KR20080066204A - Microorganism overexpressed 5'-phosphoribosyl-5-aminoimidazole(air) carboxylase and the process for producing 5'-inosinic acid using the same - Google Patents

Abstract

A Corynebacterium sp. microorganism transformed by a vector including a purKE gene encoding phosphoribosylaminoimidazole carboxylase is provided to produce 5'-inosinic acid more economically with high productivity through direct fermentation thereof. A Corynebacterium sp. microorganism is characterized in that it produces a highly concentrated 5'-inosinic acid by over-expressing 5'-phosphoribosyl-5-aminoimidazole(AIR) carboxylase. A method for over-expressing phosphoribosylaminoimidazole carboxylase comprises a step of transforming a Corynebacterium sp. microorganism by a recombinant vector including a purKE gene encoding the phosphoribosylaminoimidazole carboxylase. The Corynebacterium ammoniagenes CN01-0025(deposition no. KCCM-10826P) prepared by the method is cultured and then the 5'-inosinic acid is collected from a culture solution to produce the 5'-inosinic acid.

Description

MICROORGANISM OVEREXPRESSED 5'-PHOSPHORIBOSYL-5-AMINOIMIDAZOLE (AIR) CARBOXYLASE AND THE PROCESS FOR PRODUCING 5'-INOSINIC ACID USING THE SAME}

1 encodes phosphoribosyl aminoimidazole carboxylase Cloning process and plasmid of the purKE gene is shown.

The present invention relates to a microorganism over-expressed phosphoribosyl aminoimidazole carboxylase and a method for producing 5'-inosinic acid using the same. More specifically, the present invention relates to Corynebacterium ammoniagenes (Corynebacterium ammoniagenes) CJHB100 (KCCM-10330) phosphorobosyl aminoimidazole carboxylase (5'-phosphoribosyl-5-aminoimidazole (AIR) carboxylase) A microorganism having an overexpression of phosphoibosyl aminoimidazole carboxylase by transformation with a recombinant vector containing a purKE gene encoding to increase the production efficiency of 5'-inosinic acid, and a method for producing 5'-inosinic acid using the same will be.

5'-inosinic acid (5'-inosinic acid) is an intermediate of the nucleic acid biosynthetic metabolic system, and 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.

As a method for producing 5'-inosinic acid, a method of enzymatically decomposing ribonucleic acid extracted from yeast cells (Japanese Patent Publication No. 1614/1957, etc.), a method of chemically phosphorylating inosine produced by fermentation (Agri Biol. Chem., 36, 1511 (1972), and the like, and methods of culturing microorganisms capable of producing 5'-inosine acid and recovering 5'-inosine acid accumulated in the medium. The most widely used method among these methods is a method of producing 5'-inosinic acid using microorganisms. Among the microorganisms used to produce 5'-inosinic acid, Corynebacterium sp. Strains, for example, are produced by fermenting Corynebacterium ammoniagenes (Korean Patent Publication No. 2003-42972, etc.). ) And the like are known.

Phosphoribobosil aminoimidazole (5'-phosphoribosyl-5-aminoimidazole: AIR) is an intermediate in the purine biosynthesis pathway and is used as a precursor of thiamine biosynthesis and 5'-inosinic acid biosynthesis. At this time, the first enzyme leading to 5'-inosinic acid biosynthesis is phosphoribosyl aminoimidazole carboxylase.

Phosphoribobosil aminoimidazole carboxylase (5'-phosphoribosyl-5-aminoimidazole (AIR) carboxylase) is an enzyme belonging to the purine biosynthesis pathway, encoding the carboxy aminoimidazole ribonucleotide synthase (N5-carboxyaminoimidazole ribonucleotide synthase). The gene (hereinafter purK ; SEQ ID NO: 3) and the gene encoding the carboxyaminoimidazole ribonucleotide mutase (hereinafter referred to as purE ; SEQ ID NO: 4) are linked by the structure of the operon (hereinafter referred to as purKE ). It has all the features. Corynebacterium ammoniagenes has revealed its gene sequence and sequence structure on the genome (FEMS Microbiol Lett. 1996 Apr 1; 137 (2-3): 265-8.).

Accordingly, the present inventors, while developing a strain capable of producing a high concentration of 5'-inosinic acid from a Corynebacterium sp. Strain, while conducting a study for enhancing purine metabolism, phosphoribobosil aminoimidazole carboxylase When transformed with a vector comprising a coding gene sequence, it was found that 5'-inosinic acid is produced at a high concentration, thereby completing the present invention.

An object of the present invention is to improve the yield by transforming the Corynebacterium genus microorganisms with a recombinant vector expressing the phosphorobosil aminoimidazole carboxylase (5'-phosphoribosyl-5-aminoimidazole (AIR) carboxylase) To provide a method for producing a microorganism of the genus Corynebacterium that produces 5'-inosinic acid.

Another object of the present invention is to provide a microorganism prepared by the above method.

Still another object of the present invention is to provide a method for producing 5'-inosinic acid using the prepared microorganism.

In order to achieve the above object, the present invention by transforming Corynebacterium ammonia genes (Coynebacterium ammoniagenes ) with a recombinant vector containing a gene encoding a phosphoribosyl aminoimidazole carboxylase, by overexpressing it, The present invention relates to a method for producing Corynebacterium ammonia genes producing high concentration of 5'-inosinic acid.

The microorganism of the genus Corynebacterium in the present invention is a microorganism known to be capable of producing 5'-inosinic acid, preferably Corynebacterium ammonia genes.

The gene encoding the phosphoribosyl aminoimidazole carboxylase of the present invention, namely the purKE gene, can be obtained from a microorganism producing the enzyme. Preferably, the gene encoding phosphoribosyl aminoimidazole carboxylase can be obtained from a microorganism of the genus Corynebacterium ammonia genes. In a specific embodiment of the present invention, through chromosome sequencing of Corynebacterium ammonia genes ATCC 6872, it was confirmed that the purKE gene is present in about 2.0 kbp size.

Recombinant vector of the present invention is a gene encoding the phosphoribosyl aminoimidazole carboxylase obtained through a polymerase chain reaction ( purKE gene; SEQ ID NO: 3 and SEQ ID NO: 4) is a DNA restriction enzyme, for example BamHI and SpeI and the like And DNA T4 ligase or the like can be prepared by inserting into a vector cut with the same DNA restriction enzyme. The vector which can be used is not specifically limited, A well-known expression vector can be used. Preferably, a pECCG117 vector (Biotechnology letters vol 13, No. 10, p.721-726 (1991) or Korean Patent Publication No. 92-7401) can be used, and the recombinant vector is a phosphorobosil amino to the pECCG117 vector. The pECCG117- purKE vector prepared by inserting the purKE gene encoding imidazole carboxylase is preferred. In addition, the phosphoribosyl aminoimidazole carboxylase enzyme was encoded and included in the gene in front of the gene to induce its expression to be regulated by its own promoter of phosphoribosyl aminoimidazole carboxylase.

In a specific embodiment, the recombinant vector, for example, a microorganism strain with a linear DNA fragment using pECCG117- purKE conventional electroporation (electroporation) methods (e. G., Corynebacterium ammoniagenes to Ness (Corynebacterium ammoniagenes ) KCCM-10610, Republic of Korea Patent Publication No. 10-2006-0060404) and can be selected by culturing in a medium containing the antibiotic kanamycin (kanamycin), a selection marker. Insertion of the pECCG117- purKE vector in selected strains can be confirmed by PCR.

In another aspect, the present invention provides a purKE encoding a phosphoribosyl aminoimidazole carboxylase. The present invention relates to a microorganism of the genus Corynebacterium transformed with an expression vector containing a gene.

Microorganisms of the genus transformed Corynebacterium of the present invention refers to a microorganism that produces a high concentration of 5'-inosinic acid by the over-expression of phosphoribosyl aminoimidazole carboxylase by transformation.

In a specific embodiment of the present invention, Corynebacterium ammonia genes transformed to over-express phosphorobosil aminoimidazole carboxylase to produce 5'-inosinic acid at high concentration is referred to as' Corynebacterium ammonia genes CN01-0025. It was deposited with the accession number KCCM-10826P as of December 15, 2006 to the Korean Culture Center of Microorganisms (KCCM) in Hongje 1-dong, Seodaemun-gu, Seoul under the Treaty of Budapest.

As another aspect, the present invention relates to a method for culturing the transformed Corynebacterium microorganisms to produce 5'-inosinic acid from the culture.

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

Glucose may be used as the carbon source in the culture, and various inorganic nitrogen sources such as ammonia, ammonium chloride, and ammonium sulfate and peptone, NZ-amine, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, fish or Organic nitrogen sources such as degradation products thereof, skim soy cakes, or degradation products thereof can be used. 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 to 40 ° C. The pH of the medium is preferably maintained near neutral during the culture. Cultivation can be performed for 5 to 6 days, and the 5'-inosinic acid accumulated by direct fermentation can be recovered by a conventional method. In a specific embodiment, the Corynebacterium genus microorganism (KCCM-10826P) transformed by the present invention produces 5'-inosinic acid in higher yield than the non-transfected Corynebacterium microorganism (KCCM-10610). It was.

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  One. purKE  Gene Cloning

Corynebacterium ammoniagenes's Ness (Corynebacterium ammoniagenes ) Isolate the chromosomal gene of CJHB100 (KCCM-10330) and use the primers of SEQ ID NO: 1 and SEQ ID NO: 2 as a template to encode phosphoribosyl aminoimidazole carboxylase through polymerase chain reaction. ( purKE Genes) (SEQ ID NO: 3 and SEQ ID NO: 4). Fragments of the obtained purKE gene were digested with restriction enzymes BamHI (New England Biolabs, Beverly, MA) and SpeI (New England Biolabs, Beverly, MA). Ligase (New England Biolabs, Beverly, Mass.) Was used to conjugate the gene fragment to linear pECCG117 vector digested with BamHI and SpeI restriction enzymes. The produced vector was named pECCG117-purKE. The pECCG117-purKE vector was transformed into Corynebacterium ammonia genes (KCCM-10330) by electroporation, and CM solid medium containing 10 mg of kanamycin per liter (10 g / juice) L, yeast extract 10g / L, bactopeptone 10g / L, sodium chloride 2.5g / L, bactoa 1.7%, pH 7.0) to recover single colonies. The recovered colonies were grown in a CM liquid medium to which the same antibiotic was added, the plasmids were isolated and their size was determined first, and secondly, colony PCR was performed using primers containing both ends of the multicloning site of pECCG117. The presence or absence of transformation was confirmed through the size of the inserted DNA. Cloning Process and Cloned pECCG117- The purKE vector is shown in FIG.

The sequences of the primers used to amplify the purKE gene are as follows.

Primer purKE-5 (SEQ ID NO: 1)

5 '-GGATCCGGCCAAGAAGGCCTCAGCAACTGTCTGAACTCGCCTC-3'

Primer purKE-3 (SEQ ID NO: 2)

5 '-ACTAGTGTTTAAACGGTTGCTGCAAGCTTCAGCG-3'

The strain containing the purKE gene selected through this process was named ' Corynebacterium ammoniagenes CN01-0025', and the accession number KCCM-10826P was assigned to the Korea Microorganism Conservation Center on December 15, 2006. Deposited.

Example  2. Erlenmeyer flask Fermentation potency  exam

After dispensing 3 ml of seed medium into an 18 mm diameter test tube and autoclaving, the seed was inoculated with Corynebacterium ammonia genes CN01-0025 (KCCM-10826P) 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, autoclaved at 120 ° C. for 10 minutes, and then inoculated with 3 ml of the seed culture solution and incubated for 5 to 6 days. Culture conditions were adjusted to 200 times per minute, temperature 32 ℃, pH7.2. At this time, the result of comparing the 5'-inosinic acid accumulation in the medium with the parent strain KCCM-10610 is shown in Table 1 below, which means that the growth of the cells compared to the parent strain KCCM-10610 under the same conditions as the CN01-0025 It shows good speed and better productivity of 5'-inosinic acid produced per unit time.

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

Species medium : glucose 1%, peptone 1%, gravy 1%, yeast extract 1%, sodium chloride 0.25%, adenine 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%, glucose 6.5%.

Strain name Cell OD (5 days after culture) Productivity (g / l / hr) (after 5 days of culture) Control Group (KCCM-10610) 37.1 0.111 CN01-0025 (KCCM-10826P) 38.4 0.135

Example  3. In 5L fermenters Fermentation potency  Experiment

50 ml of the seed medium of Example 2 was dispensed into a 500 ml shaking Erlenmeyer flask and autoclaved according to a conventional method, and then inoculated with the used strain, followed by shaking culture at 30 ° C. for 24 hours to be used as a seed culture solution.

1000 ml of fermenter seed medium was placed in a 2.5 L-fermentation tank and autoclaved at 120 ° C. for 15 minutes, and then 50 ml of seed culture solution was inoculated and then cultured for 1 to 2 days. Culture conditions were adjusted to 900 revolutions per minute, temperature 28 to 34 ℃, pH 7.2.

The composition of the fermenter seed medium is as follows.

Fermenter broth : 5% glucose, 1% peptone, 2% yeast extract, 0.1% potassium phosphate, 0.1% potassium diphosphate, 0.1% magnesium sulfate, 0.5% ammonium sulfate, 80% iron sulfate, 40mg zinc sulfate / l, manganese sulfate 40 mg / l, L-cysteine 80 mg / l, calcium pantothenate 60 mg / l, thiamine hydrochloride 20 mg / l, biotin 240 μg / l, adenine 1200 mg / l, guanine 1200 mg / l, pH 7.2.

In addition, 1250 ml of the fermenter main medium was placed in a 5 L-fermentation tank and autoclaved at 120 ° C. for 15 minutes to inoculate 250 ml of the fermenter seed culture broth. Tea additional sugar was added to be 32% of the final reducing sugar by mixing fructose, glucose and molasses and incubated for 5 to 6 days. Culture conditions were adjusted to 900 revolutions per minute, temperature 30 ℃, pH 7.2.

The composition of the fermenter main medium is as follows.

Fermenter Main Medium : Calcium chloride 120mg / l, Copper sulfate 8mg / l, Magnesium sulfate 1.5%, Iron sulfate 24mg / l, Zinc sulfate 24mg / l, Manganese sulfate mg / l, L-cysteine 26.4mg / l, Sodium glutamate 0.12%, Thiamin hydrochloride 6mg / l, biotin 40μg / l, nicotinic acid 50mg / l, alanine 145mg / l, adenine 200mg / l, phosphoric acid (85%) 4.3%, fructose, glucose 35.2% (pH 7.2)

In this case, the result of comparing the 5'-inosinic acid accumulation in the medium with the parent strain KCCM-10610 is shown in Table 2 below, and in the case of the strain according to the present invention, the productivity of 5'inosinic acid was compared to the parent strain KCCM-10610 under the same conditions. Excellent.

Strain name Cell. OD Fermentation time (hr) Productivity (g / l / hr) Control Group (KCCM-10610) 200 103.5 0.64 CN01-0025 (KCCM-10826P) 203 102.7 0.71

As described above in detail, a high concentration of 5'-inosinic acid is produced by overexpression of phosphoribosyl aminoimidazole carboxylase by transformation with a vector comprising a purKE gene encoding phosphoribosyl aminoimidazole carboxylase. When 5'-inosinic acid is produced by direct fermentation using Nebacterium genus microorganism CN01-0025 (KCCM-10826P), 5'-inosinic acid can be economically produced with higher productivity.

<110> CJ Corp. <120> Microorganism overexpressed          5'-phosphoribosyl-5-aminoimidazole (AIR) carboxylase and the          process for producing 5'-inosinic acid using the same <130> PA06-0455 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> primer (purKE-5) <400> 1 ggatccggcc aagaaggcct cagcaactgt ctgaactcgc ctc 43 <210> 2 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer (purKE-3) <400> 2 actagtgttt aaacggttgc tgcaagcttc agcg 34 <210> 3 <211> 1242 <212> DNA <213> Corynebacterium ammoniagenes <400> 3 atgaaacgcg tgagtgaaca agcaggaaac ccagacggaa accctcaagc acatgttccc 60 ggcatgccgg ttatcgccgt tattggtgat ggccagctag ctcgcatgat gcaaaccgcc 120 gccattgagc tcggccaatc gctgcgcctt cttgccggcg cacgcgatgc ctctgcggca 180 caagtatgcg cggatgtagt gcttggtgat tacaccaact acgacgactt gctcaaagcc 240 gtcgacggtg ccaccgctgt cacttttgac catgagcacg tgcctaatga gcacctcacc 300 gcgttgatcg atgcaggcta taacgatcag ccacaacctg ctgcgctgat taacgcccaa 360 gacaaattgg ttatgcgcga gcgcctcgcc gagctgggcg cacccgtgcc gcgctttgcg 420 ccgattgaat ctgcccaaga tgcgtacgat ttttggacct tgacgtccgg gcaggtctgt 480 ctgaaggcgc gccggggtgg ctacgacggc aaaggcgtgt ggtttccgaa taatgaatct 540 gagctgactg ctttggtctc tgacctttcg cgccgcggcg tggccttgat ggctgaagag 600 aaggttggct ggtcccgcga gctttccgtg ctggtcgcgc ggactccctc gggcgaggtt 660 gctacttggc cgctgactga gtctgtgcag cgcaacggtg tgtgcgctga agctgtcgcg 720 ccagccccgg gagttgaccc gcagctgcag caacgcgctg agacactggg tgaaaagatt 780 gccaccgagt tgggtgtaac tggtgtgctc gcggtagagc tttttgcatt tgcgaatgag 840 tccggtgcgg aagatatcgc ggttaatgaa ctggcaatgc gcccgcacaa taccggccac 900 tggaccctag taggttctgt gacctcccaa tttgagcagc acctgcgcgc ggtgatggat 960 gagccactgg gggatacatc cacgcttgcc ccagtcaccg tgatggccaa cgtcttaggc 1020 gctgacgaag accaaagatg ccaatgggcg agcgtgccga gaagtggcgc gcccgttccc 1080 gcgaccaaag tccatctcta cggcaagggc atcgcccaag gccgtaagat tggccacgtg 1140 aacctcaccg gtgaggacga agaggcaacc cgtcgcgatg ctcgcttggc tgcggatttc 1200 ctcgtgaacg ccgcgtggtc tgataactgg tccgctaaat ag 1242 <210> 4 <211> 534 <212> DNA <213> Corynebacterium ammoniagenes <400> 4 atgactgcac cgctagttgg gctcatcatg ggctctgatt ctgattggcc aaccgttgaa 60 ccagcagctg aggttctcga cgaattcggt gttccttttg aggtgggcgt ggtctctgcg 120 caccgcacgc cggaaaagat gctggattac gccaagcaag cccacactcg cggcatcaag 180 gtgattgttg cttgtgccgg tggggcagcg cacctaccag gcatggtggc tgcagcaact 240 cctttgccag ttattggtat tccacgtgcc ttgaaagatt tggaaggtct ggactctttg 300 ctgtctatcg tgcagatgcc agctggggtt ccggttgcga ccgtgtctat cggcggcgct 360 aagaatgctg gcttgctcgc catccgtacc ctgggcgtgc agtactcaga attggttgaa 420 cgcatggccg attaccaaga aaatatggcc gattaccaac cgaaaatatg gccaaggaag 480 ttgagcaaaa agacgccaat cttcgcgcca agctcatggg ggactagacc ttag 534  

Claims (6)

A microorganism of the genus Corynebacterium, characterized by producing a high concentration of 5'-inosinic acid by overexpressing a phosphoribosyl aminoimidazole carboxylase (5'-phosphoribosyl-5-aminoimidazole (AIR) carboxylase). The microorganism according to claim 1, wherein the microorganism of the genus Corynebacterium is Corynebacterium ammonia genes. As a method of overexpressing phosphoribosyl aminoimidazole carboxylase, A method of transforming a microorganism of the genus Corynebacterium into a recombinant vector comprising a purKE gene encoding a phosphoribosyl aminoimidazole carboxylase. 4. The method of claim 3, wherein the microorganism of the genus Corynebacterium is Corynebacterium ammonia genes. Corynebacterium ammonia genes CN01-0025 (KCCM-10826P) prepared according to the method of claim 3. A method of culturing the microorganism according to claim 5, and producing 5'-inosinic acid from the culture solution.

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