using SEQ ID NOs: 31 and 32 as primers.

The amplified product was cloned into E. coli vector pCR2.1 using the TOPO cloning kit to obtain pCR-purC-A and pCR-purC-B vectors. The pCR vector is treated with restriction enzymes (purC-A: BamHI + SalI, purC-B: SalI) contained at each end of purC-A and purC-B, and each purC is removed from the pCR vector. Gene was isolated. Next, the pDZ vector treated with the restriction enzymes BamHI and SalI was cloned through three-piece conjugation, to finally prepare a pDZ-2purC recombinant vector in which two purC genes were continuously cloned. 7 is a diagram showing a vector pDZ-2purC for corynebacterium chromosome insertion.

The pDZ-2purC vector was used in the Corynebacterium ammonia genes CJXFT0301 strain, an XMP producing strain, and the Corynebacterium ammonia genes CJIP2401, an IMP producing strain, and the corynebacterium ammonia genes, which is an inosine producing strain, CJIS-H273. The strain was transformed through an electroporation method, and a second crossover process was performed to further insert one purC gene immediately next to the intrinsic purC gene on the chromosome to obtain a strain having a total number of two. Successively inserted purC genes are two purCs It was finally confirmed by PCR using primers of SEQ ID NOs: 33 and 34, which can amplify the linking sites.

The sequences of the primers used for the PCR reaction for constructing the pDZ-2purC vector and the primers used for identifying the corresponding amplification strains were as follows.

SEQ ID NO: 30 gctcggatcc cgcagtggct gttgcgctga acatgcg

SEQ ID NO: 31 gcaggtcgac cacggacata tcggtttgct tcacgcggg

SEQ ID NO: 32 gcaggtcgac cgcagtggct gttgcgctga acatgcg

SEQ ID NO: 33 gagcgcttgt ccggcaagcg tttc

SEQ ID NO: 34: ggtggttgcg gtaagaaccc ggcc

Example 7 Development of High Yield XMP Strains by Purine Biosynthesis Gene Enhancement

PDZ-2purFM, pDZ-2purDB, pDZ-2purNH, pDZ-2purSL, pDZ-2purKE and pDZ-2purC vectors prepared in the above examples were combined or all introduced into the Corynebacterium ammonia genes CJXFT0301 strain producing XMP. . The order of vector introduction was chosen arbitrarily, the introduction method and confirmation are as indicated in the above examples.

Below are the genetic traits of Corynebacterium ammonia gene strains with enhanced ability to produce XMP developed by purine biosynthetic gene enrichment.

CN02-0058 Gene Traits

: CJXFT0301 + 2purDB + 2purNH + 2purSL

CN02-0078 Gene Traits

: CJXFT0301 + 2purDB + 2purNH + 2purSL + 2purFM

CN02-0120 Gene Traits

: CJXFT0301 + 2purDB + 2purNH + 2purSL + 2purKE + 2purC + 2purFM

Example 8 Development of High Yield IMP Strains by Purine Biosynthesis Gene Enhancement

PDZ-2purFM, pDZ-2purDB, pDZ-2purNH, pDZ-2purSL, pDZ-2purKE and pDZ-2purC vectors prepared in the above examples were combined or all introduced into the Corynebacterium ammonia genes CJIP2401 strain producing IMP. . The order of vector introduction was chosen arbitrarily, the introduction method and confirmation are as indicated in the above examples.

Below are the genetic traits of Corynebacterium ammonia gene strains with enhanced ability to produce IMPs developed by purine biosynthetic gene enrichment.

CN01-0118 Gene Traits

: CJIP2401 + 2purDB + 2purNH + 2purSL + 2purKE + 2purC

CN01-0300 Gene Traits

: CJIP2401 + 2purDB + 2purNH + 2purSL + 2purKE + 2purC + 2purFM

Example 9 Development of High Yield Inosine Strains by Purine Biosynthesis Gene Enhancement

PDZ-2purFM, pDZ-2purDB, pDZ-2purNH, pDZ-2purSL, pDZ-2purKE and pDZ-2purC vectors prepared in the above examples are combined or all in the Corynebacterium ammonia genes CJIS-H273 strain producing inosine Introduced. The order of vector introduction was chosen arbitrarily, the introduction method and confirmation are as indicated in the above examples.

Below are the genetic traits of Corynebacterium ammonia gene strains with enhanced ability to produce inosine developed by purine biosynthetic gene enrichment.

CN04-0049 Gene Traits

: CJIS-H273 + 2purDB + 2purNH + 2purSL + 2purKE + 2purC

CN04-0050 Gene Traits

: CJIS-H273 + 2purDB + 2purNH + 2purSL + 2purKE + 2purC + 2purFM

Example 10: XMP Erlenmeyer flask fermentation potency test

The parent strain Corynebacterium ammonia genes CJXFT0301 strain and Corynebacterium ammonia genes CN02-0058, CN02-0078, and CN02-0120 strains prepared in the patent were prepared in a 14 ml tube containing 3 ml of the following species. Inoculation and shaking culture at 200 rpm for 20 hours at 30 ℃. 0.4 ml of the seed culture was inoculated into a 250 ml corner-baffle flask containing 32 ml of the following production medium (24 ml of fermentation medium + 8 ml of starch medium), followed by shaking culture at 230 ° C. at 230 rpm for 96 hours.

The composition of the seed medium and fermentation medium is as follows.

Species medium : glucose 30g / l, peptone 15g / l, yeast extract 15g / l, sodium chloride 2.5g / l, urea 3g / l, adenine 150mg / l, guanine 150mg / l, pH 7.2

Flask fermentation medium : glucose 80g / l, magnesium sulfate 10g / l, iron sulfate 20mg / l, zinc sulfate 10mg / l, manganese sulfate 10mg / l, adenine 30mg / l, guanine 30mg / l, biotin 100 Μg / l, copper sulfate 1mg / l, thiamine hydrochloride 5mg / l, calcium chloride 10mg / l, pH 7.2

Flask differentiation medium : 10 g / l potassium phosphate, 10 g / l potassium phosphate, 7 g / l urea, 5 g / l ammonium sulfate

After the incubation, the production of XMP was measured by the method using HPLC, and the XMP results in the culture are shown in Table 1 below.

Strain name Cell OD (4 days after culture) Productivity (g / l / hr) (after 4 days of culture) Control Group (CJXFT0301) 45.3 0.298 CN02-0058 43.3 0.327 CN02-0078 42.1 0.340 CN02-0120 43.9 0.315

Comparing the XMP accumulation in the medium with the parent strain Corynebacterium ammonia genes CJXFT0301 strain, Corynebacterium ammonia genes CN02-0058, CN02-0078 and CN02-0120 strains under the same conditions Coryne strain XMP productivity per unit time is increased by 10.6 ~ 11.4% compared to the bacterium ammonia genes CJXFT0301 strain.

In addition, the amplification gene combination (2purDB + 2purNH + 2purSL) possessed by the Corynebacterium ammonia genes CN02-0058 strain alone showed that the production yield was significantly improved compared to the parent strain. As a combination of the Nebacterium ammonia genes CN02-0078 strain, it was confirmed that only amplification of purFM gene was added compared to the Corynebacterium ammonia genes CN02-0058 strain. In addition, when the genes on all purine biosynthetic pathways were amplified, the yield improvement was somewhat low.

Example 11: IMP Erlenmeyer flask fermentation potency test

After dispensing 3 ml of the following medium into an 18 mm diameter test tube and autoclaving, the parent strain Corynebacterium ammonia genes CJIP2401 strain and Corynebacterium ammonia genes CN01-0118 and CN01-0300 produced in this patent Strains were inoculated and shake cultured at 30 ° C. for 24 hours to use as seed culture. 27 ml of the fermentation broth was dispensed into a 500 ml shake flask, sterilized under pressure at 120 ° C. for 10 minutes, and then inoculated with 3 ml of the flask culture and incubated for 5 to 6 days. Culture conditions were adjusted to 200 rpm, temperature 32 ℃, pH 7.2.

The composition of the seed medium and fermentation medium is as follows.

Species medium : glucose 1%, peptone 1%, gravy 1%, yeast extract 1%, sodium chloride 0.25%, adenine 100 mg / l, guanine 100 mg / l, pH 7.2

Flask fermentation medium : 0.1% sodium glutamate, 1% ammonium chloride, magnesium sulfate 1.2%, calcium chloride 0.01%, iron sulfate 20mg / l, manganese sulfate 20mg / l, zinc sulfate 20mg / l, copper sulfate 5mg / L, cysteine 23 mg / l, alanine 24 mg / l, nicotinic acid 8 mg / l, biotin 45 µg / l, thiamine hydrochloride 5 mg / l, adenine 30 mg / l, phosphoric acid (85%) 1.9%, glucose 4.2%, 2.4% per raw material

After the incubation, the production of IMP by the method using HPLC was measured, the results of 5'-inosinic acid in the culture is shown in Table 2 below.

Strain name Cell OD (5 days after culture) Productivity (g / l / hr) (after 5 days of culture) Control Group (CJIP2401) 31.2 0.136 CN01-0118 32.4 0.152 CN01-0300 31.5 0.145

Comparing IMP accumulation in the medium with the parent strain Corynebacterium ammonia genes CJIP2401, Corynebacterium ammonia genes CN01-0118 and CN01-0300 strains under the same conditions, the parent strain Corynebacterium ammonia crab The productivity of IMP produced per unit time compared to Ness CJIP2401 strain is increased by 10.6 ~ 11.2%.

Example 12 Inosine Erlenmeyer Flask Fermentation Titer Test

After dispensing 3 ml of the following medium into an 18 mm diameter test tube and autoclaving, the parent strain Corynebacterium ammonia genes CJIS-H273 strain and Corynebacterium ammonia genes CN04-0049, CN04 produced in the present patent The strains were inoculated and shaken for 24 hours at 30 ° C. to be used as the seed culture. 27 ml of the fermentation broth was dispensed into a 250 ml Erlenmeyer flask for shaking, and autoclaved at 120 ° C. for 10 minutes, inoculated with 3 ml of the seed culture solution, and then cultured for 5 to 6 days. Culture conditions were adjusted to 220 rpm, temperature 32 ℃, pH 7.2.

The composition of the seed medium and fermentation medium is as follows.

Species medium : glucose 5%, peptone 0.5%, gravy 0.5%, sodium chloride 0.25%, yeast extract 1%, adenine 100 mg / l, guanine 100 mg / l, pH 7.2

Flask fermentation medium : Sodium glutamate 0.1%, Ammonium chloride 1%, Magnesium sulfate 1.2%, Calcium chloride 0.01%, Iron sulfate 20mg / l, Manganese sulfate 20mg / l, Zinc sulfate 20mg / l, Copper sulfate 5mg / l, L-cysteine 23 mg / l, alanine 24 mg / l, nicotinic acid 8 mg / l, biotin 45 μg / l, thiamine hydrochloride 5 mg / l, adenine 50 mg / l, guanine 30 mg / l, phosphoric acid (85%) 1.9%, mixed with fructose, glucose and molasses to 6% as reducing sugar, pH 7.2

After the incubation, the production of inosine was measured by the method using HPLC, the results of inosine in the culture is shown in Table 3 below.

Strain name Cell OD (5 days after culture) Productivity (g / l / hr) (after 5 days of culture) Control Group (CJIS-H273) 70.1 0.100 CN04-0049 69.8 0.113 CN04-0050 68.7 0.121

Comparing the inosine accumulation in the medium with the parent strain Corynebacterium ammonia genes CJIS-H273, Corynebacterium ammonia genes CN04-0049 and CN04-0050 strains under the same conditions Inosine production per unit time is increased by 11.3 to 12.1% compared to the ammonia genes CJIS-H273 strain. In addition, unlike in the case of XMP producing strains and IMP producing strains, it was found that in the inosine producing strains, the amplified combination of all purine genes showed the greatest improvement in production yield.

1 shows a vector pDZ vector for insertion into Corynebacterium chromosome.

Figure 2 shows the vector pDZ-2purFM for insertion into Corynebacterium chromosome.

Figure 3 shows the vector pDZ-2purDB for insertion into the Corynebacterium chromosome.

Figure 4 shows a vector pDZ-2purNH for insertion into Corynebacterium chromosome.

Figure 5 shows the vector pDZ-2purSL for insertion into the Corynebacterium chromosome.

Figure 6 shows the vector pDZ-2purKE for insertion into Corynebacterium chromosome.

Figure 7 shows the vector pDZ-2purC for insertion into the Corynebacterium chromosome.

<110> CJ Cheiljedang Corporation <120> Microorganisms of corynebacterium having enhanced nucleotide or          nucleoside productivity and method of producing nucleotide or          nucleoside using the same <130> PA08-0233 <160> 44 <170> KopatentIn 1.71 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cgacgagaat tccccgaccc gcatgagatg 30 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gtatcgtcta gagcggtagc ggtggcttcg 30 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 cgacgatcta gacccgaccc gcatgagatg 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gtatcgaagc ttgcggtagc ggtggcttcg 30 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gctatcgttt cccctgaa 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 tgattctact aagtttgc 18 <210> 7 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ggcacaagct tccgcgtagt atcataagca gtc 33 <210> 8 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 cagcactagt gctggctcta gtgcgaagtc at 32 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 cagcactagt ccgcgtagta tcataagcag tc 32 <210> 10 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gccctctaga gctggctcta gtgcgaagtc at 32 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 tcgctgacaa gcacgcgttt atcggc 26 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 ggtagcgggg tcggccgcaa ggcc 24 <210> 13 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 cgggatcccg aggcgaagac gatattgagg acag 34 <210> 14 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 acgcgtcgac gtgggaaacg cagacgagaa ca 32 <210> 15 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 acgcgtcgac gaggcgaaga cgatattgag gacag 35 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 tcgatgcctg catcttgg 18 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ggcgataagg cttcgagt 18 <210> 18 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 gctcggatcc gcgatactca gccccagcaa cagcagaaaa tgaagc 46 <210> 19 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 cagcgtcgac gcagccgtcg caggcaccat cgcagcagt 39 <210> 20 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 cagcgtcgac gcgatactca gccccagcaa cagcagaaaa tgaagc 46 <210> 21 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 gctcggatcc gcagccgtcg caggcaccat cgcagcagt 39 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 acttgacctc cagcccta 18 <210> 23 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 aagaacaacg tcggcgtc 18 <210> 24 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 acgtcaggat cccctatcgt gctttgctgt 30 <210> 25 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 ctctaaggta ccattggtac tagtagccgc 30 <210> 26 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 acgtcaggta cccctatcgt gctttgctgt 30 <210> 27 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 ctctaatcta gaattggtac tagtagccgc 30 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 ccagctgggg ttccggtt 18 <210> 29 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 tttcgatgcg cttcgttt 18 <210> 30 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 gctcggatcc cgcagtggct gttgcgctga acatgcg 37 <210> 31 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 gcaggtcgac cacggacata tcggtttgct tcacgcggg 39 <210> 32 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 gcaggtcgac cgcagtggct gttgcgctga acatgcg 37 <210> 33 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 gagcgcttgt ccggcaagcg tttc 24 <210> 34 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 ggtggttgcg gtaagaaccc ggcc 24 <210> 35 <211> 1500 <212> DNA <213> Corynebacterium ammoniagenes <400> 35 gtggtgaaca ctactttccc cagcgacgtg aatttagatg accaaggcga gcaagaaccc 60 cgcgaagagt gcggtgtctt tggcgtctgg gctcctggtg aagatgttgc gacactgacc 120 tactttggtc tgttcgcatt gcagcatcgt gggcaggaag ctgcaggtat cggcgtcggt 180 gatggagacc gcctcgttgt cttcaaagac atgggcttgg tctcgaatat tttcgatgag 240 tccattttaa attccctcca tggctccgtg ggcgtggggc atacgcgcta ctcgactgcc 300 ggtggcaaag agtggtcgaa tgtccagccg atgtttaata ccacctcaaa tggggtagac 360 atcgctttgt gccacaacgg caacttggtg aactaccaag aactgcgcga tgaagcagta 420 gctctgggac tttaccgaga gaatgaaaaa tccctgtcgg attccatgat catgacagct 480 ttgctggcgc acggagtcgg ggaaggcaac tctgtctttg acgccgctaa gcaactgctg 540 ccaagcatca aaggcgcttt ttgcttgacc tttaccgatg gcaagacctt gtacgccgcg 600 cgtgacccgc acggtgtacg ccccttggtc attggccgct tggcgcaagg ctgggttgtt 660 gcttccgaaa cctgtgcgct ggatatcgtg ggcgcacagt ttatccgtga ggtagagccc 720 ggtgaactta tctctgtcaa tgaggcagga atccacagcg aaaaattcgc tgagccgaag 780 cgccagggct gcgtctttga atacgtctac ttggcacgtc cagacaccgt gatcaaaggc 840 cgcaacgttc acgcgacgcg cgtggatatt ggtcgcgcac ttgcgaaatc tcaccctgcg 900 ccagaagctg acatggtcat ccccgtgcca gaatccggaa acccggcagc tgttggctac 960 gcccgggaat cgggcctgac atttgcgcac ggcttggtca aaaacgccta cgtgggtcga 1020 accttcattc agcccaccca gaccttgcgc cagctgggta ttcgcctcaa gctcaacccc 1080 ctgcgcgagg tcatcgaggg caagtcactc gttgttgtag atgactctat tgtccgcggc 1140 aacacccaac gcgcgctggt gcgcatgctg cgtgaagcag gcgctgctga agtgcacgtg 1200 cgcattgctt caccgccagt caaatggcct tgtttctacg gcattgactt cgcctcgcct 1260 ggtgaattga ttgctaatat caagccttct gatgatcctc aggtagtaac cgatgcagtg 1320 tgcgaagcta tcggagcaga ctctttaggg tttgtatctg tagatgagat ggttgaggca 1380 acgcaccaac ctatcaattc cttgtgtacc gcttgctttg atggcaacta cgaactcgga 1440 cttccgaccg ctaaccccaa tgctgacgct gtgcgaactt tgctcagcca aaagaactga 1500                                                                         1500 <210> 36 <211> 1281 <212> DNA <213> Corynebacterium ammoniagenes <400> 36 atgcgcattc ttgtaattgg ttccggcggc cgcgagcacg ccattttgaa aggccttgcg 60 gccgaccccg ctaccaccga cctgcacgtt gcaccgggct cacctgcttt tgcatcgcta 120 gctactgtgc acgctgacta caaagaagta gcggatccgg cccgcatgct ggagttggct 180 caggacatta aacctgagct ggttgtcatc ggtccagaga ttccattggt tgccggtgtt 240 gctgacacat tgcgcgctga aggcatcgcg gtctttggac cgaacgcgga tgcagcacag 300 attgaaggct ctaaggcctt tgccaaggaa gtcatggaag ctgcaggcgt tgctaccgcg 360 cgtgcgcaga ccctaccacc gggaatgact gacgacgata ttgaacatga gctcgactac 420 ttcggcccca tgtatgtcgt caaagatgac gggctggccg caggcaaagg cgttgtggtc 480 accgctgacc gcgccgaagc acgccagcac atccacctgg tgcatgccgc tggtaaccca 540 gtgctactag aatctttctt ggacggtcca gaggtctcgc tgttttgcct tgtcgatggc 600 gaaaccgtcg tgccactgct gccagcccaa gaccacaagc gtgcctacga caatgatgaa 660 ggcccgaata ccggcggcat gggcgcatac accccgctgc cgtggttgtc tgctgaaggc 720 gtcgaccgca tcgtgcgcga ggtctgcgag ccggtagcta agcaaatggt cgagcgcgga 780 accccatact ccggcctgct gtacgcaggt ctggcatggg gccaagaagg cccctccgtc 840 attgagttca actgccgctt cggcgaccca gaaacccagc cgctgctgtc gctgttgaag 900 acgccgctgg caggggttct caacgcagtt gcaaccggaa ctctcgacga acttcctgcg 960 ctggagtggg aagacgccta tgccgtgacc gtagtgctgg cggctgcgaa ttatccagaa 1020 tctccacgca agggcgatgc catcacctcg ccagatctgg cagataccga caagattttg 1080 cacgcgggta ccgcggtaaa ggacgcagag gttatctcca atggcggtcg cgtgcttaac 1140 gtaatcggca agggtgagac cctatctgcc gcgcgcgctg ccgcgtatga ggtgctcgag 1200 aacattgagc tggcggatag cttctaccgc accgacattg gccaagctgc tgaagaaggt 1260 cgcatcagca ttgactctta g 1281 <210> 37 <211> 606 <212> DNA <213> Corynebacterium ammoniagenes <400> 37 gtgactgaat cgccttcgca agttttgaaa gcacaagacc cgcttcaagt agtggtgctg 60 gtatctggca ccggatcttt gctgcaaaat attatcgaca accaagatga ctcctatcgg 120 gttatcaagg tagtcgcgga taagccctgc ccggggatta accgagccca agatgcaggc 180 atcgacaccg aagtcgtgct tttaggctca gaccgcgcgc agtggaacaa agaccttgtc 240 gcagcggttg gtaccgccga tgttgtggtg tccgctggat ttatgaaaat cctggggcct 300 gaattcttgg ccagctttga aggccgcaca ataaatacgc atcccgcact cctgccgtcc 360 tttccgggcg cgcatggagt acgggatgcg ttggcttatg gcgtgaaagt caccggctct 420 actgtccatt ttgtggacgc gggagtcgat actggccgca tcatcgcaca acgcgcagta 480 gagattgagg cagaagatga tgaggcaagc ttgcatgagc gcatcaaaag cgtcgaacgt 540 gagcttatcg tgcaggtctt acgcgcagcg aatgttcaag accagcagct tattattgag 600 atttaa 606 <210> 38 <211> 243 <212> DNA <213> Corynebacterium ammoniagenes <400> 38 atggctcgtg ttgttgtcaa tgtcatgccc aaggctgaaa tcctcgaccc gcagggacaa 60 gctgttgtcc gtgcacttgg acgcctgggt gtaaacggag taagcgatgt ccgtcagggc 120 aagcgctttg aaatcgaagt cgatgattca gtcagcgctg aagatctaga caaggtcgca 180 gcaagcttgc tggcaaacac cgtcatcgag gactacgaag ttgtagggct ggaggtcaag 240 taa 243 <210> 39 <211> 2277 <212> DNA <213> Corynebacterium ammoniagenes <400> 39 atgactgttt ccaatgacac agtagataat gcaaaggcca ctcccgagct agaccagccg 60 tgggaagaac tcggcttaaa gcaagacgaa tacgacaaga ttgtaggcat cttgggccgc 120 cgcccaaccg atgctgagct gacggtttac tccgtgatgt ggtcggagca ctgctcttac 180 aagtcttcca agacccacct acgctacttt ggcgagacca ccactgagga aatggcgtcg 240 aagattcttg ccggtatcgg tgagaacgct ggtgtcgttg acatcggcga cggtgacgca 300 gtgaccttcc gcgtcgaatc ccacaaccac ccatccttcg tcgagcctta ccagggtgcc 360 gcgaccggtg ttggcggcat cgtccgcgac atcatggcga tgggtgcacg tccaatcgca 420 gtgatggatc agctgcgctt cggcccagct gatgccccgg ataccgcacg tgttctgccg 480 ggcgttgttt ccggcatcgg cggttacggc aactccctcg gcctgccgaa catcggcggc 540 gagaccgtct ttgatgagtc ttatgccggc aacccactgg tcaacgcact gtgcgtgggt 600 accttgcgcg tggaagacct gaagctggct tttgcttccg gtactggcaa caaggtgatg 660 ctctttggct cccgcacggg cctcgacggc atcggcggcg tatccgtttt gggttctgct 720 tccttcgaag aaggcgaaga gcgcaagctt cctgcagtcc aggtcggcga cccattcgcg 780 gaaaaagtcc tcatcgaatg ctgcctggag ctctacgctg cgggcgtcgt tgtcggtatt 840 caggaccttg gtggcggtgg cctcgcatgt gcgacctctg agctggcagc agctggcgac 900 ggcggcatgg tggtcaacct ggataatgtt ccactgcgtg cagagaacat gtccgccgca 960 gaaatcctgg cttccgaatc ccaggagcgc atgtgtgctg ttgtctcccc agataacgtg 1020 gagaagttcc gcgagatctg tgaaaagtgg gacgtaacgt gtgctgaaat cggtgaagtt 1080 accgataaga aagacaccta cctcgtgtac cacaacggtg agctggtagt agacgctccg 1140 ccatcaacta tcgatgaagg ccctgtctac gagcgcccat acgcacgccc tcagtggcag 1200 gatgagatcc agcaggctcc ggaaattgca cgtccggaat ccttggtaca ggcattcaag 1260 gacatggtgt cctccccagc tctgtcatcg cgtgcattta tcactgagca gtatgaccgc 1320 tacgtgcgcg gtaacaccgt caaggcgaag cagtctgact ccggcgttct gcgtatcaat 1380 gaggaaactt ctcgcggtgt cgcaatttct gccgatgcct ccggtcgcta caccaagctg 1440 gacccaaaca tgggtgcacg tttggcgctg gctgaggcat accgcaacgt tgctgtgacc 1500 ggcgcacgac catatgcggt gaccaactgc ttgaacttcg gttctccaga aaacaccgac 1560 gtgatgtggc aattccgcga ggccgttcac ggtctggctg acggttctaa ggaactgaat 1620 atcccagtct ccggcggtaa cgtctccttc tacaaccaga ctggtgatga gccaattctg 1680 ccgaccccag ttgttggcgt gctcggtgtc attgatgatg ttcacaaggc actggcacat 1740 gacttgggcg gcattgatga gcctgaaacc ctgattctgc ttggtgagac caaggaagaa 1800 ttcggcggct ccatctggca gcaggtctcc ggcggcggcc tgcagggtct gccaccacag 1860 gtggatctgg cgaatgaggc aaagctggcg gacttcttcg tcggcaacac ctccgttgca 1920 gcctcccacg acctctctga gggcggtctg gctatcgcgg cgtttgagat ggcgcaaaag 1980 aacaacgtcg gcgtcgacct tgatttgagc gttgtacacg aggatgcact gaccgcactg 2040 tttagtgagt ccgcatcgcg tgttctgatt tccaccgcgt ctgaccacct cgatggaatc 2100 ttgcagcgtg cttccgagct gggcattcca gctgtcgtgg taggaaccac caatgattcc 2160 ggcaacatca ccttcgctgg tgaagaagtt gctaccgctg agctgcgcga ggcatggtct 2220 gcaaccttgc caaacctgtt tggccacgct gttggcgcta attccgtagt cgaataa 2277 <210> 40 <211> 1056 <212> DNA <213> Corynebacterium ammoniagenes <400> 40 atgtctgaaa atacttacgc cgcggcaggc gtcaacattg aagaaggcga ccgcgccgtt 60 gagcttttcg ctccactggc taagcgcgct acccgtccag aggtaatggg tggactcggt 120 ggcttcgcgg gactgtttaa gctcggcgaa tacaaagagc caatccttgc agctggctcc 180 gacggcgtgg gcaccaagct cgccgttgcc caggcaatgg ataagcacga caccatcggc 240 attgacctgg ttgcaatgtg cgtcgatgac ttggtcgtgt gtggtgctga gccactattt 300 ctccaggact acatcgcagt aggcaaggtt gttccggaaa aggttgcgca gattgttgcc 360 ggtattgctg agggctgcgt gcaggcaggc tgtgcacttc ttggtggcga gaccgctgag 420 cacccgggcg taatgaatga aaaggactac gatgtttccg ccaccgctgt cggcgttgtc 480 gaagcagacg agcttctcgg accagacaag gttcgcgacg gcgatgtttt gattgccatg 540 ggctcatccg gactgcactc caatggttac tccttggcgc gccacgtctt gttagagcag 600 gcaggattgc cgctcgatgg ctacatcgat gaccttggcc gcacgcttgg tgaagagctc 660 ctggagccga cccgcatcta cgccaaggac tgcctggcgc tagtttctga gtgtgacgtt 720 gctactttct gccacgtcac cggcggtggc ttggcaggca acctcgagcg cgtgctacct 780 gaaggccttg tcgcagaggt taaccgcgca tcgtggaccc cagcagcgat tttccgcacc 840 atcgcgtctt tcggcaaggt cagcctggaa gagatggaaa agaccttcaa catgggcgtt 900 ggcatgatcg ctatcgtttc ccctgaagac cgtgaccgcg ccttggcgat gctaactgcg 960 cgccacgttg atgcatggga gctgggctcg gttcgcacca agaaggaaga cgacaccgca 1020 ggtgttgtca tgcaaggtga gcactctaac ttctaa 1056 <210> 41 <211> 1779 <212> DNA <213> Corynebacterium ammoniagenes <400> 41 atgaaacgcg tgagtgaaca agcaggaaac ccagacggaa accctcaagc acatgttccc 60 ggcatgccgg ttatcgccgt tattggtgat ggccagctag ctcgcatgat gcaaaccgcc 120 gccattgagc tcggccaatc gctgcgcctt cttgccggcg cacgcgatgc ctctgcggca 180 caagtatgcg cggatgtagt gcttggtgat tacaccaact acgacgactt gctcaaagcc 240 gtcgacggtg ccaccgctgt cacttttgac catgagcacg tgcctaatga gcacctcacc 300 gcgttgatcg atgcaggcta taacgtgcag ccacaacctg ctgcgctgat taacgcccaa 360 gacaaattgg ttatgcgcga gcgcctcgcc gagctgggcg cacccgtgcc gcgctttgcg 420 ccgattgaat ctgcccaaga tgcgtacgat ttttggacct tgacgtccgg gcaggtctgt 480 ctgaaggcgc gccggggtgg ctacgacggc aaaggcgtgt ggtttccgaa taatgaatct 540 gagctgactg ctttggtctc tgacctttcg cgccgcggcg tggccttgat ggctgaagag 600 aaggttgcgc tggtccgcga gctttccgtg ctggtcgcgc ggactccctc gggcgaggtt 660 gctacttggc cgctgactga gtctgtgcag cgcaacggtg tgtgcgctga agctgtcgcg 720 ccagccccgg gagttgaccc gcagctgcag caacgcgctg agacactggg tgaaaagatt 780 gccaccgagt tgggtgtaac tggtgtgctc gcggtagagc tttttgcatt tgcgaatgag 840 tccggtgcgg aagatatcgc ggttaatgaa ctggcaatgc gcccgcacaa taccggccac 900 tggaccctag atggttctgt gacctcccaa tttgagcagc acctgcgcgc ggtgatggat 960 gagccactgg gggatacatc cacgcttgcc ccagtcaccg tgatggccaa cgtcttaggc 1020 gctgacgaag acccaaagat gccaatgggc gagcgtgccc gagaagtggc gcgccgcttc 1080 ccgcgagcca aagtccatct ctacggcaag gggcatcgcc caggccgtaa gattggccac 1140 gtgaacctca ccggtgagga cgtagaggca acccgtcgcg atgctcgctt ggctgcggat 1200 ttcctcgtga acgccgcgtg gtctgataac tggtccgcta aatagcaaga tgtatcaaga 1260 tatataagga aagaaatgac tgcaccgcta gttgggctca tcatgggctc tgattctgat 1320 tggccaaccg ttgaaccagc agctgaggtt ctcgccgaat tcggtgttcc ttttgaggtg 1380 ggcgtggtct ctgcgcaccg cacgccggaa aagatgctgg attacgccaa gcaagcccac 1440 actcgcggca tcaaggtgat tgttgcttgt gccggtgggg cagcgcacct accaggcatg 1500 gtggctgcag caactccttt gccagttatt ggtattccac gtgccttgaa agatttggaa 1560 ggtctggact ctttgctgtc tatcgtgcag atgccagctg gggttccggt tgcgaccgtg 1620 tctatcggcg gcgctaagaa tgctggcttg ctcgccatcc gtaccctggg cgtgcagtac 1680 tcagaattgg ttgaacgcat ggccgattac caagaaaata tggccaagga agttgagcaa 1740 aaagacgcca atcttcgcgc caagctcatg ggggactag 1779 <210> 42 <211> 888 <212> DNA <213> Corynebacterium ammoniagenes <400> 42 atgcgcccac agctttctga ttatcagcac gtatcctccg gcaaagtccg cgatatctac 60 gaagtagatg acaacacttt gctcatggtg gtcaccgacc gcatctccgc ctatgacttc 120 gcactagagc cagccatccc cgataaaggc cgggttctta ccgcaaccac catgttcttc 180 ttcgacgcca tcgatttccc gaaccatttg gcaggaccca tcgatgatgc gcggattcca 240 gaagaagtat tgggccgagc gatcatcgtt aagaagctca acatgctgcc ctttgagtgc 300 gttgcccgcg gttacctcac cggttccggc ttgaaggaat acaacgctaa cggcaccgtg 360 tgcggcatcg agctgccaga aggcttggtt gaggcgtcgc gtctgccaga gccaattttc 420 accccagcca ccaaggcaga gcagggcgac cacgatgaaa acgtcagctt cgagcgcgtg 480 gtgcaggacc ttggccaaga gcgcgcagag cagcttcgcg atgaaaccct gcgcatctac 540 tccgccgccg ccaagattgc cgaagaaaag ggcatcatct tggctgatac gaagtttgaa 600 ttcggccttg attccgaagg caatctggtc ttgggcgatg aagtacttac gcctgattcc 660 tcccggtact ggccagcaga cacctacgcg gaaggcattg tgcagcccag ctttgacaag 720 cagtacgtgc gcaactggtt gacctcggag gaatccggct gggatgtgga gtcggaaacc 780 cagccgccag tgcttcccga tgacatcgtc gccgccaccc gcctgcgcta catcgaggct 840 tatgagcgct tgtccggcaa gcgtttcatc gacttcattg gcggttaa 888 <210> 43 <211> 1440 <212> DNA <213> Corynebacterium ammoniagenes <400> 43 atgaagcccg tggctgataa gaagaagatt tccaacgttt tgtcctcgcg ttacgcctct 60 gcagagttat ccaacttgtg gagcccggaa cacaaaatca tcatggagcg ccagctctgg 120 atcgcggtca tgcgcgccca gaaagacctc ggcgttgaca ttccatctga agcaattgac 180 gcatacgaag cagtcattga ccaggtagac ctaggttcca tcgcggatcg tgagcgcgtg 240 acgcgccatg acgtgaaagc tcgcatcgaa gaattcaacg cgctggctgg tcatgagcac 300 attcacaagg gcatgacctc ccgtgacctg acggagaacg tcgagcagct gcaaatctac 360 cgctccctgg agctcattcg tgataaagcc atcgcggttg cagcgcgcat tggtgagcac 420 gccgcgaaat accagacttt ggtcatggcg ggccgctctc acaacgttgc cgcgcaggcg 480 accaccttgg gtaagcgttt cgcatcggct ggtgatgaaa tgctgctcgc cattgagcgc 540 gtagaaaacc tccttgcccg ctacccactg cgcggcatca agggaccaat gggcacttcc 600 caggacatgc tggacttgat gggcggcgac gaaaacaagc tcgcagcgct cgaaaccgac 660 atcgcggacc acttgggctt tgcccgcgtg ttcaactcgg tgggccagat ttacccacgc 720 acccttgatt ttgatgcggt ttccgcgctg gttgagctgg gtgcctcgcc atcgtctctg 780 gctaccacca tccgcttgat ggccggcaac gaaaccgtca ccgaaggttt caaggaaggc 840 caagttggct cttccgcgat gccgcacaag atgaatgccc gctcctgcga acgcgtcggt 900 ggcctccagg ttatcttgcg cggctacctg actatggttg cagaccttgc tggtcagcag 960 tggaacgaag gcgacgtatt ctgctcggtg gttcgccgtg tagccctgcc ggacgctttc 1020 ttcgccatcg atggccagtt tgaaaccttc cttaccgtgt tggatgagtt cggcgcattc 1080 ccagcaatga tcgaccgcga actcgagcgc tacctgccat tcttggccac cacccgcatc 1140 cttatggctg cggtgcgcgc aggcgttggc cgcgaaaccg cacacgaagt catcaaggaa 1200 aacgcagtgg ctgttgcgct gaacatgcgc gaaaacggcg gggaacaaga cctactggag 1260 cgccttgctg ccgatgagcg cctgcccatg agcaaggaac agctcgaaga agcactcgct 1320 gacaagcacg cgtttatcgg cgctgctgaa tcccaggtag atgcagttct tagacgcatt 1380 aaggaactcg cggaccagca tccgaaggcg gccgcctaca ccccaggcga aatcttgtaa 1440                                                                         1440 <210> 44 <211> 1551 <212> DNA <213> Corynebacterium ammoniagenes <400> 44 atgagtgatg accgcaagca gatcaagcgt gcactaatta gcgtttatga caagacaggg 60 ctcgaagagc tcgctcgcac gcttgacagc gcaggcgtag agattgtgtc caccggctcc 120 accgccgcca agattgctga tcttggtatt aacgtcactc cggttgaatc tctcaccgga 180 ttcccagagt gcctcgaagg ccgcgttaag accttgcacc cacgcgtgca tgcgggcatt 240 ttggctgata cccgcaagcc ggatcacctt aatcagctgg aagagcttga gattgagcca 300 ttccagttgg tcgtggttaa cctgtaccca tttaaagaga ctgtagcttc tggcgcagac 360 ttcgatggtt gcgtcgagca gattgatatc ggcggtccat ccatggtccg tgctgctgcc 420 aagaaccacc catcggtggc ggttgttgta gacccagcgc gttacggcga catcgctgag 480 gctgtcgctc agggcggatt cgatctggcg cagcgtcgtc agctggccgc gactgcgttt 540 aagcacacgg cagattatga tgttgcagtt tctggctggt ttgcccagca gcttgccgat 600 gactctgttg cctctgctga gcttgaaggc gacgcgctgc gttatggtga gaaccctcac 660 cagcaggctt ccatcgttcg tgaaggcacg accggtgttg ctaatgcgaa gcagctgcac 720 ggtaaggaaa tgagctacaa caactaccag gacgcggatg ccgcatggcg cgcggcttgg 780 gatcatgaac gtccatgtgt agcaattatt aagcacgcta acccttgcgg tatcgctgtt 840 tctgatgagt ccatcgcagc agcacacgca gcggcacacg cctgtgaccc aatgtccgct 900 ttcggtggcg ttattgcggt caaccgcgaa gtcaccaagg aaatggcaac ccaggttgct 960 gacatcttca ccgaggtcat catcgcaccg tcctacgaag atggagccgt cgagattttg 1020 cagggcaaga agaatattcg catccttgtt gctgagcatg aagtaccagc agtagaggtc 1080 aaagaaatct ctggtggccg tctgctgcag gaagcagacg tttaccaggc tgagggcgat 1140 aaggcttcga gttggacttt ggctgccggc gaagctgcat ccgaggaaaa gctcgcggag 1200 ctggaattcg cttggcgcgc agtacgctcg gtaaagtcca acgccatctt gttggcgcat 1260 gaaggtgcaa ccgttggcgt gggtatgggc caggtcaacc gcgttgattc ggcgaagttg 1320 gctgttgacc gcgcgaatac tttggctgat tccgcagagc gtgctcgcgg ttccgtcgca 1380 gcatcggatg cgttcttccc attcgccgat ggcttgcagg tgcttatcga tgccggcgtt 1440 tccgccgttg tccagcccgg cggctccatc cgcgatgaag aagttattgc tgccgctgaa 1500 gcagccggta tcaccatgta cttcactggc acccgccact tcgcgcacta a 1551  

Claims (13)

Iii) phospholibosyl pyrophosphate amidotransferase, ii) phospholibosylamine glycine ligase, iii) phospholibosylglycine amide formyltransferase, iii) phospholibosyl formyl glycine amidine synthetase, iii) Phosphoribosylformylglycineamidine synthetase II, i) phospholibosylaminoimidazole synthetase, i) phospholibosylaminoimidazole carboxylase, i) phospholibosilaminoimidazole rosinocarboxamide synthetase A microorganism of the genus Corynebacterium, in which the production capacity of a nucleic acid-based substance is improved by enhancing two or more enzyme activities selected from the group of azeases, iii) adenylocicinate lyase, and iii) inosinic acid cyclohydrolase. The method of claim 1, Ii) phospholibosylamine glycine ligase, iii) phospholibosyl glycine amide formyl transferase, iii) phospholibosylformylglycine amidine synthetase, iii) phospholibosylformylglycine amidine synthetase II, ⅸ A microorganism of the genus Corynebacterium having improved production capacity of nucleic acid-based material by enhancing the enzymatic activity of adenyroloxylate lyase and iii) inosinic acid cyclohydrolase. The method of claim 2, In addition, microorganisms of the genus Corynebacterium having improved production capacity of nucleic acid-based substances by further enhancing the enzymatic activity of phosphoboribosylpyrophosphate amidotransferase and iii) phosphoribosylaminoimidazole synthetase. The method of claim 2, In addition, microorganisms of the genus Corynebacterium having enhanced production of nucleic acid-based substances by further enhancing the activities of iii) phosphoribosylaminoimidazole carboxylase and iii) phosphoribosylaminoimidazole rosinocarboxamide synthetase. The method of claim 1, i) phospholibosyl pyrophosphate amidotransferase, ii) phospholibosylamine glycine ligase, iii) phospholibosylglycine amide formyl transferase, iii) phospholibosyl phosphimlycine amidine synthetase, i) Phosphoribosylformylglycineamidine synthetase II, i) phospholibosylaminoimidazole synthetase, i) phospholibosylaminoimidazole carboxylase, i) phospholibosilaminoimidazole rosinocarboxamide synthetase A microorganism of the genus Corynebacterium in which the production capacity of a nucleic acid-based substance is improved by enhancing both the activities of azedes, iii) adenylolycinate lyase and iii) inosinic acid cyclohydrolase. The method of claim 1, Genes encoding the enzymes are purF, purD, purN, purS, purL, purM, purKE, purC, purB and purH Corynebacterium genus microorganisms with improved production capacity of nucleic acid-based material, characterized in that. The method of claim 6, The genes of the genus Corynebacterium improved production capacity of nucleic acid-based material, characterized in that having the nucleotide sequence of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43 and 44. The method of claim 1, The activity of the enzyme is one or more of purF, purD, purN, in addition to each of the intrinsic purF, purD, purN, purS, purL, purM, purKE, purC, purB, and purH genes possessed naturally by Corynebacterium sp. A microorganism of the genus Corynebacterium with improved production capacity of a nucleic acid-based material, characterized in that the purS, purL, purM, purKE, purC, purB and purH genes are each enhanced. The method of claim 1, The microorganism of the genus Corynebacterium is a vector pDZ-2purFM, pDZ-2purDB, pDZ-2purNH, pDZ-2purSL, pDZ-2purKE and pDZ-2purC having a cleavage map of Figures 2, 3, 4, 5, 6 and 7. Microorganisms of the genus Corynebacterium with improved production capacity of nucleic acid-based material, characterized in that transformed with two or more vectors selected from the group consisting of. The method of claim 1, The nucleic acid-based material is 5'-xanthylic acid (XMP) or 5'-inosinic acid (IMP) or inosine, Corynebacterium genus microorganisms with improved production capacity of the nucleic acid-based material, characterized in that. The method of claim 10, The microorganism of the genus Corynebacterium is Corynebacterium genus microorganisms with improved production capacity of nucleic acid-based material, characterized in that Corynebacterium ammonia genes CN01-0118 (KCCM10965P). A method for producing a nucleic acid-based substance, wherein the microorganism of the genus Corynebacterium according to any one of claims 1 to 11 is cultured, and a nucleic acid-based substance is obtained from the culture solution. The method of claim 12, The nucleic acid-based material is a method for producing a nucleic acid-based material, characterized in that 5'-xanthyl acid (XMP) or 5'-inosinic acid (IMP) or inosine.

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