BR112015026761B1 - MICROORGANISM OF THE GENUS CORYNEBACTERIUM THAT HAS AN IMPROVED CAPACITY TO PRODUCE L-ARGININE AND METHOD OF PRODUCTION OF L-ARGININE - Google Patents

Abstract

MICRO-ORGANISM CORYNEBACTERIUM SP. WITH IMPROVED L-ARGININE PRODUCTIVITY AND METHOD FOR PRODUCING L-ARGININE USING THE SAME. The present invention relates to a Corynebacterium sp., which has enhanced aspartate ammonia lyase and/or aspartate aminotransferase activity, and thus has improved L-arginine productivity; and a method of producing L-arginine using the microorganism Corynebacterium sp.

Description

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

[001] The present invention relates to a recombinant microorganism of the genus Corynebacterium with an improved ability to produce L-arginine, and method to produce L-arginine using the same.

DESCRIPTION OF THE PRIOR ART

[002] L-arginine is present in garlic or plant seed in free form. L-arginine is widely used in medicine, food and the like and is also used as an amino acid-fortified dietary supplement.

[003] Microorganisms of the genus Corynebacterium biosynthesize L-arginine through a cyclic pathway. L-arginine is synthesized from L-glutamate via N-acetylglutamate, N-acetylglutamyl phosphate, N-acetylglutamate semialdehyde, N-acetylornithine, ornithine, citrulline and argininosuccinate.

[004] Additionally, Corynebacterium glutamicum is known to be regulated by feedback inhibition due to intracellular arginine (Vehary Sakanyan, et al., Microbiology, 142:9-108, 1996), which suggests that L-arginine production in high yield is limited.

[005] Ikeda et al. reported that citrulline is accumulated in a fermentation medium during arginine fermentation ( Appl Environ Microbiol. Mar 2009; 75(6):1635-1641. Epub Jan 9, 2009 ).

[006] In the process of biosynthesis, citrulline binds to aspartate to generate argininosuccinate, which then releases fumarate to generate arginine. In this process, aspartate ammonia lyase (AspA) is an enzyme that synthesizes aspartate from fumarate and ammonia (Figure 1), and aspartate aminotransferase (AspB) is an enzyme that catalyzes the synthesis of various L-amino acids by transferring the amino group of various L-amino acids such as aspartate, glutamate and aminobutyrate in cetacids such as a-ketoglutaric acid and a-ketoisovaleric acid.

[007] Menkel et al. reported that E. coli aspA was introduced into microorganism s of the genus Corynebacterium, the microorganism s from Corynebacterium increased aspathate availability and then increased lysine production (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, March 1989, pp. 684 to 688).

[008] Consequently, the present invention provides a microorganism of the genus Corynebacterium that has an improved ability to produce L-arginine by increasing the influx of aspartate that binds to citrulline through the enhancement of expression of aspartate ammonia-lyase and/or aspartate aminotransferase.

SUMMARY OF THE INVENTION

[009] It is an object of the present invention to provide a microorganism of the genus Corynebacterium with an improved ability to produce L-arginine.

[0010] Another object of the present invention is to provide a method of producing L-arginine with the use of the above microorganism of the genus Corynebacterium.

[0011] To achieve the above objects, the present invention provides a microorganism of the genus Corynebacterium with an improved ability to produce L-arginine by enhancing the activity of aspartate ammonia lyase.

[0012] The present invention also provides a method of producing L-arginine by cultivating the above microorganism of the genus Corynebacterium.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 shows a pathway to synthesize arginine from citrulline and a pathway to synthesize aspartate from fumarate.

DETAILED DESCRIPTION OF THE INVENTION

[0014] From this point on in the present document, the present invention will be described in detail.

[0015] The present invention provides a microorganism of the genus Corynebacterium with improved ability to produce L-arginine.

[0016] As used herein, the term "ability to produce L-arginine (L-arginine productivity)" refers to the ability to accumulate L-arginine in a medium for culturing the microorganism of the genus Corynebacterium during culture. Such ability to produce L-arginine can be either the property of a natural state or the property transmitted or enhanced by artificial mutation of microorganisms of the genus Corynebacterium.

[0017] For example, in order to impart the ability to produce L-arginine, a microorganism of the genus Corynebacterium can be bred as a variant that has resistance to arginine hydroxamate; a variant that has auxotrophy to succinic acid or that has resistance to an analogous nucleic acid base; a variant deficient in the ability to metabolize arginine and which has resistance to an antagonistic arginine and canavanine and auxotrophy to lysine; a variant that has resistance to arginine, arginine hydroxamate, homoarginine, D-arginine and canavanine, or resistance to arginine hydroxamate and 6-azauracil; or a variant that has canavanine resistance.

[0018] Additionally, the ability to produce L-arginine can be imparted by modifying a microorganism of the genus Corynebacterium so as to increase the level of expression of genes encoding enzymes to biosynthesize L-arginine. Examples of enzymes to biosynthesize L-arginine may include N-acetylglutamyl phosphate reductase (ArgC), ornithine acetyl transferase (ArgJ), N-acetylglutamate kinase (ArgB), acetylornithine transaminase (ArgD), ornithine carbamoyl transferase (ArgF), arginine acid succinic synthetase (ArgG), arginine succinic acid lyase (ArgH) and carbomyl phosphate synthetase. These enzymes are placed in the Arg operon (argCJBDFRGH) and are regulated by an argR-encoded arginine repressor ( J Bacteriol. 2002 Dec; 184(23):6602-6614 .). Thus, an ability to produce L-arginine can be imparted by attenuating an arginine repressor (US2002-0045223) or overexpressing at least one of the genes related to biosynthesis.

[0019] In the present invention, the microorganism of the genus Corynebacterium with the ability to produce L-arginine is not specifically limited as long as it can produce L-arginine. Specifically, it can be Corynebacterium glutamicum which has the ability to produce L-arginine. More specifically, it may be Corynebacterium glutamicum KCCM10741 (Patent Registration Number KR 10-0791659) or Corynebacterium glutamicum ATCC21831), but this is not a limitation thereof.

[0020] In one aspect, the present invention provides a microorganism of the genus Corynebacterium with enhanced ability to produce L-arginine by enhancing aspartate ammonia lyase activity.

[0021] As used herein, the term "aspartate ammonia lyase (AspA)" refers to an enzyme that functions to synthesize aspartate from fumarate and ammonia, and aspartate synthesis can be increased by improving the expression of aspartate ammonia lyase, and thus L-arginine productivity can be increased by increased aspartate.

[0022] In the present invention, the aspartate ammonia lyase can be an enzyme derived from any microorganism, as long as it has the activity of aspartate ammonia lyase. Specifically, aspartate ammonia lyase may be an enzyme derived from a microorganism of the genus Escherichia or the genus Corynebacterium. More specifically, it can be an enzyme derived from E. coli or Corynebacterium glutamicum. Specifically, the aspartate ammonia lyase may have an amino acid sequence represented by SEQ ID NO: 21 or 22. Corynebacterium-derived aspartate ammonia lyase having the amino acid sequence represented by SEQ ID NO: 21 is encoded by aspA (NCgl1446) that has a nucleotide sequence represented by SEQ ID NO: 23, and the Escherichia-derived aspartate ammonia lyase that has an amino acid sequence represented by SEQ ID NO: 22 is encoded by aspA (NCBI GENE ID: 12933698) which has a nucleotide sequence represented by SEQ ID NO: 24.

[0023] A protein that has a homology of at least 80%, specifically at least 90%, more specifically at least 95%, and even more specifically at least 97% to the amino acid sequence of ammonia aspartate -lyase described above is also included in the scope of the present invention, as long as it has the aspartate ammonia lyase activity described in the present invention.

[0024] As used herein, the term "homology" refers to the identity between two amino acid sequences and can be determined by the method well known to the person skilled in the art, using the BLAST algorithm (see Karlin and Altschul, Pro Natl. Acad. Sci. USA, 90, 5873 (1993)) or FASTA (see Pearson, Methods Enzymol., 183, 63 (1990)) to compute the parameter such as score, identity, and similarity. Based on the BLAST algorithm, a program called BLASTN or BLASTX was developed (see http://www.ncbi.nlm.nih.gov).

[0025] In another aspect, the present invention provides a microorganism of the genus Corynebacterium, wherein the activity of aspartate aminotransferase is further enhanced.

[0026] As used herein, the term "aspartate aminotransferase" which may be derived from a microorganism of the genus Corynebacterium has an amino acid sequence represented by SEQ ID NO: 25 and is encoded by aspB (NCgl0237) which has the nucleotide sequence represented by SEQ ID NO: 26, but is not limited thereto.

[0027] A protein that has a homology of at least 80%, specifically at least 90%, more specifically at least 95%, and even more specifically at least 97% to the amino acid sequence of aspartate aminotransferase described above is also included in the scope of the present invention, as long as it has the aspartate aminotransferase activity described in the present invention. The homology to the amino acid sequence can be determined by, for example, BLAST algorithm (see Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) or FASTA (see Pearson, Methods Enzymol., 183, 63 (1990)). Based on this algorithm, a program called BLASTN or BLASTX was developed (see http://www.ncbi.nlm.nih.gov).

[0028] In yet another aspect, the present invention provides a microorganism of the genus Corynebacterium, wherein the activity of aspartate ammonia lyase and aspartate aminotransferase are both enhanced.

[0029] The inventive microorganism of the genus Corynebacterium with an ability to produce L-arginine is characterized by being modified by improving (or increasing) the activity of aspartate ammonia lyase and/or aspartate aminotransferase, for endogenous activity .

[0030] As used in this document, the term "endogenous activity" refers to the activity of a microorganism of the genus Corynebacterium with the capacity to produce L-arginine that has not been subjected to any manipulation or genetic modification to regulate the activities of the enzymes described above. Additionally, the term "enhanced" or "increased" means that the productivity of L-arginine has been improved compared to the endogenous activity of a microorganism of the genus Corynebacterium with an ability to produce L-arginine.

[0031] The enhancement (or increase) of enzyme activity in the present invention can be accomplished using various methods well known in the art. Examples of such methods include a method of increasing the copy number of a nucleotide sequence encoding aspartate ammonia lyase and/or aspartate aminotransferase by introducing a polynucleotide encoding aspartate ammonia lyase and/or aspartate aminotransferase into a vector system , a method of replacing the promoter with a strong promoter, a method of introducing a mutation in the promoter, and a method based on genetic mutation, but are not limited thereto.

[0032] In a specific embodiment of the present invention, in order to improve the activity of aspartate ammonia lyase and/or aspartate aminotransferase in the microorganism of the genus Corynebacterium with the capacity to produce L-arginine, a method can be used in which a copy number of the gene encoding aspartate ammonia lyase and/or aspartate aminotransferase is increased by introducing the gene encoding aspartate ammonia lyase and/or aspartate aminotransferase that received aspartate into a vector and transforming the microorganism of the genus Corynebacterium with the vector.

[0033] As used herein, the term "transform" means to introduce a vector containing a polynucleotide encoding a target protein into a host cell so that the target protein can be expressed in the host cell. The introduced polynucleotide can be located inside or outside the host cell chromosome, as long as it can be expressed in the host cell. The polynucleotide can be introduced in any form, as long as it can be expressed in the host cell.

[0034] In one embodiment of the present invention, the transformed microorganism was prepared by introducing the recombinant vector that contains the genes described above, and then underwent isolation of the strain that produces L-arginine that contains 2 copies of each of the genes up the chromosome through a second crossover.

[0035] As used herein, the term "vector" refers to a DNA construct that contains the nucleotide sequence of a polynucleotide encoding the target protein that is operatively linked to a suitable regulatory sequence so that it has the ability to express the target protein in a suitable host. The regulatory sequence may include a promoter to initiate transcription, any sequence operable to regulate such transcription, a sequence e which encodes the appropriate mRNA ribosome binding sites, and sequences which regulate the termination of transcription and translation. After being transformed and a suitable host, the vector can be replicated or perform its function independently of the host genome, or it can be integrated into the genome itself.

[0036] The vector which is used in the present invention is not specifically limited as long as it can be replicated in a host. The vector can be any vector known in the art. Examples of vectors that are commonly used may include natural or recombinant plasmids, cosmids, viruses, and bacteriophages.

[0037] The Corynebacterium glutamicum strain (KCCM11351P) transformed by the method of the present invention is produced by obtaining aspA that encodes aspartate ammonia-lyase from the Corynebacterium glutamicum chromosome that produces L-arginine ATCC21831 by PCR, inserting the obtained aspA into a vector, and then introducing the vector into Corynebacterium glutamicum which produces L-arginine KCCM10741P, and then is a transformed microorganism that has increased expression of aspA. It was concluded that the KCCM11351P strain can produce L-arginine in high production by overexpression of aspA.

[0038] In yet another aspect, the present invention also provides a method of producing L-arginine by culturing the transformed Corynebacterium microorganism.

[0039] In the inventive method of producing L-arginine, the process of cultivating the transformed microorganism that overexpresses L-arginine can be carried out in suitable culture media and conditions known in the art. This culture process can be easily adjusted by anyone skilled in the art depending on a selected strain. Examples of the culture process include, but are not limited to, batch culture, continuous culture, and batch feed culture. Such culture processes are disclosed, for example, in "Biochemical Engineering" (James M. Lee, Prentice-Hall International Editions, pp. 138 to 176, 1991).

[0040] The medium which is used in the present invention contains sugar or glucose in a natural state as a main source of carbon. Additionally, molasses which contain a large amount of natural sugar can also serve as a carbon source. Additionally, adequate amounts of various carbon sources can be used, and specifically, purified glucose can be used. Examples of a nitrogen source that can be used in the present invention include organic nitrogen sources such as peptone, yeast extract, beef broth, malt extract, cornstarch and soy flour; and inorganic sources of nitrogen such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. Specifically, peptone can be used. These nitrogen sources can be used alone or in combination. The medium may contain, as a source of phosphorus, potassium dehydrogen phosphate, dipotassium hydrogen phosphate and corresponding sodium-containing salts. Additionally, the medium may contain a metal salt such as magnesium sulfate or iron sulfate. Additionally, the medium may contain appropriate amino acids, vitamins and precursors. Such media or precursors can be added in a batch or continuous manner. However, examples of a culture medium composition are not limited to these.

[0041] A compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to a culture medium during culture in a suitable way to adjust the pH of the culture medium. In addition, an antifoaming agent such as fatty acid polyglycol ester can be added during culturing to inhibit bubbling. Additionally, in order to maintain an aerobic condition of the culture medium, oxygen or gas containing oxygen (eg air) can be injected into the culture medium. The temperature of the culture medium can be generally between 20°C and 45°C, and specifically between 25°C and 40°C. Culture can be carried out until a desired amount of L-arginine is produced. Specifically, the culture time can be from 10 to 160 hours.

[0042] The separation of L-arginine from the culture medium can be carried out by a conventional method known in the art. The method may include centrifugation, filtration, ion exchange chromatography and crystallization. For example, L-arginine can be separated by centrifuging the culture medium at low speed to remove biomass, and then separating the remaining supernatant by ion exchange chromatography.

[0043] Hereinafter, the present invention will be described in further details with reference to examples. It should be understood, however, that these examples are for illustrative purposes and are not intended to limit the scope of the present invention.

EXAMPLES EXAMPLE 1: EXAMINATION OF THE EFFECT OF ASPA ON L-ARGININE PRODUCTION

[0044] In order to examine whether aspA is a significant gene in L-arginine production, a quote-deleted vector was constructed and transformed into the L-arginine-producing strain Corynebacterium glutamicum KCCM10741P (KR Patent Application Number 0791659), and , then the L-arginine productivity of the transformed strain was evaluated.

(1) DEFICIENT VECTOR CONSTRUCTION OF ASPA

[0045] In order to eliminate the aspA gene encoding aspartate ammonia lyase (Ncgl1446) from the chromosome, the chromosome of Corynebacterium glutamicum ATCC21831 purchased from the American Type Culture Collection (ATCC) was extracted using a Genomic tip (QIAGEN), and crossover PCR was performed using chromosome as a template.

[0046] Specifically, a fragment of about 798 bp that has an XmaI restriction enzyme site in the 5' region was amplified by PCR with Pfu polymerase using primers from SEQ ID NOs: 1 and 2 per 30 cycles, each consisting of denaturing at 94 °C for 1 min, annealing at 58 °C for 30 seconds, and polymerization at 72 °C for 60 seconds. Then, a fragment of about 829 bp that has an XbaI restriction enzyme site in the 3' region was amplified by PCR using primers from SEQ ID NOs: 3 and 4 in the same manner as described above. The resulting DNA fragments were isolated using the GeneAllR ExpinTM GEL SV kit (Seoul, South Korea) and were then used as a crossover PCR template.

[0047] In order to obtain a DNA fragment containing deleted aspA, crossover PCR was performed using the two DNA fragments prepared above as a template and primers of SEQ ID NOs: 1 and 4. Specifically, a fragment of about 1583 bp was amplified by the PCR method described above. The amplified fragment was treated with XmaI and XbaI restriction enzymes, and then ligated with a pD vector treated with the same enzymes, thereby constructing the pDKO1446 vector.

(2) PREPARATION OF RECOMBINANT STRAIN

[0048] The pDKO1446 vector constructed as described above on the L-arginine producing strain Corynebacterium glutamicum KCCM10741P and subjected to a second treatment, which thereby obtained an L-arginine producing strain containing an aspA deleted in the chromosome. The strain obtained was called "KCCM10741ΔaspA".

(3) PRODUCTIVITY EXAMINATION OF L-ARGININE FROM RECOMBINANT STRAIN

[0049] In order to examine the effect of an aspA deletion on L-arginine productivity, the above produced recombinant Corynebacterium glutamicum KCCM10741ΔaspA which is a strain that produces L-arginine was cultured as follows.

[0050] As a control group, the host cell, Corynebacterium glutamicum KCCM10741P, was used. Specifically, a circuit of the strain was inoculated into a 250 ml baffle corner flask containing 25 ml of the following production medium, and was cultured at 30 °C for 48 hours at 200 rpm. After the end of the culture, L-arginine production was measured by HPLC, and the results are shown in TABLE 1 below.

PRODUCTION MEANS

[0051] 6% glucose, 3% ammonium sulfate, 0.1% potassium dehydrogenated phosphate, 0.2% magnesium sulfate heptahydrate, 1.5% milocin (CSL), 1% NaCl, 0.5% yeast extract, 100 mg/L biotin, pH 7.2. TABLE 1: L-ARGININE PRODUCTIVITY COMPARISON

[0052] As can be seen in Table 1 above, the strain containing a deleted aspA had reduced ability to produce L-arginine, and aspA was a significant gene in the production of L-arginine.

EXAMPLE 2: VECTOR CONSTRUCTION TO INTRODUCE CORYNEBACTERIUM-DERIVED ASPA

[0053] In order to construct a vector containing two copies of aspA (Ncgl1446 encoding Corynebacterium-derived aspartate ammonia lyase, PCR was performed using the chromosome of Corynebacterium glutamicum ATCC21831 as a template and each of a primer set of SEQ ID NOs: 5 and 6 and a primer set of SEQ ID NOs: 7 and 8, thereby obtaining DNA fragments, each containing aspA.

[0054] Specifically, a fragment of about 1893 bp that has an XmaI restriction enzyme site in the 5' region and a BamHI restriction enzyme site in the 3' region was amplified by PCR with Pfu polymerase with the use of primers from SEQ ID NOs: 5 and 6. The PCR was performed for 30 cycles, each consisting of denaturation at 94 °C for 1 min, annealing at 58 °C for 30 seconds and polymerization at 72 °C for 2 min. Meanwhile, a fragment of about 1885 bp that has a BamHI restriction enzyme site in the 5' region and an XbaI restriction enzyme site in the 3' region was extended using the primers of SEQ ID NOs: 7 and 8 in the same manner as described above.

[0055] The DNA fragments obtained were separated using the GeneAllR ExpinTM GEL SV kit (Seoul, South Korea). Then, the first fragment out of the above two fragments was treated with XmaI and BamHI, and the second fragment was treated with BamHI and XbaI. Additionally, a pD vector was treated with XmaI and XbaI. The two DNA fragments and the pD vector, treated with the restriction enzymes, were ligated, and thus formed a pD1446-2X vector.

EXAMPLE 3: PREPARATION OF RECOMBINANT STRAINS THAT HAVE INCREASED ASPA EXPRESSION

[0056] The pD1446-2X vector constructed in EXAMPLE 2 was transformed into each of the strains that produce L-arginines, Corynebacterium glutamicum KCCM10741P and ATCC21831, and then subjected to a second crossover, thereby obtaining the strain that produces L-arginines that contain 2 copies of aspA on the chromosome. The strains obtained were called "KCCM10741P::aspA_2X" and "ATCC21831::aspA_2X", respectively.

EXAMPLE 4: VECTOR CONSTRUCTION TO INTRODUCE CORYNEBACTERIUM-DERIVATIVE aspB

[0057] In order to construct a vector containing 2 copies of aspB (Ncgl0237) encoding aspartate aminotransferase, PCR was performed using the chromosome of Corynebacterium glutamicum ATCC21831 as a template and each of a primer set of SEQ ID NOs: 9 and 10 and a primer set of SEQ ID NOs: 11 and 12, thereby obtaining DNA fragments, each containing aspB.

[0058] Specifically, a fragment of about 1692 bp that has an XmaI restriction enzyme site in the 5' region and an NdeI restriction enzyme site in the 3' region was amplified by PCR with Pfu polymerase with the use of primers from SEQ ID NOs: 9 and 10. The PCR was performed for 30 cycles, each consisting of denaturation at 94 °C for 1 min, annealing at 58 °C for 30 seconds and polymerization at 72 °C for 2 min. Meanwhile, a second fragment of about 1643 bp that has a NdeI restriction enzyme site in the 5' region and a SpeI restriction enzyme site in the 3' region was amplified by the same PCR method as described above.

[0059] The DNA fragments obtained were isolated using the GeneAllR ExpinTM GEL SV kit (Seoul, South Korea). Then, the first fragment of the above two fragments was treated with XmaI and NdeI, and the second fragment was treated with NdeI and SpeI. Additionally, a pD vector was treated with XmaI and XbaI. The pD vector and the DNA fragments, which were treated with the restriction enzymes, were ligated into 3 pieces, thus building a pD0237-2X vector.

EXAMPLE 5: PREPARATION OF STRAINS THAT HAVE INCREASED EXPRESSION OF aspB

[0060] The pD0237-2X vector constructed in EXAMPLE 4 was transformed into each strain that produces L-arginines, Corynebacterium glutamicum KCCM10741P and ATCC21831, and then subjected to a second crossover, which thereby obtained the strain that produces L - arginines that contain 2 copies of aspB in the chromosome. The strains obtained were called "KCCM10741P::aspB_2X" and "ATCC21831::aspB_2X", respectively.

EXAMPLE 6: PREPARATION OF STRAINS THAT HAVE INCREASED EXPRESSION OF aspA AND aspB

[0061] The pD0237-2X vector constructed in EXAMPLE 4 was transformed into each of the recombinant strains (KCCM10741P::aspA_2X and ATCC21831::aspA_2X) prepared in EXAMPLE 3, and were then subjected to a second crossover, which, thus In this way, he obtained the strain that produces L-arginines that contains 2 copies of aspA and aspB in the chromosome. The strains obtained were called "KCCM10741P::aspA_2X::aspB_2X" and "ATCC21831::aspA_2X::aspB_- 2X".

EXAMPLE 7: EVALUATION OF L-ARGININE PRODUCTIVITY

[0062] In order to examine the effect of an increase in aspA, aspB, or both aspA and aspB on L-arginine productivity, each of the strains that produce L-arginines, Corynebacterium glutamicum KCCM10741P::aspA_2X, ATCC21831::aspA_2X, KCCM10741- P::aspB_2X, ATCC21831::aspB_2X, KCCM10741P::aspA_2-X::aspB_2X and ATCC21831::aspA_2X::aspB_2X, prepared in EXAMPLES 3, 5 and 6, was cultured in the following manner.

[0063] As a control group, host cells from each of Corynebacterium glutamicum KCCM10741P and ATCC21831 were used. Specifically, one circuit of each of the strains was inoculated into a 250 ml baffle corner flask containing 25 ml of the production medium described in EXAMPLE 1, and the inoculated strains were grown at 30°C for 48 hours at 200 rpm. After the end of the culture, the production of L-arginine was measured, and the measurement results are shown in Table 2 below. TABLE 2: COMPARISON OF ARGININE PRODUCTIVITY BETWEEN STRAINS

[0064] As can be seen from Table 2 above, L-arginine production was increased in both types of Corynebacterium glutamicum strains when 2 copies of aspA were introduced into the strains. Particularly, the L-arginine productivity of KCCM10741P::aspA_2X was remarkably increased by 30% compared to the control group. KCCM10741P::aspA_2X has been deposited with the Korean Culture Center of Microorganism (KCCM), an international depository authority located at 361-221, Hongje 1-dong Seodaemun-gu, Seoul, South Korea, on January 21, 2013 under registration number membership KCCM11351P.

EXAMPLE 8: VECTOR CONSTRUCTION TO INTRODUCE a quote derived from the GENUS E. coll'

[0065] To introduce aspA (NCBI-GeneID: 12933698), a gene encoding aspartate ammonia lyase derived from the genus E. coli, into the chromosome of Corynebacterium glutamicum with an ability to produce L-arginine, an Ncgl1221 site known as an exporter of glutamate, was used (yggB: Appl Environ Microbiol. 2007 Jul;73(14):4491-8 ).

[0066] In order to construct a vector that has an E. coli aspA introduced into the Ncgl1221 locus, a pDKO1221 vector that contains a localized gene splice of Ncgl1221 was constructed.

[0067] In order to obtain a DNA fragment containing a localized gene disjunction of Ncgl1221, the chromosome of Corynebacterium glutamicum ATCC21831 was extracted, and crossover PCR was performed using the chromosome as a template. Specifically, a fragment of about 789 bp that has an EcoRI restriction enzyme site in the 5' region was amplified by PCR with Pfu polymerase using primers and SEQ ID NOs: 13 and 14. The PCR was performed for 30 cycles, each consisting of denaturing at 94 °C for 1 min, annealing at 58 °C for 30 seconds, and polymerization at 72 °C for 60 seconds. Meanwhile, a fragment of about 835 bp that has a PstI restriction enzyme site in the 3' region was amplified by PCR using primers of SEQ ID NOS: 15 and 16 in the same manner as described above. The DNA fragments obtained were isolated using the GeneAllR ExpinTM GEL SV Kit (Seoul, South Korea) and were then used as a template for crossover PCR.

[0068] To obtain a DNA fragment containing the localized gene disjunction of Ncgl1221, crossover PCR was performed using the two DNA fragments obtained above as a template and primers from SEQ ID NOs: 13 and 16. Specifically , a fragment of about 1602 bp was amplified by the PCR method described above. The amplified fragment was treated with restriction enzymes EcoRI and PstI, and then ligated with a pD vector treated with the same restriction enzymes, thus forming a pDKO1221 vector.

[0069] Using the constructed pDKO1221 vector, a vector that has an E. coli aspA introduced into it was constructed. To obtain a cj7 promoter (Patent No. KR 10-0620092) that operates in Corynebacterium glutamicum, a fragment of about 524 bp that has a BamHI restriction enzyme site in the 5' region was amplified by PCR with Pfu polymerase with the use of p117 pcj7-gfp with a template and primers from SEQ ID NOs: 17 and 18. PCR was performed for 30 cycles, each consisting of denaturation at 94 °C for 1 min, annealing at 58 °C for 30 seconds and polymerization at 72 °C for 30 seconds. E. coli-derived aspA was extracted from E. coli chromosome W3110 using a Genomics Edge System (QIAGEN), and a fragment of about 1607 bp that has an XbaI restriction enzyme site in region 3 ' was extended using the chromosome as a template and primers of SEQID NOs: 19 and 20. The DNA fragments obtained were isolated using the GeneAllR ExpinTM GEL SV Kit (Seoul, South Korea) and were then used as a template for crossover PCR.

[0070] To obtain an aspA DNA fragment derived from E. coli, crossover PCR was performed using the two DNA fragments obtained above as a template and primers from SEQ ID NOs: 17 and 20. Specifically, a fragment of about 2095 bp was amplified by the PCR method described above. The amplified fragment was treated with BamHI and XbaI restriction enzymes, and then ligated with a pDKO1221 vector treated with the same restriction enzymes, thereby constructing a pDKO1221-EC_aspA vector.

EXAMPLE 9: PREPARATION OF RECOMBINANT STRAIN THAT HAS INCREASED EXPRESSION OF E. coli aspA

[0071] The pDKO1221-EC_aspA vector constructed as described above was transformed into each of the strains that produce L-arginines Corynebacterium glutamicum KCCM10741P and ATCC21831 and subjected to a second crossover, thus obtaining the strain that produces L-arginines, being that each contains an E. coli -derived aspA gene on the chromosome. The strains obtained were called "KCCM10741PΔNcgl1221-EC_aspA" and "ATCC21831ΔNcgl1221-EC_aspA", respectively. Additionally, the pDKO1221 vector constructed as described above was transformed into each of the L-arginine-producing strains Corynebacterium glutamicum KCCM10741P and ATCC21831 and subjected to a second crossover, thereby obtaining strains KCCM10741PΔNcgl1221 and ATCC21831ΔNcgl1221, each of which contains an NCgl1221 deleted from the chromosome.

EXAMPLE 10: EVALUATION OF L-ARGININE PRODUCTIVITY FROM RECOMBINANT STRAINS THAT HAVE INCREASED EXPRESSION OF E. coli aspA

[0072] In order to evaluate the effect of introducing E. coli aspA on L-arginine productivity, the strain producing L-arginines KCCM10741PΔNcgl1221-EC_aspA and ATCC21831ΔNcgl1221-EC_aspA prepared in EXAMPLE 9 was cultured as follows.

[0073] As a control group, host cells of each of Corynebacterium glutamicum KCCM10741P, ATCC21831, KCCM10741PΔNcgl1221 and ATCC21831ΔNcgl1221 were cultured. Specifically, one circuit of each strain was inoculated into a 250 ml baffle corner flask containing 25 ml of the production medium described above and cultured at 30°C for 48 hours at 200 rpm. After the end of the culture, L-arginine production was measured and the results are shown in Table 3 below. TABLE 3: L-ARGININE PRODUCTIVITY COMPARISON

[0074] As can be seen in TABLE 3 above, L-arginine production in both types of Corynebacterium glutamicum was increased when E. coli aspA was introduced into the strains.

[0075] As described above, the present invention provides the recombinant microorganism of the genus Corynebacterium, which has enhanced aspartate ammonia lyase activity and/or enhanced aspartate aminotransferase activity, and thus has an enhanced ability to produce L-arginine . The recombinant microorganism of the genus Corynebacterium can produce L-arginine in high production, and thus is industrially useful. From the foregoing, persons skilled in the art will observe that the present invention can be implemented in other specific forms without departing from the technical spirit or the essential characteristics thereof. Accordingly, it is to be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention is to be defined by the appended claims rather than the detailed description, and it should be noted that all modifications or alterations derived from the meaning and scope of the claims and their equivalents are encompassed by the scope of the present invention.

MEMBERSHIP NUMBER

[0076] Depositing Authority: Korean Culture Center of Microorganisms

[0077] Membership Number: KCCM11351P

[0078] Date of Deposit: January 21, 2013

Claims (6)

1. A microorganism of the genus Corynebacterium, characterized in that it has an enhanced ability to produce L-arginine, which has an enhanced activity of aspartate ammonia lyase and aspartate aminotransferase, wherein the aspartate aminotransferase is derived from a microorganism of the genus Corynebacterium , wherein the aspartate ammonia lyase has an amino acid sequence represented by SEQ ID NO: 21 or 22 and wherein the aspartate aminotransferase has an amino acid sequence represented by SEQ ID NO: 25. 2. Microorganism, according to claim 1, characterized by the fact that the aspartate ammonia-lyase is derived from a microorganism of the genus Corynebacterium or a microorganism of the genus Escherichia coli. 3. Microorganism, according to claim 1, characterized in that the microorganism of the genus Corynebacterium is Corynebacterium glutamicum. 4. Method for producing L-arginine, characterized in that it comprises the steps of: cultivating the microorganism of the genus Corynebacterium with an enhanced ability to produce L-arginine, which has an enhanced activity of aspartate ammonia lyase and aspartate aminotransferase in a culture medium; and recovering L-arginine from the culture medium which is obtained by culturing, wherein the aspartate ammonia lyase has an amino acid sequence represented by SEQ ID NO: 21 or 22 and wherein the aspartate aminotransferase has an amino acid sequence represented by SEQ ID NO: 25. 5. Method according to claim 4, characterized in that the aspartate ammonia-lyase is derived from a microorganism of the genus Corynebacterium or a microorganism of the genus Escherichia. 6. Method according to claim 4, characterized in that the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.