KR20090069572A - Microorganisms of corynebacterium having an inactivated gene encoding abc-transpoter and processes for the preparation of 5'-inosinic acid using the same - Google Patents

Abstract

A Corynebacterium sp. microorganism which produces 5'-inosinic acid is provided to inactivate a gene coding ABC-tranporter and massively produce 5'-inosinic acid. A Corynebacterium sp. microorganism in which a gene coding ABC-transporter is inactivated accumulates a 5'-inosinic acid in medium. A gene coding a protein which does not have ABC-transporter activation of Corynebacterium sp. microorganism has sequence of the sequence number 2(SEQ ID NO:2). A method for manufacturing the Corynebacterium sp. microorganism comprises: a step of preparing a recombinant vector containing the gene coding the protein which does not have ABC-transporter activation of Corynebacterium sp. microorganism; a step of substituting the gene coding ABC-transporter of Corynebacterium sp. microorganism and inactivating.

Description

MICROORGANISMS OF CORYNEBACTERIUM HAVING AN INACTIVATED GENE ENCODING ABC-TRANSPOTER AND PROCESSES FOR THE PREPARATION OF 5'-INOSINIC ACID USING THE SAME}

The present invention is a microorganism of the genus Corynebacterium that produces 5'-inosinic acid, in which a gene encoding an ABC-transporter (hereinafter referred to as "novA gene") is inactivated. It relates to a microorganism of the genus Corynebacterium and a method for producing 5'-inosinic acid using the same. The present invention also relates to a gene encoding the ABC-transporter of a microorganism of the genus Corynebacterium and a gene for inactivating the ABC-transporter by replacing the gene.

5'-inosinic acid is an intermediate of the nucleic acid biosynthetic metabolic system, which plays a physiologically important role in the body of animals and plants, and is used in various fields such as food, medicine, and various medical uses. In particular, when used together with sodium glutamate It is one of nucleic acid-based seasonings that has been spotlighted as a seasoning seasoning because of its synergistic effect. Currently, 5'-inosinic acid is known to be produced by fermenting a strain of the genus Corynebacterium, for example Corynebacterium ammoniagenes (Korean Patent Publication No. 2003-42972, etc.).

Many attempts have been made to improve the 5'-inosinic acid production method using such Corynebacterium strains. Among them, there was a study to improve 5'-inosinic acid-producing Corynebacterium strain by destroying or attenuating specific genes using recombinant DNA technology. There was also a study to improve the 5'-inosinic acid production capacity by applying random mutations using NTG. However, even by the conventional method described above, there is still a need for strains with improved production capacity of 5'-inosinic acid.

Random mutations using conventional NTG can cause unnecessary mutations, delay growth and weaken stress tolerance. Moreover, it is difficult to identify accurate genetic information without knowing the exact mutation position.

L-aspartate, one of the precursors of 5'-inosinic acid, is a central metabolite of nitrogen metabolism in cells, and acts as a unit or regulator of cell composition or protein synthesis. As a cell structural unit, it is used for the synthesis of nucleic acids, amino acids, or lipids of a cell, and as a unit for protein synthesis, it is used as a structure or a main functional group of a protein.

L-aspartate has been used in large quantities in proteins to encode proteins encoded by non-essential genes that are not essential for cell growth, maintenance and regulation. Thus, removing or inactivating a gene encoding a non-essential protein with high L-aspartate content can reduce or eliminate the unnecessary consumption of large amounts of L-aspartate required for product synthesis of that gene. In other words, in terms of reducing the consumption of unnecessary L- aspartate, it is thought that it will work advantageously for the production of 5'-inosinic acid under the same conditions.

Accordingly, the present inventors have developed a microorganism of the genus Corynebacterium having an increased production yield of 5'-inosinic acid by inactivating the novA gene encoding ABC-transporter, a non-essential protein having a high content of L-aspartate.

The novA gene, a gene encoding the ABC-transporter of a microorganism of the corynebacterium, has not been reported yet, and no variant strain inactivating the novA gene has been reported.

An object of the present invention is to provide a microorganism of the genus Corynebacterium producing 5'-inosinic acid, wherein the gene encoding the ABC-transporter is inactivated.

In addition, an object of the present invention is to provide a method for producing 5'-inosinic acid using the microorganism.

Still another object of the present invention is to provide a gene encoding the ABC-transporter of a microorganism of the genus Corynebacterium.

Still another object of the present invention is to provide a gene which inactivates the gene by being substituted with a gene encoding the ABC-transporter of the microorganism of the genus Corynebacterium.

The present invention is to solve the problems of the prior art, and provides a microorganism inactivated novA gene encoding the ABC-transporter of the genus Corynebacterium and a method for producing 5'-inosinic acid using the same.

The present invention also provides the novA gene and a gene substituted therewith to inactivate the novA gene.

The microorganism of the genus Corynebacterium in which the gene encoding the ABC-transporter according to the present invention is inactivated is a microorganism that directly accumulates 5'-inosinic acid in a culture medium with high yield and high concentration. Allows production of 5'-inosinic acid in yield.

In order to achieve the object of the present invention, the present invention provides the novA gene of Corynebacterium genus.

The microorganism of the genus Corynebacterium in the present invention is a microorganism known to be able to produce 5'-inosinic acid, preferably Corynebacterium ammoniagenes.

In one embodiment of the present invention, the inventors performed chromosome sequencing of Corynebacterium ammoniagenes ATCC 6872 with a microorganism producing 5'-inosinic acid, and the gene of novA encoding the ABC-transporter. DNA base sequence was analyzed. As a result, it was found that the novA gene of Corynebacterium sp. Microorganism has a nucleotide sequence of SEQ ID NO: 1 of about 1 kb.

The novA gene of the microorganism of the genus Corynebacterium of the present invention is at least 90%, preferably at least 95%, more preferably at least 90% of the gene of SEQ ID NO: 1 or the gene of SEQ ID NO: 1 by conventional substitution, deletion and / or insertion. Preferably, the gene has a homology of 98% or more. Preferably, the present invention includes a gene having 90% homology to the gene of SEQ ID NO: 1 or the gene of SEQ ID NO: 1 according to another embodiment of the present invention.

A gene encoding a protein that does not have ABC-transporter activity of a microorganism of the genus Corynebacterium of the present invention is a gene having a nucleotide sequence of SEQ ID NO: 2 for inactivating the gene encoding the ABC-transporter, Corynebacter The gene encoding the ABC-transporter of the bacteria of the genus Leeum is removed from the front 248bp and the rear part 237bp, meaning the truncated novA, that is, from 249bp to 735bp of SEQ ID NO: 1.

A gene encoding a protein that does not have ABC-transporter activity of a microorganism of the genus Corynebacterium of the present invention is about 80 to the gene of SEQ ID NO: 2 or the gene of SEQ ID NO: 2 by conventional substitution, deletion and / or insertion A gene having a homology of at least%, preferably at least 90%, more preferably at least 95%, most preferably at least 98%. Preferably, the present invention provides a gene having 80% homology to the gene of SEQ ID NO: 2 or the gene of SEQ ID NO: 2 according to another embodiment of the present invention.

The present invention also provides a microorganism of the genus Corynebacterium in which the novA gene is inactivated.

The microorganism according to the present invention is a microorganism belonging to the genus Corynebacterium in which the gene of SEQ ID NO: 1 is inactivated, and the sequence of SEQ ID NO: 1 and 90 by the normal substitution, deletion, and / or insertion of the gene of SEQ ID NO: Genes having a homology of at least%, preferably at least 95%, more preferably at least 98%, include microorganisms of the genus Corynebacterium.

Preferably, the Corynebacterium sp. Microorganism of the Corynebacterium sp. Microorganism having at least 90% homology to the gene of SEQ ID NO: 1 or the gene of SEQ ID NO: 1 is provided.

In addition, the microorganism of the present invention is preferably a microorganism in which the gene of SEQ ID NO: 1 or a gene having 90% or more homology to the gene of SEQ ID NO: 1 is inactivated by replacement.

The substitution is preferably made by a gene of SEQ ID NO: 2 encoding a protein that does not have ABC-transporter activity of Corynebacterium ammoniagenes, and also a conventional substitution for the gene of SEQ ID NO: 2 , Deletion, and / or insertion by a gene having at least about 80%, preferably at least 90%, more preferably at least 95% and most preferably at least 98% homology with the gene of SEQ ID NO: 2. It includes.

Preferably, the microorganism of the present invention is a microorganism of the genus Corynebacterium that produces 5'-inosinic acid as a recombinant vector comprising the gene of SEQ ID NO: 2 or a gene having a homology with 80% or more of the sequence of SEQ ID NO: 2 A microorganism obtained by transforming and selecting strains in which ABC-transporter is inactivated.

The recombinant vector is a polymerase chain reaction of a gene having at least 80% homology to the gene of SEQ ID NO: 2 or the gene of SEQ ID NO: 2 encoding a protein that does not have ABC-transporter activity of a microorganism of the genus Corynebacterium Amplified by Polymerase Chaine Reaction (PCR), and the linear DNA fragments can be prepared by inserting them into a known expression vector pCR2.1 vector (TOPO TA Cloning Kit Invitrogen, USA).

The vector which can be used is not specifically limited, A well-known expression vector can be used. The recombinant vector thus prepared may be delivered to Escherichia coli using a conventional electroporation method, and the recombinant vector may be collected according to a known method (Quiagen plasmid preparation KIT). Therefore, the recombinant vector is preferably pTOPO KO-novA prepared by inserting the gene of SEQ ID NO: 2 in the pCR2.1 vector.

Microbial strains (eg, Corynebacterium ammoniagenes CJHB100 (KCCM-10330), Republic of Korea Patent Publication No. 2003-42972 using the conventional electroporation method To pass). The microbial mutant is transformed into a pTOPO KO-novA vector by homologous recombination at a site on a microbial chromosome that is homologous to a homologous region of a microbial chromosome located in a recombinant vector, for example, the pTOPO KO-novA vector. The novA gene may be inactivated by substitution with a nucleotide, and then cultured in a medium containing the antibiotic Kanamycin, a selection marker, may be selected. The insertion of pTOPO KO-novA into the genome by homologous recombination in the selected strains can be confirmed by PCR. The cloning process of the gene is briefly shown in FIG.

More preferably, the microorganism of the present invention is Corynebacterium ammoniagenes CN01-0208 (KCCM 10907P).

The present invention also provides a method of preparing a recombinant vector comprising a gene encoding a protein that does not have ABC-transporter activity of a microorganism of the genus Corynebacterium; Inactivating the novA gene by transforming a microorganism of the genus Corynebacterium producing 5'-inosinic acid with the recombinant vector; Selecting and culturing the transformed strain; And it provides a method for producing 5'-inosinic acid comprising the step of separating the 5'-inosinic acid from the culture.

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

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

When the 5'-inosinic acid is prepared according to the preparation method of the present invention, the 5'-inosinic acid can be produced in high yield by inactivating the novA gene encoding the ABC-transporter, an enzyme that degrades nucleic acids. .

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. novA  Gene of SEQ ID NO: 2 which inactivates the gene Cloning

The pTOPO KO-novA vector was constructed as briefly shown in FIG. The pCR2.1 vector contains the KmR gene. The gene fragment of SEQ ID NO: 2 obtained through polymerase chain reaction was conjugated to the pCR2.1 vector using TOPO KIT.

Conjugated DNA was transformed to E. coli GM2525 (Escherichia coli genetic stock center, Yale University, New Haven, Connecticut) by electroporation, and kanamycin per liter of 25 mg of LB containing antibiotics Single colonies grown on solid medium (10 g / L yeast extract, 10 g / L bactotriptone, 10 g / L sodium chloride, 1.7% bactoa, pH 7.0) were recovered. The recovered colonies were separated from the plasmid cultured in LB medium with the same antibiotic added, and the size of the plasmid was determined first. Secondly, the DNA fragments of 487 bp were separated by double cutting with Xba I and BamHI. Recombination plasmid pTOPO KO-novA (4.5 kb) containing the novA gene fragment was constructed by confirming that the gene was released.

The primers used herein are as follows.

Primer novA-F (SEQ ID NO: 3): 5 '-ggtggctcgccacatcgcaac-3'

Primer novA-B (SEQ ID NO: 4): 5 '-agcttgttcagagactaggcc-3'

Example  2. Insertion of Recombinant Plasmids Chromosome novA  Gene Deactivated  Screening of Strains

CM solid medium to which 0.05 mg / L gentamicin was added by transforming to a 5'-inosine production strain CJHB100 (KCCM-10330) using recombinant plasmid pTOPO KO-novA isolated from Escherichia coli GM2525 (10 g / L yeast extract, 10 g / L bactotryptone, 2.5 g / L sodium chloride, 1.7% bactoa, 100 mg / L adenine, 100 mg / L guanine, pH 7.0) and incubated at 32 ° C. for 96 hours. . Through a single colony polymerase chain reaction, recombinant strains inserted at specific positions of chromosomes of Corynebacterium ammoniagenes CJHB100 (KCCM-10330) were selected. The primers used herein are as follows, and the process of inactivating the novA gene by inserting pTOPO KO-novA into the chromosomal DNA is shown in FIG.

Primer novA-FC (SEQ ID NO: 5): 5 '-gggtttaggccgeagtgacc-3'

Primer novA-FB (SEQ ID NO: 6): 5 '-cattcgccttcgcatcactgatc-3'

The microorganisms were designated as Corynebacterium ammoniagenes CN01-0208, and were deposited at the Korea Microbiological Conservation Center on December 20, 2007. Accession number: KCCM 10907P).

Example  3. Erlenmeyer flask Fermentation potency  exam

3 ml of seed medium was dispensed into an 18 mm diameter test tube, autoclaved and inoculated with Corynebacterium ammoniagenes CN01-0208 (KCCM 10907P) 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 for 3 minutes, sterilized at 120 ° C. for 10 minutes, inoculated with 3 ml of the seed culture solution, and then incubated for 5 to 6 days. The rotation speed was adjusted to 200 times per minute at a temperature of 32 ° C. and pH 7.2. 5'-inosinic acid accumulation in the medium was about 7% higher than the parent strain CJHB100 (KCCM-10330). The composition of the seed medium and fermentation medium is as follows.

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

Flask fermentation medium: 0.1% sodium glutamate, 1% ammonium chloride, magnesium sulfate 1.2%, calcium chloride 0.01%, iron sulfate 20mg / l, manganese sulfate 20mg / l, zinc sulfate 20mg / l, copper sulfate 5mg / l, L- Cysteine 23mg / l, Alanine 24mg / l, Nicotinic Acid 8mg / l, Biotin 45µg / l, Thiamine Hydrochloride 5mg / l, Adenine 30mg / l, Phosphoric Acid (85%) 1.9%, Fructose, Glucose and Molasses as reducing sugars Use to add 8%

Example  4. In 5-L fermenters Fermentation potency  Experiment

50 ml of the seed medium of Example 3 was dispensed into a 500 ml shaking Erlenmeyer flask and autoclaved, and then inoculated with Corynebacterium ammoniagenes CN01-0208 (KCCM 10907P) and shaken at 30 ° C. for 24 hours. 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 to inoculate 50 ml of seed culture solution, and then cultured for 1-2 days. Rotation speed was adjusted to 900 times per minute, temperature 28 ~ 34 ℃, pH7.2.

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 broth broth. Then, when the reducing sugar in the culture became 2%, the primary, secondary, tertiary, Fourth additional sugar was mixed with fructose, glucose and molasses and added to the final reducing sugars to 32% and incubated for 5-6 days. Rotation speed was adjusted to 900 times per minute, temperature 32 ℃, pH 7.2. At this time, 5'-inosin acid accumulation in the medium was improved by 5% compared to the parent strain CJHB100 (KCCM-10330). The composition of the fermenter seed broth and the fermenter main broth are as follows.

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

Fermenter Main Medium: 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%, Thiamine Hydrochloride 6mg / l, Biotin 40µg / l, Nicotinic Acid 50mg / l, Alanine 145mg / l, Adenine 200mg / l, Phosphoric Acid (85%) 4.3%, Fructose, Glucose and Molasses were added to 32% as reducing sugar Usage (pH7.2)

1 shows the cloning process of genes that inactivate novA.

Figure 2 shows the process of inactivating novA by inserting pTOPO KO-novA into the chromosomal DNA.

<110> CJ cheiljedang <120> Microorganisms of Corynebacterium having an inactivated gene          encoding ABC-transporter and processes for the preparation of          5'-inosinic acid using the same <130> PA07-0342 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 972 <212> DNA <213> Corynebacterium ammoniagenes <400> 1 atgccagata tgagcttttt cgagaacatt gcagcggcct tggatgccga gggtattgag 60 tctcgcgtca atgatgacat tatgtttgtg ccgattaccg cggacttgga gattcagttc 120 gtggaaattg atcaactgct tcccgcagcg aatgtctaca ttgcagcagc tgatgtcgac 180 gaagacgacg aagactttga agcagtacta gtgtcggtgg ctttttccgt tgaagatgca 240 gtagaagcgg tggctcgcca catcgcaacc gaccaggtgg tcaccgtctt gcgcgatttg 300 ctggaaggca ctgatgagcg catcgcagag ttggaatttg tgcaagatga attcaacccg 360 aacctagtcg tagcagaagt agcgaatgat tcagagctgc gcgtgctcgt tgaaacagtc 420 gacggcgttc cgtcggctat cgtgcgcttt ttgtcttttg cctacactga agatgaattt 480 gatgatgccg aagattcttc gcagatcgat gactacgatg atgcaaccat ccaagaactg 540 tgggatgtgg atgcagaaga aggcgtcgat tctgaggagc cattgagcga cgatgaatac 600 cagctgctgt tgcagtcttc gcttttcgac ggagcagaac cggtcattga gattcccgca 660 gaaaccctcg agctgggaac ctacatcgac ttcgaccgtc tttttgacgt tcttggccta 720 gtctctgaac aagctttgga ttgggagtca cagttagtgc ctttcgattc tgatgagttt 780 gatgagccag atgtctatga catctatggt gaagacactt tggatgaaga actcgaaagt 840 gagcttgaag gatacgaaga cggctttgac gatgaggatg acgagggtga cgacctcgaa 900 atccacgaac tatcgggctc gcgtacaggt gctgattctg aagactcaga tgatgctgaa 960 cgcgataact ag 972 <210> 2 <211> 487 <212> DNA <213> Corynebacterium ammoniagenes <400> 2 ggtggctcgc cacatcgcaa ccgaccaggt ggtcaccgtc ttgcgcgatt tgctggaagg 60 cactgatgag cgcatcgcag agttggaatt tgtgcaagat gaattcaacc cgaacctagt 120 cgtagcagaa gtagcgaatg attcagagct gcgcgtgctc gttgaaacag tcgacggcgt 180 tccgtcggct atcgtgcgct ttttgtcttt tgcctacact gaagatgaat ttgatgatgc 240 cgaagattct tcgcagatcg atgactacga tgatgcaacc atccaagaac tgtgggatgt 300 ggatgcagaa gaaggcgtcg attctgagga gccattgagc gacgatgaat accagctgct 360 gttgcagtct tcgcttttcg acggagcaga accggtcatt gagattcccg cagaaaccct 420 cgagctggga acctacatcg acttcgaccg tctttttgac gttcttggcc tagtctctga 480 acaagct 487 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer novA-F <400> 3 ggtggctcgc cacatcgcaa c 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer novA-B <400> 4 agcttgttca gagactaggc c 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer novA-FC <400> 5 gggtttaggc cgtagtgacc 20 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer novA-FB <400> 6 cattcgcctt cgcatcactg atc 23  

Claims (9)

A microorganism of the genus Corynebacterium producing 5'-inosinic acid, the microorganism of the genus Corynebacterium in which the gene encoding the ABC-transporter of the microorganism of the genus Corynebacterium is inactivated. The method of claim 1, The inactivation is a microorganism of the genus Corynebacterium, characterized in that by substitution by a gene encoding a protein having no ABC-transporter activity. The method of claim 2, The substitution is a microorganism of the genus Corynebacterium, characterized in that the substitution by a gene having a nucleotide sequence of SEQ ID NO: 2 coding for a protein that does not have ABC-transporter activity of the microorganism of the genus Corynebacterium. The method of claim 1, The microorganism is Corynebacterium ammonia genes (Corynebacterium ammoniagenes) CN01-0208 (KCCM 10907P) characterized in that the genus Corynebacterium. Preparing a recombinant vector comprising a gene encoding a protein that does not have ABC-transporter activity of a microorganism of the genus Corynebacterium; Transforming and inactivating the gene encoding the ABC-transporter of the Corynebacterium microorganism by transforming the microorganism of the Corynebacterium microorganism with the recombinant vector; And Selecting the transformed strain; Corynebacterium genus microorganisms manufacturing method comprising a. The method of claim 5, The recombinant vector is a vector pTOPO KO-novA prepared by conjugating the gene fragment of SEQ ID NO: 2 to the pCR2.1 vector, the method for producing a microorganism of the genus Corynebacterium. A method for producing 5'-inosinic acid comprising culturing the microorganism according to any one of claims 1 to 4, and separating 5'-inosinic acid from the culture solution. Gene of SEQ ID NO: 1 The gene of SEQ ID NO: 2.

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