Microorganism of genus <i>Corynebacterium </i>producing L-arginine and method for producing L-arginine using the same

Abstract

A microorganism of the genus Corynebacterium producing L-arginine, and a method for producing L-arginine using the same.

Claims

1. A microorganism obtained from Corynebacterium glutamicum that produces L-arginine, wherein the microorganism is modified by inactivation of an endogenous protein comprising the amino acid sequence of SEQ ID NO: 1, and wherein the modified microorganism has increased production of L-arginine.

2. The microorganism of claim 1, wherein the protein is encoded by a gene consisting of a nucleotide sequence of SEQ ID NO: 2.

3. A method for producing L-arginine, comprising: culturing the microorganism of claim 1 in a medium.

4. The method for producing L-arginine of claim 3, further comprising: recovering L-arginine from the microorganism or the medium.

5. A method for producing L-arginine, comprising: culturing the microorganism of claim 2 in a medium.

6. The method for producing L-arginine of claim 5, further comprising: recovering L-arginine from the microorganism or the medium.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Hereinbelow, the present disclosure will be described in detail with accompanying exemplary embodiments. However, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present disclosure.

Example 1: Construction of Random Mutant Library Using Transposon

(2) To obtain a strain having increased L-arginine productivity, a vector library was constructed in the following manner.

(3) First, using Corynebacterium glutamicum KCCM10741P (Korean Patent No. 0791659) as a parent strain, a plasmid obtained using the EZ-Tn5™ <R6Kγori/KAN-2>Tnp Transposome™ Kit (Epicentre) was transformed into the parent strain by an electric pulse method (Appl. Microbiol. Biotechnol. (1999) 52:541-545). Then, the strain was spread on a complex medium plate containing kanamycin (25 mg/L), and thereby about 20,000 colonies were obtained.

(4) <Complex Medium Plate (pH 7.0)>

(5) 10 g glucose, 10 g peptone, 5 g beef extract, 5 g yeast extract, 18.5 g brain heart infusion, 2.5 g NaCl, 2 g urea, 91 g sorbitol, 20 g agar (per liter of distilled water)

Example 2: Random Mutant Library Screening Using Transposon

(6) Each of about 20,000 colonies obtained in Example 1 was inoculated onto 300 μL of the following selective medium and cultured in a 96-deep-well plate at 30° C. at 1,000 rpm for about 24 hours.

(7) <Selective Medium (pH 8.0)>

(8) 10 g glucose, 5.5 g ammonium sulfate, 1.2 g MgSO.sub.47H.sub.2O, 0.8 g KH.sub.2PO.sub.4, 16.4 g K.sub.2HPO.sub.4, 100 μg biotin, 1 mg thiamine HCl, 2 mg calcium-pantothenate, 2 mg nicotinamide (per liter of distilled water)

(9) To analyze the amount of L-arginine produced in the culture, the ninhydrin method was used (Moore, S., Stein, W. H., Photometric ninhydrin method for use in the chromatography of amino acids. J. Biol. Chem. 1948, 176, 367-388).

(10) After completion of the culturing, 10 μL of the culture supernatant was reacted with 190 μL of a ninhydrin reaction solution at 65° C. for 30 minutes, and then the absorbance at a wavelength of 570 nm was measured with a spectrophotometer. Based on the results of the measurement, about 60 colonies showing higher absorbance than the Corynebacterium glutamicum KCCM10741P strain used as the control were selected as mutant strains. It was confirmed that other colonies showed absorbance similar to or lower than that of the Corynebacterium glutamicum KCCM10741P strain used as the control.

(11) About 60 strains selected as described above were cultured again in the same manner as described above, and then subjected to the ninhydrin reaction. As a result, the top ten mutant strains having increased L-arginine productivity compared to the Corynebacterium glutamicum KCCM10741P strain used as the parent strain were selected.

Example 3: Analysis of L-Arginine Productivity of Selected Random Mutant Strains

(12) In order to finally select strains whose L-arginine productivity was reproducibly increased from the ten mutants selected in Example 2, flask culture was performed using the following medium. After completing of the culturing, the concentration of L-arginine in the culture was analyzed by HPLC. The concentration of L-arginine produced by each of the mutant strains is shown in Table 1 below.

(13) <Production Medium (pH 7.0)>

(14) 6% glucose, 3% ammonium sulfate, 0.1% monopotassium phosphate, 0.2% magnesium sulfate heptahydrate, 1.5% corn steep liquor (CSL), 1% NaCl, 0.5% yeast extract, 100 mg/L biotin, 3% CaCO.sub.3, (per liter of distilled water)

(15) TABLE-US-00001 TABLE 1 Concentrations of L-arginine produced by 10 selected random mutant strains L-Arginine (g/L) Strains Batch 1 Batch 2 Batch 3 Average Control KCCM10741P 3 3.1 3.1 3.07 1 KCCM10741P/mt-1 2.8 3 2.7 2.83 2 KCCM10741P/mt-2 3.1 3 3.2 3.10 3 KCCM10741P/mt-3 3.3 3.2 3.4 3.30 4 KCCM10741P/mt-4 2.5 2.2 2.1 2.27 5 KCCM10741P/mt-5 2.9 3 3.2 3.03 6 KCCM10741P/mt-6 3.5 3.2 3.2 3.30 7 KCCM10741P/mt-7 3.2 3.3 3.3 3.27 8 KCCM10741P/mt-8 3.4 3 3.2 3.20 9 KCCM10741P/mt-9 2.7 2.7 3 2.80 10 KCCM10741P/mt-10 3.6 3.9 3.5 3.67

(16) Among the 10 selected mutant strains, KCCM10741P/mt-10 was finally selected as a strain whose L-arginine productivity was significantly increased.

Example 4: Identification of Causes of Increased L-Arginine Productivity of Finally Selected Strain

(17) In this Example, an experiment was performed on the mutant strain finally selected in Example 3 in order to identify genes deleted by random insertion of the transposon.

(18) Genomic DNA was extracted from KCCM10741P/mt-10, digested, and then ligated, and the ligation product was transformed into E. coli DH5α. The transformed E. coli cells were plated on an LB solid medium containing kanamycin (25 mg/L). Twenty transformed colonies were selected, and then plasmids containing an unknown gene portion were obtained. The nucleotide sequences were analyzed using primer 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4) of the EZ-Tn5™ <R6Kγori/KAN-2>Tnp Transposome™ Kit. As a result, it was confirmed that the deleted and inactivated gene had the nucleotide sequence of SEQ ID NO: 2 encoding the amino acid sequence of SEQ ID NO: 1

(19) TABLE-US-00002 Primer 1 (SEQ ID NO: 3):  ACCTACAACAAAGCTCTCATCAACC Primer 2 (SEQ ID NO: 4): CTACCCTGTGGAACACCTACATCT

(20) Accordingly, in order to confirm whether the protein including the amino acid sequence of SEQ ID NO: 1 has an effect on the ability to produce L-arginine upon inactivation of the protein, the gene above was selected as a candidate gene for deletion.

Example 5: Construction of Recombinant Vector for Deletion of the Gene Comprising the Nucleotide Sequence of SEQ ID NO: 2

(21) In this Example, in order to confirm the influence of the inactivation of the gene comprising the nucleotide sequence of SEQ ID NO: 2 and that of the production of L-arginine, a recombinant plasmid for deleting the gene, selected in Example 4, on the chromosome of the Corynebacterium L-arginine-producing strain was constructed.

(22) First, primers 3 to 6 shown in Table 2 were synthesized in order to prepare a recombinant vector capable of deleting the gene on the chromosome of the microorganism of the genus Corynebacterium.

(23) TABLE-US-00003 TABLE 2  Primers used Nucleotide sequences Primer 3 (SEQ ID NO: 5) CCGCTCGAGACATCGAAATCGTA AGGGTA Primer 4 (SEQ ID NO: 6) CAGCATTGACAAGCAGTTCT Primer 5 (SEQ ID NO: 7) AGAACTGCTTGTCAATGCTGGGC CCTTTCCCAGGTGGCAT Primer 6 (SEQ ID NO: 8) CCGCTCGAGAAGGCCACCGCTGC AGACCG

(24) Specifically, in order to delete the ORF region (SEQ ID NO: 2) of the gene, primer 3 (SEQ ID NO: 5), primer 4 (SEQ ID NO: 6), primer 5 (SEQ ID NO: 7), and primer 6 (SEQ ID NO: 8) were synthesized so that an XhoI restriction enzyme site is present at both the 5′ terminus and the 3′ terminus. PCR was carried out with the chromosomal DNA of Corynebacterium glutamicum KCCM10741P as a template using primers 3 to 6 above. As a result, it was confirmed that each of the DNA fragments corresponding to the front and rear portions of the part where the protein encoded by the gene is encoded was amplified by 500 bp, respectively. Herein, after denaturation at 95° C. for 5 minutes, PCR was carried out for a total of 30 cycles under the following conditions: denaturation at 94° C. for 30 seconds, annealing at 56° C. for 30 seconds, and polymerization at 72° C. for 1 minute. Thereafter, the polymerization reaction was carried out at 72° C. for 7 minutes. Next, a pDZ vector (Korean Patent No. 10-0924065), which is not replicable in Corynebacterium glutamicum, was treated with an XhoI restriction enzyme, and then the obtained PCR product was fusion-cloned. The fusion cloning was carried out using an In-Fusion® HD Cloning Kit (Clontech). Thereafter, the resultant was transformed into E. coli DH5α, and then plated on an LB solid medium containing kanamycin (25 mg/L).

(25) A colony transformed with a plasmid having the desired gene inserted therein was selected by PCR, and then the plasmid was isolated using a plasmid extraction technique. The plasmid was named “pDZ-ΔRS1”.

Example 6: Construction of Corynebacterium glutamicum KCCM10741P with the Deletion of the Gene Comprising the Nucleotide Sequence of SEQ ID NO: 2, and Evaluation of L-Arginine Productivity of the Constructed Strain

(26) Based on the L-arginine-producing strain of the genus Corynebacterium, which is the KCCM10741P strain, a strain in which the gene comprising the nucleotide sequence of SEQ ID NO: 2 is deleted was constructed, and the L-arginine productivity of the constructed strain was evaluated.

(27) Specifically, the recombinant plasmid pDZ-ΔRS1 prepared in Example 5 was transformed into Corynebacterium glutamicum KCCM10741P, which is an L-arginine-producing strain, by homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999).

(28) Next, the transformant was grown on a solid medium plate containing 4% sucrose to allow a second recombination to take place. After completion of the second recombination, deletion of the gene on the chromosome of the transformed Corynebacterium glutamicum strain was confirmed by PCR using primer 3 and primer 6. The recombinant strain was named “Corynebacterium glutamicum KCCM10741P-RS1”.

(29) In order to analyze the L-arginine productivity, the constructed Corynebacterium glutamicum KCCM10741P-RS1 strain and the parent strain Corynebacterium glutamicum KCCM10741P were cultured in the following manner.

(30) Each of the parent strain Corynebacterium glutamicum KCCM10741P and the Corynebacterium glutamicum KCCM10741P-RS1 strain constructed in Example 6 was inoculated into a 250 mL corner-baffled flask containing 25 mL of the following seed medium and shake-cultured at 200 rpm at 30° C. for 20 hours. Next, 1 mL of each of the seed cultures was inoculated into a 250 mL corner-baffle flask containing 24 mL of the following production medium and shake-cultured at 200 rpm at 30° C. for 72 hours. The composition of the seed medium and the composition of the production medium were as follows.

(31) <Seed Medium (pH 7.0)>

(32) 20 g glucose, 10 g peptone, 5 g yeast extract, 1.5 g urea, 4 g KH.sub.2PO.sub.4, 8 g K.sub.2HPO.sub.4, 0.5 g MgSO.sub.4.Math.7H.sub.2O, 100 μg biotin, 1 mg thiamine HCl, 2 mg calcium pantothenate, 2 mg nicotinamide (per liter of distilled water)

(33) <Production Medium (pH 7.0)>

(34) 6% glucose, 3% ammonium sulfate, 0.1% monopotassium phosphate, 0.2% magnesium sulfate heptahydrate, 1.5% corn steep liquor (CSL), 1% NaCl, 0.5% yeast extract, 100 mg/L biotin (per liter of distilled water)

(35) After completion of the culturing, the amount of L-arginine produced was measured by HPLC, and the concentration of L-arginine analyzed is shown in Table 3 below.

(36) TABLE-US-00004 TABLE 3 Analysis of L-arginine productivity of Corynebacterium glutamicum KCCM10741P in which the gene comprising the nucleotide sequence of SEQ ID NO: 2 is deleted L-Arginine (g/L) Strains Batch 1 Batch 2 Batch 3 Average KCCM10741P 3.0 3.1 3.1 3.06 KCCM10741P-RS1 3.8 3.9 3.8 3.83

(37) Based on the results above, it was confirmed that KCCM10741P-RS1, in which the gene is deleted from Corynebacterium glutamicum KCCM10741P (i.e., the L-arginine-producing strain), had the L-arginine productivity which is increased by 25% on average compared to the parent strain.

(38) The KCCM10741P-RS1 strain was named “CA06-2830”, and was deposited to the Korean Culture Center of Microorganisms (KCCM), which is an international depositary authority under the Budapest Treaty, on Dec. 15, 2017, and assigned Accession No. KCCM12187P.

(39) Therefore, it was confirmed that the L-arginine productivity could be improved by deleting the gene comprising the nucleotide sequence of SEQ ID NO: 2 from the microorganism of the genus Corynebacterium.

Example 7: Construction of Recombinant Vector for Attenuating the Gene

(40) Comprising the Nucleotide Sequence of SEQ ID NO: 2

(41) In order to construct a recombinant vector capable of attenuating the gene comprising the nucleotide sequence of SEQ ID NO: 2 on the chromosome of the strain of the genus Corynebacterium, the initiation codon of the gene was replaced from ATG to TTG to attenuate the gene. Further, in order to construct a fragment therefor, primer 3 and primers 7 to 9 shown in Table 4 below were synthesized.

(42) TABLE-US-00005 TABLE 4  Gene Primers used Nucleotide sequences SEQ ID Primer 3  CCGCTCGAGACATCGAAATCGTA NO: 2 (SEQ ID NO: 5) AGGGTA Primer 7  GATCCACAGTCCCAATATTCTCG (SEQ ID NO: 9) CTTCTTC Primer 8  GAAGAAGCGAGAATATTGGGAC (SEQ ID NO: 10) TGTGGATC Primer 9  CCGCTCGAGCAGCATTGACAAGC (SEQ ID NO: 11) AGTTCT

(43) In order to amplify the ORF region (SEQ ID NO: 2) of the gene, primer 3 (SEQ ID NO: 5), primer 7 (SEQ ID NO: 9), primer 8 (SEQ ID NO: 10), and primer 9 (SEQ ID NO: 11) were synthesized so that an XhoI restriction enzyme site is present at both the 5′ terminus and the 3′ terminus. PCR was carried out with the chromosomal DNA of Corynebacterium glutamicum KCCM10741P as a template using primer 3, primer 7, primer 8, and primer 9. Herein, after denaturation at 95° C. for 5 minutes, PCR was carried out for a total of 30 cycles under the following conditions: denaturation at 94° C. for 30 seconds, annealing at 56° C. for 30 seconds, and polymerization at 72° C. for 1 minute. Thereafter, the polymerization reaction was carried out at 72° C. for 7 minutes. Next, a pDZ vector (Korean Patent No. 10-0924065), which is not replicable in Corynebacterium glutamicum, was treated with an XhoI restriction enzyme, and then the obtained PCR product was fusion-cloned. The fusion cloning was carried out using an In-Fusion® HD Cloning Kit (Clontech). Thereafter, the resultant was transformed into E. coli DH5α, and then plated on an LB solid medium containing kanamycin (25 mg/L).

(44) A colony transformed with a plasmid having the desired gene inserted therein was selected by PCR, and then the plasmid was isolated using a plasmid extraction technique. The plasmid was named “pDZ-ΔRS2”.

Example 8: Construction of Corynebacterium glutamicum KCCM10741P in which the Gene Comprising the Nucleotide Sequence of SEQ ID NO: 2 is Attenuated, and Evaluation of L-Arginine Productivity of the Constructed Strain

(45) The recombinant plasmid pDZ-ΔRS2 prepared in Example 7 was transformed into Corynebacterium glutamicum KCCM10741P, which is an L-arginine-producing strain, by homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999).

(46) Next, the transformant was grown on a solid medium plate containing 4% sucrose to allow a second recombination to take place. The nucleotide sequence of the transformed Corynebacterium glutamicum strain obtained after the second recombination was analyzed using primer 3 and primer 9 to identify the strain in which the initiation codon of the gene comprising the nucleotide sequence of SEQ ID NO: 2 is substituted with TTG. The recombinant strain was named “Corynebacterium glutamicum KCCM10741P-R52”.

(47) Each of the parent strain Corynebacterium glutamicum KCCM10741P and the Corynebacterium glutamicum KCCM10741P-RS2 strain constructed above was cultured in the same manner as in Example 6 in order to analyze the L-arginine productivity. Thereafter, the amount of L-arginine produced was measured by HPLC, and the concentration of L-arginine analyzed is shown in Table 5 below.

(48) TABLE-US-00006 TABLE 5 Analysis of L-arginine productivity of Corynebacterium glutamicum KCCM10741P in which the gene comprising the nucleotide sequence of SEQ ID NO: 2 is attenuated L-Arginine (g/L) Strains Batch 1 Batch 2 Batch 3 Average KCCM10741P 4.2 4.2 4.1 4.17 KCCM10741P-RS2 4.6 4.8 4.6 4.67

(49) As shown in the results above, it was confirmed that when the gene comprising the nucleotide sequence of SEQ ID NO: 2 was attenuated in the L-arginine-producing strain KCCM10741P, the L-arginine productivity of the strain was increased by 12% on average.

(50) Therefore, it was confirmed that the L-arginine productivity could be improved by attenuating the expression of the gene comprising the nucleotide sequence of SEQ ID NO: 2 in the microorganism of the genus Corynebacterium.

(51) From the above results, it can be seen that in the strain in which the gene comprising the nucleotide sequence of SEQ ID NO: 2 is deleted or attenuated, the L-arginine productivity is increased, and this suggests that L-arginine can be mass-produced in the microorganism when the activity of the protein encoded by the gene is inactivated.

(52) While the present disclosure has been described with reference to the particular illustrative embodiments, it will be understood by those skilled in the art to which the present disclosure pertains that the present disclosure may be embodied in other specific forms without departing from the technical spirit or essential characteristics of the present disclosure. Therefore, the embodiments described above are considered to be illustrative in all respects and not restrictive. Furthermore, the scope of the present disclosure is defined by the appended claims rather than the detailed description, and it should be understood that all modifications or variations derived from the meanings and scope of the present disclosure and equivalents thereof are included in the scope of the appended claims.

(53) Accession Number

(54) Depositary Institution: Korean Culture Center of Microorganisms (KCCM) (International Depositary Authority)

(55) Accession Number: KCCM12187P

(56) Date of Deposit: Dec. 15, 2017