KR100977440B1 - Novel gene isolated from Corynebaterium, inosine-producing Corynebacterium transformed by the same gene and method of producing inosine using the same - Google Patents

Abstract

The present invention provides a novel gene of SEQ ID NO: 1 isolated from a Corynebacterium microorganism, a recombinant vector comprising the gene, a Corynebacterium microorganism producing the inosine transformed with the recombinant vector, and It relates to a method for producing inosine using a microorganism of the genus Corynebacterium.

Corynebacterium ammonia genes, inosine

Description

Novel gene isolated from Corynebaterium, inosine-producing Corynebacterium transformed by the same, Novel gene isolated from Corynebacterium microorganism, Corynebacterium microorganism producing transgenic inosine gene and method of producing inosine using the same}

The present invention is a novel gene of SEQ ID NO: 1 isolated from Corynebacterium microorganisms, Corynebacterium microorganisms producing inosine transformed with the gene and Inosine using the Corynebacterium microorganism It provides a production method of

Inosine is an important substrate for the chemical synthesis of 5'-inosinic acid, which has been spotlighted as a seasoning seasoning, and the synthesis by enzyme transfer reaction. Inosine is an intermediate of nucleic acid biosynthetic metabolism and has a physiological significance in the body of animals and plants, and is widely used in various fields such as food and medicine.

Inosine is conventionally mainly used in Bacillus (Agric. Biol. Chem., 46, 2347 (1982); Korean Patent No. 27280) or Corynebacterium ammonia genes (Agric. Biol. Chem., 42, 399 (1978) The fermentation method using a microorganism such as) or the thermal decomposition method of 5'-inosinic acid (Japanese Patent Application No. 43-3320). However, in the case of pyrolysis, a large amount of heat is required to decompose 5′-inosinic acid, which is low in practicality. In the case of direct fermentation, various genotype strains using E. coli have been developed and studied (Biosci Biotechnol Biochem. 2001 Mar; 65 (3): 570-8) The production cost is high due to the low titer of inosine production strain. Previous inosine research has also focused on E. coli and Bacillus.

Therefore, there is still a need to develop a strain capable of producing inosine at a higher yield and accumulating at a higher concentration, and a method for producing inosine using the strain.

Therefore, the inventors of the present invention during the continuous research to produce inosine at high concentration / high yield by direct fermentation method using microorganisms, Lac-I family transcription factor in Corynebacterium ammonia genes The present invention was completed by finding a novel gene having a nucleotide sequence of SEQ ID NO: 1 and increasing the expression of this gene and confirming that inosine accumulates at a high concentration.

Accordingly, an object of the present invention is a novel gene of SEQ ID NO: 1 isolated from Corynebacterium ammonia genes (hereinafter referred to as 'Ncam2271'), a recombinant vector comprising the gene and inosine transformed by the vector To provide a microorganism of the genus Corynebacterium.

Still another object of the present invention is to provide a method of producing inosine at high concentration and high yield by culturing the microorganism.

In order to achieve the above object, the present invention provides a novel gene having a sequence represented by SEQ ID NO: 1 isolated from Corynebacterium ammonia genes.

According to one embodiment of the invention, chromosomal sequencing of the genus Corynebacterium microorganisms known as inosine-producing microorganisms, for example, Corynebacterium ammonia genes ATCC 6872, Corynebacterium ammonia crab While investigating Nes's transcriptional regulator protein, a novel gene with a nucleotide sequence of SEQ ID NO: 1 of about 1,119 bp was found. The gene is not listed in NCBI and its function is unknown, but it was estimated to be Lac-I family transcription factor through NCBI Blast search. Thus, the genes of the present invention are novel genes that are believed to act as Lac-I family transcription factors.

The present invention also provides a recombinant vector comprising the novel gene of SEQ ID NO: 1 isolated from Corynebacterium ammonia genes.

According to one embodiment of the present invention, usable vectors are not particularly limited, and known expression vectors can be used, and preferably, pHC131T-gfp vector (Korean Patent Application No. 10-2007-005832) can be used. have. Therefore, the recombinant vector according to the present invention may be a pHC131T-Ncam2271 vector prepared by inserting the gene of SEQ ID NO: 1 into the pHC131T-gfp vector.

According to one embodiment of the present invention, a recombinant vector comprising the novel gene of SEQ ID NO: 1 is obtained by polymerase chain reaction (PCR) gene of SEQ ID NO: 1 DNA restriction enzymes, such as SmaI and XbaI, etc. And a DNA T4 ligase or the like can be prepared into the vector treated with the restriction enzymes EcoRV and XbaI.

The present invention also provides a microorganism of the genus Corynebacterium having increased inosine production capacity of the expression of the novel gene of SEQ ID NO: 1 isolated from Corynebacterium ammonia genes.

According to one embodiment of the invention, the microorganism of the present invention is a microorganism of the genus Corynebacterium whose expression is increased by modification of a regulatory factor that regulates the expression of the gene represented by SEQ ID NO: 1 or by an increase in the number of gene copies. Can be.

According to one embodiment of the invention, the microorganism of the present invention is a genus of Corynebacterium having the ability to produce inosine transformed with a recombinant vector comprising a novel gene of SEQ ID NO: 1 isolated from Corynebacterium ammonia genes It may be a microorganism.

Corynebacterium microorganisms of the present invention include Corynebacterium glutamicum, Corynebacterium epiphyllium, Corynebacterium diphtheria, and Corynebacterium ammonia genes, preferably Corynebacterium Corynebacterium ammonia genes CN04-0027 (KCCM-10905) having ammonia genes and most preferably blocking inosine degradation pathways and having adenine leaky auxotroph and guanine leaky requirements The parent strain is Corynebacterium ammonia genes CN04-0094 (KCCM-10906) produced by transformation with a recombinant vector containing a novel gene of SEQ ID NO: 1.

According to one embodiment of the present invention, the genus microorganism of Corynebacterium producing inosine is a conventional electroporation method using a recombinant vector comprising a novel gene of SEQ ID NO: 1, for example pHC131T-Ncam2271 vector. It can be selected by culturing in a medium containing the antibiotic kanamycine, which is transformed using the selection marker.

The present invention also provides a method for producing inosine, comprising culturing a microorganism of the genus Corynebacterium having an increased inosine production capacity, and separating inosine from the culture medium. .

In the inosine production method according to the present invention, the microorganism of the genus Corynebacterium having inosine production capacity is transformed with a recombinant vector comprising a novel gene of SEQ ID NO: 1 isolated from Corynebacterium ammonia genes to inosine production capacity It is an increased Corynebacterium microorganism, and may be preferably Corynebacterium ammonia genes CN04-0094 (KCCM-10906).

Inosine production method according to the invention comprises the step of culturing the microorganism of the genus Corynebacterium having increased inosine production capacity of the expression of the novel gene of SEQ ID NO: 1. According to one embodiment of the invention, the microorganism is cultured under aerobic conditions in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins and the like while controlling the temperature, pH and the like. As a carbon source, carbohydrates such as glucose, fructose, sterilized pretreated molasses, ie, molasses converted into reducing sugars, and the like can be used. As a nitrogen source, various inorganic nitrogen sources such as ammonia, ammonium chloride and ammonium sulfate and peptone, NZ-amine, meat Organic nitrogen sources such as extracts, yeast extracts, corn steep liquors, casein hydrolysates, fish or degradation products thereof, skim soybean 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 20-40 ° C. The pH of the medium is preferably maintained at pH 6 to 8 during the culture, the culture can be carried out for 5 to 6 days and the inosine accumulated by the direct fermentation method can be recovered by a conventional method.

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.

Microorganisms of the genus Corynebacterium that produce inosine transformed with a recombinant vector comprising the novel gene of SEQ ID NO: 1 according to the present invention is a microorganism that directly accumulates inosine in a high concentration in a culture medium. It can be produced in high yield.

Example  One. Ncam2271  Gene Acquisition and Identification

Transcription factor-related genes were searched for the entire genome of Corynebacterium ammonia genes ATCC6872. The Blast search of NCBI using the open reading frame (ORF) sequence of Corynebacterium ammonia genes ATCC6872 yielded many candidate genes identified as transcription factors, one of which was sequence number 1 for Ncam2271. It was confirmed to have. Secured Ncam2271 was identified as putative Lac-I family transcriptional factor.

Table 1. Ncam2271 Blast search results

NCBI GI Product Organism Matched range Identity
% e-value Query% Subject% One 38234533 Putative LacI-family transcriptional regulator Corynebacterium diphtheriae NCTC 13129 94.4 97.6 56 1e-106 2 68535394 Putative transcriptional regulator (LacI family) Corynebacterium jeikeium K411 94.1 100.0 55 1e-104 3 25029067 Putative regulatory protein Corynebacterium efficiens YS-314 91.1 96.7 58 1e-101 4 19553826 Transcriptional regulator Corynebacterium glutamicum R 91.3 96.7 54 1e-95 5 62391466 Transcriptional regulator, LacI family Corynebacterium glutamicum ATCC 13032 91.3 97.2 54 2e-95

Example  2. Ncam2271  Gene Cloning  And transformation Inosine  Production strain production

1 shows a process for cloning a novel gene of SEQ ID NO: 1.

(1) Preparation of Recombinant Vector pHC131T

To clone the novel gene of SEQ ID NO: 1, a recombinant vector pHC131T comprising a CJ1 promoter and a rrnB ₁ B ₂ terminator for inducing overexpression of the gene was prepared.

First, the CJ1 promoter uses pECCG117-CJ1 (Korean Patent Publication No. 2006-0068505) as a template, using the primers of SEQ ID NO: 4 and SEQ ID NO: 5, denaturation of 30 seconds at 94 ° C, annealing of 30 seconds at 55 ° C, and 68 A cycle consisting of an extension of 30 seconds at < RTI ID = 0.0 > C < / RTI >

The rrnB ₁ B ₂ terminator uses genomic DNA of Escherichia coli K-12 W3110 as a template and performs PCR and electrophoresis under the same conditions as those of the CJ1 promoter using primers of SEQ ID NO: 6 and SEQ ID NO: 7 to 411 bp. Obtained as a fragment.

Plasmid pECCG117 (Domestic Publication No. 92-7401) was treated with restriction enzymes EcoRV and KpnI, followed by electrophoresis on a 0.8% agarose gel to elute a band of about 5.9 kb, and the CJ1 promoter was subjected to the same restriction enzyme EcoRV. After treatment with KpnI and separation using a Quiaquick PCR Purification Kit (Quiagen), the two DNA fragments were linked using a Quick Ligation Kit (NEB) to obtain a recombinant plasmid pECCG117-CJ1. Thereafter, the obtained pECCG117-CJ1 was treated with XbaI and NotI, followed by electrophoresis on a 1% agarose gel to elute a band of about 6.2 kb in size. The rrnB ₁ B ₂ terminator obtained by PCR was treated with XbaI and NotI, followed by electrophoresis on 1.0% agarose gel to elute a band of about 0.4 kb in size.

The pECCG117-CJ1 and rrnB ₁ B ₂ terminator fragments were linked to obtain the recombinant plasmid pECCG117-CJ1-rrnB ₁ B ₂ (about 6.6 kb), which was named pHC131T.

(2) Cloning of the Ncam2271 Gene

In order to obtain the gene fragment of SEQ ID NO: 1, the polymerase chain reaction using the genomic DNA of Corynebacterium ammonia genes ATCC6872 was performed using the primers of SEQ ID NO: 2 and SEQ ID NO: 3. The gene fragment of SEQ ID NO: 1 thus obtained was cleaved with restriction enzymes SmaI (New England Biolabs, Beverly, MA) and XbaI (New England Biolabs, Beverly, MA). The gene fragment was conjugated with EcoRV and XbaI restriction enzymes using T4 ligase (New England Biolabs, Beverly, Mass.) To prepare a linearized pHC131T vector to prepare pHC131T-Ncam2271.

2 shows recombinant vector pHC131T-Ncam2271 comprising the gene of SEQ ID NO: 1, Ncam2271. After the recombinant vector pHC131T-Ncam2271 prepared as described above was transformed into inosine producing strain Corynebacterium ammonia genes CN04-0027 (KCCM-10905) by electroporation (Appl. Microbiol. Biotechnol. (1999) 52 : Using the transformation method of 541-545), and selected from Corynebacterium ammonia genes CN04-0094 transformed with the above synthesis vector in a selection medium containing 25 mg / L of kanamycin (kanamycin).

Example  3. Erlenmeyer flask Fermentation potency  exam

3 ml of the seed medium having the composition shown in Table 2 was dispensed into a test tube having a diameter of 18 mm, autoclaved, and then inoculated with Corynebacterium ammonia genes CN04-0027 and CN04-0094 (KCCM-10906) at a temperature of 30 ° C. The culture was shaken for 24 hours and used as a seed culture solution. 27 ml of the fermentation broth having the composition shown in Table 2 was dispensed into a 250 ml Erlenmeyer flask for shaking, autoclaved at a temperature of 120 ° C. for 10 minutes, and inoculated with 3 ml of the culture medium, followed by incubation for 5 to 6 days. Culture conditions of the shake incubator was adjusted to 220 revolutions per minute (rpm), temperature 32 ℃ and pH 7.2. The result of comparing the accumulation amount of inosine in the medium to the parental strain is shown in Table 3 below. Corynebacterium ammonia genes CN04-0094 transformed by the recombinant vector containing the gene of SEQ ID NO: 1 according to the present invention. In the case of the inosine accumulation can be seen that about 15% improved. The Corynebacterium ammonia genes CN04-0094 was deposited to the Korean Culture Center of Microorganisms as of December 20, 2007 (Accession No. KCCM 10906).

TABLE 2

Badge type Badge composition Nutrition medium Peptone 1%, Juicy 1%, Sodium Chloride 0.25%, Yeast Extract 1%, Adenine 100mg / L, Guanine 100mg / L, Agar 2%, pH 7.2 Minimum badge Glucose 2%, Sodium sulfate 0.3%, Potassium phosphate 0.1%, Dipotassium phosphate 0.3%, Magnesium sulfate 0.3%, Calcium chloride 10mg / L, Iron sulfate 10mg / L, Zinc sulfate 1mg / L, Manganese chloride 3.6mg / L , L-cysteine 20mg / L, calcium pantothenate 10mg / L, thiamine hydrochloride 5mg / L, biotin 30μg / L, adenine 20mg / L, guanine 20mg / L, agar 2%, pH 7.2 Species Glucose 5%, Peptone 0.5%, Juicy 0.5%, Sodium Chloride 0.25%, Yeast Extract 1%, Adenine 100mg / L, Guanine 100mg / L, pH 7.2 flask
Fermentation medium Sodium glutamate 0.1%, ammonium chloride 1%, magnesium sulfate 1.2%, calcium chloride 0.01%, iron sulfate 20mg / L, manganese sulfate 20mg / L, zinc sulfate 20mg / L, copper sulfate 5mg / L, L-cysteine 23mg / L, alanine 24mg / L, Nicotinic Acid 8mg / L, Biotin 45ug / L, Thiamine Hydrochloride 5mg / L, Adenine 30mg / L, Phosphoric Acid (85%) 1.9%, Fructose, Glucose and Molasses are mixed and added to 6% as reducing sugar. , pH 7.2

TABLE 3

Inosine concentration (g / l) % Increase compared to control Control Group (CN04-0027) 5.1 - CN04-0094 (CN04-0027-pHC131T) 5.9 15

From the above description, those skilled in the art can understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the examples and experimental examples described herein are exemplary in all respects and are not intended to limit the scope of the present invention. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the appended claims and the equivalent concept rather than the detailed description are included in the scope of the present invention.

1 shows the cloning process of Ncam2271 gene.

2 shows recombinant vector pHC131-Ncam2271.

<110> CJ Corporation <120> Novel gene isolated from Corynebacterium, inosine-producing          Corynebacterium transformed by the same gene and method of          producing inosine using the sameh <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 1119 <212> DNA <213> Corynebacterium ammoniagenes <220> <221> gene (222) (1) .. (1119) <223> putative Lac-I transcription factor <400> 1 atggttaaac gcagtccggt tcgtaaaact cttgcttccc tcgcggcaga attaggggtc 60 tcgcgaacga ctgtttctaa tgcttataac cgtcccgatc agctctctcc tgagctgcgc 120 gagcgcatct tgacggctgc tcgcgcgcat ggctattcgg ggccggatcc gatggccagg 180 agtctacgta cgcgtcgtgt gggctccatt ggtgtagtgc tgacggagga tttgtcctat 240 gccttcgaag atgctgcgtc ggtggatttc ttagcaggta tggcgcaagg gctagcgggt 300 aaaaatagca cgttgacgtt gattccggca ggcccagata ccgctgaagg caccggcgcg 360 gaggaacttt tgggccgcgc ggttgtggat ggttttgtgg tctattccgt cggcggcgat 420 gacccgtatc tagcggccgc tagtaaccgc ggcctgccgg tggtggtctg tgaccagccg 480 cgcgataccg ggcagccttt tgttggcatt gatgatttca cagcgattgc tccggcggcg 540 caagcgctcg tcgatgccgg gcacaccaaa gtcggcatcc tagccattcg tctgcgtcat 600 gaacgcctcg atggcgtggt ggaccgaggg aacctcgccg cagcagagtt tgatacccag 660 cgactgcgcg tgcagggagc gttaaaagtc ttcgccgatg ccggtatcga tgcggataag 720 attcccgtgg tcactcgcca catcaacact cgcgacacgg cgcgggaagc agccgaggaa 780 ctgctgagca ataacccgga attaaccgca gtgctgtgca caacggactc catggcgctg 840 ggtgtcttgg acttcgcgga gcgccaagga attcccattc ctggggatct ttccgtgacc 900 ggcttcgacg gtatcgaatc cgctcgcctg cgtggattga ccacagtgat tcaacccaac 960 ttcaataaag gcgctgaagc gggccggatg ctcgccgagc ttatcgatgc ctccgtcgcc 1020 aaccttgcct atcccactag cgaagacgga aagccactgc gcgtgctgtt gaagacccaa 1080 ctcgcgccgg gcaagacgat tgcccgccca cgccactag 1119 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer for Ncam2271 <400> 2 gggatggtta aacgcagtcc gg 22 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer for Ncam2271 <400> 3 gctctagact agtggcgtgg gcgggcaa 28 <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer for CJ1 promoter <400> 4 cgggtaccac cgcgggctta ttccattaca t 31 <210> 5 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer for CJ1 promoter <400> 5 acgcgatatc ttaatctcct agattgggtt tc 32 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer for rrnB1B2 terminator <400> 6 gctctagagc tgttttggcg gatgaga 27 <210> 7 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer for rrnB1B2 promoter <400> 7 ataagaatgc ggccgccgca aaaaggccat ccgtcag 37  

Claims (8)

A gene derived from Corynebacterium ammonia genes having the sequence represented by SEQ ID NO: 1. A recombinant vector comprising the gene of claim 1. The recombinant vector of claim 2, wherein the recombinant vector is prepared based on pHC131T. The expression of the gene having the sequence represented by SEQ ID NO: 1 is increased by an increase in the number of copies of the gene having the sequence represented by SEQ ID NO: 1 or by the modification of a regulatory factor that regulates the expression of the gene having the sequence represented by SEQ ID NO: 1, Corynebacterium microorganism having inosine production ability. The Corynebacterium microorganism according to claim 4, wherein the Corynebacterium microorganism is transformed with a recombinant vector comprising a gene having a sequence represented by SEQ ID NO: 1. The corynebacterium microorganism according to claim 5, wherein the corynebacterium microorganism is corynebacterium ammonia genes. The Corynebacterium microorganism according to claim 5, wherein the Corynebacterium microorganism is Corynebacterium ammonia Genes CN04-0094 (KCCM-10906). A method for producing inosine, comprising culturing the genus Corynebacterium microorganism according to any one of claims 4 to 7, and separating inosine from the culture.

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