Mutant of <i>Corynebacterium glutamicum </i>with enhanced l-glutamic acid productivity and method for preparing l-glutamic acid using the same

Abstract

The present invention relates to a Corynebacterium glutamicum mutant strain having increased L-glutamic acid productivity, a method for constructing the same, and a method of producing L-glutamic acid using the same. The Corynebacterium glutamicum mutant strain is a strain into which a mechanosensitive ion channel gene derived from a Corynebacterium sp. strain has been introduced, and thus it can produce L-glutamic acid in an improved yield due to enhancement of glutamic acid release. Therefore, when the mutant strain is used, it is possible to more effectively produce L-glutamic acid.

Claims

1. A Corynebacterium glutamicum mutant strain having improved L-glutamic acid productivity compared to its parent strain, wherein the Corynebacterium glutamicum mutant strain contains a mechanosensitive ion channel gene derived from one strain selected from the group consisting of Corynebacterium deserti, Corynebacterium crudilactis, and Corynebacterium callunae, and wherein the mechanosensitive ion channel gene is encoded by any one of the nucleotide sequences of SEQ ID NOs: 1 to 5.

2. A method for constructing the Corynebacterium glutamicum mutant strain of claim 1, the method comprising a step of introducing into a parent C. glutamicum strain a mechanosensitive ion channel gene derived from one strain selected from the group consisting of Corynebacterium deserti, Corynebacterium crudilactis, and Corynebacterium callunae, wherein the mechanosensitive ion channel gene is encoded by any one of the nucleotide sequences of SEQ ID NOs: 1 to 5.

3. A method for producing L-glutamic acid, the method comprising steps of: (i) culturing the Corynebacterium glutamicum mutant strain of claim 1 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.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the structure of a pKmscS1 vector containing a mechanosensitive ion channel gene derived from a Corynebacterium deserti strain.

(2) 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

(3) 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

(4) 1-1. Construction of Vector into which Mechanosensitive Ion Channel Gene Derived from Corynebacterium deserti has been Introduced

(5) 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.

(6) 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.

(7) 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).

(8) TABLE-US-00001 TABLE1 SEQID Primer Nucleotidesequence(5.fwdarw.3) NO Primer1 CGCGGATCCTCTGCCTTGCTTGCCTTGGT 6 Primer2 CGGCAGTCCTAAAATCATGAGCCAAGATT 7 AGCGCTG Primer3 CAGCGCTAATCTTGGCTCATGATTTTAGG 8 ACTGCCG Primer4 ACGTCTGTAATCAGCGTCTTATGGGATGG 9 ACGTTGG Primer5 CCAACGTCCATCCCATAAGACGCTGATTA 10 CAGACGT Primer6 CGCGGATCCCCGTTGCCTGGGAGAGAAAG 11 Primer7 GGTGGTGAGTTCCTGGTT 12 Primer8 GTCAACTTCGCCTTCCTG 13
1-2. Construction of Vector into which Mechanosensitive Ion Channel Gene Derived from Corynebacterium Crudilactis has been Introduced

(9) 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.

(10) TABLE-US-00002 TABLE2 SEQID Primer Nucleotidesequence(5.fwdarw.3) NO Primer1 CGCGGATCCTCTGCCTTGCTTGCCTTGGT 6 Primer2 GCGTTCACCTAAAATCATGAGCCAAGATT 14 AGCGCTG Primer3 CAGCGCTAATCTTGGCTCATGATTTTAGG 15 TGAACGC Primer4 ACGTCTGTAATCAGCGTCTTATGGGGTGG 16 ACATTGG Primer5 CCAATGTCCACCCCATAAGACGCTGATTA 17 CAGACGT Primer6 CGCGGATCCCCGTTGCCTGGGAGAGAAAG 11 Primer7 GGTGGTGAGTTCCTGGTT 12 Primer8 GTCAACTTCGCCTTCCTG 13
1-3. Construction of Vector into which the Mechanosensitive Ion Channel Gene Derived from Corynebacterium callunae has been Introduced

(11) 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.

(12) TABLE-US-00003 TABLE3 SEQID Primer Nucleotidesequence(5.fwdarw.3) NO Primer1 CGCGGATCCTCTGCCTTGCTTGCCTTGGT 6 Primer2 ACCTCTCTATGACCTCTAGAGAGCCAAGA 18 TTAGCGCTGAA Primer3 TTCAGCGCTAATCTTGGCTCTCTAGAGGT 19 CATAGAGAGGT Primer4 ACACGTCTGTAATCAGCGTCATCCCTACT 20 GGGTGGACGTA Primer5 TACGTCCACCCAGTAGGGATGACGCTGAT 21 TACAGACGTGT Primer6 CGCGGATCCCCGTTGCCTGGGAGAGAAAG 11 Primer7 GGTGGTGAGTTCCTGGTT 12 Primer8 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

(13) 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.

(14) 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).

(15) TABLE-US-00004 TABLE4 Nucleotidesequence SEQID Primer (5.fwdarw.3) NO Primer1 CGCGGATCCGGCAGCTCTCAAAGT 22 Primer2 GCGATGATGGATTGCGCGCCtgcACCAA 23 TGGCCGCAGAGGCAA Primer3 TTGCCTCTGCGGCCATTGGTgcaGGCGC 24 GCAATCCATCATCGC Primer4 CGCGGATCCCAGCGATATCTTCTTGGGC 25 Primer5 GGTGGTGAGTTCCTGGTT 12 Primer6 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

(16) 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.

(17) TABLE-US-00005 TABLE5 Nucleotidesequence SEQID Primer (5.fwdarw.3) NO Primer1 CGCGGATCCGGCAGCTCTCAAAGT 22 Primer2 TTGCCTCTGCGGCCATTGGTgttGGCGC 26 GCAATCCATCATCGC Primer3 GCGATGATGGATTGCGCGCCaacACCAA 27 TGGCCGCAGAGGCAA Primer4 CGCGGATCCCAGCGATATCTTCTTGGGC 25 Primer5 GGTGGTGAGTTCCTGGTT 12 Primer6 GTCAACTTCGCCTTCCTG 13
1-6. Transformation of Corynebacterium glutamicum KCTC11558BP Strain and Construction of Mutant Strains

(18) 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.

(19) 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.sub.610 value of 0.3, and then cultured for at 18 C. and 180 rpm for 12 to 16 hours until the OD.sub.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 F. 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 g/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

(20) 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.

(21) 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.

(22) TABLE-US-00006 TABLE 6 Component Content Glucose 5 g/L Yeast extract 10 g/L Urea 3 g/L KH.sub.2PO.sub.4 1 g/L Biotin 2 g/L Soybean hydrolysate 0.1% v/v Leucine 50 mg/L Agar 20 g/L

(23) TABLE-US-00007 TABLE 7 Component Content Glucose 70 g/L MgSO.sub.47H.sub.2O 0.4 g/L Urea 2 g/L KH.sub.2PO.sub.4 1 g/L Soybean hydrolysate 1.5% v/v (NH.sub.4).sub.2SO.sub.4 5 g/L FeSO.sub.47H.sub.2O 10 mg/L MnSO.sub.45H.sub.2O 10 mg/L Thiamin-HCl 200 g/L Biotin 2 g/L Calcium carbonate 50 g/L

(24) TABLE-US-00008 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

(25) 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

(26) 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.

(27) 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.

(28) TABLE-US-00009 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

(29) 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.

(30) 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.

(31) 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.