BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a microorganism belonging to the genus Corynebacterium producing 5’-inosinic acid  in which the expression of genes encoding purine biosynthesis related enzymes is increased higher than the intrinsic expression  and a method for producing 5’-inosinic acid  comprising culturing the microorganism of the genus Corynebacterium with improved 5""-inosinic acid productivity.

2. Description of the Related Art
One of the nucleotide compounds  5’-inosinic acid is an intermediate material of the metabolic system of nucleotide biosynthesis  which is used in a variety of fields such as foods  medicines  and other various medical areas and functions to play an important role in animal and plant physiology. In particular  5’-inosinic acid is a nucleotide seasoning  which has drawn much attention as a savory seasoning  because it has synergistic effects when used with monosodium glutamate (MSG).
So far well known processes for producing 5’-inosinic acid include a process of enzymatically decomposing ribonucleic acid extracted from yeast cells (Japanese Published Examined Patent Application No. 1614/1957  etc)  a process of chemically phosphorylating inosine produced by fermentation (Agric. Biol. Chem.  36  1511(1972)  etc) and a process of culturing a microorganism capable of producing 5’-inosinic acid and recovering inosine monophosphate (IMP) accumulated in the medium. Currently  the processes of producing 5’-inosinic acid using microorganisms are mostly used. The strains of the genus Corynebacterium are widely used as a microorganism for the production of 5’-inosinic acid  and for example  a method for producing 5’-inosinic acid by culturing Corynebacterium ammoniagenes is disclosed (Korean Patent Publication No. 2003-0042972).
To improve a production yield of 5’-inosinic acid by a microorganism  studies have been made to develop strains by increasing or decreasing activity or expression of the enzymes involved in the biosynthetic or degradative pathway of 5’-inosinic acid. Korean Patent No. 785248 discloses a microorganism in which a purC gene encoding phosphoribosylaminoimidazole succinocarboxamide synthetase is overexpressed in the purine biosynthetic pathway and a method for producing 5’-inosinic acid using the same. In addition  Korean Patent No. 857379 discloses a Corynebacterium ammoniagenes strain in which the purKE -encoded phosphoribosylaminoimidazole carboxylase is overexpressed and a method for producing high concentration of IMP in a high yield using the same.
However  there is still a need to develop a strain capable of producing 5’-inosinic acid in a higher yield and a method for producing 5’-inosinic acid using the same.
Therefore  the present inventors have conducted studies to develop a strain capable of producing 5’-inosinic acid with high productivity. As a result  they found that the productivity of 5’-inosinic acid can be improved by simultaneously increasing activities of the major enzymes involved in the purine biosynthesis pathway higher than the intrinsic activity  thereby completing the present invention.

SUMMARY OF THE INVENTION
An object of the present invention is to provide a microorganism of the genus Corynebacterium having improved 5’-inosinic acid productivity.
Another object of the present invention is to provide a method for producing 5’-inosinic acid using the microorganism of the genus Corynebacterium having improved 5’-inosinic acid productivity.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a pDZ vector for chromosomal insertion into the microorganism of the genus Corynebacterium;
FIG. 2 shows a pDZ-2purFM vector for chromosomal insertion into the microorganism of the genus Corynebacterium;
FIG. 3 shows a pDZ-2purNH vector for chromosomal insertion into the microorganism of the genus Corynebacterium;
FIG. 4 shows a pDZ-2purSL vector for chromosomal insertion into the microorganism of the genus Corynebacterium;
FIG. 5 shows a pDZ-2purKE vector for chromosomal insertion into the microorganism of the genus Corynebacterium;
FIG. 6 shows a pDZ-2purC vector for chromosomal insertion into the microorganism of the genus Corynebacterium; and
FIG. 7 shows a pDZ-2prs vector for chromosomal insertion into the microorganism of the genus Corynebacterium.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
In order to achieve the above objects  the present invention provides a microorganism belonging to the genus Corynebacterium producing 5’-inosinic acid  in which the expression of genes encoding purine biosynthesis related enzymes is increased higher than the intrinsic expression.
The microorganism of the genus Corynebacterium of the present invention has more improved 5’-inosinic acid productivity than a parental strain  because the expression of genes encoding purine biosynthesis related enzymes is increased higher than the intrinsic expression.
As used herein  the term “purine biosynthesis related enzyme” means an enzyme that catalyzes the reaction involved in the purine biosynthesis pathway producing a purine base as a final product  and includes phosphoribosylpyrophosphate amidotransferase  phosphoribosylglycinamide formyltransferase  phosphoribosylformylglycinamidin synthetase  phosphoribosylformylglycinamidin synthetase II  phosphoribosylaminoimidazole synthetase  phosphoribosylaminoimidazole carboxylase  phosphoribosyl aminoimidazole succinocarboxamide synthetase  inosinic acid cyclohydrolase  ribosephosphate pyrophosphokinase or the like.
In a specific embodiment of the present invention  the purine biosynthesis related enzymes may be a combination of one or more enzymes selected from the group consisting of phosphoribosylpyrophosphate amidotransferase  phosphoribosylglycinamide formyltransferase  phosphoribosylformylglycinamidin synthetase  phosphoribosylformylglycinamidin synthetase II  phosphoribosylaminoimidazole synthetase  phosphoribosylaminoimidazole carboxylase  phosphoribosyl aminoimidazole succinocarboxamide synthetase and inosinic acid cyclohydrolase  and ribosephosphate pyrophosphokinase.
In a specific embodiment of the present invention  the gene encoding purine biosynthesis related enzymes  of which expression is increased higher than the intrinsic expression  are a combination of a purN gene of SEQ ID NO. 36  which codes for phosphoribosylglycinamide formyltransferase  a purS gene of SEQ ID NO. 37  which codes for phosphoribosylformylglycinamidin synthetase  a purL gene of SEQ ID NO. 38  which codes for phosphoribosylformylglycinamidin synthetase II  a purKE gene of SEQ ID NO. 40  which codes for phosphoribosylaminoimidazole carboxylase  a purC of SEQ ID NO. 41  which codes for phosphoribosyl aminoimidazole succinocarboxamide synthetase  a purH gene of SEQ ID NO. 42  which codes for inosinic acid cyclohydrolase  and a prs gene of SEQ ID NO. 43  which codes for ribosephosphate pyrophosphokinase.
In a specific embodiment of the present invention  the gene encoding purine biosynthesis related enzymes  of which expression is increased higher than the intrinsic expression  are a combination of a purF gene of SEQ ID NO. 35  which codes for phosphoribosylpyrophosphate amidotransferase  a purN gene of SEQ ID NO. 36  which codes for phosphoribosylglycinamide formyltransferase  a purS gene of SEQ ID NO. 37  which codes for phosphoribosylformylglycinamidin synthetase  a purL gene of SEQ ID NO. 38  which codes for phosphoribosylformylglycinamidin synthetase II  a purM gene of SEQ ID NO. 39  which codes for phosphoribosylaminoimidazole synthetase  a purKE gene of SEQ ID NO. 40  which codes for phosphoribosylaminoimidazole carboxylase  a purC of SEQ ID NO. 41  which codes for phosphoribosyl aminoimidazole succinocarboxamide synthetase  a purH gene of SEQ ID NO. 42  which codes for inosinic acid cyclohydrolase  and a prs gene of SEQ ID NO. 43  which codes for ribosephosphate pyrophosphokinase.
As used herein  the term “increased higher than the intrinsic expression” means that the gene expression level is higher than that in naturally expressed in a microorganism or higher than that expressed in a parental strain  and includes an increase in the number (copy number) of the genes encoding corresponding enzyme and the expression level increased thereby or an increase in the expression level by mutation or an increase in the expression level by both of them.
In a specific embodiment of the present invention  the increase in the expression level of the gene encoding purine biosynthesis related enzyme includes the increase in the copy number of the gene by additionally introducing the corresponding foreign gene into a strain or by amplifying the intrinsic gene  or the increase in transcription efficiency or translation efficiency by mutation in the transcription or translation regulatory sequence  but is not limited thereto. The amplification of the intrinsic gene may be easily performed by a method known in the art  for example  by cultivation under a suitable selection pressure.
In a specific embodiment of the present invention  the expression level of the gene encoding purine biosynthesis related enzyme may be increased by additionally introducing the gene encoding purine biosynthesis related enzyme into a cell or by amplifying the intrinsic gene encoding the purine biosynthesis related enzyme.
In a specific embodiment of the present invention  the gene encoding purine biosynthesis related enzyme  of which the expression level is increased higher than the intrinsic expression  may exist as two or more copies in the microorganism of the genus Corynebacterium having improved 5’-inosinic acid productivity by introducing one or more copies into a cell  in addition to the corresponding intrinsic gene.
In a specific embodiment of the present invention  the gene encoding purine biosynthesis related enzyme is introduced into the microorganism of the genus Corynebacterium having improved 5’-inosinic acid productivity by transformation using a recombinant vector containing two copies of the corresponding gene that are consecutively arranged.
In a specific embodiment of the present invention  the recombinant vector used for preparation of the microorganism of the genus Corynebacterium having improved 5’-inosinic acid productivity may be selected from the group consisting of pDZ-2purFM  pDZ-2purNH  pDZ-2purSL  pDZ-2purKE  pDZ-2purC  and pDZ-2prs recombinant vectors  which have the cleavage maps of FIGs. 2 to 7 respectively  depending on the genes introduced.
In a specific embodiment of the present invention  the microorganism of the genus Corynebacterium having improved 5’-inosinic acid productivity may be derived from Corynebacterium microorganisms capable of producing 5’-inosinic acid. For example  the microorganism of the genus Corynebacterium having improved 5’-inosinic acid productivity according to the present invention may be derived from Corynebacterium ammoniagenes ATCC6872  Corynebacterium thermoaminogenes FERM BP-1539  Corynebacterium glutamicum ATCC13032  Brevibacterium flavum ATCC14067  Brevibacterium lactofermentum ATCC13869  and strains prepared therefrom.
In a specific embodiment of the present invention  the microorganism of the genus Corynebacterium having improved 5’-inosinic acid productivity may include two or more copies of the gene encoding purine biosynthesis related enzyme.
In a specific embodiment of the present invention  the microorganism of the genus Corynebacterium having improved 5’-inosinic acid productivity may be Corynebacterium ammoniagenes  and more preferably a transformed Corynebacterium ammoniagenes  in which the activity of a combination of the prs gene and one or more genes selected from the group consisting of purF  purN  purS  purL  purM  purKE  purC  and purH is increased to produce high concentration of 5’-inosinic acid.
In a specific embodiment of the present invention  the microorganism of the genus Corynebacterium having improved 5’-inosinic acid productivity may be a strain  in which the 5’-inosinic acid-producing Corynebacterium ammoniagenes CJIP2401 (KCCM-10610) strain is introduced with each of the pDZ-2purFM  pDZ-2purNH  pDZ-2purSL  pDZ-2purKE  pDZ-2purC  and pDZ-2prs recombinant vectors having the cleavage maps of FIGs. 2  3  4  5  6  and 7 in order or in combination  and one of two copies of the introduced purF  purN  purS  purL  purM  purKE  purC  purH and prs genes are substituted for the corresponding intrinsic genes by homologous recombination  and thus each two copies of the purF  purN  purS  purL  purM  purKE  purC  purH  and prs genes are inserted into the strain.
In a specific embodiment of the present invention  the microorganism of the genus Corynebacterium having improved 5’-inosinic acid productivity may be Corynebacterium ammoniagenes containing two copies of the genes encoding purine biosynthesis related enzymes that are a combination of the purN gene of SEQ ID NO. 36  which codes for phosphoribosylglycinamide formyltransferase  the purS gene of SEQ ID NO. 37  which codes for phosphoribosylformylglycinamidin synthetase  the purL gene of SEQ ID NO. 38  which codes for phosphoribosylformylglycinamidin synthetase II  the purKE gene of SEQ ID NO. 40  which codes for phosphoribosylaminoimidazole carboxylase  the purC of SEQ ID NO. 41  which codes for phosphoribosyl aminoimidazole succinocarboxamide synthetase  the purH gene of SEQ ID NO. 42  which codes for inosinic acid cyclohydrolase  and the prs gene of SEQ ID NO. 43  which codes for ribosephosphate pyrophosphokinase  and preferably Corynebacterium ammoniagenes CN01-0120.
In a specific embodiment of the present invention  the microorganism of the genus Corynebacterium having improved 5’-inosinic acid productivity may be Corynebacterium ammoniagenes containing two copies of the genes encoding purine biosynthesis related enzymes that are a combination of the purF of SEQ ID NO. 35  which codes for phosphoribosylpyrophosphate amidotransferase  the purN gene of SEQ ID NO. 36  which codes for phosphoribosylglycinamide formyltransferase  the purS gene of SEQ ID NO. 37  which codes for phosphoribosylformylglycinamidin synthetase  the purL gene of SEQ ID NO. 38  which codes for phosphoribosylformylglycinamidin synthetase II  the purM of SEQ ID NO. 39  which codes for phosphoribosylaminoimidazole synthetase  the purKE gene of SEQ ID NO. 40  which codes for phosphoribosylaminoimidazole carboxylase  the purC of SEQ ID NO. 41  which codes for phosphoribosyl aminoimidazole succinocarboxamide synthetase  the purH gene of SEQ ID NO. 42  which codes for inosinic acid cyclohydrolase  and the prs gene of SEQ ID NO. 43  which codes for ribosephosphate pyrophosphokinase  and preferably Corynebacterium ammoniagenes CN01-0316 (KCCM 10992P).
Further  the present invention provides a method for producing 5’-inosinic acid  comprising the steps of culturing the microorganism belonging to the genus Corynebacterium producing 5’-inosinic acid  in which the expression of a gene encoding purine biosynthesis related enzyme is increased higher than the intrinsic expression  and recovering 5’-inosinic acid from the culture medium.
In the method for producing 5’-inosinic acid of the present invention  the medium and other culture conditions used for the cultivation of the microorganism of the genus Corynebacterium may be the same as those typically used in the cultivation of the microorganism of the genus Corynebacterium  and easily selected and adjusted by those skilled in the art. In addition  the cultivation may be performed by any cultivation method known to those skilled in the art  for example  batch  continuous  and fed-batch culture  but is not limited thereto.
In a specific embodiment of the present invention  the microorganism belonging to the genus Corynebacterium producing 5’-inosinic acid may be Corynebacterium ammoniagenes.
In a specific embodiment of the present invention  the microorganism belonging to the genus Corynebacterium producing 5’-inosinic acid may be Corynebacterium ammoniagenes CN01-0120 or Corynebacterium ammoniagenes CN01-0316 (KCCM 10992P).
In a specific embodiment of the present invention  culturing the microorganism of the genus Corynebacterium is performed by culturing the strain in a conventional medium containing suitable carbon sources  nitrogen sources  amino acids  vitamins or the like under aerobic conditions by adjusting temperature  pH or the like.
As a carbon source  carbohydrates such as glucose and fructose may be used. As a nitrogen source  various inorganic nitrogen sources such as ammonia  ammonium chloride  and ammonium sulphate may be used  and organic nitrogen sources such as peptone  NZ-amine  beef extract  yeast extract  corn steep liquor  casein hydrolysate  fish or fish meal  and defatted soybean cake or meal may be used. Examples of the inorganic compounds include potassium monohydrogen phosphate  potassium dihydrogen phosphate  magnesium sulfate  ferrous sulfate  manganese sulfate  and calcium carbonate. When needed  vitamins and auxotrophic bases may be used.
The cultivation is performed under aerobic conditions  for example  by shaking culture or stirring culture  preferably at a temperature of 28 to 36°C. During the cultivation  the pH is preferably maintained within the range of pH 6 to 8. The cultivation may be performed for 4 to 6 days.

Hereinafter  the present invention will be described in more detail with reference to Examples. However  these Examples are for illustrative purposes only  and the invention is not intended to be limited by these Examples.

Example 1. Insertion of genes encoding purine biosynthesis related enzymes using vector (pDZ) for chromosomal insertion and Development of strain producing high yield of 5’-inosinic acid thereby
In order to insert a foreign gene into the chromosome of Corynebacterium ammoniagenes strain  a pDZ-based recombinant vector containing two consecutive copies of the corresponding gene was used. The pDZ vector is a vector for chromosomal insertion into the microorganism of the genus Corynebacterium  and was prepared by the method disclosed in Korean Patent Publication No. 2008-0025355 incorporated by reference herein. FIG. 1 is a schematic diagram showing the structure of the pDZ vector.
In the following (1) to (6)  recombinant vectors were prepared  in which the recombinant vectors function to insert the gene encoding purine biosynthesis related enzyme into the chromosome of the microorganism of the genus Corynebacterium to obtain two copies of each gene. Transformation by each recombinant vector and selection of transformants were performed as follows.
The 5’-inosinic acid-producing strain  Corynebacterium ammoniagenes CJIP2401 (KCCM-10610) was transformed with the pDZ recombinant vector containing the desired gene encoding purine biosynthesis related enzyme by electroporation  and then strains  in which the gene carried by the vector is inserted into their chromosome by homologous recombination   were selected on a selection medium containing 25 mg/l of kanamycin. The successful chromosomal insertion of the vector was confirmed by the color of the colonies on a solid medium (1% beef extract  1% yeast extract  1% peptone  0.25% sodium chloride  1% adenine  1% guanine  1.5% agarose) containing X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactoside). That is  blue colonies were selected as a transformant  in which the vector was inserted into the chromosome. The strain  in which the vector was inserted into its chromosome via a first crossover  was shaking-cultured (30°C  8 hours) in a nutrient medium (1% glucose  1% beef extract  1% yeast extract  1% peptone  0.25% sodium chloride  1% adenine  1% guanine). Then  the cultured strain was serially diluted from 10-4 to 10-10  and the diluted culture was plated on a solid medium containing X-gal. Most colonies exhibited blue color  but white colonies also existed at a low level. By selecting the white colonies  strains in which the sequence of the vector was removed from the chromosome via a second crossover were selected. The selected strain was identified as a final strain by a susceptibility test for kanamycin and a gene sequence analysis by PCR.

(1) Cloning of purFM gene and Construction of recombinant vector (pDZ-2purFM)
The purF and purM genes are located close to each other on the chromosome of the microorganism of the genus Corynebacterium  and thus a purFM vector containing both of the genes and the promoter region was constructed in order to express both of the genes at the same time.
The chromosome was isolated from Corynebacterium ammoniagenes CJIP2401 producing 5’-inosinic acid  and Polymerase Chain Reaction (PCR) was performed using the chromosome as a template in order to obtain purFM  namely  a fragment containing the consecutively arranged purF and purM. PfuUltra™ High-Fidelity DNA Polymerase (Stratagene) was used as a polymerase  and Polymerase Chain Reaction was performed with 30 cycles of denaturing at 96°C for 30 sec  annealing at 53°C for 30 sec  and polymerization at 72°C for 2 min. As a result  two purFM genes containing the promoter region (purFM-A  purFM-B) were obtained. The purFM-A was amplified using the primers of SEQ ID NOs. 1 and 2  and the purFM-B was amplified using the primers of SEQ ID NOs. 3 and 4. The amplification products were cloned into an E.coli vector pCR2.1 using a TOPO Cloning Kit (Invitrogen) so as to obtain pCR-purFM-A and pCR-purFM-B vectors  respectively. The pCR vectors were treated with restriction enzymes contained in each end of the purFM-A and purFM-B (purFM-A: EcoRI+XbaI  purFM-B: XbaI+HindIII)  and each purFM gene was separated from the pCR vectors. Thereafter  the pDZ vector treated with restriction enzymes  EcoRI and HindIII was cloned by 3-piece ligation so as to construct a pDZ-2purFM recombinant vector where two purFM genes are consecutively cloned. FIG. 2 shows a pDZ-2purFM vector for chromosomal insertion into Corynebacterium.
The 5’-inosinic acid-producing strain  Corynebacterium ammoniagenes CJIP2401 was transformed with the pDZ-2purFM vector by electroporation  and one purFM gene is additionally inserted next to the intrinsic purFM gene on the chromosome via a second crossover  so as to obtain a strain having total two copies. The consecutively inserted purFM genes were identified by PCR using the primers of SEQ ID NOs. 5 and 6 which are able to amplify the regions of connecting two purFM genes.

(2) Cloning of purNH gene and Construction of recombinant vector (pDZ-2purNH)  preparation of purNH-inserted strain
The purN and purH genes are located close to each other on the chromosome of the microorganism of the genus Corynebacterium  and thus a purNH vector containing the promoter region was constructed in order to express both of the genes at the same time.
The chromosome was isolated from Corynebacterium ammoniagenes CJIP2401 producing 5’-inosinic acid  and Polymerase Chain Reaction (PCR) was performed using the chromosome as a template in order to obtain purNH  namely  a fragment containing the consecutively arranged purN and purH. PfuUltra™ High-Fidelity DNA Polymerase (Stratagene) was used as a polymerase  and Polymerase Chain Reaction was performed with 30 cycles of denaturing at 96°C for 30 sec  annealing at 53°C for 30 sec  and polymerization at 72°C for 2 min. As a result  two purNH genes containing the promoter region (purNH-A  purNH-B) were obtained. The purNH-A was amplified using the primers of SEQ ID NOs. 7 and 8  and the purNH-B was amplified using the primers of SEQ ID NOs. 8 and 9. The amplification products were cloned into an E.colivector pCR2.1 using a TOPO Cloning Kit (Invitrogen) so as to obtain pCR-purNH-A and pCR-purNH-B vectors  respectively. The pCR vectors were treated with restriction enzymes contained in each end of the purNH-A and purNH-B (purNH-A: BamHI+SalI  purNH-B: SalI)  and each purNH gene was separated from the pCR vectors. Thereafter  the pDZ vector treated with restriction enzymes  BamHI and SalI was cloned by 3-piece ligation so as to construct a pDZ-2purNH recombinant vector where two purNH genes are consecutively cloned. FIG. 3 shows a pDZ-2purNH vector for chromosomal insertion into Corynebacterium.
The 5’-inosinic acid-producing strain  Corynebacterium ammoniagenes CJIP2401 was transformed with the pDZ-2purNH vector by electroporation  and one purNH gene is additionally inserted next to the intrinsic purNH gene on the chromosome via a second crossover  so as to obtain a strain having total two copies. The consecutively inserted purNH genes were identified by PCR using the primers of SEQ ID NOs. 10 and 11 which are able to amplify the regions of connecting two purNH genes.

(3) Cloning of purSL gene and Construction of recombinant vector (pDZ-2purSL)  preparation of purSL-inserted strain
The purS and purL genes are located close to each other on the chromosome of the microorganism of the genus Corynebacterium  and thus a purSL vector containing the promoter region was constructed in order to express both of the genes at the same time.
The chromosome was isolated from Corynebacterium ammoniagenes CJIP2401 producing 5’-inosinic acid  and Polymerase Chain Reaction (PCR) was performed using the chromosome as a template in order to obtain purSL  namely  a fragment containing the consecutively arranged purS and purL. PfuUltra™ High-Fidelity DNA Polymerase (Stratagene) was used as a polymerase  and Polymerase Chain Reaction was performed with 30 cycles of denaturing at 96°C for 30 sec  annealing at 53°C for 30 sec  and polymerization at 72°C for 2 min. As a result  two purSL genes containing the promoter region (purSL-A  purSL-B) were obtained. The purSL-A was amplified using the primers of SEQ ID NOs. 12 and 13  and the purSL-B was amplified using the primers of SEQ ID NOs. 14 and 15. The amplification products were cloned into an E.coli vector pCR2.1 using a TOPO Cloning Kit (Invitrogen) so as to obtain pCR-purSL-A and pCR-purSL-B vectors  respectively. The pCR vectors were treated with restriction enzymes contained in each end of the purSL-A and purSL-B (purSL-A: BamHI+SalI  purSL-B: SalI+BamHI)  and each purSL gene was separated from the pCR vectors. Thereafter  the pDZ vector treated with restriction enzyme  BamHI was cloned by 3-piece ligation so as to construct a pDZ-2purSL recombinant vector where two purSL genes are consecutively cloned. FIG. 4 shows a pDZ-2purSL vector for chromosomal insertion into Corynebacterium.
The 5’-inosinic acid-producing strain  Corynebacterium ammoniagenes CJIP2401 was transformed with the pDZ-2purSL vector by electroporation  and one purSL gene is additionally inserted next to the intrinsic purSL gene on the chromosome via a second crossover  so as to obtain a strain having total two copies. The consecutively inserted purSL genes were identified by PCR using the primers of SEQ ID NOs. 16 and 17 which are able to amplify the regions of connecting two purSL genes.

(4) Cloning of purKE gene and Construction of recombinant vector (pDZ-2purKE)  preparation of purKE-inserted strain
The purK and purE genes are located close to each other on the chromosome of the microorganism of the genus Corynebacterium  and thus a purKE vector containing the promoter region was constructed in order to express both of the genes at the same time.
The chromosome was isolated from Corynebacterium ammoniagenes CJIP2401 producing 5’-inosinic acid  and Polymerase Chain Reaction (PCR) was performed using the chromosome as a template in order to obtain purKE  namely  a fragment containing the consecutively arranged purK and purE. PfuUltra™ High-Fidelity DNA Polymerase (Stratagene) was used as a polymerase  and Polymerase Chain Reaction was performed with 30 cycles of denaturing at 96°C for 30 sec  annealing at 53°C for 30 sec  and polymerization at 72°C for 2 min. As a result  two purKE genes containing the promoter region (purKE-A  purKE-B) were obtained. The purKE-A was amplified using the primers of SEQ ID NOs. 18 and 19  and the purKE-B was amplified using the primers of SEQ ID NOs. 20 and 21. The amplification products were cloned into an E.coli vector pCR2.1 using a TOPO Cloning Kit (Invitrogen) so as to obtain pCR-purKE-A and pCR-purKE-B vectors  respectively. The pCR vectors were treated with restriction enzymes contained in each end of the purKE-A and purKE-B (purKE-A: BamHI+KpnI  purKE-B: KpnI+XbaI)  and each purKE gene was separated from the pCR vectors. Thereafter  the pDZ vector treated with restriction enzymes  BamHI and XbaI was cloned by 3-piece ligation so as to construct a pDZ-2purKE recombinant vector where two purKE genes are consecutively cloned. FIG. 5 shows a pDZ-2purKE vector for chromosomal insertion into Corynebacterium.
The 5’-inosinic acid-producing strain  Corynebacterium ammoniagenes CJIP2401 was transformed with the pDZ-2purKE vector by electroporation  and one purKE gene is additionally inserted next to the intrinsic purKE gene on the chromosome via a second crossover  so as to obtain a strain having total two copies. The consecutively inserted purKE genes were identified by PCR using the primers of SEQ ID NOs. 22 and 23 which are able to amplify the regions of connecting two purKE genes.

(5) Cloning of purC gene and Construction of recombinant vector (pDZ-2purC)  preparation of purC-inserted strain
The chromosome was isolated from Corynebacterium ammoniagenes CJIP2401  and Polymerase Chain Reaction (PCR) was performed using the chromosome as a template in order to obtain purC. PfuUltra ™ High-Fidelity DNA Polymerase was used as a polymerase  and Polymerase Chain Reaction was performed with 30 cycles of denaturing at 96°C for 30 sec  annealing at 53°C for 30 sec  and polymerization at 72°C for 2 min. As a result  two purC genes containing the promoter region (purC-A  purC-B) were obtained. The purC-A was amplified using the primers of SEQ ID NOs. 24 and 25  and the purC-B was amplified using the primers of SEQ ID NOs. 25 and 26. The amplification products were cloned into an E.coli vector pCR2.1 using a TOPO Cloning Kit so as to obtain pCR-purC-A and pCR-purC-B vectors  respectively. The pCR vectors were treated with restriction enzymes contained in each end of the purC-A and purC-B (purC-A: BamHI+SalI  purC-B: SalI)  and each purC gene was separated from the pCR vectors. Thereafter  the pDZ vector treated with restriction enzymes  BamHI and SalI was cloned by 3-piece ligation so as to construct a pDZ-2purC recombinant vector where two purC genes are consecutively cloned. FIG. 6 shows a pDZ-2purC vector for chromosomal insertion into Corynebacterium.
The 5’-inosinic acid-producing strain  Corynebacterium ammoniagenes CJIP2401 was transformed with the pDZ-2purC vector by electroporation  and one purC gene is additionally inserted next to the intrinsic purC gene on the chromosome via a second crossover  so as to obtain a strain having total two copies. The consecutively inserted purC genes were identified by PCR using the primers of SEQ ID NOs. 27 and 28 which are able to amplify the regions of connecting two purC genes.

(6) Cloning of prs gene and Construction of recombinant vector (pDZ-2prs)  preparation of prs-inserted strain
The chromosome was isolated from Corynebacterium ammoniagenes CJIP2401  and Polymerase Chain Reaction (PCR) was performed using the chromosome as a template in order to obtain prs. PfuUltra™ High-Fidelity DNA Polymerase was used as a polymerase  and Polymerase Chain Reaction was performed with 30 cycles of denaturing at 96°C for 30 sec  annealing at 53°C for 30 sec  and polymerization at 72°C for 2 min. As a result  two prs genes containing the promoter region (prs-A  prs-B) were obtained. The prs-A was amplified using the primers of SEQ ID NOs. 29 and 30  and the prs-B was amplified using the primers of SEQ ID NOs. 31 and 32. The amplification products were cloned into an E.coli vector pCR2.1 using a TOPO Cloning Kit so as to obtain pCR-prs-A and pCR-prs-B vectors  respectively. The pCR vectors were treated with restriction enzymes contained in each end of the prs-A and prs-B (prs-A: BamHI+SpeI  prs-B: SpeI+PstI)  and each prs gene was separated from the pCR vectors. Thereafter  the pDZ vector treated with restriction enzymes  BamHI and PstI was cloned by 3-piece ligation so as to construct a pDZ-2prs recombinant vector where two prs genes are consecutively cloned. FIG. 7 shows a pDZ-2prs vector for chromosomal insertion into Corynebacterium.
The 5’-inosinic acid-producing strain  Corynebacterium ammoniagenes CJIP2401 was transformed with the pDZ-2prs vector by electroporation  and one prs gene is additionally inserted next to the intrinsic prs gene on the chromosome via a second crossover  so as to obtain a strain having total two copies. The consecutively inserted prs genes were identified by PCR using the primers of SEQ ID NOs. 33 and 34 which are able to amplify the regions of connecting two prs genes.

(7) Development of strain producing high yield of 5’-inosinic acid by enhancement of purine biosynthesis
Combinations of pDZ-2purFM  pDZ-2purNH  pDZ-2purSL  pDZ-2purKE  pDZ-2purC  and pDZ-2prs vectors constructed in (1) to (6) were introduced into the 5’-inosinic acid-producing Corynebacterium ammoniagenes CJIP2401. The introduction order of the vectors was randomly selected  and introduction method and identification are the same as the above.
The Corynebacterium ammoniagenes CJIP2401 was used as a parental strain  and transformed with a combination of pDZ-2purNH  pDZ-2purSL  pDZ-2purKE  pDZ-2purC  and pDZ-2prs  and a combination of pDZ-2purNH  pDZ-2purSL  pDZ-2purKE  pDZ-2purC  pDZ-2purFM and pDZ-2prs to obtain Corynebacterium ammoniagenes CN01-0120 (2purNH + 2purSL + 2purKE + 2purC + 2prs) and Corynebacterium ammoniagenes CN01-0316 (2purNH + 2purSL + 2purKE + 2purC + 2purFM + 2prs)  which contain two copies of the genes encoding the major enzymes involved in the purine biosynthetic pathway.

Example 2. Fermentation titer test of recombinant Corynebacterium ammoniagenes
Each 3 ml of the seed medium with the following composition was distributed into test tubes having a diameter of 18 mm  and sterilized under pressure. Then  the parental strain Corynebacterium ammoniagenes CJIP2401  and the Corynebacterium ammoniagenes CN01-0120 and Corynebacterium ammoniagenes CN01-0316 prepared in Example 1 were inoculated  and shaking-cultured at 30°C for 24 hours to be used as a seed culture. Each 27 ml of the fermentation medium with the following composition was distributed into 500 ml Erlenmeyer shake flasks and sterilized under pressure at 120°C for 10 minutes  and each 3 ml of the seed culture was inoculated thereto and shaking-cultured for 5 to 6 days. The cultivation was carried out under the conditions of 200 rpm  32°C  and pH 7.2

The seed medium and the fermentation medium have the following compositions.
Seed medium: 1% glucose  1% peptone  1% beef extract  1% yeast extract  0.25% sodium chloride  100 mg/l adenine  100 mg/l guanine  pH7.2
Flask fermentation medium: 0.1% sodium glutamate  1% ammonium chloride  1.2% magnesium sulfate  0.01% calcium chloride  20 mg/l iron sulfate  20 mg/l manganese sulfate  20 mg/l zinc sulfate  5 mg/l copper sulfate  23 mg/l L-cysteine  24 mg/l alanine  8 mg/l nicotinic acid  45 µg/l biotin  5 mg/l thiamine hydrochloride  30 mg/l adenine  1.9% phosphoric acid (85%)  4.2% glucose  and 2.4% raw sugar

After completion of the cultivation  the productivity of 5’-inosinic acid was measured by HPLC  and the accumulation amount of 5’-inosinic acid in the culture medium is shown in the following Table.

[Table 1]
Strain name Cell OD (5 days after culture) Productivity (g/l/hr) (5 days after culture)
Control group (CJIP2401) 31.2 0.136
CN01-0120 31.8 0.155
CN01-0316 31.3 0.149

The accumulation amount of 5’-inosinic acid in the culture medium was compared with that of the parental strain  Corynebacterium ammoniagenes CJIP2401. As a result  in Corynebacterium ammoniagenes CN01-0120 and Corynebacterium ammoniagenes CN01-0316  their 5’-inosinic acid productivity per hour was found to be increased to 10.9 - 11.4% under the same conditions  compared to the parental strain  Corynebacterium ammoniagenes CJIP2401.
Corynebacterium ammoniagenes CN01-0316 having improved 5’-inosinic acid productivity by increasing the activity of purine biosynthesis related enzymes was deposited in the Korean Culture Center of Microorganisms (KCCM) located at Hongje 1-dong  Seodaemun-gu  Seoul  with the Accession No. KCCM 10992P on Feb. 19  2009 under the Budapest treaty.

Effect of the invention
The microorganism belonging to the genus Corynebacterium producing 5’-inosinic acid according to the present invention  in which the expression of gene encoding purine biosynthesis related enzymes is increased higher than the intrinsic expression  can be used to produce 5’-inosinic acid in a high concentration and a high yield  thereby reducing production costs.

WE CLAIM:

1. A microorganism belonging to the genus Corynebacterium producing 5’-inosinic acid  in which the expression level of genes encoding purine biosynthesis related enzymes is increased higher than the intrinsic expression level  wherein the purine biosynthesis related enzymes are a combination of ribosephosphate pyrophosphokinase and one or more enzymes selected from the group consisting of phosphoribosylpyrophosphate amidotransferase  phosphoribosylglycinamide formyltransferase  phosphoribosylformylglycinamidin synthetase  phosphoribosylformylglycinamidin synthetase II  phosphoribosylaminoimidazole synthetase  phosphoribosylaminoimidazole carboxylase  phosphoribosyl aminoimidazole succinocarboxamide synthetase  and inosinic acid cyclohydrolase.

2. The microorganism belonging to the genus Corynebacterium according to claim 1  wherein the genes encoding purine biosynthesis related enzymes are a combination of a purN gene of SEQ ID NO. 36  which codes for phosphoribosylglycinamide formyltransferase  a purS gene of SEQ ID NO. 37  which codes for phosphoribosylformylglycinamidin synthetase  a purL gene of SEQ ID NO. 38  which codes for phosphoribosylformylglycinamidin synthetase II  a purKE gene of SEQ ID NO. 40  which codes for phosphoribosylaminoimidazole carboxylase  a purC of SEQ ID NO. 41  which codes for phosphoribosyl aminoimidazole succinocarboxamide synthetase  a purH gene of SEQ ID NO. 42  which codes for inosinic acid cyclohydrolase  and a prs gene of SEQ ID NO. 43  which codes for ribosephosphate pyrophosphokinase.

3. The microorganism belonging to the genus Corynebacterium according to claim 1  wherein the genes encoding purine biosynthesis related enzymes are a combination of a purF gene of SEQ ID NO. 35  which codes for phosphoribosylpyrophosphate amidotransferase  a purN gene of SEQ ID NO. 36  which codes for phosphoribosylglycinamide formyltransferase  a purS gene of SEQ ID NO. 37  which codes for phosphoribosylformylglycinamidin synthetase  a purL gene of SEQ ID NO. 38  which codes for phosphoribosylformylglycinamidin synthetase II  a purM gene of SEQ ID NO. 39  which codes for phosphoribosylaminoimidazole synthetase  a purKE gene of SEQ ID NO. 40  which codes for phosphoribosylaminoimidazole carboxylase  a purC of SEQ ID NO. 41  which codes for phosphoribosyl aminoimidazole succinocarboxamide synthetase  a purH gene of SEQ ID NO. 42  which codes for inosinic acid cyclohydrolase  and a prs gene of SEQ ID NO. 43  which codes for ribosephosphate pyrophosphokinase.

4. The microorganism belonging to the genus Corynebacterium according to claim 1  wherein the expression level of the gene encoding purine biosynthesis related enzyme is increased by additionally introducing the gene encoding purine biosynthesis related enzyme into a cell or by amplifying the intrinsic gene encoding purine biosynthesis related enzyme.

5. The microorganism belonging to the genus Corynebacterium according to claim 4  wherein the gene encoding purine biosynthesis related enzyme exists as two or more copies by introducing one or more copies into a cell  in addition to the corresponding intrinsic gene.

6. The microorganism belonging to the genus Corynebacterium according to claim 5  wherein introduction of the gene encoding purine biosynthesis related enzyme into the cell is performed by transformation using a recombinant vector containing two copies of the corresponding gene that are consecutively arranged.

7. The microorganism belonging to the genus Corynebacterium according to claim 6  wherein the recombinant vector is selected from the group consisting of pDZ-2purFM  pDZ-2purNH  pDZ-2purSL  pDZ-2purKE  pDZ-2purC  and pDZ-2prs that have the cleavage maps of FIGs. 2 to 7  respectively.

8. The microorganism belonging to the genus Corynebacterium according to claim 1  wherein the microorganism of the genus Corynebacterium is Corynebacterium ammoniagenes.

9. The microorganism belonging to the genus Corynebacterium according to claim 2  wherein the microorganism of the genus Corynebacterium is Corynebacterium ammoniagenes CN01-0120.

10. The microorganism belonging to the genus Corynebacterium according to claim 3  wherein the microorganism of the genus Corynebacterium is Corynebacterium ammoniagenes CN01-0316 (KCCM 10992P).

11. A method for producing 5’-inosinic acid  comprising culturing the microorganism belonging to the genus Corynebacterium according to any one of claims 1 to 10  and recovering 5’-inosinic acid from the culture medium.