Patent Publication Number: US-2023136217-A1

Title: Mutant of corynebacterium glutamicum with enhanced l-glutamic acid productivity and method for preparing l-glutamic acid using the same

Description:
TECHNICAL FIELD 
     The present invention relates to a  Corynebacterium glutamicum  mutant strain having improved L-glutamic acid productivity and a method of producing L-glutamic acid using the same, and particularly, to a  Corynebacterium glutamicum  mutant strain having increased L-glutamic acid productivity due to a mechanosensitive ion channel gene derived from a  Corynebacterium  sp. strain, which has been introduced therein, a method for constructing the same, and a method of producing L-glutamic acid using the same. 
     BACKGROUND ART 
     L-glutamic acid is a representative amino acid that is produced by microbial fermentation. Monosodium L-glutamate (MSG) may increase the preference of foods such as meat, fish, chicken, vegetables, sauces, soups and seasonings by balancing and harmonizing the overall taste of the food, may enhance the taste of low-salt foods having a salt content reduced up to 30%, and thus is widely used as a household seasoning and a seasoning for the production of processed food. 
     In brief, regarding the pathway of L-glutamic acid fermentation, glucose mainly undergoes the glycolytic pathway (EMP), but a portion thereof is metabolized into two pyruvic acid molecules through the hexose monophosphate pathway (HMP). Among these molecules, one molecule combines with CO 2  to form oxaloacetic acid, and the other molecule combines with acetyl CoA from pyruvic acid to form citric acid. Then, oxaloacetic acid and citric acid enter the citric acid cycle (TCA cycle) to form α-ketoglutaric acid. Here, since the TCA cycle lacks the metabolic pathway for the oxidation of α-ketoglutaric acid to succinic acid and isocitrate dehydrogenase and glutamate dehydrogenase are closely involved therein, reductive amino acid synthesis by the oxidation of α-ketoglutaric acid efficiently occurs, thus producing L-glutamic acid. 
     L-glutamic acid is usually produced by fermentation using mainly  Brevibacterium  sp. or  Corynebacterium  sp. strains and mutant strains thereof. In order to increase the production of L-glutamic acid by microbial culture, a method of regulating the expression of genes involved in L-glutamic acid biosynthesis has been used. For example, a method of increasing the copy number of the gene or regulating the activity of the enzyme in the biosynthesis pathway by modifying the promoter of the gene may be mainly used. Specifically, it was disclosed that L-glutamic acid production was increased by amplifying a specific gene such as pyc gene or fasR (U.S. Pat. No. 6,852,516) or manipulating the promoter region of each of gdh, gltA, icd, pdh and argG genes (U.S. Pat. No. 6,962,805). As such, most of the genes involved in the glutamic acid biosynthesis pathway are already known, and thus it is required to identify a new gene capable of increasing glutamic acid production and to develop a new glutamic acid-producing strain by applying this gene. 
     Accordingly, the present inventors have identified a new gene that helps L-glutamic acid release, and have constructed a mutant strain by introducing the gene into an L-glutamic acid-producing strain. Also, the present inventors have found that the mutant strain has improved L-glutamic acid productivity, thereby completing the present invention. 
     DISCLOSURE 
     Technical Problem 
     An object of the present invention is to provide a  Corynebacterium glutamicum  mutant strain containing a mechanosensitive ion channel gene derived from a  Corynebacterium  sp. strain and having improved L-glutamic acid productivity. 
     Another object of the present invention is to provide a method for constructing a  Corynebacterium glutamicum  mutant strain, the method comprising a step of introducing a mechanosensitive ion channel gene derived from a  Corynebacterium  sp. strain. 
     Still another object of the present invention is to provide a method for producing L-glutamic acid, the method comprising steps of: (i) culturing the  Corynebacterium glutamicum  mutant strain in an L-glutamic acid production medium; and (ii) recovering L-glutamic acid from the cultured mutant strain or the medium in which the mutant strain has been cultured. 
     Technical Solution 
     One aspect of the present invention provides a  Corynebacterium glutamicum  mutant strain containing a mechanosensitive ion channel gene derived from a  Corynebacterium  sp. strain and having improved L-glutamic acid productivity. 
     The present inventors have made efforts to develop a novel  Corynebacterium glutamicum  mutant strain having improved L-glutamic acid productivity, and as a result, have found that, when a mechanosensitive ion channel gene derived from a  Corynebacterium  sp. strain is introduced into an L-glutamic acid-producing  Corynebacterium glutamicum  strain, it helps L-glutamic acid release, thereby significantly improving the L-glutamic acid productivity of the mutant strain. 
     In the present invention, the “ Corynebacterium  sp. strain” is a strain from which a mechanosensitive ion channel gene to be introduced into a  Corynebacterium glutamicum  strain is derived, and may include any  Corynebacterium  sp. microorganism. Specifically, the  Corynebacterium  sp. strain may be  Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium callunae, Corynebacterium suranareeae, Corynebacterium lubricantis, Corynebacterium doosanense, Corynebacterium efficiens, Corynebacterium uterequi, Corynebacterium stationis, Corynebacterium pacaense, Corynebacterium singulare, Corynebacterium humireducens, Corynebacterium marinum, Corynebacterium halotolerans, Corynebacterium spheniscorum, Corynebacterium freiburgense, Corynebacterium striatum, Corynebacterium canis, Corynebacterium ammoniagenes, Corynebacterium renale, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium caspium, Corynebacterium testudinoris, Corynebacaterium pseudopelargi , or  Corynebacterium flavescens.    
     In the present invention, the “mechanosensitive ion channel” is a channel present in the cell membranes of bacteria as well as eukaryotes, and is involved in osmotic homeostasis. The gene encoding this mechanosensitive ion channel may be derived from a  Corynebacterium  sp. strain. 
     Preferably, the gene may be a gene having the nucleotide sequence of SEQ ID NO: 1 derived from  Corynebacterium deserti , the nucleotide sequence of SEQ ID NO: 2 derived from  Corynebacterium  crudilactis, or the nucleotide sequence of SEQ ID NO: 3 derived from  Corynebacterium callunae . In addition, the gene may also comprise a nucleotide sequence having a homology of 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, most preferably 95% or more, to the nucleotide sequence of each of SEQ ID NOs: 1 to 3. “Homology of . . . % or more” is determined by comparing two optimally aligned sequences over a comparison region, wherein the portion of the polynucleotide sequence in the comparison region may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. In addition, the gene encoding the mechanosensitive ion channel may have a mutation that occurred naturally or non-naturally (e.g., genetic manipulation, radiation, chemical treatment, etc.), and the function of the gene may be weakened or enhanced by this mutation. 
     According to one embodiment of the present invention, the mechanosensitive ion channel gene may be encoded by any one of the nucleotide sequences of SEQ ID NOs: 1 to 5. 
     The nucleotide sequence of SEQ ID NO: 1 may be the nucleotide sequence of a mechanosensitive ion channel gene derived from  Corynebacterium deserti , the nucleotide sequence of SEQ ID NO: 2 may be the nucleotide sequence of a mechanosensitive ion channel gene derived from  Corynebacterium  crudilactis, and the nucleotide sequences of SEQ ID NOs: 3 to 5 may be the nucleotide sequences of mechanosensitive ion channel genes derived from  Corynebacterium callunae . Here, the nucleotide sequence of each of SEQ ID NOs: 4 and 5 may encode an amino acid sequence having a substitution of alanine (ALA) or valine (VAL) for leucine (LEU) at position 107 in the amino acid sequence of the mechanosensitive ion channel gene derived from  Corynebacterium callunae.    
     The present inventors have presumed that a nucleotide sequence having a homology of 70% or more to the mechanosensitive ion channel gene involved in the release of glutamic acid from  Corynebacterium glutamicum  (a homology of 74% for  Corynebacterium deserti,  72% for  Corynebacterium  crudilactis, and 70% for  Corynebacterium callunae ) plays the same role in glutamic acid release, and have found that the mutant strain containing the mechanosensitive ion channel gene according to the present invention has significantly improved glutamic acid productivity compared to other glutamic acid-producing strains. 
     In the present invention, the strain having “increased productivity” means that the strain has L-glutamic acid productivity of the strain is higher than that of a parent strain thereof. The term “parent strain” refers to a wild-type strain or mutant strain to be mutated, and includes a strain that is to be mutated directly or to be transformed with a recombinant vector or the like. In the present invention, the parent strain may be a wild-type  Corynebacterium glutamicum  strain or a strain mutated from the wild-type strain. Preferably, the parent strain may be a  Corynebacterium glutamicum  KCTC 11558BP strain. 
     The amount of L-glutamic acid produced by the  Corynebacterium glutamicum  mutant strain having increased L-glutamic acid productivity according to an embodiment of the present invention may be at least 5%, specifically 5 to 20% higher than the amount of L-glutamic acid produced by the parent strain. That is, the  Corynebacterium glutamicum  mutant strain can produce 36 to 50 g of L-glutamic acid, preferably 38 to 45 g of L-glutamic acid, per liter of a culture of the strain. 
     Another aspect of the present invention is to provide a method for constructing the  Corynebacterium glutamicum  mutant strain, the method comprising a step of introducing a mechanosensitive ion channel gene derived from a  Corynebacterium  sp. strain. 
     The above-described step is a step of transforming the parent strain with a vector containing a polynucleotide encoding the mechanosensitive ion channel gene. 
     In the present invention, the term “vector” refers to any vehicle for the cloning of and/or transfer of a nucleic acid into a host cell. The vector may be a replicon to which another DNA fragment may be attached so as to bring about the replication of the attached fragment. The term “replicon” refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control. In the present disclosure, the vector is not particularly limited as long as it may replicate in a host, and any vector known in the art may be used. The vector used for the construction of the recombinant vector may be a plasmid, cosmid, virus or bacteriophage in a native state or a recombinant state. Example of a phage vector or cosmid vector that may be used include pWE15, M13, λEMBL3, λEMBL4, λFIXII, λDASHII, λZAPII, λgt10, Δgt11, Charon4A, and Charon21A, and examples of a plasmid vector that may be used include a pDZ vector, and pBR-based, pUC-based, pBluescriptll-based, pGEM-based, pTZ-based, pCL-based and pET-based vectors. A vector that may be used is not particularly limited, and a known expression vector may be used, but is not limited thereto. 
     In the present disclosure, the term “transformation” means introducing a gene into a host cell so that the gene may be expressed in the host cell. For the transformed gene, it does not matter whether the gene is inserted into the chromosome of the host cell or located outside of the chromosome, as long as the gene may be expressed in the host cell. In the present invention, an example of the transformation method may be an electroporation method (van der Rest et al., Appl. Microbiol. Biotechnol., 52, 541-545, 1999) or the like. 
     Still another object of the present invention is to provide a method for producing L-glutamic acid, the method comprising steps of: (i) culturing the  Corynebacterium glutamicum  mutant strain in an L-glutamic acid production medium; and (ii) recovering L-glutamic acid from the cultured mutant strain or the medium in which the mutant strain has been cultured. 
     The culturing may be performed using a suitable medium and culture conditions known in the art, and any person skilled in the art may easily adjust and use the medium and the culture conditions. Specifically, the medium may be a liquid medium, but is not limited thereto. Examples of the culturing method include, but are not limited to, batch culture, continuous culture, fed-batch culture, or a combination thereof. 
     According to one embodiment of the present invention, the medium should meet the requirements of a specific strain in a proper manner, and may be appropriately modified by a person skilled in the art. For the culture medium for the  Corynebacterium  sp. strain, reference may be made to, but not limited to, a known document (Manual of Methods for General Bacteriology, American Society for Bacteriology, Wash. D.C., USA, 1981). 
     According to one embodiment of the present invention, the medium may contain various carbon sources, nitrogen sources, and trace element components. Examples of carbon sources that may be used include: sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances may be used individually or as a mixture, but are not limited thereto. Examples of nitrogen sources that may be used include peptone, yeast extract, meat extract, malt extract, corn steep liquor, soybean meal, urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. The nitrogen sources may also be used individually or as a mixture, but are not limited thereto. Examples of phosphorus sources that may be used include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. In addition, the culture medium may contain, but is not limited to, metal salts such as magnesium sulfate or iron sulfate, which are required for growth. In addition, the culture medium may contain essential growth substances such as amino acids and vitamins. Moreover, suitable precursors may be used in the culture medium. The medium or individual components may be added to the culture medium batchwise or in a continuous manner by a suitable method during culturing, without being limited thereto. 
     According to one embodiment of the present invention, the pH of the culture medium may be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the microorganism culture medium in an appropriate manner during the culturing. In addition, during the culturing, foaming may be suppressed using an anti-foaming agent such as a fatty acid polyglycol ester. Additionally, to keep the culture medium in an aerobic condition, oxygen or an oxygen-containing gas (for example, air) may be injected into the culture medium. The temperature of the culture medium may be generally 20° C. to 45° C., for example, 25° C. to 40° C. The culturing may be continued until a desired amount of a useful substance is produced. For example, the culturing time may be 10 hours to 160 hours. 
     According to one embodiment of the present invention, in the step of recovering L-glutamic acid from the cultured mutant strain and the culture medium, the produced L-glutamic acid may be collected or recovered from the medium using a suitable method known in the art depending on the culture method. Examples of the method for collecting or recovering L-glutamic acid include, but are not limited to, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity and size exclusion). 
     According to one embodiment of the present invention, the step of recovering L-glutamic acid may be performed by centrifuging the culture at a low speed to remove biomass and separating the obtained supernatant through ion-exchange chromatography. 
     According to one embodiment of the present disclosure, the step of recovering L-glutamic acid may include a process of purifying L-glutamic acid. 
     Advantageous Effects 
     Since the  Corynebacterium glutamicum  mutant strain according to one embodiment of the present invention is a strain into which the mechanosensitive ion channel gene derived from a  Corynebacterium  sp. strain has been introduced, it can produce L-glutamic acid in an improved yield due to enhancement of glutamic acid release. Thus, when the mutant strain is used, it is possible to more effectively produce L-glutamic acid. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG.  1    shows the structure of a pKmscS1 vector containing a mechanosensitive ion channel gene derived from a  Corynebacterium deserti  strain. 
         FIG.  2    shows the structure of a pKmscS3-L107A vector containing a mechanosensitive ion channel gene encoding an amino acid sequence having a substitution of alanine (ALA) for the amino acid residue leucine (LEU) at position 107 in the amino acid sequence of a mechanosensitive ion channel gene derived from a  Corynebacterium callunae  strain. 
     
    
    
     MODE FOR INVENTION 
     Hereinafter, the present invention will be described in more detail. However, these descriptions are provided for illustrative purposes only to aid in the understanding of the present invention, and the scope of the present invention is not limited by these illustrative descriptions. 
     Example 1. Construction of  Corynebacterium glutamicum  Mutant Strains 
     1-1. Construction of Vector into which Mechanosensitive Ion Channel Gene Derived from  Corynebacterium deserti  has been Introduced 
     In order to introduce the mechanosensitive ion channel gene mscS1, chromosomal DNA was isolated and purified from  Corynebacterium deserti  GIMN1.010. Then, using the purified DNA as a template, PCR amplification was performed using a set of primers 3 and 4 in Table 1 below for 30 cycles, each consisting of 95° C. for 30 sec, 58° C. for 30 sec, and 72° C. for 2 min. 
     The amplified gene was amplified by PCR using a set of primers 1 and 2 and a set of primers 5 and 6 shown in Table 1 below, which recognized the position within the vector into which the gene was to be inserted, for 30 cycles, each consisting of 95° C. for 30 sec, 58° C. for 30 sec, and 72° C. for 2 min. Bands of about 1,500 bp, 1,600 bp and 1,500 bp, respectively, were confirmed by electrophoresis of the PCR products. 
     Each purified PCR product (mscS1 gene and vector) was then used as a template for crossover polymerase chain reaction (PCR) and amplified again by a crossover PCR technique (Bacteriol., 179: 6228-6237, 1997) using a set of primers 1 and 6 shown in Table 1 below. Next, the 4.6-kb PCR product was purified, digested with BamHI restriction enzyme (Takara, Japan), and then cloned into a pK19mobSacB vector (Gene, 145: 69-73, 1994) digested with the same restriction enzyme, thereby constructing a pKmscS1 vector for introduction of the mscS1 gene ( FIG.  1   ). 
     
       
         
           
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                   
                   
                 SEQ ID 
               
               
                 Primer 
                 Nucleotide sequence (5′ → 3′) 
                 NO 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 Primer 1 
                 CGC GGATCC TCTGCCTTGCTTGCCTTGGT 
                 6 
               
               
                   
               
               
                 Primer 2 
                 CGGCAGTCCTAAAATCATGAGCCAAGATT 
                 7 
               
               
                   
                 AGCGCTG 
                   
               
               
                   
               
               
                 Primer 3 
                 CAGCGCTAATCTTGGCTCATGATTTTAGG 
                 8 
               
               
                   
                 ACTGCCG 
                   
               
               
                   
               
               
                 Primer 4 
                 ACGTCTGTAATCAGCGTCTTATGGGATGG 
                 9 
               
               
                   
                 ACGTTGG 
                   
               
               
                   
               
               
                 Primer 5 
                 CCAACGTCCATCCCATAAGACGCTGATTA 
                 10 
               
               
                   
                 CAGACGT 
                   
               
               
                   
               
               
                 Primer 6 
                 CGC GGATCC CCGTTGCCTGGGAGAGAAAG 
                 11 
               
               
                   
               
               
                 Primer 7 
                 GGTGGTGAGTTCCTGGTT 
                 12 
               
               
                   
               
               
                 Primer 8 
                 GTCAACTTCGCCTTCCTG 
                 13 
               
               
                   
               
            
           
         
       
     
     1-2. Construction of Vector into which Mechanosensitive Ion Channel Gene Derived from  Corynebacterium  Crudilactis has been Introduced 
     A pKmscS2 vector for introduction of the mscS2 gene was constructed in the same manner as in Example 1-1, except that C. crudilactis strain JZ16 was used instead of  Corynebacterium deserti  and the primers shown in Table 2 below were used. 
     
       
         
           
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                   
                   
                 SEQ ID 
               
               
                 Primer 
                 Nucleotide sequence (5′ → 3′) 
                 NO 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 Primer 1 
                 CGC GGATCC TCTGCCTTGCTTGCCTTGGT 
                 6 
               
               
                   
               
               
                 Primer 2 
                 GCGTTCACCTAAAATCATGAGCCAAGATT 
                 14 
               
               
                   
                 AGCGCTG 
                   
               
               
                   
               
               
                 Primer 3 
                 CAGCGCTAATCTTGGCTCATGATTTTAGG 
                 15 
               
               
                   
                 TGAACGC 
                   
               
               
                   
               
               
                 Primer 4 
                 ACGTCTGTAATCAGCGTCTTATGGGGTGG 
                 16 
               
               
                   
                 ACATTGG 
                   
               
               
                   
               
               
                 Primer 5 
                 CCAATGTCCACCCCATAAGACGCTGATTA 
                 17 
               
               
                   
                 CAGACGT 
                   
               
               
                   
               
               
                 Primer 6 
                 CGC GGATCC CCGTTGCCTGGGAGAGAAAG 
                 11 
               
               
                   
               
               
                 Primer 7 
                 GGTGGTGAGTTCCTGGTT 
                 12 
               
               
                   
               
               
                 Primer 8 
                 GTCAACTTCGCCTTCCTG 
                 13 
               
               
                   
               
            
           
         
       
     
     1-3. Construction of Vector into which the Mechanosensitive Ion Channel Gene Derived from  Corynebacterium callunae  has been Introduced 
     A pKmscS3 vector for introduction of the mscS3 gene was constructed in the same manner as in Example 1-1, except that  C. callunae  DSM 20147 was used instead of  Corynebacterium deserti  and the primers shown in Table 3 below were used. 
     
       
         
           
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                   
                   
                 SEQ ID 
               
               
                 Primer 
                 Nucleotide sequence (5′ → 3′) 
                 NO 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 Primer 1 
                 CGC GGATCC TCTGCCTTGCTTGCCTTGGT 
                 6 
               
               
                   
               
               
                 Primer 2 
                 ACCTCTCTATGACCTCTAGAGAGCCAAGA 
                 18 
               
               
                   
                 TTAGCGCTGAA 
                   
               
               
                   
               
               
                 Primer 3 
                 TTCAGCGCTAATCTTGGCTCTCTAGAGGT 
                 19 
               
               
                   
                 CATAGAGAGGT 
                   
               
               
                   
               
               
                 Primer 4 
                 ACACGTCTGTAATCAGCGTCATCCCTACT 
                 20 
               
               
                   
                 GGGTGGACGTA 
                   
               
               
                   
               
               
                 Primer 5 
                 TACGTCCACCCAGTAGGGATGACGCTGAT 
                 21 
               
               
                   
                 TACAGACGTGT 
                   
               
               
                   
               
               
                 Primer 6 
                 CGC GGATCC CCGTTGCCTGGGAGAGAAAG 
                 11 
               
               
                   
               
               
                 Primer 7 
                 GGTGGTGAGTTCCTGGTT 
                 12 
               
               
                   
               
               
                 Primer 8 
                 GTCAACTTCGCCTTCCTG 
                 13 
               
               
                   
               
            
           
         
       
     
     1-4. Construction of Vector into which Amino Acid Residue Mutation of Mechanosensitive Ion Channel Gene Derived from  Corynebacterium callunae  has been Introduced 
     In order to substitute the amino acid residue at position 107 in the amino acid sequence of the mechanosensitive ion channel gene mscS3 and introduce the gene, chromosomal DNA was isolated and purified from  Corynebacterium callunae  DSM 20147. Then, using the purified DNA as a template, PCR amplification was performed using a set of primers 1 and 2 and a set of primers 3 and 4 shown in Table 4 below for 30 cycles, each consisting of 95° C. for 30 sec, 58° C. for 30 sec, and 72° C. for 2 min. 
     Each purified PCR product (mscS3 gene and vector) was then used as a template for crossover polymerase chain reaction (PCR) and amplified again by a crossover PCR technique (Bacteriol., 179: 6228-6237, 1997) using a set of primers 1 and 4 shown in Table 4 below. Next, the 718-bp PCR product was purified, digested with BamHI restriction enzyme (Takara, Japan), and then cloned into a pK19mobSacB vector (Gene, 145: 69-73, 1994) digested with the same restriction enzyme, thereby constructing a pKmscS3-L107A vector for introduction of a substitution mutation of alanine for the leucine residue at position 107 in the amino acid sequence of the mscS3 gene ( FIG.  2   ). 
     
       
         
           
               
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                   
                 Nucleotide sequence 
                 SEQ ID 
               
               
                 Primer 
                 (5′ → 3′) 
                 NO 
               
               
                   
               
             
            
               
                 Primer 1 
                 CGC GGATCC GGCAGCTCTCAAAGT 
                 22 
               
               
                   
               
               
                 Primer 2 
                 GCGATGATGGATTGCGCGCC tgc ACCAA 
                 23 
               
               
                   
                 TGGCCGCAGAGGCAA 
                   
               
               
                   
               
               
                 Primer 3 
                 TTGCCTCTGCGGCCATTGGT gca GGCGC 
                 24 
               
               
                   
                 GCAATCCATCATCGC 
                   
               
               
                   
               
               
                 Primer 4 
                 CGC GGATCC CAGCGATATCTTCTTGGGC 
                 25 
               
               
                   
               
               
                 Primer 5 
                 GGTGGTGAGTTCCTGGTT 
                 12 
               
               
                   
               
               
                 Primer 6 
                 GTCAACTTCGCCTTCCTG 
                 13 
               
               
                   
               
            
           
         
       
     
     1-5. Construction of Vector into which Amino Acid Residue Mutation in Mechanosensitive Ion Channel Gene Derived from  Corynebacterium callunae  has been Introduced 
     A pKmscS3-L107V vector for introduction of a substitution mutation of valine for the leucine residue at position 107 in the amino acid sequence of the mscS3 gene was constructed in the same manner as in Example 1-4, except that the primers shown in Table 5 below were used instead of the primers in Example 1-4. 
     
       
         
           
               
               
               
             
               
                 TABLE 5 
               
               
                   
               
               
                   
                 Nucleotide sequence 
                 SEQ ID 
               
               
                 Primer 
                 (5′ → 3′) 
                 NO 
               
               
                   
               
             
            
               
                 Primer 1 
                 CGC GGATCC GGCAGCTCTCAAAGT 
                 22 
               
               
                   
               
               
                 Primer 2 
                 TTGCCTCTGCGGCCATTGGT gtt GGCGC 
                 26 
               
               
                   
                 GCAATCCATCATCGC 
                   
               
               
                   
               
               
                 Primer 3 
                 GCGATGATGGATTGCGCGCC aac ACCAA 
                 27 
               
               
                   
                 TGGCCGCAGAGGCAA 
                   
               
               
                   
               
               
                 Primer 4 
                 CGC GGATCC CAGCGATATCTTCTTGGGC 
                 25 
               
               
                   
               
               
                 Primer 5 
                 GGTGGTGAGTTCCTGGTT 
                 12 
               
               
                   
               
               
                 Primer 6 
                 GTCAACTTCGCCTTCCTG 
                 13 
               
               
                   
               
            
           
         
       
     
     1-6. Transformation of  Corynebacterium glutamicum  KCTC11558BP Strain and Construction of Mutant Strains 
     As a method for transformation of the  Corynebacterium glutamicum  KCTC 11558BP strain, an electrocompetent cell preparation method, a modification of the method of van der Rest, was used. 
     First, the  Corynebacterium glutamicum  KCTC 11558BP strain was cultured in 100 ml of a 2YT medium (16 g/l tryptone, 10 g/l yeast extract, 5 g/l sodium chloride) supplemented with 2% glucose, and 1 mg/ml of isonicotinic acid hydrazine and 2.5% glycine were added to the same medium free of glucose. Then, the seed culture medium was inoculated to reach an OD 610  value of 0.3, and then cultured for at 18° C. and 180 rpm for 12 to 16 hours until the OD 610  value reached 1.2 to 1.4. After keeping on ice for 30 minutes, centrifugation was performed at 4,000 rpm at 4° C. for 15 minutes. Thereafter, the supernatant was discarded and the precipitated  Corynebacterium glutamicum  KCTC 11558BP strain was washed 4 times with a 10% glycerol solution and finally re-suspended in 0.5 ml of a 10% glycerol solution, thereby preparing competent cells. Electroporation was performed using a Bio-Rad electroporator. The prepared competent cells and each of the constructed pKmscS1, pKmscS2, pKmscS3, pKmscS3-L107A and pKmscS3-L107V vectors were placed in an electroporation cuvette (0.2 mm), followed by electroporation under the conditions of 2.5 kV, 200 n and 12.5 μF. Immediately after completion of the electroporation, 1 ml of a regeneration medium (containing 18.5 g/l brain heart infusion powder, and 0.5 M sorbitol) was added to the cells which were then heat-treated at 46° C. for 6 minutes. Next, the cells were cooled at room temperature, transferred into a 15-ml cap tube, incubated at 30° C. for 2 hours, and plated on a selection medium (containing 5 g/l tryptone, 5 g/l NaCl, 2.5 g/l yeast extract, 18.5 g/l brain heart infusion powder, 15 g/l agar, 91 g/l sorbitol, and 20 μg/l kanamycin). The cells were cultured at 30° C. for 72 hours, and the generated colonies were cultured in BHI medium until a stationary phase to induce secondary recombination. Then, the cells were diluted to 10-5 to 10-7 cells, and plated on an antibiotic-free plate (containing 10% sucrose), and strains having no kanamycin resistance and capable of growing in the medium containing 10% sucrose were selected. The selected colonies were confirmed to be  Corynebacterium glutamicum  mutant strains (IS1, IS2 and IS3) into which the mscS1, mscS2 and mscS3 genes have been introduced, respectively, using a set of primers 7 and 8 shown in each of Tables 1 to 3 above. In addition, the selected colonies were confirmed to be  Corynebacterium glutamicum  mutant strains (IS3-A and IS3-V) into which the mscS3-L107A and mscS3-L107V genes have been introduced, respectively, using a set of primers 5 and 6 shown in each of Tables 4 and 5 above. 
     Experimental Example 1. Comparison of L-Glutamic Acid Productivity Between Mutant Strains 
     L-glutamic acid productivity was compared between the mutant strains (IS1, IS2 and IS3), which were constructed in Example 1 above and into which the mscS1, mscS2 and mscS3 genes have been introduced, respectively, and the patent strain  Corynebacterium glutamicum  KCTC 11558BP strain. 
     Each of the mutant strains and the parent strain was plated on an active plate medium (pH 7.5) having the composition shown in Table 6 below and was cultured at 30° C. for 24 hours. Thereafter, 10 mL of a flask medium (pH 7.6) having the composition shown in Table 7 below was placed in a 100-ml flask and inoculated with a loop of each of the strains cultured in the plate medium, followed by culturing at 30° C. and 200 rpm for 48 hours. After completion of culturing, the amount of L-glutamic acid in each of the cultures was measured, and the results are shown in Table 8 below. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 6 
               
               
                   
                   
               
               
                   
                 Component 
                 Content 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Glucose 
                 5 
                 g/L 
               
               
                   
                 Yeast extract 
                 10 
                 g/L 
               
               
                   
                 Urea 
                 3 
                 g/L 
               
               
                   
                 KH 2 PO 4   
                 1 
                 g/L 
               
               
                   
                 Biotin 
                 2 
                 μg/L 
               
               
                   
                 Soybean hydrolysate 
                 0.1% 
                 v/v 
               
               
                   
                 Leucine 
                 50 
                 mg/L 
               
               
                   
                 Agar 
                 20 
                 g/L 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
                 TABLE 7 
               
               
                   
                   
               
               
                   
                 Component 
                 Content 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Glucose 
                 70 
                 g/L 
               
               
                   
                 MgSO 4 •7H 2 O 
                 0.4 
                 g/L 
               
               
                   
                 Urea 
                 2 
                 g/L 
               
               
                   
                 KH 2 PO 4   
                 1 
                 g/L 
               
               
                   
                 Soybean hydrolysate 
                 1.5% 
                 v/v 
               
               
                   
                 (NH 4 ) 2 SO 4   
                 5 
                 g/L 
               
               
                   
                 FeSO 4 •7H 2 O 
                 10 
                 mg/L 
               
               
                   
                 MnSO 4 •5H 2 O 
                 10 
                 mg/L 
               
               
                   
                 Thiamin-HCl 
                 200 
                 μg/L 
               
               
                   
                 Biotin 
                 2 
                 μg/L 
               
               
                   
                 Calcium carbonate 
                 50 
                 g/L 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
             
               
                   
                 TABLE 8 
               
               
                   
                   
               
               
                   
                 Glutamic acid production (g/L) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 Parent strain (KCTC 11558BP) 
                 35.3 
               
               
                 Mutant strain (IS1) 
                 38.2 
               
               
                 Mutant strain (IS2) 
                 37.9 
               
               
                 Mutant strain (IS3) 
                 38.4 
               
               
                   
               
            
           
         
       
     
     As shown in Table 8 above, it was confirmed that the L-glutamic acid productivities of the  Corynebacterium glutamicum  mutant strains IS1, IS2 and IS3 into which the C.  deserti -derived mscS1 gene, the C. crudilactis-derived mscS2 gene and the  C. callunae -derived mscS3 gene have been introduced, respectively, increased by about 8%, 7% and 9%, respectively, compared to the L-glutamic acid productivity of the parent strain  Corynebacterium glutamicum  KCTC 11558BP strain into which none of the genes has been introduced. 
     Experimental Example 2. Comparison of L-Glutamic Acid Productivity with Those of Mutant Strains Having Amino Acid Residue Substitution 
     L-glutamic acid productivity was compared between the mutant strains IS3, IS3-A and IS3-V, which were constructed in Example 1 above and into which the mscS3, mscS3-L107A and mscS3-L107V genes have been introduced, respectively, and the patent strain  Corynebacterium glutamicum  KCTC 11558BP strain. 
     The strains were cultured in the same manner as in Example 1, the amount of L-glutamic acid in each of the cultures was measured, and the results are shown in Table 9 below. 
     
       
         
           
               
               
             
               
                   
                 TABLE 9 
               
               
                   
                   
               
               
                   
                 Glutamic acid production (g/L) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 Parent strain (KCTC 11558BP) 
                 35.3 
               
               
                 Mutant strain (IS3) 
                 38.4 
               
               
                 Mutant strain (IS3-A) 
                 39.7 
               
               
                 Mutant strain (IS3-V) 
                 36.8 
               
               
                   
               
            
           
         
       
     
     As shown in Table 9 above, it was confirmed that the L-glutamic acid productivities of the  Corynebacterium glutamicum  mutant strains IS3, IS3-A and IS3-V increased by about 9%, 12% and 4%, respectively, compared to that of the patent strain  Corynebacterium glutamicum  KCTC 11558BP strain. 
     In particular, it could be seen that, in the case (IS3-A) in which the amino acid residue leucine at position 107 was substituted with alanine, the L-glutamic acid productivity increased compared to that in the case (IS3) in which the amino acid residue leucine was not substituted or in the case (IS3-V) in which the amino acid residue leucine was substituted with valine, suggesting that the amino acid residue at position 107 in the amino acid sequence of the mscS3 gene derived from  C. callunae  is an important position involved in glutamic acid productivity. 
     So far, the present invention has been described with reference to the embodiments. Those of ordinary skill in the art to which the present invention pertains will appreciate that the present invention may be embodied in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view, not from a restrictive point of view. The scope of the present invention is defined by the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.