KR20090080654A - A microorganism of corynebacterium genus having enhanced xanthosine 5'-monophosphate productivity and method of producing xanthosine 5'-monophosphate using the same - Google Patents

Abstract

A method for producing a xanthosine 5'-monophosphate by using Corynebacterium sp. microorganism is provided to enhance the activation of enzyme related to the purine biosynthesis and inactivate an enzyme which resolves nucleic acid. A method for improving the productivity of xanthosine 5'-monophosphate from Corynebacterium sp. microorganism is enhancing the activation of phosphoribosylglycinamide formyltransferase and inosinic acid cyclohydrolase. Two or more purNH genes which encode the enzymes are contained inside chromosome. The purNH gene as gene sequence of the sequence number 12. A method for producing a xanthosine 5'-monophosphate comprises: a step of culturing the Corynebacterium sp. microorganism to produce the xanthosine 5'-monophosphate; and a step of collecting the xanthosine 5'-monophosphate from a cell or medium.

Description

MICROORGANISM OF CORYNEBACTERIUM GENUS HAVING ENHANCED XANTHOSINE 5'-MONOPHOSPHATE PRODUCTIVITY AND METHOD OF PRODUCING XANTHOSINE 5ATE-MONOPHOSPH SAME}

The present invention relates to the 5'-phosphoribosylglycinamide formyltransferase 1 and inosine acid cyclohydrolase (hereinafter referred to as IMP cyclohydrolase) enzymes involved in purine biosynthesis. X'anthocine acid (hereinafter referred to as 5'-XMP) by further inactivating 5'-nucleotidase, which enhances the nucleic acid and preferably degrades the nucleic acid. The present invention relates to a microorganism of the genus Corynebacterium with improved production capacity and a method of producing 5'-xanthyl acid using the same.

5'-XMP is an intermediate product of the purine nucleotide biosynthesis metabolic system and is an important material as a raw material for producing 5'-guanylic acid (hereinafter referred to as 5'-GMP). 5'-GMP, along with inosinic acid (hereinafter referred to as IMP), is widely used as a food seasoning additive. 5'-GMP is known to taste mushrooms by itself, but is known to enhance the flavor of mainly sodium L-glutamate (MSG). This property is particularly strong when used with IMP.

Known methods for producing 5'-GMP include (1) enzymatic digestion of ribonucleic acid (RNA) extracted from yeast cells, (2) direct fermentation of 5'-GMP by microbial fermentation, and (3) Chemical phosphorylation of guanosine produced by microbial fermentation, (4) Enzymatic phosphorylation of guanosine produced by microbial fermentation, (5) 5'-XMP produced by microbial fermentation using coryneform microorganisms The method of converting into 5'-GMP, (6) The method of converting 5'-XMP produced by microbial fermentation into 5'-GMP using Escherichia coli. The method of (1) has a problem in the supply and demand of raw materials, and the method of (2) has a disadvantage in that the yield is low due to the problem of 5'-GMP cell membrane permeability, and other methods are mainly used industrially. have.

If 5'-GMP is produced by converting 5'-XMP to 5'-GMP, the strategy to enhance productivity of 5'-XMP is needed. Therefore, conventionally known Corynebacterium strains and 5'-XMP production method using the 5'-XMP biosynthesis-related genes have been known. For example, Korean Patent No. 10-1991-018061 discloses a lysozyme, a lysozyme that is resistant to guanosine analogs and is a cell wall degrading enzyme that is resistant to adenosine and guanine semiconducting xanthyl acid aminase in order to increase the yield of 5'-XMP. A highly susceptible strain was prepared and disclosed. Korean Patent No. 10-2001-000513 discloses 5'-XMP producing Corynebacterium ammoniagenes, which is irradiated with ultraviolet rays, N-methyl-N'-nitro-N-nitrosoguanidine (N- Analogues to valines that affect the biosynthesis of 5'-XMP by modifying the traits of the parent strains by treating the mutant traits according to conventional methods, such as methyl-N'-nitro-N-nitrosoguanidine (NTG) Norvaline resistant strains) were selected, and as a result, a 5'-XMP production method using microorganisms that directly accumulate 5'-XMP in culture medium at high yield and high concentration was disclosed. In addition, Korean Patent 10-2003-0091342 In the reported to reduce the concentration of the nucleic acid by breaking the nucleic acid from the microorganism (Escherichia coli and Salmonella: Cellular and Molecular Biology: 2 nd edition: 561 ~ 579) Corynebacterium the ushA gene A method for producing high yield of inosinic acid by inactivation in inosinic acid producing strains was disclosed.

Conventionally, however, genes encoding phosphoribosylglycineamide formyltransferase and IMP cyclohydrolase enzymes, respectively, involved in the purine biosynthesis pathway involved in biosynthesis of 5'-XMP to produce high yield 5'-XMP producers. Examples of microorganisms which have enhanced phosphorus purN , purH (hereinafter referred to as purNH ) to improve 5'-XMP yield have not been disclosed. In addition, there is no mention of a mutant strain that inactivated the ushA gene encoding 5'-nucleotidase, which degrades nucleic acids. In addition, a method for producing high yield 5'-XMP from a strain in which the purNH gene is enhanced and the ushA gene is inactivated is not disclosed.

Therefore, the present inventors produce 5'-XMP and produce 5'-GMP using the method of converting it to 5'-GMP, since 5'-XMP is required as much as 5'-GMP needs. In order to enhance the productivity, it was determined that it is necessary to maximize the productivity of 5'-XMP by enhancing the activity of enzymes involved in the purine biosynthetic pathway and inactivating enzymes that degrade nucleic acids. We enhance the activity of the gene purNH , which encodes phosphoribosylglycineamide formyltransferase and IMP cyclohydrolase enzymes involved in the purine biosynthetic pathway, and encodes a 5'-nucleotidase that degrades nucleic acids. By inactivating the ushA gene, it was found that the productivity of 5'-XMP was increased, thus completing the present invention.

Accordingly, an object of the present invention is to introduce a corynebacte having improved production capacity of 5'-XMP by introducing into the chromosome a purNH gene encoding phosphoribosylglycineamide formyltransferase and an IMP cyclohydrolase enzyme, thereby enhancing the activity of the enzyme. To provide microorganisms in theium.

It is also an object of the present invention to provide a microorganism of the genus Corynebacterium, which has an enhanced ability to produce 5'-XMP, which inactivates the activity of the ushA gene encoding 5'-nucleotidase.

Another object of the present invention is to provide a method for producing 5'-XMP by culturing the microorganism of the genus Corynebacterium improved production capacity of the 5'-XMP.

In order to achieve the above object, the present invention provides a purNH gene encoding the phosphoribosylglycine amide formyl transferase and IMP cyclohydrolase enzyme is present in the chromosome by two or more copies to enhance the activity of the enzyme It provides microorganisms of the genus Corynebacterium with improved production capacity of 5'-XMP.

The present invention also provides a microorganism of the genus Corynebacterium having improved production capacity of 5'-XMP by inactivating the microorganism of the genus Corynebacterium by inserting one base into the ushA gene encoding 5'-nucleotidase. to provide.

The present invention also provides a method of producing 5'-XMP by culturing the microorganism of the genus Corynebacterium improved production capacity of the 5'-XMP.

The microorganisms of the genus Corynebacterium according to the present invention have increased activity of phosphoribosylglycineamide formyltransferase and IMP cyclohydrolase than the intrinsic activity of microorganisms of corynebacterium, and 5'-nucleo Tidase is inactivated to improve 5'-XMP production capacity compared to the microorganisms used to produce 5'-XMP. Therefore, the use of the microorganism can increase the productivity by producing 5'-XMP with high efficiency.

In the present invention, two or more copies of the purNH gene encoding the phosphoribosylglycine amide formyltransferase and the IMP cyclohydrolase enzyme are present in the chromosome to enhance the activity of the enzyme, thereby enhancing the activity of the 5'-XMP. Provides microorganisms in the genus Nebacterium.

The biosynthetic pathway to make 5'-XMP is very complex and there is a series of reactions involving additional amino acids and coenzymes. In the present invention, phosphoribosylglycine amide formyltransferase and IMP cyclohydrolase enzyme are both enzymes involved in the synthesis of purines involved in the synthesis of 5'-XMP.

Phosphoribosylglycineamide formyltransferase plays an important role in the biosynthesis of purines and 10-formylterra to produce N2-formyl-NL (5-phospho-D-ribosyl) glycineamide and tetrahydrofolic acid. It is an enzyme that catalyzes the transfer of the formyl group of hydrofolic acid (N10-formyltetrahydrofolate) to N1- (5-phospho-D-ribosyl) glycinamide. Tetrahydrofolic acid plays an important role in the synthesis of purine, thymidine, DNA and the like, and is essential for the synthesis of 5'-XMP. In addition, IMP cyclohydrolase catalyzes the last step of purine biosynthesis and is an enzyme that catalyzes 5-formylaminoimidazole-4-carboxamide ribonucleotide with IMP.

The base sequence of the gene encoding the phosphoribosyl glycine amide formyl transferase and IMP cyclohydrolase is already known, preferably the gene encoding the enzyme is purNH gene derived from Corynebacterium ammonia genes ( GenBank Accession No. AB003159; SEQ ID NO: 12).

The method of enhancing the activity of the enzyme in the present invention includes increasing the enzyme activity by increasing the number of gene copies and mutation. In the present invention, the increase in the number of gene copies includes not only an increase in the number of copies due to the introduction of a foreign gene, but also an amplification of an endogenous gene. In the case of foreign gene introduction, the gene is introduced into a vector capable of functioning in a microorganism of the genus Corynebacterium to increase the gene copy number. The vector usable in the present invention is not particularly limited, and known expression vectors may be used. In the case of endogenous gene amplification, the endogenous gene in the present invention means a gene that the microorganism of Corynebacterium has in its natural state. 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 a preferred embodiment of the invention, the activity of the genus Corynebacterium microorganism is achieved by introducing new purNH gene least one copy in addition to the intrinsic purNH gene that has a natural state, preferably purNH gene of two copies of the enzyme By homologous recombination, one more copy of the purNH gene is inserted next to the intrinsic purNH gene, and finally two copies of the purNH gene are amplified and mutated. Enzyme activity is enhanced.

In the present invention, a vector usable in the introduction of a foreign gene preferably uses a vector pDZ for chromosome insertion. According to one aspect of the invention, the vector used for the gene introduction is a pDZ- purNH vector comprising the purNH gene.

The present invention also provides a microorganism of the genus Corynebacterium with an improved production capacity of 5'-XMP which further inactivated the activity of the ushA gene encoding 5'-nucleotidase.

In the present invention, 5'-nucleotidase is an enzyme that hydrolyzes phosphate bonds with sugars of nucleotides, thereby degrading the nucleic acid to reduce the concentration of the nucleic acid. The base sequence of the gene ushA encoding the enzyme can be obtained from Korean Patent No. 10-2003-0091342. Preferably, the ushA gene derived from Corynebacterium ammonia genes has the base sequence of SEQ ID NO: 13.

In the present invention, the inactivation includes the activity of the ushA gene inactivated by substitution, deletion, insertion or the like. More preferably, the ushA gene is inactivated by frame shift due to base insertion in the chromosome, and the base may be any base. Substitution, deletion and / or insertion of such genes can be readily made by conventional methods known in the art.

Corynebacterium genus microorganism into which the gene is introduced in the present invention may include any of Corynebacterium microorganisms. For example, the microorganisms of the genus Corynebacterium include Corynebacterium ammonia genes KCCM-10530, Corynebacterium ammonia genes ATCC6872, Corynebacterium thermoaminogenes (FERM BP-1539), Coryne Bacterium glutamicum ATCC13032, Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum ATCC 13869 and strains prepared therefrom, preferably corynebacterium ammonia Gennes KCCM-10530.

According to one aspect of the present invention, the Corynebacterium spp. Microorganisms having the cleavage maps of FIGS. 1 and 2, respectively, may be introduced into the Corynebacterium spp . Vector pDZ- purNH and pDZ- ushA sequentially or simultaneously. . The microorganism of the genus Corynebacterium has at least one copy of the purNH gene inserted into the chromosome to enhance the activity of the phosphoribosylglycine amide formyltransferase and the IMP cyclohydrolase enzyme, and more preferably the ushA gene is further added to the chromosome. It is inactivated by the frame shift due to the base insertion.

In a preferred embodiment of the present invention, two copies of the purNH gene are introduced into Corynebacterium ammonia genes KCCM-10530 with vectors pDZ- purNH and pDZ- ushA having a cleavage map of FIGS. Was inserted into the chromosome and one base was inserted into the ushA gene to obtain an inactivated strain. The prepared strains were corynebacterium ammonia genes KCJ-0001 (Accession Number: KCCM10913P), KCCM-0002 (Accession Number: KCCM10914P).

In another aspect, the present invention provides a method of directly accumulating 5'-XMP in high yield in a culture medium by direct fermentation using the microorganism of the present invention. More specifically, the step of producing a 5'-XMP in cells or cultures by culturing the microorganism of the genus Corynebacterium produced in accordance with the present invention improved 5'-XMP; And it relates to a method for producing 5'-XMP in high yield, characterized in that it comprises the step of recovering 5'-XMP from the cell or culture.

In the method of producing 5'-XMP of the present invention, the abiotic culturing process may be carried out according to a suitable medium and culture conditions known in the art. This culture process can be used by those skilled in the art easily adjusted according to the strain selected. Examples of the culture method include, but are not limited to, batch, continuous and fed-batch cultures.

The medium used for cultivation should suitably meet the requirements of the particular strain. The medium used in the present invention contains glycerol as part or all of the carbon source. Other suitable amounts of carbon source can be used in various ways. Particularly preferred carbon source is glucose. Examples of nitrogen sources that can be used include organic nitrogen sources and urea such as peptone, yeast extract, gravy, malt extract, corn steep liquor, and soybean wheat, inorganic nitrogen sources such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate Included. These nitrogen sources may be used alone or in combination. The medium may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate and the corresponding sodium-containing salts as a person. It may also include metal salts such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, appropriate precursors, and the like can be included. These media or precursors may be added batchwise or continuously to the culture.

During the culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture. In addition, during the culture, antifoaming agents such as fatty acid polyglycol esters can be used to suppress foaming. In addition, to maintain the aerobic state of the culture, oxygen or oxygen-containing gas is injected into the culture or nitrogen, hydrogen, or carbon dioxide gas is injected without gas injection or to maintain anaerobic and unaerobic conditions. The temperature of the culture is usually 20 to 45 ° C, preferably 25 to 40 ° C. The incubation period can continue the production of the desired useful substance, preferably 10 to 160 hours.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are only for carrying out the present invention by way of example, but the scope of the present invention is not limited to these examples.

Reference Example  1: Vector for chromosome insertion pDZ Gene insertion method using

In this embodiment, the chromosome insertion of the gene was performed using the chromosome insertion vector pDZ. In order to insert nucleotides into the chromosome of Corynebacterium, a pDZ vector having a nucleotide to be inserted into a sequence having homology with the insertion site on the chromosome is prepared. In order to insert the extra gene into the chromosome, a pDZ vector containing two copies of the gene of interest was produced, and the strain to be introduced was transformed with the electropulse method, followed by kanamicin 25mg / L. Strains inserted by homology with nucleotides on the chromosome in the medium were selected.

Successful chromosome insertion of the vector was made possible by checking whether it was blue in a solid medium containing X-gal (5-bromo-4-chloro-3-indolyl-B-D-galactosid).

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

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

Example  1: 5'- XMP Production strain Corynebacterium Ammonia Genes KTCC From -10530 purNH Cloning  And recombinant vectors for chromosomal insertion ( pDZ - purNH Production

In this example, the base sequence information (NCBI accession number _ AB003159) of the purNH gene was obtained based on NIH GenBank, and two pairs of primers (SEQ ID NOs: 1-3) were synthesized based on this.

PCR was performed using Corynebacterium KCCM-10530 as a template and oligonucleotides of SEQ ID NOs: 7 to 10 as primers. The polymerase was PfuUltra ^™ high-reliability DNA polymerase (stratagene), PCR conditions were denaturation 95 ℃, 30 seconds; Annealing 53 ° C., 30 seconds; And 25 degreeC of the polymerization reaction 72 degreeC and 2 minutes was repeated.

As a result, two pairs of purNH genes ( purNH- A, purNH- B) of 2.7 kb were obtained. purNH- A was amplified using SEQ ID NOs: 1 and 2 as primers, and purNH -B was amplified using SEQ ID NOs: 2 and 3 as primers. The amplification product was cloned into E. coli vector pCR2.1 using TOPO cloning kit (Invitrogen) to obtain pCR- purNH -A and pCR- purNH -B vectors.

SEQ ID NO: 1 cgg gat ccc gag gcg aag acg ata ttg agg aca g

SEQ ID NO: 2 acg cgt cga cgt ggg aaa cgc aga cga gaa ca

SEQ ID NO: 3 acg cgt cga cga ggc gaa gac gat att gag gac ag

For the pCR vector with a restriction enzyme comprising at each end of purNH-A, B-purNH processes the (purNH -A:: BamHI + salI , purNH -B SalI + salI), each -A purNH from the pCR vectors, purNH- B gene was isolated. Next, a restriction enzyme by BamHI and SalI for the processing of three pieces joined to the pDZ vector, finally purNH 2 copies are continuously pDZ- purNH the obtained recombinant vector by cloning. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows pDZ- purNH for corynebacterium chromosome insertion.

Example  2 : purNH   Insert strain development

Manufactured pDZ- purNH The vector was transformed into Corynebacterium ammonia gene KCCM-10530 strain. As described in Reference Example 1, a 5'-XMP-producing strain that increased the number of copies to two by inserting an additional copy of the purNH gene right next to the purNH gene on the chromosome through the second crossover process, was described as Corynebacte. Leeum's ammonia Ness (Corynebacterium ammoniagenes ) was named KCJ-0001 and deposited on January 8, 2008 at the Korea Microbiological Conservation Center, an affiliated organization of the Korean spawn association, a relief deposit organization located at 361-221, Hongje 1-dong, Seodaemun-gu, Seoul, Korea (KCCM-10913P). .

The continuously inserted purNH gene was finally confirmed by PCR using primers (SEQ ID NOs. 4 and 5) capable of amplifying two copies of the purNH linkage.

SEQ ID NO: 4 tcg atg cct gca tct tgg

SEQ ID NO: 5 ggc gat aag gct tcg agt

Example  3: 5'- XMP Production strain Corynebacterium Ammonia Genes KTCC From -10530 ushA Cloning  And recombinant vectors for chromosome insertion ( pDZ - ushA Production

In this example, a gene of ushA , a gene encoding 5'-nucleotidase, was secured by PCR using chromosomal DNA of Corynebacterium ammonia gene KCCM-10530 having 5'-XMP production capacity. .

Based on the domestic patent 10-2003-0091342 to obtain the nucleotide sequence information of the ushA gene, based on this two pairs of primers (SEQ ID NO: 6 to 9) was synthesized. One base was inserted at both ends of primers 7 and 8 to prepare a primer so that one base was inserted into the ushA gene of the chromosome.

PCR was performed using Corynebacterium ammonia genes KCCM-10530 as a template and oligonucleotides of SEQ ID NOs: 6 to 9 as primers. The polymerase is PfuUltra ^™ high-trust DNA polymerase (stratagene) and PCR conditions were denatured 95 ° C., 30 seconds; Annealing 53 ° C., 30 seconds; And repeating the polymerization reaction 72 ° C. for 1 minute 25 times.

As a result, two pairs of 400 bp ushA genes ( ushA -A, ushA -B) were obtained. ushA- A was amplified using SEQ ID NOs: 6 and 7 as primers, and ushA -B was amplified using SEQ ID NOs: 8 and 9 as primers. The amplification products were cloned into E. coli vector pCR2.1 using TOPO cloning kit (Invitrogen) to obtain pCR- ushA -A and pCR- ushA -B vectors.

SEQ ID NO: 6 g acg gcc agt gaa ttc gac cgc ggc tat gac gat ttg ctc

SEQ ID NO: 7 agt gca gtg tct aga acc atc acg ttc gat gat gcc ctg c

SEQ ID NO: 8 cgt gat ggt tct aga cac tgc act ggg cgc agg ggc atg a

SEQ ID NO: 9 a tgc ctg cag gtc gac gta cgc cac cag cat tca taa tgc

The pCR vector is treated with restriction enzymes ( ushA- A: EcoRI + xbaI, ushA- B: xbaI + salI) contained at each end of ushA- A and ushA- B to separate each ushA gene from the pCR vector. It was. Next, through three-piece conjugation to the pDZ vector treated with restriction enzymes EcoRI and SalI, one nucleotide was finally inserted into the ushA gene to make a cloned pDZ- ushA . 2 is a diagram showing a pDZ- ushA vector for corynebacterium chromosome insertion.

Example  4 : purNH  Insert and ushA  Construction of Gene Inactivation Strains

The produced pDZ- ushA vector was transformed into a strain of Corynebacterium ammonia gene KCJ-0001 (KCCM-10913P), followed by a second crossover process using the method of Reference Example 1, and one base was added to the ushA gene on the chromosome. After making the inserted and inactivated producer, it was named Corynebacterium Ammonia Genes KCJ-0002 and deposited him on January 8, 2008 at 361-221 Hongje 1-dong, Seodaemun-gu, Seoul, Korea. It was deposited in the Korea Microbiological Conservation Center, an affiliated organization of the Korean spawn association (KCCM-10914P).

Nucleotide variation of the gene was finally confirmed by PCR using the primers of SEQ ID NOs: 10 and 11 including the mutated part as a primer sequence.

SEQ ID NO: 10 tcg aac gtg atg gtc

SEQ ID NO: 11 gta cgc cac cag cat tca taa tgc

Example  5: purNH  Insert and ushA  5'- of gene inactivated strain XMP  production

Corynebacterial ammonia genes KCJ-0001 (KCCM-10913P) and KCJ-0002 (KCCM-10914P), which were finally produced in Examples 2 and 4, were produced to produce 5'-XMP The culture was carried out by the method. A 14 ml tube containing 3 ml of the following species medium was inoculated with Corynebacterium ammonia genes strains KCCM-10530, KCJ-0001 (KCCM-10913P) and KCJ-0002 (KCCM-10914P) and 200 for 20 hours at 30 ° C. Shake culture was performed at rpm.

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 this medium + 8 ml of starch medium) and shake-cultured at 230 ° C. at 230 rpm for 96 hours. After the incubation, the yield of 5'-xanthyl acid was measured by HPLC. 5'-XMP results in cultures for Corynebacterium ammonia genes KCCM-10530, KCJ-0001 (KCCM-10913P) and KCJ-0002 (KCCM-10914P) are shown in Table 1 below.

Strain name KCCM-10530 KCJ-0001 (KCCM-10913P) KCJ-0002 (KCCM-10914P) 5'-XMP (g / l) 28.6 29.7 30.3

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

Production medium (main 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 100μg / l , Copper sulfate 1mg / l, thiamine hydrochloride 5mg / l, calcium chloride 10mg / l, pH 7.2

Production medium (starch medium): 10 g / l potassium phosphate, 10 g / l potassium phosphate, 7 g / l urea, 5 g / l ammonium sulfate

As shown in Table 1, KCJ-0001 (KCCM-10913P) and KCJ-0002 (KCCM-10914P) was confirmed that the production of 5'-XMP increased 1.7 g / l compared to the parent strain KCCM-10530.

1 shows the structure of a pDZ- purNH vector prepared by cloning the purNH gene in a pDZ vector.

Figure 2 shows the structure of the pDZ- ushA vector produced by cloning the ushA -A and ushA -B genes in the pDZ vector.

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Artificial Sequence <220> <223> primer <400> 7 agtgcagtgt ctagaaccat cacgttcgat gatgccctgc 40 <210> 8 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 cgtgatggtt ctagacactg cactgggcgc aggggcatg 39 <210> 9 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 atgcctgcag gtcgacgtac gccaccagca ttcataatgc 40 <210> 10 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 tcgaacgtga tggtc 15 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gtacgccacc agcattcata atgc 24 <210> 12 <211> 2900 <212> DNA <213> Corynebacterium ammoniagenes <400> 12 cagtggacta cattgatggt gaggtcgaag aagagacatc cgttgccaac gagggtggct 60 cccagggtga ggcagtctcc gttggagcaa tgtcggagga gctgatcgat aatttggaag 120 atggttcctt ccccgatgag gtgccaggcg aagacgatat tgaggacagg atcggtactg 180 ctacgcatga gccaagcgat ggagatgcca ataacgagcc cagcgtcgaa gacgttgaca 240 tcgacaatgc agagcaaacc gcggaagggg tattgccaaa ctctgacaac ggagcccaag 300 aagctgcgga aaatgacaca gaagcttctg gggtcgagca cgctgatgat ggccacgatg 360 gccaacagca aagcgatgag gacgataaaa caggcaaaga tacccaataa gaggggataa 420 gttgtcctct tttccgtggt ggaggaaaaa ttggcggcat cgtttcgtcc tcgaaagggc 480 tacagactag tctttaacac gtgactgaat cgccttcgca agttttgaaa gcacaagacc 540 cgcttcaagt agtggtgctg gtatctggca ccggatcttt gctgcaaaat attatcgaca 600 accaagatga ctcctatcgg gttatcaagg tagtcgcgga taagccctgc ccggggatta 660 accgagccca agatgcaggc atcgacaccg aagtcgtgct tttaggctca gaccgcgcgc 720 agtggaacaa agaccttgtc gcagcggttg gtaccgccga tgttgtggtg tccgctggat 780 ttatgaaaat cctggggcct gaattcttgg ccagctttga aggccgcaca ataaatacgc 840 atcccgcact cctgccgtcc tttccgggcg cgcatggagt acgggatgcg ttggcttatg 900 gcgtgaaagt caccggctct actgtccatt ttgtggacgc gggagtcgat actggccgca 960 tcatcgcaca acgcgcagta gagattgagg cagaagatga tgaggcaagc ttgcatgagc 1020 gcatcaaaag cgtcgaacgt gagcttatcg tgcaggtctt acgcgcagcg aatgttcaag 1080 accagcaact tattattgag atttaaacca gatttcccac agtcaggaag gcaaggcttt 1140 catccatgag tgatgaccgc aagcagatca agcgtgcact aattagcgtt tatgacaaga 1200 cagggctcga agagctcgct cgcacgcttg acagcgcagg cgtagagatt gtgtccaccg 1260 gctccaccgc cgccaagatt gctgatcttg gtattaacgt cactccggtt gaatctctca 1320 ccggattccc agagtgcctc gaaggccgcg ttaagacctt gcacccacgc gtgcatgcgg 1380 gcattttggc tgatacccgc aagccggatc accttaatca gctggaagag cttgagattg 1440 agccattcca gttggtcgtg gttaacctgt acccatttaa agagactgta gcttctggcg 1500 cagacttcga tggttgcgtc gagcagattg atatcggcgg tccatccatg gtccgtgctg 1560 ctgccaagaa ccacccatcg gtggcggttg ttgtagaccc agcgcgttac ggcgacatcg 1620 ctgaggctgt cgctcagggc ggattcgatc tggcgcagcg tcgtcagctg gccgcgactg 1680 cgtttaagca cacggcagat tatgatgttg cagtttctgg ctggtttgcc cagcagcttg 1740 ccgatgactc tgttgcctct gctgagcttg aaggcgacgc gctgcgttat ggtgagaacc 1800 ctcaccagca ggcttccatc gttcgtgaag gcacgaccgg tgttgctaat gcgaagcagc 1860 tgcacggtaa ggaaatgagc tacaacaact accaggacgc ggatgccgca tggcgcgcgg 1920 cttgggatca tgaacgtcca tgtgtagcaa ttattaagca cgctaaccct tgcggtatcg 1980 ctgtttctga tgagtccatc gcagcagcac acgcagcggc acacgcctgt gacccaatgt 2040 ccgctttcgg tggcgttatt gcggtcaacc gcgaagtcac caaggaaatg gcaacccagg 2100 ttgctgacat cttcaccgag gtcatcatcg caccgtccta cgaagatgga gccgtcgaga 2160 ttttgcaggg caagaagaat attcgcatcc ttgttgttga gcatgaagta ccagcagtag 2220 aggtcaaaga aatctctggt ggccgtctgc tgcaggaagc agacgtttac caggctgagg 2280 gcgataaggc ttcgagttgg actttggctg ccggcgaagc tgcatccgag gaaaagctcg 2340 cggagctgga attcgcttgg cgcgcagtac gctcggtaaa gtccaacgcc atcttgttgg 2400 cgcatgaagg tgcaaccgtt ggcgtgggta tgggccaggt caaccgcgtt gattcggcga 2460 agttggctgt tgaccgcgcg aatactttgg ctgattccgc agagcgtgct cgcggttccg 2520 tcgcagcatc ggatgcgttc ttcccattcg ccgatggctt gcaggtgctt atcgatgccg 2580 gcgtttccgc cgttgtccag cccggcggct ccatccgcga tgaagaagtt attgctgccg 2640 ctgaagcagc cggtatcacc atgtacttca ctggcacccg ccacttcgcg cactaaagct 2700 ttcctccgct tcgcgtccgg gaagctttag cccggaagag atagcaaaag gggcctgttc 2760 acccgcaatt ttaacggtgt gaacaggccc cttggctttc tagagctcga cttttaaata 2820 tcgagcttta agcgtcgatg ttctcgtctg cgtttcccag ccacggcgcg atgacctggt 2880 ctgacttctt cggacaacaa 2900 <210> 13 <211> 2615 <212> DNA <213> Corynebacterium ammoniagenes <400> 13 taccaacgcg gataagctca agagcgaacc agagcgcatg gcgcaggttg agtgggaaca 60 aggaaagtcg tcttccggtg ctttcgctgc aaccttgcag ctagaagcgc tcgataggca 120 aggcctgctc tttgaggtca ccaaggtatt taccgaagcg aagctgaacg tattatccat 180 gcgctcggtt cgaggtgaag accatattgc gaccctgcag tttacttttt cggtctcaga 240 taccaagcag cttggttctt tgatgacaac gttgcgtaat actgaaggcg tattcgacgt 300 ctatcgcgtg actgcttagt tgccccttct gcggggtagc taagccacta gcaggtggcg 360 tgaagttaaa gctgtggtga attattttca tcgacgcggt aaatgcggag gtggtgcaaa 420 accattgcac tgcttagata atttaaaggc gatataaaga ctcctgtaag taatttctca 480 ttattctcac aggagaaccc gtgtctaagt ttcgccgttt tggccgtctg ctggctgcaa 540 ccaccgtatc cgccaccatc gtagttggcg cgcccgtagc ctttggtcaa gaagataatg 600 ttgttgattt ctcagtcacc aatatcactg acttccatgg ccacctttct acgcaggagg 660 ctgacgctcc tgatgccaac tcagagatgg gggcggccaa gctcaaggaa ctgattgagt 720 acgttaatca ggaccaggaa tacatcatga ctacctccgg cgataacgtc ggcggtagcg 780 ctttcgtctc ggcaatcgct gatgatgagc caaccctaga tgtcctcaac atgctcggcg 840 ttgacgtctc tgcggtaggt aaccatgaat tcgaccgcgg ctatgacgat ttgctcggtc 900 gtattgctga aaaatccgcc tacccattgc tgggcgcgaa tgtcacctta aacggtgaac 960 cgatgttgga tgcttcctac gttcaagaaa ttgacggcgt caaggtggga tttgtcggta 1020 ctgtgaccac cttgactaag gacaaggtcg caccatccgc agttgaaggt gtggagtttt 1080 ctgaccccgt cgaggctacg aataaggaat ctgaccgtct gaaggaatct ggcgaagcag 1140 acgttgttgt agcgctcatg cacgaggacg cgcagcagta cgctgctggc ttcaacaaca 1200 atgtggatat ccttttcgga ggcgactctc accagcgctc gcagggcatc atcgaacgtg 1260 atggtgcgca gccactgcac tgggcgcagg ggcatgaata tggcaaggtt ctccaagacg 1320 ccgatatttc ctttaacaag gacaccggcg aaattgagtc cgtcgaaatc acccagtacg 1380 accgctcgat gcctgaggtt gaagcactag cccctgatgc agaaattgcc gctcgcgtgg 1440 cagccgctga ggcagaagct gaagagcttg gagctaaagt tgtcggccag atggaccgcg 1500 ctactttccg tggtcaagat gagggcgcag gagcggggag caaccgcggt gttgaatcta 1560 cgttgaactc gctaattgct gatgccaacc gcgcatcagt tgctaaggcg actggtgcgg 1620 acgtggattt gggcattatg aatgctggtg gcgtacgtgc tgaccttcct gcaggcgatg 1680 tcactttcca ggatgtgctt accgtgcagc ctttcggcaa ttcgattgca tacggcactt 1740 tgaccggcca agatattttg gatgctttgg aagctcagtg gcagccagga tcgtctcgtc 1800 cacgcttggc tttgggtcta tctgctggat ttgagtacgc ctacgaccca actgcagagc 1860 aaggccagcg cgttatctcc gcgaccttgg atggcgaaga aattgaccca tcagcggaat 1920 acactgttgc aacttccacg ttcctcttcg atggtggcga caacttcgca tctctggcca 1980 atgtccaaaa cctcactgac gtgggctaca tggactactc agttctcaat gactacatca 2040 aagatggcgc agaggtacag gaaggccagt ctgatatcgg tatcagcacc gaaggcaccc 2100 tggcagctgg tgaagaagtt accttcaacc tcacctcgct taactacact atggaagaag 2160 atccacaagc caccacagcg accgtcaccg tgggcgacgt gcaagaaacc gccgatatcg 2220 acgctcagtg gaatgctgaa gaagatccgc agaagaatga atttggccgt gctagcgtaa 2280 cgctgactct gccagaggac attgaggaaa ccgacttggt taccgtaacc accgatgccg 2340 gcaccgagat tactgtgccg ctgagggcct tgggcaccaa cgagggcacg gacggcggct 2400 ccaatcctgg cagcagcgca cctggcacag gtaagggctc ttctcggggt gcttcggctg 2460 ctgcaggaat cgctggagtg ttggcagcga ttgcgggcat cgccgcgttt attggtctga 2520 acggtcagtt tgatcagttc atcccagcta acatgcagcg cgctcttggt gacctgcgca 2580 gtcagctcaa cgaggttagc cacatcaaat tctag 2615  

Claims (13)

Phosphoribosylglycinamide formyltransferase and inosine acid cyclohydrolase enzymes that encode A microorganism of the genus Corynebacterium, wherein purNH gene is present in two or more copies in a chromosome, thereby enhancing the activity of the enzyme, thereby improving the production capacity of 5'-xanthyl acid. The method of claim 1, The purNH gene is a microorganism of the genus Corynebacterium, characterized in that it has a nucleotide sequence of SEQ ID NO: 12. The method of claim 1, The activity of the enzyme in the microorganism is Corynebacterium genus Corynebacterium microorganism, characterized in that the reinforcement being embedded with one or more copy of the introduced gene in addition to the purNH purNH gene in a natural state. The method of claim 1, And a microorganism of the genus Corynebacterium, characterized in that the activity of the ushA gene encoding 5'-nucleotidase is inactivated. The method of claim 4, wherein Microorganism of the genus Corynebacterium, characterized in that the ushA gene has a nucleotide sequence of SEQ ID NO: 13. The method of claim 4, wherein Microorganism of the genus Corynebacterium, characterized in that the activity of the ushA gene is inactivated by substitution, deletion, insertion. The method of claim 4, wherein The microorganism of the genus Corynebacterium, characterized in that the activity of the ushA gene is inactivated by inserting one base into the ushA gene of the chromosome. The method of claim 1, The genus Corynebacterium microorganisms Corynebacterium ammonia Ness's (Corynebacterium ammoniagenes ) Microorganism of the genus Corynebacterium, characterized in that KCCM-10530. The method of claim 1, The genus Corynebacterium microorganism is Corynebacterium ammoniagenes gene's (Corynebacterium ammoniagenes ) Microorganism of the genus Corynebacterium, characterized in that KCJ-0001 (Accession Number: KCCM-10913P). The method of claim 1, The genus Corynebacterium microorganisms Corynebacterium ammonia Ness's (Corynebacterium ammoniagenes ) Microorganism of the genus Corynebacterium, characterized in that KCJ-0002 (Accession Number: KCCM-10914P). A vector comprising a purNH gene encoding a phosphoribosylglycinamide formyltransferase and an inosine acid cyclohydrolase enzyme. A vector comprising a ushA gene encoding a 5'-nucleotidase having any base inserted therein. Culturing the microorganism of the genus Corynebacterium according to claim 1 to produce 5'-xanthyl acid in a cell or culture; And Recovering 5'-xanthyl acid from the cell or culture; Including, the method of producing 5'-xanthyl acid.

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