# Patent

## Title
Microorganism producing glutamic acid in high yield and a process of producing glutamic acid using the same

## Abstract

Disclosed herein are mutant strains, KCCM-10784P and KCCM-10785P, which are obtained through gene manipulation of Corynebacterium glutamicum KFCC-11074, and a process of producing L-glutamic acid using the mutant strains. The mutant strains are capable of producing L-Glutamic acid at high yield.

## Background
TECHNICAL FIELD
The present invention relates to a microorganism producing L-glutamic acid at high yield and a process of producing L-glutamic acid using the microorganism. More particularly, the present invention relates to a mutant strain of Corynebacterium glutamicum KFCC-11074, which has resistance to kanamycin and/or chloramphenicol and produces L-glutamic acid at high yield, and a process of producing L-glutamic acid using the mutant strain.
BACKGROUND ART
L-glutamic acid is a representative amino acid produced by fermentation. The annual worldwide production of L-glutamic acid is estimated to be more than one million tons, which is comparable to compounds commonly used in the chemical industry. L-glutamic acid has been widely used in pharmaceuticals, food, animal feedstuffs, and other products.
L-glutamic acid has conventionally been produced by fermentation mainly using so-called coryneform L-glutamic acid-producing bacteria belonging to the genus Brevibacterium, Corynebacterium or Microbacterium, or variants thereof (“Amino Acid Fermentation”, Gakkai Shuppan Center, pp. 195-215, 1986). Production of L-glutamic acid by fermentation using other strains includes methods using a microorganism belonging to the genus Bacillus, Streptomyces or Penicillium (U.S. Pat. No. 3,220,929); a microorganism belonging to the genus Pseudomonas, Arthrobacter, Serratia or Candida (U.S. Pat. No. 3,563,857); a microorganism such as a bacterium belonging to the genus Bacillus, Pseudomonas, Serratia, or Aerobacter aerogenes (currently Enterobacter aerogenes) (Examined Japanese Patent Publication (KOKOKU) No. 32-9393 (1957)); and a mutant strain of Escherichia coli (Japanese Patent Laid-open Application (KOKAI) No. 5-244970 (1993)).
Many studies have been conducted to improve the productivity of L-glutamic acid by changing the medium composition and developing a resistance strain. For example, a strain having resistance to β-fluoropyruvate was developed in order to increase the supply of pyruvate, which is used as an intermediate in the glutamic acid-producing metabolic pathway.
The productivity of L-glutamic acid has been considerably improved through the aforementioned methods. However, to meet increasing demand in the future, a less expensive and more effective process for producing L-glutamic acid needs to be developed.
In this regard, the inventors of this application conducted intensive and thorough research into the development of a bacterial strain producing L-glutamic acid at higher yields. The research resulted in the finding that, when cg2624 and cg2115 genes are knocked out, so that they are not expressed, the knock-out mutant has increased glycerol utilization and produces higher concentrations of L-glutamic acid even with lower biomass compared to a parental strain having an ability to produce L-glutamic acid, thereby leading to the present invention.
DISCLOSURE OF INVENTION
Technical Problem
It is therefore an object of the present invention to provide a mutant strain of Corynebacterium glutamicum KFCC-11074, which produces L-glutamic acid at high yield.
It is another object of the present invention to provide a process of preparing the mutant strain.
It is a further object of the present invention to provide a process of producing L-glutamic acid at high yield using the mutant strain.
Technical Solution
In one aspect, the present invention provides a mutant strain of Corynebacterium glutamicum KFCC-11074, which is capable of producing L-glutamic acid at high yield.
Corynebacterium glutamicum KFCC-11074 is an L-glutamic acid-producing strain, which was obtained prior to the present invention by treating a parental strain, Corynebacterium glutamicum KFCC 10656, with a mutagen such as UV irradiation, N-methyl-N′-nitro-N-nitrosoguanidine (NTG), and is capable of growing on a medium containing β-fluoropyruvate. C. glutamicum KFCC-11074 is disclosed in Korean Pat. Application No. 1999-09675, the entire disclosure of which is incorporated herein by reference.
A mutant strain of C. glutamicum KFCC-11074 according to the present invention is induced by knocking out cg2624 and/or cg2115 genes so that they are not expressed in C. glutamicum KFCC-11074. The present inventors intended to utilize glycerol as a carbon source, expecting that glutamic acid productivity can be increased when the carbon source is more effectively utilized and the metabolism thereof via pathways other than glutamic acid synthesis is reduced. In order to confer the ability to utilize glycerol to C. glutamicum KFCC-11074, C. glutamicum KFCC-11074 was treated with NTG and smeared onto a minimal medium containing glycerol. Among emerged colonies, a rapidly growing colonial population was selected and subjected to DNA array analysis along with the parental strain. The selected single colony exhibited an about 2-fold decrease in expressed levels of cg2624 and cg2115 genes compared to the parental strain. The present inventors expected that, if the KFCC-11074 strain is mutated so as not to express cg2624 and/or cg2115 genes, the strain is able to utilize glycerol as a carbon source and thus may have improved glycerol utilization and increased glutamic acid productivity, and this supposition was confirmed through experiments. Thus, in a preferred aspect, the present invention provides a mutant strain of C. glutamicum KFCC-11074, KCCM-10784P, in which the cg2624 gene is not expressed. As well, in a further preferred aspect, the present invention provides a C. glutamicum KFCC-11074 mutant strain, KCCM-10785P, in which neither cg2624 nor cg2115 genes are expressed. KCCM-10784P and KCCM-10785 mutant strains were deposited at the Korean Culture Center of Microorganisms (KCCM) on Sep. 28, 2006. The mutant strains according to the present invention were primarily selected on a kanamycin-containing medium because the parental strain KFCC-11074 has resistance to kanamycin. The mutant strain KCCM-10785P(IBT03) further has a chloramphenicol resistance gene.
In another aspect, the present invention provides a process of preparing the mutant strains KCCM-10784P(IBT02) and KCCM-0785P(IBT03).
In a preferred aspect, the mutant strains may be prepared by knocking out the cg2624 gene and/or the cg2115 gene in the parental strain KFCC-11074, which has the ability to produce glutamic acid. Thus, in a detailed aspect, the present invention provides a vector for knocking out the cg2624 gene and/or the cg2115 gene.
As used herein, the term “vector” has commonly known meanings. The term “vector” refers to an extrachromosomal element that may contain a gene, which does not participate in the cellular central metabolism, and is usually a circular double-stranded DNA. The element may be a linear or circular, single-stranded or double-stranded DNA or RNA, which contains a self-replication sequence, a genome integration sequence, or a phage nucleotide sequence. In general, the vector includes sequences suitable for directing gene transcription and translation, a selectable marker, and a sequence directing self-replication or chromosomal integration. A suitable vector contains a 5′ region including a transcription initiation site of a gene, and a 3′ region for controlling the transcription termination of the gene.
The “suitable regulatory sequence” refers to a sequence capable of controlling the transcription and translation of a polynucleotide (a coding sequence). Examples of such regulatory sequences include a ribosome binding sequence (RBS), a promoter and a terminator. Any promoter capable of initiating the transcription of a gene carried by the vector may be used. Non-limiting examples of such promoters include CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces), lac, trp, λPL, λPR, T7, tac and trc (useful for expression in E. coli). The terminator may be derived from various genes of preferred host cells, or may not be required.
In a preferred aspect, the vector includes a polynucleotide encoding a portion of the cg2624 and/or cg2115 genes of Corynebacterium glutamicum in order to knock out cg2624 and/or cg2115 genes. In a preferred embodiment, a polynucleotide containing a partial sequence of the cg2624 gene has the sequence of SEQ ID No. 1, and a polynucleotide containing a partial sequence of the cg2115 gene has the sequence of SEQ ID No. 2. However, it will be apparent to those skilled in the art that the partial sequence of cg2624 and/or cg2115 genes for knockout thereof is not limited to the above sequences, and includes any polynucleotides containing a certain partial sequence of the genes, as long as they are capable of knocking out cg2624 and/or cg2115 genes in the genome of a transformed strain through homologous recombination.
In addition to the partial sequence of cg2624 and/or cg2115 genes, a complete sequence of the genes may be used. In this case, the complete sequence may be partially altered through substitution so as to knock out cg2624 and/or cg2115 genes, leading to the lack of production of a normal translational product thereof. Such a knock-out gene may be constructed using a method known to those skilled in the art.
In an embodiment of the present invention, a vector for knocking out the cg2624 gene was constructed, and included the polynucleotide represented by SEQ ID No. 1. This vector was designated “pCJ200”. In an embodiment of the present invention, a knock-out vector for the cg2115 gene was also constructed, and included the polynucleotides of SEQ ID Nos. 2 and 3. The polynucleotide of SEQ ID No. 3 is a nucleotide sequence encoding a cmr gene. The knock-out vector containing the polynucleotides of SEQ ID Nos. 2 and 3 was designated “pCJ201”.
When a vector harboring a partial polynucleotide of the cg2624 and/or cg2115 genes is transformed into a Corynebacterium species, the polynucleotide carried by the vector is integrated into the host chromosome through homologous recombination so that it knocks out cg2624 and/or cg2115 genes on the host chromosome. Since the vector has a pUC origin, which acts only in E. coli, it is unable to replicate in Corynebacterium and can replicate only when integrated into the host chromosome. Accordingly, the vector of the present invention may be used to stably integrate the vector itself or an exogenous gene carried by the vector into the chromosome of a microorganism of the genus Corynebacterium. 
Thus, when the pCJ200 is introduced into a strain, the chromosomal cg2624 gene is knocked out. When the pCJ201 is introduced into a strain, the chromosomal cg2624 and cg2115 genes do not act in the strain, i.e., they are knocked out. The resulting transformed mutant strains do not express mRNA of the genes. In this way, an IBT02 strain, the cg2624 gene of which was disrupted, and an IBT03 strain, the cg2115 and cg2624 genes of which were both disrupted, were obtained and deposited at the Korean Culture Center of Microorganisms (KCCM). The mutant strains displayed decreased OD values and glutamic acid productivity increased by about 20% and about 37%, respectively, compared to a parental strain (see Examples of the present invention).
In a further aspect, the present invention provides a process of producing L-glutamic acid by culturing the mutant strain. In detail, the process comprises knocking out cg2624 and/or cg2115 genes of C. glutamicum KFCC-11074 as a parental strain and culturing the gene knock-out strain. In a preferred aspect, the knockout of cg2624 and/or cg2115 genes, as described above, may be induced by introducing a vector containing a partial sequence of the genes into C. glutamicum KFCC-11074 to integrate the partial sequence of the cg2624 gene and/or the partial sequence of the cg2115 gene into the KFCC-11074 strain through homologous recombination, preventing the expression of the genes. L-glutamic acid may be produced by culturing the thus-prepared mutant strain in a proper medium.
In a detailed embodiment of the present invention, the vector (pCJ200) containing a partial sequence of the cg2624 gene was transformed into Corynebacterium, thereby obtaining an IBT02 strain the cg2624 gene of which was disrupted. The vector (pCJ201), containing a cmr gene and a partial sequences of the cg2115 gene, was transformed into the IBT02 strain, thereby obtaining an IBT03 strain the cg2624 and cg2115 genes of which were both disrupted. The thus obtained KFCC-11704 mutant strains, KFCC-000(IBT02) and KFCC-000(IBT03), were found to have increased glutamic acid productivity compared to the parental strain KFCC-11704 (Table 1).
Advantageous Effects
In accordance with the present invention, a cg2624/cg2115 gene knock-out mutant strain produces glutamic acid at higher yields than a parental strain. Thus, such gene manipulation is useful for increasing the amount of metabolites while controlling the biomass of a microorganism of the genus Corynebacterium. 



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a cleavage map of a pCJ200 vector for disrupting the cg2624 gene in Corynebacterium. 
FIG. 2 is a cleavage map of a pCJ201 vector for disrupting the cg2115 gene in Corynebacterium. 
FIG. 3 is a photograph of agarose gel electrophoresis of PCR samples showing that the pCJ200 vector is integrated into the chromosome of Corynebacterium. 
FIG. 4 is a photograph of agarose gel electrophoresis of PCR samples showing that the pCJ201 vector is integrated into the chromosome of Corynebacterium. 



BEST MODE FOR CARRYING OUT THE INVENTION
A better understanding of the present invention may be obtained through the following examples, which are set forth to illustrate, but are not to be construed as the limit of the present invention.
Example 1
Mutagenesis for Selection of Strain Deficient in cg2624 and cg2115 Genes
In order to confer glycerol utilization to a parental strain C. glutamicum KFCC-11074, the parental strain was treated with NTG, and smeared onto a minimal medium containing glycerol [10 g/L of glycerol, 5 g/L of ammonium sulfate, 2 g/L of urea, 1 g/L of potassium phosphate monobasic, 2 g/L potassium phosphate dibasic, 0.4 g/L of magnesium sulfate, 0.5 g/L of sodium chloride, 200 μg/L of biotin, 3 mg/L of thiamine, 1 mg/L of pantothenic acid, 5 mg/L of NCA, and 1 ml/L of trace elements (10 mg/L of calcium chloride, 270 mg/L of copper sulfate, 1 g/L of iron chloride, 10 mg/L of manganese chloride, 40 mg/L of ammonium molybdate, 90 mg/L of borax, 10 mg/L of zinc sulfate)]. Among emergent colonies, a colonial population growing rapidly was selected and designated “IBT01”. The IBT01 strain was subjected to DNA array analysis along with the parental strain. The IBT01 strain exhibited an about 2-fold decrease in expressed levels of cg2624 and cg2115 genes compared to the parental strain.
Example 2
Cloning of cg2624 Gene
The nucleotide sequence of the cg2624 gene of Corynebacterium glutamicum was obtained by searching the nucleotide database at NCBI, and was used to design oligonucleotide primers having the nucleotide sequences of SEQ ID Nos. 6 and 7.
Genomic DNA was extracted from C. glutamicum. PCR was carried out using the genomic DNA as a template with the oligonucleotide primers having the nucleotide sequences of SEQ ID Nos. 6 and 7 in order to amplify a partial nucleotide sequence (SEQ ID No. 1) of the cg2624 gene.
The amplified partial nucleotide sequence of the cg2624 gene was cloned into pCR2.1-TOPO (a vector in a TOPO TA Cloning kit) using a TOPO TA Cloning kit (Invitrogen, USA), thereby constructing pCR2.1-TOPO-cg2624(pCJ200).
Example 3
Cloning of cg2115 Gene
The nucleotide sequence of the cg2115 gene of Corynebacterium glutamicum was obtained by searching the nucleotide database at NCBI, and was used to design oligonucleotide primers having the nucleotide sequences of SEQ ID Nos. 8 and 9. Genomic DNA was extracted from C. glutamicum. PCR was carried out using the genomic DNA as a template with the oligonucleotide primers having the nucleotide sequences of SEQ ID Nos. 8 and 9 in order to amplify the partial nucleotide sequence (SEQ ID No. 2) of the cg2115 gene.
The amplified partial nucleotide sequence of the cg2115 gene was cloned into pCR2.1-TOPO using a TOPO TA Cloning kit (Invitrogen, USA), thereby obtaining pCR2.1-TOPO-cg2115.
Then, the nucleotide sequence of a chloramphenicol resistance gene was obtained by searching the nucleotide database at NCBI, and was used for designing oligonucleotide primers having the nucleotide sequences of SEQ ID Nos. 10 and 11. PCR was carried out using a pACYC-duet vector DNA as a template with the oligonucleotide primers having the nucleotide sequences of SEQ ID Nos. 10 and 11 in order to amplify a chloramphenicol resistance gene (cmr) of the cg2115 gene.
The amplified chloramphenicol resistance gene (cmr) was inserted into a pGEM-T vector (Promega), thereby obtaining pGEM-T-cmr. The pCR2.1-TOPO-cg2115 vector was digested with NsiI and SacI to excise the partial sequence of cg2115 gene, which was purified and ligated with the pGEM-T-cmr vector, predigested with NsiI and SacI, thereby constructing pGEM-T-cmr-cg2115(pCJ201).
Example 4
Transformation of Corynebacterium with pCR2.1-Topo-cg2624
C. glutamicum KFCC-11074 was cultured in No. 2 medium (10 g/L of polypeptone, 5 g/L of yeast extract, 5 g/L of ammonium sulfate, 1.5 g/L of urea, 4 g/L of potassium phosphate monobasic, 8 g/L of potassium phosphate dibasic, 0.5 g/L of magnesium sulfate, 100 μg/L of biotin, 1 mg/L of thiamine, 2 mg/L of pantothenic acid, 2 mg/L of NCA, 20 g/L of glucose) at 30° C. for 12 hrs. Thereafter, the resulting culture was inoculated into No. 2 EPO medium (No. 2 medium plus 4 g/L of isoniazid, 25 g/L of glycine, 1 g/L of Tween 80) until the culture solution reached an OD value of 0.3, and then cultured at 30° C. until the culture solution reached an OD value of 1.0. Then, the culture was placed on ice for 10 min and centrifuged at 1500×g for 5 min. The cell pellet was washed with 50 ml of pre-cooled 10% glycerol four times, and resuspended in 0.5 ml of 10% glycerol. 100 μl of the cell suspension was transferred into a 1.5-ml micro centrifuge tube.
The pCJ200 vector prepared in Example 1 was added to the competent cells, and transferred into a 2-mm electroporation cuvette on ice. Electroporation was carried out at 1.5 kV, 25 μF and 600Ω. Immediately after electroporation, 1 ml of BHIS medium (37 g/L of brain heart infusion, 91 g/L of sorbitol) was added to the cuvette. The cuvette was incubated at 46° C. for 6 min and then cooled on ice. Then, the cells were smeared onto an active BHIS medium (10 g/L of meat extract, 10 g/L of polypeptone, 5 g/L of yeast extract, 5 g/L of sodium chloride, 18.5 g/L of brain heart infusion, 91 g/L of sorbitol, 20 g/L of agar) supplemented with 25 μg/ml of kanamycin.
Example 5
Evaluation of Integration of the pCJ200 Vector into the Chromosome of Corynebacterium 
Genomic DNA was isolated from the Corynebacterium transformant prepared in Example 4. PCR was carried out using the genomic DNA as a template with a pair of oligonucleotide primers having the nucleotide sequences of SEQ ID Nos. 6 and 7 (M13F and M13R) in order to determine whether the pCJ200 vector was integrated into the host chromosome. PCR products were electrophoresed. A band corresponding to the pCJ200 vector was observed, confirming that the vector was integrated into the host chromosome.
FIG. 3 is a photograph of agarose gel electrophoresis showing the results of PCR using the genomic DNA, isolated from Corynebacterium cells transformed with the pCJ200 vector, as a template with a pair of oligonucleotide primers having nucleotide sequences of SEQ ID Nos. 6 and 7 (M13F and M13R). As shown in FIG. 3, all of five selected clones (lanes 1 to 5) were found to contain the pCJ200 vector within their chromosome. The transformant disrupted in the cg2624 gene through homologous recombination was designated “IBT02” and deposited at the Korean Culture Center of Microorganisms.
Example 6
Transformation of Corynebacterium with pCJ201
The transformant IBT02, prepared in Example 5, was further transformed with the pCJ201 vector. The IBT02 strain was transformed according to the same procedure as in Example 4, and smeared onto an active BHIS medium supplemented with 7 μg/ml of chloramphenicol.
Example 7
Evaluation of Integration of the pCJ201 Vector into the Chromosome of Corynebacterium 
Genomic DNA was isolated from the Corynebacterium transformant prepared in Example 6. PCR was carried out, using the genomic DNA as a template, with a pair of oligonucleotide primers having the nucleotide sequences of SEQ ID Nos. 12 and 13 in order to determine whether the pCJ201 vector was integrated into the host chromosome. PCR products were electrophoresed. The oligonucleotides of SEQ ID Nos. 12 and 13 had a partial sequence of the pGEM-T vector.
FIG. 4 is a photograph of agarose gel electrophoresis showing the results of PCR using the genomic DNA isolated from Corynebacterium cells transformed with the pCJ201 vector, as a template with oligonucleotide primers having nucleotide sequences of SEQ ID Nos. 12 and 13. As shown in FIG. 4, all of three selected clones (lanes 1 to 3) were found to contain the pCJ201 vector within their chromosome. The transformant that was disrupted in both the cg2624 and cg2115 genes was designated “IBT03” and deposited at the KCCM.
Example 8
Evaluation of Glutamic Acid Productivity of the Mutant Strains
The IBT01, IBT02 and IBT03 mutant strains, prepared in Examples 1, 5 and 6, respectively, and the parental strain C. glutamicum KFCC-11074 were evaluated for glutamic acid productivity. This test was carried out in a flask. Each strain was grown on an active plate (10 g/L of meat extract, 5 g/L of yeast extract, 10 g/L of polypeptone, 5 g/L of sodium chloride, 20 g/L of agar) at 30° C. for 12 hrs. One loop for each strain was inoculated in a 250-ml flask containing 40 ml of a flask titer medium (3% glucose, 1% molasses, 0.04% magnesium sulfate, 0.1% potassium phosphate dibasic, 0.3% ammonium sulfate, 0.001% iron sulfate, 0.001% manganese sulfate, 500 μg/L biotin, 2 mg/L thiamine hydrochloride, 0.1% urea, pH 7.1), and was grown at 30° C. for 40 hrs. The culture was evaluated for glutamic acid productivity. As shown in Table 1, the IBT01 strain displayed a decreased OD value and an increased glutamic acid yield compared to the parental strain. Also, the IBT02 and IBT03 strains displayed a decreased OD value and glutamic acid yields increased by 20% and 37%, respectively, compared to the parental strain. These results indicate that the IBT02 and IBT03 mutant strains produce L-glutamic acid at higher yields than the parental strain.







TABLE 1







Glutamic Acid Productivity












24 h

40 h














Strains
OD
GA (g/L)
OD
GA (g/L)

















KFCC-11074
18.8
10.2
20.8
11.5



IBT01
12.2
9.1
15.4
11.9



IBT02
12.2
9.5
13.5
13.7



IBT03
5.8
8.4
8.1
15.8

## Claims

1. A mutant strain of Corynebacterium glutamicum KFCC-11074, in which cg2624 gene is knocked out and which produces L-glutamic acid in high yields.

2. A mutant strain of Corynebacterium glutamicum KFCC-11074, in which both cg2624 and cg2115 genes are knocked out and which produces L-glutamic acid in high yields.

3. The mutant strain according to claim 1, which is Corynebacterium glutamicum, in which the cg2624 gene is not expressed.

4. The mutant strain according to claim 2, which is Corynebacterium glutamicum, in which the cg2624 and cg2115 genes are not expressed.

5. The mutant strain according to claim 2, which has resistance to chloramphenicol.

6. A method of producing a mutant strain having increased glycerol utilization and producing L-glutamic acid in high yields, comprising knocking out cg2624 gene and/or cg2115 gene of Corynebacterium glutamicum KFCC-11074.

7. A method of producing L-glutamic acid in high yields by culturing the mutant strain of any one of claims 1 to 5.