MUTANT STRAIN HAVING ENHANCED L-GLUTAMIC ACID PRODUCING ABILITY, AND L-GLUTAMIC ACID PREPARATION METHOD USING SAME

Abstract

The present disclosure relates to a mutant strain having enhanced L-glutamic acid productivity and a method of producing L-glutamic acid using the same. The mutant strain according to one embodiment of the present disclosure has reduced production of citramalate as a by-product due to weakening or inactivation of the activity of citramalate synthase and has excellent L-glutamic acid productivity. The strain having an additional mutation in the YggB protein may 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.

Claims

1. A Corynebacterium sp. mutant strain in which an activity of citramalate synthase has been weakened or inactivated and which has enhanced L-glutamic acid productivity.

2. The Corynebacterium sp. mutant strain of claim 1, wherein the citramalate synthase is encoded by 2-isopropylmalate synthase (leuA) gene.

3. The Corynebacterium sp. mutant strain of claim 1, which has an additional mutation caused by substitution of alanine or valine for at least one leucine selected from the group consisting of leucine at amino acid position 93 and leucine at amino acid position 109 in the amino acid sequence of a YggB protein represented by SEQ ID NO: 15.

4. The Corynebacterium sp. mutant strain of claim 1, wherein the strain is Corynebacterium glutamicum.

5. A method for producing L-glutamic acid, the method comprising steps of: (a) culturing the Corynebacterium sp. mutant strain according to claim 1 in a medium; and (b) recovering L-glutamic acid from the cultured mutant strain or the medium.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0047] FIG. 1 shows the synthetic pathway of citramalate.

[0048] FIG. 2 shows a pKLA vector constructed to disrupt the leuA gene in the Corynebacterium glutamicum KCTC 11558BP strain.

[0049] FIG. 3 shows the positions of primers 1 and 4 in a leuA gene-deleted region.

[0050] FIG. 4a shows the structure of YggB protein.

[0051] FIG. 4b shows the pore structure of the YggB protein having a substitution of alanine for leucine at position 93 in the amino acid sequence of the YggB protein.

[0052] FIG. 4c shows the pore structure of the YggB protein having a substitution of alanine for leucine at position 109 in the amino acid sequence of the YggB protein.

[0053] FIG. 5. shows a pKY93 vector constructed to substitute another amino acid for the amino acid at position 93 in the amino acid sequence of the YggB protein in an I10 strain.

MODE FOR INVENTION

[0054] Hereinafter, one or more embodiments will be described in more detail with reference to examples. However, these examples serve to illustratively describe one or more embodiments, and the scope of the present disclosure is not limited to these examples.

Example 1. Intracellular Metabolite Analysis of Strain after Completion of Fermentation

[0055] The Corynebacterium glutamicum KCTC 11558BP strain, which is a mutant strain obtained by chemically treating an strain with nitrosoguanidine (NTG) that causes DNA mutation, has molasses resistance, and thus may produce L-glutamic acid in high yield by using inexpensive molasses as a main carbon source (Korean Patent Application Publication No. 10-2011-0034718).

[0056] Corynebacterium glutamicum 16 (hereinafter referred to as 16) and Corynebacterium glutamicum KCTC 11558BP (hereinafter referred to as KCTC 11558BP) having the ability to produce glutamic acid were analyzed for the amount of metabolites and L-glutamic acid in the cells.

[0057] Each of KCTC 11558BP and 16 was inoculated into 25 ml of CM liquid medium (containing, per liter, 1% glucose, 1% polypeptone, 0.5% yeast extract, 0.5% beef extract, 0.25% NaCl, 0.2% urea, 50% NaOH 100 l, pH 6.8), and then cultured with shaking at 200 rpm at 30° C. When cell growth reached an exponential phase during culture, cells were separated from the culture medium by rapid vacuum filtration (Durapore HV, 0.45 m; Millipore, Billerica, Mass.). The filter having the cells adsorbed thereon was washed twice with 10 ml of cooling water, and then immersed in methanol containing 5 M morpholine ethanesulfonic acid and 5 M methionine sulfone for 10 minutes to stop the reaction. The extract obtained therefrom was mixed with the same amount of chloroform and a 0.4-fold volume of water, and then only the aqueous phase was applied to a spin column to remove protein contaminants. The relative amount of citramalate in the filtered extract was analyzed using capillary electrophoresis mass spectrometry, and the results are shown in Table 1 below.

[0058] In addition, L-glutamic acid production was analyzed by performing high performance liquid chromatography (HPLC; Agilent, USA) using a C18 column (Apollo C18, 5 μm, 150 mm×4.6 mm). The results are shown in Table 1 below. An analysis buffer containing 8.85 g/L of KH.sub.2PO.sub.4, 1.652 g/L of tetrabutyl ammonium hydroxide solution, 10 ml/L of acetonitrile and 12.5 mg/L of sodium azide and having a pH of 3.15 (adjusted with phosphoric acid) was used after filtration through a 0.45 μm membrane-filter (HA).

[0059] As shown in Table 1 below, it was confirmed that the amount of citramalate produced by KCTC 11558BP increased by about 3 times compared to that produced by the control 16.

TABLE-US-00001 TABLE 1 I6 (control) KCTC 11558BP L-glutamic acid 34   37.5 production (g/L) Citramalate (relative 130.2 330.1 amount)

Example 2. Construction of leuA Gene-Deleted Corynebacterium glutamicum Mutant Strain

[0060] FIG. 1 shows the synthetic pathway of citramalate. Citramalate is produced by the citramalate synthase (cimA) gene in E. coli, but the cimA gene does not exist in Corynebacterium glutamicum. However, since it is known that the leuA gene having high homology to the cimA gene plays the role of citramalate synthase in Corynebacterium glutamicum, the present inventors have attempted to reduce the production of citramalate as a by-product by disrupting 2-isopropylmalate synthase (leuA) and save the substrate acetyl-CoA which is used in the production of L-glutamic acid, thereby further increasing L-glutamic acid productivity. The nucleotide sequence of the leuA gene is set forth in SEQ ID NO: 14.

Example 2-1. Construction of Vector for Disruption of leuA Gene

[0061] Corynebacterium glutamicum KCTC 11558BP having L-glutamic acid-producing ability was used for disruption of the leuA gene (NCg10245) in Corynebacterium glutamicum.

[0062] The chromosomal DNA of the Corynebacterium glutamicum KCTC 11558BP strain was isolated. Using the isolated DNA as a template, PCR was performed using each of a set of primers 1 and 2 and a set of primers 3 and 4 for 30 cycles, each consisting of 95° C. for 30 sec, 57° C. for 30 sec, and 72° C. for 60 sec. The obtained PCR products (H1 and H2 in FIG. 2) were purified.

[0063] The purified PCR products were then used as a template for crossover polymerase chain reaction (PCR) and amplified again using a set of primers 1 and 4.

[0064] The 1600-bp crossover PCR product for deleting the leuA gene was purified, digested with Xbal and Sall restriction enzymes (Takara, Japan), and then cloned into a pK19mobSacB vector (Gene, 145: 69-73, 1994) digested with the same restriction enzymes, thereby constructing a vector for disruption of the leuA gene. The constructed vector was named pKLA. FIG. 2 shows the pKLA vector constructed to disrupt the leuA gene in the Corynebacterium glutamicum KCTC 11558BP strain.

TABLE-US-00002 TABLE 2 Primer SEQ ID name Sequence (5′.fwdarw.3′) NO Primer 1 5′-TCTAGACCCTTCCCGGAAACCCCCAC-3′ SEQ ID  NO: 1 Primer 2 5′-GGGGTTTCGATCTTGGCAGG-3′ SEQ ID  NO: 2 Primer 3 5′-ACCGCGCGCTGGACGTCAAC-3′ SEQ ID  NO: 3 Primer 4 5′-GTCGACTGGCCGAAACGCTTAGCGCT-3′ SEQ ID  NO: 4

Example 2-2. Transformation of Corynebacterium glutamicum Strain and Construction of leuA-Deleted Mutant Strain

[0065] Using the method of van der Rest (Appl. Microbiol. Biotechnol., 52, 541-545, 1999), the vector pKLA constructed in Example 2-1 was introduced by electroporation into the Corynebacterium glutamicum KCTC 11558BP strain (hereinafter referred to as KCTC 11558BP) made electrocompetent by an electrocompetent cell preparation.

[0066] Specifically, the KCTC 11558BP strain was first 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 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. Electroporation was performed using a Bio-Rad electroporator. The competent cells prepared by the above-described method were placed in an electroporation cuvette (0.2 mm), and the pKLA vector constructed in Example 2-1 was added thereto, followed by electroporation under the conditions of 2.5 kV, 200Ω and 12.5 ρF. Immediately after completion of the electroporation, 1 ml of a regeneration medium (18.5 g/l brain heart infusion, 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 (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, 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.sup.−5 to 10.sup.−7 cells, and plated on an antibiotic-free plate (containing 10% sucrose), and a strain having no kanamycin resistance and grown in the medium containing 10% sucrose was selected. The selected strain was confirmed to be an leuA gene-deleted Corynebacterium glutamicum strain using a set of primers 1 and 4 described in Table 2 above, and the leuA gene-deleted strain was name “I10”. FIG. 3 shows the positions of primers 1 and 4 in the leuA gene-deleted region.

Example 2-3. Confirmation of Auxotrophy of leuA-Deleted Strain (110)

[0067] The leuA-deleted strain (hereinafter referred to as I10) constructed in Example 2-2 becomes a leucine auxotrophic strain due to deletion of the leuA gene. To confirm this fact, the strain was plated in each of a minimal medium prepared to have the composition shown in Table 3 below and the minimal medium supplemented with leucine and then was cultured at 30° C. for 24 hours, and the degree of growth thereof was measured. The results are shown in Table 4 below.

TABLE-US-00003 TABLE 3 Composition of minimal medium Glucose 20 g/L MgSO.sub.4. 7H.sub.2O 0.5 g/L FeSO.sub.4. 7H.sub.2O 20 mg/L MnSO.sub.4. 5H.sub.2O 20 mg/L Urea 2.5 g/L (NH.sub.4) .sub.2SO.sub.4 5 g/L KH.sub.2PO.sub.4 1.5 g/L K.sub.2HPO.sub.4 1.5 g/L Thiamine-HCl 200 μg/L Biotin 200 μg/L Nicotinic acid 200 μg/L Vitamin B2 200 μg/L Cyanocobalamine 100 μg/L

[0068] As shown in Table 4 below, the growth of the 110 strain decreased rapidly compared to the growth of the parent strain Corynebacterium glutamicum KCTC 11558BP strain in the minimal medium not supplemented with leucine, and was restored in the minimal medium supplemented with leucine. Thus, the deletion of the leuA gene in the 110 strain was confirmed.

TABLE-US-00004 TABLE 4 KCTC 11558BP I10 Minimal medium ++++ + Minimal medium + ++++ ++++ 50 ppm leucine

Example 3. Analysis of L-Glutamic Acid and Citramalate Production in leuA Gene-Deleted Corynebacterium glutamicum Strain (I10)

[0069] Using an active plate medium prepared to have the composition shown in Table 5 below, each of the leuA-deleted strain (hereinafter referred to as I10) and Corynebacterium glutamicum KCTC 11558BP (hereinafter referred to as KCTC 11558BP) was cultured at 30° C. for 24 hours. Thereafter, 10 ml of an L-glutamic acid flask medium prepared to have the composition shown in Table 6 below was placed in a 100-ml flask and inoculated with a loop of each of the cultured strains which were then cultured at 30° C. and 200 rpm for 48 hours. After completion of culture, the cultures were used to perform the analysis of L-glutamic acid production and intracellular metabolites. The analysis of L-glutamic acid production and relative citramalate amount was performed in the same manner as described in Example 1.

[0070] Table 7 below shows the L-glutamic acid production and relative citramalate amount in each of I10 and KCTC 11558BP. As shown in Table 7, it could be confirmed that, in the I10 strain, the L-glutamic acid production increased and the relative citramalate amount significantly decreased.

TABLE-US-00005 TABLE 5 Composition of active plate medium Glucose 5 g/L Yeast extract 10 g/L Urea 3 g/L KH.sub.2PO.sub.4 1 g/L Biotin 2 μ/L Soybean hydrolyzate 0.1% v/v Leucine 50 mg/L Agar 20 g/L pH 7.5

TABLE-US-00006 TABLE 6 Composition of L-glutamic acid flask medium Glucose 70 g/L MgSO.sub.4. 7H.sub.2O 0.4 g/L Urea 2 g/L KH.sub.2PO.sub.4 1 g/L Soybean hydrolyzate 1.5% v/v (NH.sub.4) .sub.2SO.sub.4 5 g/L FeSO.sub.4. 7H.sub.2O 10 mg/L MnSO.sub.4. 5H.sub.2O 10 mg/L Thiamine-HCl 200 μg/L Biotin 2 μg/L Calcium carbonate 50 g/L pH 7.6

TABLE-US-00007 TABLE 7 KCTC 11558BP I10 L-glutamic acid  37.5 39.7 production (g/L) Citramalate (relative 320.4  8.5 amount)

Example 4. Construction of YggB-Mutated Strain

[0071] Since it was confirmed from the results of metabolite analysis of the leuA-deleted strain (I10) that a lot of L-glutamic acid was accumulated inside the cells, the present inventors attempted to enhance the release of L-glutamic acid.

[0072] It is known that YggB is a mechanosensitive channel homolog and plays an important role in the release of L-glutamic acid in Corynebacterium glutamicum. The amino acid sequence of YggB protein is set forth in SEQ ID NO: 15. FIG. 4a shows the structure of the YggB protein. The structure of the YggB protein was analyzed to find a mutation capable of enhancing the release of L-glutamic acid.

[0073] FIG. 4b shows the pore structure of the YggB protein having a substitution of alanine for leucine at position 93 in the amino acid sequence of the YggB protein, and FIG. 4c shows the pore structure of the YggB protein having a substitution of alanine for leucine at position 109 in the amino acid sequence of the YggB protein.

Example 4-1. Construction of Strain Having Substitution of Amino Acid at Position 93 in Amino Acid Sequence of YggB Protein

[0074] As shown in FIG. 4b, it was assumed that, when leucine at position 93 in the amino acid sequence of the YggB protein was substituted with alanine in order to enlarge the L-glutamic acid channel, the methyl group involved in the narrow portion inside the pore would be removed, so that the diameter of the pore would increase. Based on this assumption, a mutant strain was constructed.

[0075] The chromosomal DNA of the Il0 strain constructed in Example 2-2 was isolated. Using the isolated DNA as a template, PCR was performed using each of a set of primers 5 and 6 and a set of primers 7 and 8 for 30 cycles, each consisting of 95° C. for 30 sec, 57° C. for 30 sec, and 72° C. for 60 sec. The obtained PCR products were purified. The purified PCR products were used as a template and amplified by crossover PCR using primers 5 and 8. The amplified product was sequenced to confirm mutation, and then digested with Xbal and Sall restriction enzymes (Takara, Japan). A pK19mobSacB vector was digested with the same restriction enzymes and ligated with the amplified crossover PCR product, thereby constructing a vector. The constructed vector was named pKY93. The primer sequences used are shown in Table 8 below. FIG. 5 shows the pKY93 vector for substituting the amino acid at position 93 in the amino acid sequence of the YggB protein in the 110 strain.

TABLE-US-00008 TABLE 8 SEQ ID Primer Sequence (5′.fwdarw.3′) NO Primer  5′-TCTAGATTGAGAAGCTGCCACATTCAC-3′ SEQ ID 5 NO: 5 Primer  5′- SEQ ID 6 GCAGCGCCCGCGGCAGAGAAACCAAAAGCC-3′ NO: 6 Primer  5′- SEQ ID 7 CTCTGCCGCGGGCGCTGCGATTCCGGCAAC-3′ NO: 7 Primer  5′- SEQ ID 8 GTCGACGATCTGGTTTTGCTGTTTCTTCCGG-3′ NO: 8 Primer  5′- SEQ ID 9 GACTGCGCACCAGCACCAATGGCAGCTGAC-3′ NO: 9 Primer  5′- SEQ ID 10 GGTGCTGGTGCGCAGTCGATTGTTGCGGAC-3′ NO: 10 Primer  5′-TCTAGAGGAATCAGGA SEQ ID 11 TTCTCACAAAGTTCAGGC-3′ NO: 11 Primer  5′- SEQ ID 12 CGGAATCGCAGTGCCCGCGAGAGAGAAACC-3′ NO: 12 Primer  5′- SEQ ID 13 CGCGGGCACTGCGATTCCGGCAACCATTGC-3′ NO: 13

[0076] In the same manner as in Example 2-2, the pKY93 vector was introduced into the 110 strain, and the transformed strain was selected and named 110-93. The 110-93 mutant strain had a substitution of alanine for leucine at amino acid position 93 in the YggB protein.

Example 4-2. Construction of Strain Having Substitution of Amino Acid at Position 109 in Amino Acid Sequence of YggB Protein

[0077] As shown in FIG. 4c, it was assumed that, when leucine at position 109 in the amino acid sequence of the YggB protein was substituted with alanine in order to enlarge the L-glutamic acid channel, the methyl group involved in the narrow portion inside the pore would be removed, so that the diameter of the pore would increase. Based on this assumption, a mutant strain was constructed.

[0078] The chromosomal DNA of the I10 strain constructed in Example 2-2 was isolated. Using the isolated DNA as a template, PCR was performed using each of a set of primers 5 and 9 and a set of primers 10 and 8 for 30 cycles, each consisting of 95° C. for 30 sec, 57° C. for 30 sec, and 72° C. for 60 sec. The obtained PCR products were purified. The purified PCR products were used as a template and amplified by crossover PCR using primers 5 and 8. The amplified product was sequenced to confirm mutation, and then digested with Xbal and Sall restriction enzymes (Takara, Japan). A pK19mobSacB vector was digested with the same restriction enzymes and ligated with the amplified crossover PCR product, thereby constructing a vector. The constructed vector was named pKY109. The primer sequences used are shown in Table 8 above.

[0079] In the same manner as in Example 2-2, the pKY109 vector was introduced into the I10 strain, and the transformed strain was selected and named 110-109. The 110-109 mutant strain had a substitution of alanine for leucine at amino acid position 109 in the YggB protein.

Example 5. Confirmation of L-Glutamic Acid Productivities of Mutant Strains

[0080] The amount of L-glutamic acid produced by each of the I10, 110-93 and 110-109 strains at the flask scale and the time required for production of L-glutamic acid were measured in the same manner as in Examples 1 and 3, and the results are shown in Table 9 below.

TABLE-US-00009 TABLE 9 I10 I10-93 I10-109 L-glutamic acid 39 42.5 45.2 production (g/L) Time (hr) 48 48   44  

[0081] As shown in Table 9 above, the L-glutamic acid production in each of the 110-93 and 110-109 mutant strains in which enlargement of the pore of the YggB protein was induced increased compared to that in the I10 strain. In particular, in the case of the 110-109 strain, the L-glutamic acid production significantly increased compared to that in the I10 strain, and the fermentation time was also shortened from 48 hours to 44 hours, indicating that the 110-109 strain may efficiently produce L-glutamic acid.