KR100576341B1 - Microorganisms of Corynebacterium having an enactivated gene encoding 5'-nucleotidase and processes for the preparation of 5'-inosinic acid using the same - Google Patents

Abstract

The present invention has as Corynebacterium (Corynebacterium) in microorganism producing 5'-inosinate, gene or SEQ ID NO: 1 of the sequence homology of 90% or more of SEQ ID NO: 1 of the microorganism of the genus Corynebacterium (Corynebacterium) Provided are microorganisms of the genus Corynebacterium in which a gene encoding 5'-nucleotidase is inactivated, and a method of preparing 5'-inosinic acid using the same. The present invention also provides a gene encoding a 5'-nucleotidase of a Corynebacterium sp. Microorganism and a gene for inactivating the 5'-nucleotidase by replacing the gene.

Corynebacterium ammonia genes, 5'-inosinic acid, 5'-nucleotidase

Description

Microorganisms of Corynebacterium having an enactivated gene encoding 5'-nucleotidase and processes for the preparation of 5'-inosinic acid using the same}

1 shows the cloning process of genes that inactivate ushA.

Figure 2 shows the process of inactivating ushA by inserting pBSF-ushA into chromosomal DNA.

The present invention provides a Corynebacterium (Corynebacterium) in the microorganism producing 5'-inosinate (5'-inosinic acid), 5'-nucleotidase gene coding for (5'-nucleotidase) (hereinafter "ushA gene The present invention relates to a microorganism of the genus Corynebacterium and to a method for producing 5'-inosine acid using the same. The present invention also relates to a gene encoding 5'-nucleotidase of a Corynebacterium sp. Microorganism and a gene for inactivating 5'-nucleotidase by substituting the gene.

5'-inosinic acid is an intermediate of the nucleic acid biosynthetic metabolic system, which 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. In particular, when used together with sodium glutamate It is one of nucleic acid-based seasonings that has been spotlighted as a seasoning seasoning because of its synergistic effect. Currently, 5'-inosinic acid is known to be produced by fermenting a strain of the genus Corynebacterium , for example, Corynebacterium ammoniagenes (Korean Patent Publication No. 2003-42972, etc.). ).

On the other hand, the ushA gene has been reported to act to reduce nucleic acid concentration by decomposing nucleic acids in microorganisms (Esherichia coli and Salmonella : Cellular and Molecular Biology: 2nd edition: 561-579). Therefore, even when culturing the strain of Corynebacterium ( Coynebacterium ) to produce 5'-inosinic acid, the ushA gene is expressed during fermentation of the strain, resulting in the production of 5'-nucleotidase, resulting in 5'-inosinic acid. The problem is that the yield of is lowered.

Therefore, when inhibiting or inactivating the expression of the ushA gene in the Corynebacterium genus strains, it is expected that 5'-inosinic acid can be produced in high yield. However, to date, the sequence of the ushA gene in some other microorganisms has been reported, but the ushA gene of the Corynebacterium sp. Microorganism has not been reported yet, and the mutant which inactivated ushA has also been reported. none.

The present invention is to solve the problems of the prior art, and provides a microorganism in which the ushA gene of Corynebacterium sp. Microorganisms are inactivated and a method of preparing 5'-inosinic acid using the same. The present invention also provides a gene inactivates the ushA gene is substituted with the ushA gene and its.

Therefore, the present invention is a Corynebacterium genus microorganism producing 5'-inosinic acid, the gene encoding the 5'-nucleotidase (5'-nucleotidase) inactivated Corynebacterium ( Coynebacterium) ) To provide microorganisms of the genus.

It is also an object of the present invention to provide a method for producing 5'-inosine acid using the microorganism.

It is also an object of the present invention to provide a gene encoding a 5'-nucleotidase of a microorganism of the genus Corynebacterium .

It is also an object of the present invention to provide a gene for inactivating the ushA gene by replacing a gene encoding the 5'-nucleotidase of a Corynebacterium sp. Microorganism.

According to an aspect of the present invention, a Corynebacterium microorganism producing 5'-inosinic acid ( Corynebacterium ), a microorganism having a homology with 90% or more of the sequence of SEQ ID NO: 1 or SEQ ID NO: 1 Corynebacterium ( the microorganism of the genus Corynebacterium) the gene coding for the 5'-nucleotidase (5'-nucleotidase) in a microorganism inactivated nose Rhine tumefaciens (Corynebacterium) is provided.

The present inventors have known for Corynebacterium (Corynebacterium) in microorganisms for example, in a microorganism to produce a 5'-inosinate, Corynebacterium ammoniagenes was carried out to Ness chromosomal sequence analysis of (Corynebacterium ammoniagenes) ATCC 6872, 5 ' The DNA base sequence of the gene encoding the nucleotidase was analyzed. As a result, it was found that the ushA gene of Corynebacterium sp. Microorganism has a nucleotide sequence of SEQ ID NO: 1 of about 2 kb.

Accordingly, the microorganism according to the present invention is a microorganism belonging to the genus Corynebacterium ( Cyneynebacterium ) in which the gene of SEQ ID NO: 1, SEQ ID NO: by the normal substitution, deletion, and / or insertion for the gene of SEQ ID NO: of SEQ ID NO: 1 and less than 90%, preferably with at least 95%, more preferably homology of 98% or more Corynebacterium (Corynebacterium) activating gene is non-coding for 5'-nucleotidase of the microorganism of the genus Microorganisms of the genus Corynebacterium .

Microorganism of the present invention has a sequence that is at least 90% homologous to the gene or SEQ ID NO: 1 of the Sequence ID No. 1 Corynebacterium (Corynebacterium) the gene coding for the 5'-nucleotidase of the microorganism of the genus substitution (replacement Microorganisms inactivated by) are preferred. Preferably, the substitution is made by a gene of SEQ ID NO: 2 of about 1.1 kb, and at least about 80% of the gene of SEQ ID NO: 2 by conventional substitutions, deletions, and / or insertions of the gene of SEQ ID NO: 2, Preferably at least 90%, more preferably at least 95%, most preferably at least 98%, and having 5'-nucleotidase (ushA) of Corynebacterium ammoniagenes It consists of a gene encoding a protein having no activity.

Preferably, the microorganism of the present invention has at least 80% homology with the gene of SEQ ID NO: 2 or the sequence of SEQ ID NO: 2, and has the 5'-nucleotidase activity of the microorganism of the genus Corynebacterium ( Coynebacterium ) that transformation of the Corynebacterium (Corynebacterium) in the microorganism producing 5'-inosinate a recombinant vector comprising a gene encoding the protein and, 5'-nucleotidase is an I microorganism obtained by screening an inactivated strain .

The recombinant vector has a homology of 80% or more with the gene of SEQ ID NO: 2 or the sequence of SEQ ID NO: 2, and encodes a protein that does not have 5'-nucleotidase activity of a microorganism of the genus Corynebacterium . Both ends of the DNA fragments amplified by the polymerase chaine reaction (PCR) of the gene were cut with DNA restriction enzymes such as Xba I and Bam HI, and the DNA was digested with the same DNA restriction enzyme. It can be prepared by inserting using T4 ligase and the like. The vector which can be used is not specifically limited, A well-known expression vector can be used. Preferably, pBSG vectors developed by the present inventors can be used (Accession No .: KCCM-10532). The plasmid vector thus prepared can be delivered to Escherichia coli using a conventional electroporation method, and recombinant vectors can be collected according to a known method (Quiagen plasmid preparation KIT). Therefore, the recombinant vector is preferably pBSG-ushA prepared by inserting the gene of SEQ ID NO: 2 in the pBSG vector.

The microbial strain (eg, Corynebacterium ammoniagenes CJHB100 (KCCM-10330) using the conventional electroporation method, the linear DNA fragments, Korean Patent Publication No. 2003-42972 forwarding the call), and the recombinant vector, for example, the ushA gene of pBSG-ushA vector microbial chromosome and the homology arm and the homologous microbial chromosomal region the pBSG-ushA vector a microorganism strain chromosome by homologous recombination in on which is located After replacing the cells to inactivate the ushA gene, microbial mutants can be selected by culturing in a medium containing the antibiotic gentamycine, a selection marker. Insertion of pBSG-ushA into the genome by homologous recombination in selected mutant strains can be confirmed by PCR. The cloning process of the gene is briefly shown in FIG.

More preferably, the microorganism of the present invention is Corynebacterium ammoniagenes CJWS01 (KCCM-10528).

According to another aspect of the present invention, there is provided a method for producing 5'-inosinic acid, comprising culturing the microorganism of the present invention and separating 5'-inosinic acid from the culture solution.

The microorganism of the present invention can be cultured while controlling the temperature, pH and the like under aerobic conditions in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins and the like.

As the carbon source, carbohydrates such as glucose, fructose, sterilized pretreated molasses (ie, molasses converted to reducing sugars) and the like can be used. As nitrogen sources, various inorganic nitrogen sources such as ammonia, ammonium chloride and ammonium sulfate and peptone, NZ-amine Organic nitrogen sources may be used, such as meat extracts, yeast extracts, corn steep liquors, casein hydrolysates, fish or their degradation products, skim soy cakes, or their degradation products. 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 stirring, preferably at a temperature of 20-40 ° C. The pH of the medium is preferably maintained near the neutral during the cultivation, the culturing can be carried out for 5 to 6 days and the 5-inosinic acid accumulated by the direct fermentation method can be recovered by a conventional method.

When the 5'-inosinic acid is prepared according to the preparation method of the present invention, the ushA gene, which is an enzyme that degrades nucleic acids, is inactivated, thereby producing 5'-inosinic acid with high yield.

According to a further aspect of the present invention, a gene with a gene or SEQ ID NO: 1 of the sequence homology of 90% or more of the SEQ ID NO: 1 encoding a Corynebacterium (Corynebacterium) 5'- nucleotidase of the microorganism of the genus Is provided.

According to another aspect of the invention, the gene of SEQ ID NO: 2 or more than 80% homology with the sequence of SEQ ID NO: 2, and having the 5'-nucleotidase activity of the genus Corynebacterium ( Coynebacterium ) Genes that encode proteins are provided.

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 1. Cloning of the ushA Gene

BSG-ushA vectors were constructed as briefly shown in FIG. pBSG vectors include the Gm ^R and sacB genes.

The gene fragment of SEQ ID NO: 2 obtained through the polymerase chain reaction was digested with restriction enzymes Xba I (New England Biolabs, Beverly, MA) and Bam HI (New England Biolabs, Beverly, MA). The gene fragment was conjugated to linear pBSG vectors digested with Xba I and Bam HI restriction enzymes using ligase (New England Biolabs, Beverly, Mass.). The conjugated DNA was transformed into the E. coli GM2525 (Esherichia coli genetic stock center, Yale University, New Haven, Connecticut) by electroporation, so that gentamycin per liter of 5 mg of LB containing antibiotics. Single colonies grown on solid medium (10 g / L yeast extract, 10 g / L bactotriptone, 10 g / L sodium chloride, 1.7% bactoa, pH 7.0) were recovered. The recovered colonies were separated from plasmids in cells cultured in LB medium containing the same antibiotic, and the size of the plasmids was determined first. Secondly , double-cutting with Xba I and Bam HI was performed to obtain 1107 bp DNA fragments. Recombination plasmid pBSG-ushA (5.5 kb) containing the ushA gene fragment was prepared by confirming that it comes out.

The primers used herein are as follows.

Primer ushA-F (SEQ ID NO: 3): 5`-ACGGCGTCAAGGTGGGATTT-3`

Primer ushA-B (SEQ ID NO: 4): 5`-GGGTGCCTTCGGTGCTGATAC-3`

Example 2 Analysis of gentamycin (hereinafter referred to as Gm) minimum inhibitory concentration of parent strain CJHB100 (KCCM-10330)

To prepare the concentration at 10 ⁴ CFU / ul at the first inoculation, 12 hours incubation of Corynebacterium ammoniagenes CJHB100 (KCCM-10330) in 3 ml of medium (10 ^{8-10 7} CFU / μl) l) and prepared agar nutrient medium (peptone 1%, gravy 1%, sodium chloride 0.25%, yeast extract 1%, agar 2% pH 7.2) to confirm the minimum inhibitory concentration of gentamicin. 20 ml of agar medium were added to the plates to 5, 4, 3, 1.5, 1, 0.8, 0.4, 0.1, 0.05, 0.01, 0.005 and 0.001 mg / l, respectively. The plate was prepared by diluting Corynebacterium ammoniagenes CJHB100 (KCCM-10330) grown in 3 ml of seed medium by 10 ⁶ fold. Stained plate was incubated for 96 hours at 32 ℃, strains did not grow above 0.005mg / L of gentamicin, and from this result, strains inactivated ushA gene was selected, 10 than the minimum inhibitory concentration of 0.005mg / L A fold higher 0.05 mg / L was used.

Example 3 Selection of Strains with Inactivated ushA Gene on Chromosome by Insertion of Recombinant Plasmid

CM solid medium to which 0.05 mg / L gentamicin was added was transformed to a 5'-inosinic acid-producing strain, Corynebacterium ammoniagenes CJHB100 (KCCM-10330) using a recombinant plasmid pBSG-ushA isolated from Escherichia coli GM2525. Yeast extract 10 g / L, bacto tryptone 10 g / L, sodium chloride 2.5 g / L, bactoa 1.7%, adenine 100 mg / L, guanine 100 mg / L, pH 7.0) and incubated for 96 hours at 32 ℃. Through a single colony polymerase chain reaction, a recombinant strain inserted at a specific position of a chromosome of ammonia genes ( Corynebacterium ammoniagenes ) CJHB100 (KCCM-10330) was selected. Primers used herein are as follows, the process of inactivating the ushA gene by inserting pBSG-ushA into the chromosomal DNA is shown in FIG.

Primer ushA-FC (SEQ ID NO: 5): 5'-GCTGGGCGCGAATGTCACCTTA-3 '

Primer ushA-FB (SEQ ID NO: 6): 5'-GAAAGACCGCCCGCTGTTGAGAC-3 '

The inactivated ushA gene on the chromosome selected through the above procedure was named Corynebacterium ammoniagenes CJWS01 and deposited on November 17, 2003 at the Korea Microbiological Conservation Center (Accession Number: KCCM- 10528).

Example 4. Erlenmeyer flask fermentation titer test

3 ml of seed medium was dispensed into a 18 mm diameter test tube, autoclaved and inoculated with Corynebacterium ammoniagenes CJWS01 (KCCM-10528) and shaken at 30 ° C. for 24 hours to use as a seed culture solution. 27 ml of the fermentation broth was dispensed into a 500 ml shake flask, inoculated under autoclave at 120 ° C. for 10 minutes, inoculated with 3 ml of the seed culture solution, and incubated for 5 to 6 days. The rotation speed was adjusted to 200 times per minute at a temperature of 32 ° C. and pH 7.2. 5'-inosinic acid accumulation in the medium was about 5% higher than the parent strain CJHB100 (KCCM-10330). The composition of the seed medium and fermentation medium is as follows.

Species medium: glucose 5%, peptone 0.5%, broth 0.5%, yeast extract 1%, sodium chloride 0.25%, adenine 100mg / l, guanine 100mg / l, pH7.2

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, L- Cysteine 23mg / l, Alanine 24mg / l, Nicotinic Acid 8mg / l, Biotin 45µg / l, Thiamine Hydrochloride 5mg / l, Adenine 30mg / l, Phosphoric Acid (85%) 1.9%, Fructose, Glucose and Molasses as reducing sugars Use to add 8%

Example 5. Fermentation Titer Test in 5-L Fermenter

50 ml of the seed medium of Example 43 was dispensed into a 500 ml shaking Erlenmeyer flask and autoclaved, and then inoculated with Corynebacterium ammoniagenes CJWS01 (KCCM-10528) and shaken at 30 ° C. for 24 hours for seed. Used as a culture. 1000 ml of fermenter seed medium was placed in a 2.5 L-fermentation tank and autoclaved at 120 ° C. for 15 minutes to inoculate 50 ml of seed culture solution, and then cultured for 1-2 days. The rotation speed was adjusted to 900 times per minute, the temperature of 28 ~ 34 ℃, pH7.2. In addition, 1250 ml of the fermenter broth medium was placed in a 5L-fermentation tank and autoclaved at 120 ° C. for 15 minutes to inoculate 250 ml of the fermenter broth broth. Then, when the reducing sugar became 2% in the culture medium, the primary, secondary and tertiary Fourth additional sugar was mixed with fructose, glucose and molasses, and added to 32% as the final reducing sugar, followed by incubation for 5 to 6 days. Rotation speed was adjusted to 900 times per minute, temperature 32 ℃, pH7.2. At this time, 5'-inosin acid accumulation in the medium was improved by 4% over the parent strain CJHB100 (KCCM-10330). The composition of the fermenter seed broth and the fermenter main broth are as follows.

Fermenter species medium: glucose 5.4%, peptone 1%, yeast extract 2%, potassium potassium phosphate 0.1%, potassium diphosphate 0.1%, magnesium sulfate 0.1%, ammonium sulfate 0.5%, iron sulfate 80mg / l, zinc sulfate 40mg / l, manganese sulfate 40mg / l, L-cysteine 80mg / l, calcium pantothenate 60mg / l, thiamine hydrochloride 20mg / l, biotin 240µg / l, adenine 1200mg / l, guanine 1200mg / l (pH7.2)

Fermenter Main Medium: 120 mg / l calcium chloride, 8 mg / l copper sulfate, 1.5% magnesium sulfate, 24 mg / l iron sulfate, 24 mg / l zinc sulfate, 20 mg / l manganese sulfate, 26.4 mg / l L-cysteine, 0.12% sodium glutamate, Thiamine Hydrochloride 6mg / l, Biotin 40µg / l, Nicotinic Acid 50mg / l, Alanine 145mg / l, Adenine 200mg / l, Phosphoric Acid (85%) 4.3%, Fructose, Glucose and Molasses were added to 32% as reducing sugar Usage (pH7.2)

The microorganism of the genus Corynebacterium in which the gene encoding the 5'-nucleotidase according to the present invention is inactivated is a microorganism that directly accumulates 5'-inosinic acid in a culture medium with high yield and high concentration. The production method of the present invention using the same allows to produce 5'-inosinic acid in high yield.

<110> CJ Corp. <120> Microoranisms of Corynebacterium having an enactivated gene          encoding 5'-nucleotidase and processes for the preparation of          5'-inosinic acid using the same <130> PN052456 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 1929 <212> DNA <213> Corynebacterium ammoniagenes <400> 1 atgggggcgg ccaagctcaa ggaactgatt gagtacgtta atcaggacca ggaatacatc 60 atgactacct ccggcgataa cgtcggcggt agcgctttcg tctcggcaat cgctgatgat 120 gagccaaccc tagatgtcct caacatgctc ggcgttgacg tctctgcggt aggtaaccat 180 gaattcgacc gcggctatga cgatttgctc ggtcgtattg ctgaaaaatc cgcctaccca 240 ttgctgggcg cgaatgtcac cttaaacggt gaaccgatgt tggatgcttc ctacgttcaa 300 gaaattgacg gcgtcaaggt gggatttgtc ggtactgtga ccaccttgac taaggacaag 360 gtcgcaccat ccgcagttga aggtgtggag ttttctgacc ccgtcgaggc tacgaataag 420 gaatctgacc gtctgaagga atctggcgaa gcagacgttg ttgtagcgct catgcacgag 480 gacgcgcagc agtacgctgc tggcttcaac aacaatgtgg atatcctttt cggaggcgac 540 tctcaccagc gctcgcaggg catcatcgaa cgtgatggtg cgcagccact gcactgggcg 600 caggggcatg aatatggcaa ggttctccaa gacgccgata tttcctttaa caaggacacc 660 ggcgaaattg agtccgtcga aatcacccag tacgaccgct cgatgcctga ggttgaagca 720 ctagcccctg atgcagaaat tgccgctcgc gtggcagccg ctgaggcaga agctgaagag 780 cttggagcta aagttgtcgg ccagatggac cgcgctactt tccgtggtca agatgagggc 840 gcaggagcgg ggagcaaccg cggtgttgaa tctacgttga actcgctaat tgctgatgcc 900 aaccgcgcat cagttgctaa ggcgactggt gcggacgtgg atttgggcat tatgaatgct 960 ggtggcgtac gtgctgacct tcctgcaggc gatgtcactt tccaggatgt gcttaccgtg 1020 cagcctttcg gcaattcgat tgcatacggc actttgaccg gccaagatat tttggatgct 1080 ttggaagctc agtggcagcc aggatcgtct cgtccacgct tggctttggg tctatctgct 1140 ggatttgagt acgcctacga cccaactgca gagcaaggcc agcgcgttat ctccgcgacc 1200 ttggatggcg aagaaattga cccatcagcg gaatacactg ttgcaacttc cacgttcctc 1260 ttcgatggtg gcgacaactt cgcatctctg gccaatgtcc aaaacctcac tgacgtgggc 1320 tacatggact actcagttct caatgactac atcaaagatg gcgcagaggt acaggaaggc 1380 cagtctgata tcggtatcag caccgaaggc accctggcag ctggtgaaga agttaccttc 1440 aacctcacct cgcttaacta cactatggaa gaagatccac aagccaccac agcgaccgtc 1500 accgtgggcg acgtgcaaga aaccgccgat atcgacgctc agtggaatgc tgaagaagat 1560 ccgcagaaga atgaatttgg ccgtgctagc gtaacgctga ctctgccaga ggacattgag 1620 gaaaccgact tggttaccgt aaccaccgat gccggcaccg agattactgt gccgctgagg 1680 gccttgggca ccaacgaggg cacggacggc ggctccaatc ctggcagcag cgcacctggc 1740 acaggtaagg gctcttctcg gggtgcttcg gctgctgcag gaatcgctgg agtgttggca 1800 gcgattgcgg gcatcgccgc gtttattggt ctgaacggtc agtttgatca gttcatccca 1860 gctaacatgc agcgcgctct tggtgacctg cgcagtcagc tcaacgaggt tagccacatc 1920 aaattctag 1929 <210> 2 <211> 1066 <212> DNA <213> Corynebacterium ammoniagenes <400> 2 gtcggtactg tgaccacctt gactaaggac aaggtcgcac catccgcagt tgaaggtgtg 60 gagttttctg accccgtcga ggctacgaat aaggaatctg accgtctgaa ggaatctggc 120 gaagcagacg ttgttgtagc gctcatgcac gaggacgcgc agcagtacgc tgctggcttc 180 aacaacaatg tggatatcct tttcggaggc gactctcacc agcgctcgca gggcatcatc 240 gaacgtgatg gtgcgcagcc actgcactgg gcgcaggggc atgaatatgg caaggttctc 300 caagacgccg atatttcctt taacaaggac accggcgaaa ttgagtccgt cgaaatcacc 360 cagtacgacc gctcgatgcc tgaggttgaa gcactagccc ctgatgcaga aattgccgct 420 cgcgtggcag ccgctgaggc agaagctgaa gagcttggag ctaaagttgt cggccagatg 480 gaccgcgcta ctttccgtgg tcaagatgag ggcgcaggag cggggagcaa ccgcggtgtt 540 gaatctacgt tgaactcgct aattgctgat gccaaccgcg catcagttgc taaggcgact 600 ggtgcggacg tggatttggg cattatgaat gctggtggcg tacgtgctga ccttcctgca 660 ggcgatgtca ctttccagga tgtgcttacc gtgcagcctt tcggcaattc gattgcatac 720 ggcactttga ccggccaaga tattttggat gctttggaag ctcagtggca gccaggatcg 780 tctcgtccac gcttggcttt gggtctatct gctggatttg agtacgccta cgacccaact 840 gcagagcaag gccagcgcgt tatctccgcg accttggatg gcgaagaaat tgacccatca 900 gcggaataca ctgttgcaac ttccacgttc ctcttcgatg gtggcgacaa cttcgcatct 960 ctggccaatg tccaaaacct cactgacgtg ggctacatgg actactcagt tctcaatgac 1020 tacatcaaag atggcgcaga ggtacaggaa ggccagtctg atatcg 1066 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 acggcgtcaa ggtgggattt 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 gggtgccttc ggtgctgata c 21 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 gctgggcgcg aatgtcacct ta 22 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 gaaagaccgc ccgctgttga gac 23

Claims (9)

Corynebacterium (Corynebacterium) a microorganism of the genus, SEQ ID NO: 1 gene is substituted, deleted, or inserted by the active Corey fire Corynebacterium (Corynebacterium) in the microorganism producing 5'-inosinate. The microorganism according to claim 1, wherein the gene of SEQ ID NO: 1 is inactivated by substitution by the gene of SEQ ID NO: 2. delete The method of claim 2, wherein the recombinant vector pBSG-ushA comprising the gene of SEQ ID NO: 2 transforms the microorganism of the genus Corynebacterium ( Corynebacterium ) producing 5'-inosinic acid, 5'- nucleotidase Microorganism obtained by selecting the activated strain. delete The microorganism according to claim 4, which is Corynebacterium ammoniagenes CJWS01 (KCCM-10528). A method for producing 5'-inosine acid, comprising culturing the microorganism according to any one of claims 1, 2, 4, and 6, and separating 5'-inosinic acid from the culture solution. The gene of SEQ ID NO: 1. The gene of SEQ ID NO: 2.

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