Acetohydroxy acid synthase variant and a microorganism comprising the same

Abstract

The present disclosure relates to a novel acetohydroxy acid synthase, a microorganism comprising the same, or a method for producing an L-branched-chain amino acid using the same.

Claims

1. A polynucleotide encoding an acetohydroxy acid synthase variant having acetohydroxy acid synthase activity, wherein the acetohydroxy acid synthase variant comprises an acetohydroxy acid synthase large subunit (acetolactate synthase large subunit; IlvB protein) having at least 95% sequence identity with the polypeptide of SEQ ID NO: 1, wherein said large subunit comprises a substitution at the position corresponding to position 96 of the polypeptide of SEQ ID NO: 1, and wherein the amino acid at the position corresponding to position 96 of the polypeptide of SEQ ID NO: 1 is a serine, cysteine, or alanine.

2. The polynucleotide according to claim 1, wherein the acetohydroxy acid synthase large subunit further comprises a substitution at the position corresponding to position 503 of the polypeptide of SEQ ID NO:1, and wherein the amino acid at the position corresponding to position 503 of the polypeptide of SEQ ID NO:1 is a glutamine.

3. A microorganism of the genus Corynebacterium producing an L-branched-chain amino acid, wherein the microorganism comprises an acetohydroxy acid synthase variant or a polynucleotide encoding the acetohydroxy acid synthase variant, wherein the acetohydroxy acid synthase variant comprises an acetohydroxy acid synthase large subunit (acetolactate synthase large subunit; IlvB protein) having at least 95% sequence identity with the polypeptide of SEQ ID NO: 1, wherein said large subunit comprises a substitution at the position corresponding to position 96 of the polypeptide of SEQ ID NO: 1, and wherein the amino acid at the position corresponding to position 96 of the polypeptide of SEQ ID NO: 1 is a serine, cysteine, or alanine.

4. The microorganism according to claim 3, wherein the acetohydroxy acid synthase large subunit further comprises a substitution at the position corresponding to position 503 of the polypeptide of SEQ ID NO:1, and wherein the amino acid at the position corresponding to position 503 of the polypeptide of SEQ ID NO:1 is a glutamine.

5. The microorganism according to claim 3, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.

6. The microorganism according to claim 3, wherein the L-branched-chain amino acid is L-valine or L-leucine.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Hereinafter, the present disclosure will be described in more detail with reference to the following Examples. However, these Examples are for illustrative purposes only, and the present disclosure is not intended to be limited by these Examples.

Example 1: Preparation of Library of DNA Encoding Modified Acetohydroxy Acid Synthase Using Artificial Mutagenesis

(2) In this Example, a vector library for primary crossover-insertion within the chromosome for obtaining acetohydroxy acid synthase variants was prepared by the following method. Error-prone PCR was performed for ilvB gene (SEQ ID NO: 2) encoding acetohydroxy acid synthase (SEQ ID NO: 1) derived from Corynebacterium glutamicum ATCC14067, and thereby ilvB gene variants (2,395 bp) of ilvB gene variants randomly introduced with a mutation(modification) of nucleotide substitution were obtained. The error-prone PCR was performed using the GenemorphII Random Mutagenesis Kit (Stratagene), using the genomic DNA of Corynebacterium glutamicum ATCC14067 as a template along with primer 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4).

(3) TABLE-US-00001 primer 1 (SEQ ID NO: 3): 5′-AACCG GTATC GACAA TCCAA T-3′ primer 2 (SEQ ID NO: 4): 5′-GGGTC TCTCC TTATG CCTC-3′

(4) The error-prone PCR was performed such that modifications can be introduced into the amplified gene fragment at a ratio of 0 to 3.5 mutations per 1 kb of the amplified gene fragment. PCR was performed for a total of 30 cycles as follows: denaturation at 96° C. for 30 sec, annealing at 53° C. for 30 sec, and polymerization at 72° C. for 2 min.

(5) The amplified gene fragments were connected to the pCR2.1-TOPO vector (hereinafter, “pCR2.1”) using the pCR2.1-TOPO TA Cloning Kit (Invitrogen), transformed into E. coli DH5a, and plated on a solid LB medium containing kanamycin (25 mg/L). 20 of the transformed colonies were selected, and their nucleotide sequences were analyzed after obtaining their plasmids. As a result, it was confirmed that modifications were introduced at different locations at a frequency of 2.1 mutations/kb. Plasmids were extracted from about 20,000 transformed E. coli colonies, and they were named “pCR2.1-ilvB(mt) library”.

(6) Additionally, a plasmid including the wild-type ilvB gene to be used as a control was prepared. PCR was performed using the genomic DNA of Corynebacterium glutamicum ATCC14067 as a template along with primer 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4), under the same conditions described above. For the polymerase, PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used, and the prepared plasmid was named “pCR2.1-ilvB(WT)”.

Example 2: Preparation of ilvB-Deficient Strain

(7) An ilvB-deficient strain for the introduction of the pCR2.1-ilvB(mt) library was prepared using the KCCM11201P strain (KR Patent No. 10-1117022) as the parent strain.

(8) To prepare an ilvB-deficient vector, PCR was performed using the chromosomal DNA of the wild-type Corynebacterium glutamicum ATCC14067 as a template and a primer set of primer 3 (SEQ ID NO: 5) and primer 4 (SEQ ID NO: 6) and a primer set of primer 5 (SEQ ID NO: 7) and primer 6 (SEQ ID NO: 8).

(9) TABLE-US-00002 primer 3 (SEQ ID NO: 5): 5′-GCGTC TAGAG ACTTG CACGA GGAAA CG-3′ primer 4 (SEQ ID NO: 6): 5′-CAGCC AAGTC CCTCA GAATT GATGT AGCAA TTATC C-3′ primer 5 (SEQ ID NO: 7): 5′-GGATA ATTGC TACAT CAATT CTGAG GGACT TGGCT G-3′ primer 6 (SEQ ID NO: 8): 5′-GCGTC TAGAA CCACA GAGTC TGGAG CC-3′

(10) PCR was performed as follows: denaturation at 95° C. for 5 min; 30 cycles of denaturation at 95° C. for 30 sec, annealing at 55° C. for 30 sec, and polymerization at 72° C. for 30 sec; and polymerization at 72° C. for 7 min.

(11) As a result, a 731 bp DNA fragment (SEQ ID NO: 9), which includes the upstream region of the promoter of ilvB gene, and a 712 bp DNA fragment (SEQ ID NO: 10), which includes the 3′ terminus of the ilvB gene, were obtained.

(12) PCR was performed using the amplified DNA fragments (SEQ ID NOS: 9 and 10) and a primer set of primer 3 (SEQ ID NO: 5) and primer 6 (SEQ ID NO: 8). PCR was performed as follows: denaturation at 95° C. for 5 min; 30 cycles of denaturation at 95° C. for 30 sec, annealing at 55° C. for 30 sec, and polymerization at 72° C. for 60 sec; and polymerization at 72° C. for 7 min.

(13) As a result, a 1,407 bp DNA fragment (SEQ ID NO: 11, hereinafter “ilvB fragment”), in which a DNA fragment including the upstream region of the promoter of ilvB gene and a DNA fragment including the 3′ terminus of the ilvB gene are linked, was amplified.

(14) The pDZ vector (KR Patent No. 10-0924065), which cannot replicate in Corynebacterium glutamicum, and the ilvB gene fragment amplified above were each treated with restriction enzyme XbaI, ligated using a DNA ligase, and cloned. The obtained plasmid was named “pDZ-ilvB”.

(15) The pDZ-ilvB was transformed into Corynebacterium glutamicum KCCM11201P by the electroporation method (Appl. Microbiol. Biothcenol. (1999) 52: 541-545), and the transformed strains were obtained in selection media containing kanamycin (25 mg/L) and 2 mM each of L-valine, L-leucine, and L-isoleucine. The strain, in which the gene is inactivated by the ilvB gene fragment inserted into the genome during the secondary crossover process, was obtained, and the strain was named KCCM11201PilvB.

Example 3: Preparation of Library of Modified Strains of Acetohydroxy Acid Synthase and Selection of Strains with Increased Ability of Producing L-Amino Acids

(16) The above-prepared KCCM11201PilvB strain was transformed by homologous recombination using the above-prepared pCR2.1-ilvB(mt) library, and the transformant was plated on a complex plate medium containing kanamycin (25 mg/L) and about 10,000 colonies were obtained therefrom. The colonies were named KCCM11201PilvB/pCR2.1-ilvB(mt)-1 to KCCM11201PilvB/pCR2.1-ilvB(mt)-10000.

(17) Additionally, the above-prepared pCR2.1-ilvB(WT) vector was transformed into the KCCM11201PilvB strain to prepare a control strain and the strain was named KCCM11201PilvB/pCR2.1-ilvB(WT).

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

(19) Glucose (10 g), Peptone (10 g), Beef Extract (5 g), Yeast Extract (5 g), Brain Heart Infusion (18.5 g), NaCl (2.5 g), Urea (2 g), Sorbitol (91 g), Agar (20 g) (based on 1 L of distilled water)

(20) About 25,000 colonies obtained above were each inoculated into a selective medium (300 μL) containing the components described below and cultured in a 96-deep well plate at 32° C. at a rate of 1,000 rpm for 24 hours. The amounts of L-amino acids produced in the culture were analyzed by the ninhydrin method (J. Biol. Chem. 1948. 176: 367-388). Upon completion of the cultivation, 10 μL of the culture supernatant and 190 μL of a ninhydrin reaction solution were reacted at 65° C. for 30 minutes. The absorbance was measured at wavelength 570 nm using a spectrophotometer and was compared to that of the control, i.e., KCCM11201PilvB/pCR2.1-ilvB(WT), and about 213 modified strains showing an absorbance with an at least 10% increase were selected. Other colonies showed similar or reduced absorbance compared to that of the control.

(21) <Selective Medium (pH 8.0)>

(22) Glucose (10 g), (NH.sub.4).sub.2SO.sub.4 (5.5 g), MgSO.sub.4.7H.sub.2O (1.2 g), KH.sub.2PO.sub.4 (0.8 g), K.sub.2HPO.sub.4 (16.4 g), Biotin (100 μg), Thiamine HCl (1,000 μg), Calcium-Pantothenic Acid (2,000 μg), and Nicotinamide (2,000 μg) (based on 1 L of distilled water)

(23) The above method was repeatedly performed for the selected 213 strains, and the top 60 kinds of strains with an improved ability of producing L-amino acids compared to that of KCCM11201PilvB/pCR2.1-ilvB(WT) were selected.

Example 4: Confirmation of L-Valine-Producing Ability of Strains Selected from the Library of Modified Strains of Acetohydroxy Acid Synthase

(24) The 60 kinds of strains selected in Example 3 were analyzed with respect to their L-valine-producing abilities after culturing them by the following method.

(25) Each of the strains was inoculated into a 250 mL corner-baffle flask containing 25 mL of a production medium, respectively, and cultured in a shaking incubator (200 rpm) at 30° C. for 20 hours. Then, each of the 250 mL corner-baffle flasks containing 24 mL of the culture, which contained the components described below, was inoculated with 1 mL of a seed culture broth, and cultured with shaking (200 rpm) at 30° C. for 72 hours. The concentration of L-valine in each culture was analyzed by HPLC.

(26) <Production Medium (pH 7.0)>

(27) Glucose (100 g), (NH.sub.4).sub.2SO.sub.4 (40 g), Soybean Protein (2.5 g), Corn Steep Solids (5 g), Urea (3 g), KH.sub.2PO.sub.4 (1 g), MgSO.sub.4.7H.sub.2O (0.5 g), Biotin (100 μg), Thiamine HCl (1,000 μg), Calcium-Pantothenic Acid (2,000 μg), Nicotinamide (3,000 μg), and CaCO.sub.3 (30 g) (based on 1 L of distilled water)

(28) Among the selected 60 kinds of strains, 2 kinds of strains showing an increase in L-valine concentration were selected, and the cultivation and analysis were performed repeatedly. The analysis results of the L-valine concentration are shown in Table 1 below. The remaining 58 kinds of strains actually showed a decrease in L-valine concentration.

(29) TABLE-US-00003 TABLE 1 Concentration of L-Valine Produced by Two Selected Strains of KCCM11201PilvB/pCR2.1-ilvB(mt) L-Valine (g/L) Strain Batch 1 Batch 2 Batch 3 Mean Control KCCM11201PilvB/pCR2.1- 2.7 2.9 2.9 2.8 ilvB(WT) 1 KCCM11201PilvB/pCR2.1- 3.1 3.5 3.4 3.3 ilvB(mt)-5602 2 KCCM11201PilvB/pCR2.1- 2.9 3.3 3.1 3.1 ilvB(mt)-7131

(30) As a result of the analysis of the L-valine concentration of the 2 selected strains, it was confirmed that the L-valine yield of the two strains was increased by 20.7% at maximum compared to that of the control strain, KCCM11201PilvB/pCR2.1-ilvB(WT).

Example 5: Confirmation of ilvB Gene Modification in Strains Selected from a Library of Modified Strains of Acetohydroxy Acid Synthase

(31) To confirm the random modifications introduced into the acetohydroxy acid synthase of the 2 selected strains in Example 4, the nucleotide sequences of ilvB gene were analyzed. For determining the nucleotide sequences, PCR was performed using a primer set of primer 7 (SEQ ID NO: 12) and primer 8 (SEQ ID NO: 13).

(32) TABLE-US-00004 primer 7 (SEQ ID NO: 12): 5′-CGCTT GATAA TACGC ATG-3′ primer 8 (SEQ ID NO: 13): 5′-GAACA TACCT GATAC GCG-3′

(33) The obtained modified ilvB gene fragments were each subjected to nucleotide sequence analysis, and the results were compared to the nucleotide sequence of wild-type ilvB gene (i.e., SEQ ID NO: 2). As a result, the nucleotide sequences of modified ilvB gene were confirmed, and the amino acid sequences of modified acetohydroxy acid synthase proteins were confirmed. The information of the selected two kinds of modified acetohydroxy acid synthase proteins is shown in Table 2 below.

(34) TABLE-US-00005 TABLE 2 Information of Selected Two Kinds of Modified Acetohydroxy Acid Synthase Proteins of KCCM11201P/pCR2.1-ilvB(mt) Amino Acid Modification of Acetohydroxy Acid Strain Synthase KCCM11201PilvB/pCR2.1-ilvB(mt)-5602 W503Q KCCM11201PilvB/pCR2.1-ilvB(mt)-7131 T96S

Example 6: Preparation of Vector for Introducing Modification in Acetohydroxy Acid Synthase

(35) To confirm the effects of the modified acetohydroxy acid synthase proteins which were confirmed in Example 5, a vector capable of introducing the modified acetohydroxy acid synthase proteins onto the chromosome was prepared.

(36) Based on the confirmed nucleotide sequences, a primer set of the primer 9 (SEQ ID NO: 14) and the primer 10 (SEQ ID NO: 15) and a primer set of the primer 11 (SEQ ID NO: 16) and the primer 12 (SEQ ID NO: 17), in which an XbaI restriction site was inserted at the 5′ end, were synthesized. Then, PCR was performed using each of the selected two kinds of chromosomal DNAs as a template using these primer sets, and thereby the modified ilvB gene fragments were amplified. PCR was performed as follows: denaturation at 94° C. for 5 min; 30 cycles of denaturation at 94° C. for 30 sec, annealing at 56° C. for 30 sec, and polymerization at 72° C. for 2 min; and polymerization at 72° C. for 7 min.

(37) TABLE-US-00006 primer 9 (SEQ ID NO: 14): 5′-CGCTC TAGAC AAGCA GGTTG AGGTT CC-3′ primer 10 (SEQ ID NO: 15): 5′-CGCTC TAGAC ACGAG GTTGA ATGCG CG-3′ primer 11 (SEQ ID NO: 16): 5′-CGCTC TAGAC CCTCG ACAAC ACTCA CC-3′ primer 12 (SEQ ID NO: 17): 5′-CGCTC TAGAT GCCAT CAAGG TGGTG AC-3′

(38) The two kinds of gene fragments amplified by PCR were treated with XbaI to obtain the respective DNA fragments, and linked these fragments to the pDZ vector for chromosomal introduction, which includes an XbaI restriction site therein, transformed into E. coli DH5α, and the transformants were spread on an LB solid medium containing kanamycin (25 mg/L).

(39) The colonies transformed with a vector inserted with a target gene were selected by PCR, and the plasmids were obtained by a commonly known plasmid extraction method. These plasmids were named pDZ-ilvB(W503Q) and pDZ-ilvB(T96S), each according to the modifications inserted into the ilvB gene.

Example 7: Preparation of KCCM11201P-Derived Strains with Modification in Acetohydroxy Acid Synthase and Comparison of their L-Valine-Producing Abilities

(40) The two kinds of vectors introduced with novel modifications prepared in Example 6 were each transformed into the Corynebacterium glutamicum KCCM11201P, which is a strain producing L-valine, by a two-step homologous chromosome recombination. Then, the strains introduced with the ilvB gene modification on the chromosome were selected by the analysis of nucleotide sequences. The strains introduced with the ilvB gene modification were named KCCM11201P::ilvB(W503Q) and KCCM11201P::ilvB(T96S). Additionally, the pDZ-ilvB(T96S) vector, between the vectors introduced with the above modification, was transformed into the KCCM11201P::ilvB(W503Q) strain prepared above. Then, the strains into which the two kinds of modifications on the chromosome were introduced were named KCCM11201P::ilvB(W503Q/T96S).

(41) The strains were cultured in the same manner as in Example 4, and the L-valine concentrations were analyzed from the cultured strains (Table 3).

(42) TABLE-US-00007 TABLE 3 Concentration of L-Valine Produced by KCCM11201P-Derived Strains Introduced with Modified Acetohydroxy Acid Synthase (g/L) Strain Batch 1 Batch 2 Batch 3 Mean Control KCCM11201P 2.9 2.8 2.8 2.8 1 KCCM11201P::ilvB 3.3 3.2 3.3 3.3 (W503Q) 2 KCCM11201P::ilvB 3.2 3.0 3.1 3.1 (T96S) 3 KCCM11201P::ilvB 3.3 3.4 3.4 3.4 (W503Q/T96S)

(43) As a result, two novel strains introduced with modifications (KCCM11201P::ilvB(W503Q) and KCCM11201P::ilvB(T96S)) showed a maximum increase of 17.8% in L-valine-producing ability compared to the parent strain, and the strain introduced with both modifications (KCCM11201P::ilvB(W503Q/T96S) showed an increase of 21.4% in L-valine-producing ability compared to the parent strain.

(44) Accordingly, considering that acetohydroxy acid synthase is the first enzyme in the biosynthesis pathways of L-branched-chain amino acids, the acetohydroxy acid synthase large subunit variants of the present disclosure are expected to have an effect on the production increase of L-isoleucine and L-leucine as well as L-valine.

(45) The present inventors have named the strains with an improved ability of L-valine production (i.e., KCCM11201P::ilvB(W503Q) and KCCM11201P::ilvB(T96S)) as Corynebacterium glutamicum KCJ-0793 and Corynebacterium glutamicum KCJ-0796, and deposited them with the Korean Culture Center of Microorganisms (KCCM) on Jan. 25, 2016, under the Accession Numbers KCCM11809P and KCCM11810P.

Example 8: Preparation of Overexpression Vector for L-Valine Biosynthesis Containing DNA Encoding Modified Acetohydroxy Acid Synthase

(46) As a control group, an overexpression vector for L-valine biosynthesis was prepared from Corynebacterium glutamicum KCCM11201P, which is a strain producing L-valine. Additionally, overexpression vectors for L-valine biosynthesis, in which DNAs encoding acetohydroxy acid synthase modified from each of KCCM11201P::ilvB(W503Q) and KCCM11201P::ilvB(T96S) prepared in Example 7 are included, were prepared.

(47) For the preparation of the above vectors, the primer 13 (SEQ ID NO: 18), in which a BamHI restriction site was inserted at the 5′ end, and the primer 14 (SEQ ID NO: 19), in which an XbaI restriction site was inserted at the 3′ end, were synthesized. Using the primer set, PCR was performed using each of the chromosomal DNAs of Corynebacterium glutamicum KCCM11201P (i.e., a strain producing L-valine) and the strains prepared in Example 7 (i.e., KCCM11201P::ilvB(W503Q) and KCCM11201P::ilvB(T96S)) as a template, and thereby two kinds of modified ilvBN gene fragments were amplified. PCR was performed as follows: denaturation at 94° C. for 5 min; 30 cycles of denaturation at 94° C. for 30 sec, annealing at 56° C. for 30 sec, and polymerization at 72° C. for 4 min; and polymerization at 72° C. for 7 min.

(48) TABLE-US-00008 primer 13 (SEQ ID NO: 18): 5′-CGAGG ATCCA ACCGG TATCG ACAAT CCAAT-3′ primer 14 (SEQ ID NO: 19): 5′-CTGTC TAGAA ATCGT GGGAG TTAAA CTCGC-3′

(49) The two kinds of gene fragments amplified by PCR were treated with BamHI and XbaI to obtain their respective DNA fragments. These DNA fragments were linked to the pECCG117 overexpression vector having BamHI and XbaI restriction sites, transformed into E. coli DH5α, and plated on a solid LB medium containing kanamycin (25 mg/L).

(50) The colonies transformed with a vector inserted with a target gene were selected by PCR and the plasmids were obtained by a commonly known plasmid extraction method. These plasmid were named pECCG117-ilvBN, pECCG117-ilvB(W503Q)N, and pECCG117-ilvB(T96S)N, each according to the modifications inserted into the ilvB gene.

Example 9: Preparation of Overexpression Vector for L-Valine Biosynthesis Containing DNA Encoding Modified Acetohydroxy Acid Synthase in which an Amino Acid is Substituted with Another Amino Acid at the Same Position

(51) In the modified acetohydroxy acid synthase proteins confirmed in Example 5, to confirm the effects of position in modification, vectors were prepared in which the 96.sup.th amino acid is substituted with an amino acid other than threonine or serine, and the 503.sup.rd amino acid is substituted with an amino acid other than tryptophan or glutamine.

(52) Specifically, overexpression vectors for L-valine biosynthesis, in which a modification where the 503.sup.rd amino acid of acetohydroxy acid synthase is substituted with asparagine or leucine or a modification where the 96.sup.th amino acid is substituted with alanine or cysteine, were prepared from Corynebacterium glutamicum KCCM11201P, which is a strain producing L-valine. The substituted amino acids are only examples of representative amino acids that can be substituted, and the amino acids are not limited thereto.

(53) For the preparation of these vectors, first, PCR was performed using the chromosomal DNA of Corynebacterium glutamicum KCCM11201P as a template and a primer set of the primer 13 (SEQ ID NO: 18) and the primer 15 (SEQ ID NO: 20) and a primer set of the primer 16 (SEQ ID NO: 21) and the primer 14 (SEQ ID NO: 19), and thereby an about 2,041 bp DNA fragment having a BamHI restriction site at the 5′ end and a 1,055 bp DNA fragment having an XbaI restriction site at the 3′ end were amplified. PCR was performed as follows: denaturation at 94° C. for 5 min; 30 cycles of denaturation at 94° C. for 30 sec, annealing at 56° C. for 30 sec, and polymerization at 72° C. for 2 min; and polymerization at 72° C. for 7 min.

(54) TABLE-US-00009 primer 15 (SEQ ID NO: 20): 5′-CTTCA TAGAA TAGGG TCTGG TTTTG GCGAA CCATG CCCAG-3′ primer 16 (SEQ ID NO: 21): 5′-CTGGG CATGG TTCGC CAAAA CCAGA CCCTA TTCTA TGAAG-3′

(55) Then, PCR was performed using the two amplified DNA fragments as a template and a primer set of the primer 13 (SEQ ID NO: 18) and the primer 14 (SEQ ID NO: 19). PCR was performed as follows: denaturation at 94° C. for 5 min; 30 cycles of denaturation at 94° C. for 30 sec, annealing at 56° C. for 30 sec, and polymerization at 72° C. for 4 min; and polymerization at 72° C. for 7 min.

(56) As a result, an ilvBN gene fragment in which a modification where the 503.sup.rd amino acid of acetohydroxy acid synthase is substituted with asparagine was obtained.

(57) In the same manner, PCR was performed using the chromosomal DNA of Corynebacterium glutamicum KCCM11201P as a template and a primer set of the primer 13 (SEQ ID NO: 18) and the primer 17 (SEQ ID NO: 22) and a primer set of the primer 18 (SEQ ID NO: 23) and the primer 14 (SEQ ID NO: 19), and thereby an about 2,041 bp DNA fragment having a BamHI restriction site at the 5′ end and a 1,055 bp DNA fragment having an XbaI restriction site at the 3′ end were amplified.

(58) TABLE-US-00010 primer 17 (SEQ ID NO: 22): 5′-CTTCA TAGAA TAGGG TCTGC AGTTG GCGAA CCATG CCCAG-3′ primer 18 (SEQ ID NO: 23): 5′-CTGGG CATGG TTCGC CAACT GCAGA CCCTA TTCTA TGAAG-3′

(59) Then, PCR was performed using the two amplified DNA fragments as a template and a primer set of the primer 13 (SEQ ID NO: 18) and the primer 14 (SEQ ID NO: 19).

(60) As a result, an ilvBN gene fragment in which a modification where the 503.sup.rd amino acid of acetohydroxy acid synthase is substituted with leucine was obtained.

(61) In the same manner, PCR was performed using the chromosomal DNA of Corynebacterium glutamicum KCCM11201P as a template and a primer set of the primer 13 (SEQ ID NO: 18) and the primer 19 (SEQ ID NO: 24) and a primer set of the primer 20 (SEQ ID NO: 25) and the primer 14 (SEQ ID NO: 19), and thereby an about 819 bp DNA fragment having a BamHI restriction site at the 5′ end and a 2,276 bp DNA fragment having an XbaI restriction site at the 3′ end were amplified.

(62) TABLE-US-00011 primer 19 (SEQ ID NO: 24): 5′-GGTTG CGCCT GGGCC AGATG CTGCA ATGCA GACGC CAAC-3′ primer 20 (SEQ ID NO: 25): 5′-GTTGG CGTCT GCATT GCAGC ATCTG GCCCA GGCGC AACC-3′

(63) Then, PCR was performed using the two amplified DNA fragments as a template and a primer set of the primer 13 (SEQ ID NO: 18) and the primer 14 (SEQ ID NO: 19).

(64) As a result, an ilvBN gene fragment in which a modification where the 96.sup.th amino acid of acetohydroxy acid synthase is substituted with alanine was obtained.

(65) In the same manner, PCR was performed using the chromosomal DNA of Corynebacterium glutamicum KCCM11201P as a template and a primer set of the primer 13 (SEQ ID NO: 18) and the primer 21 (SEQ ID NO: 26) and a primer set of the primer 22 (SEQ ID NO: 27) and the primer 14 (SEQ ID NO: 19), and thereby an about 819 bp DNA fragment having a BamHI restriction site at the 5′ end and a 2,276 bp DNA fragment having an XbaI restriction site at the 3′ end were amplified.

(66) TABLE-US-00012 primer 21 (SEQ ID NO: 26): 5′-GGTTG CGCCT GGGCC AGAGC ATGCA ATGCA GACGC CAAC-3′ primer 22 (SEQ ID NO: 27): 5′-GTTGG CGTCT GCATT GCATG CTCTG GCCCA GGCGC AACC-3′

(67) Then, PCR was performed using the two amplified DNA fragments as a template and a primer set of the primer 13 (SEQ ID NO: 18) and the primer 14 (SEQ ID NO: 19).

(68) As a result, an ilvBN gene fragment in which a modification where the 96.sup.th amino acid of acetohydroxy acid synthase is substituted with cysteine was obtained.

(69) Using the same method as in Example 8, the four kinds PCR-amplified modified gene fragments were treated with restriction enzymes BamHI and XbaI, and thereby the respective DNA fragments were obtained. These DNA fragments were each linked to the overexpression vector pECCG117 having BamHI and XbaI restriction sites, transformed into E. coli DH5a, and plated on a solid LB medium containing kanamycin (25 mg/L).

(70) The colonies transformed with a vector inserted with a target gene were selected by PCR, and the plasmids were obtained by a commonly known plasmid extraction method. These plasmid were each named pECCG117-ilvB(W503N)N, pECCG117-ilvB(W503L)N, pECCG117-ilvB(T96A)N, and pECCG117-ilvB(T96C)N, each according to the sequence of modifications inserted into the ilvB gene.

Example 10: Preparation of Strains in which Wild-Type-Derived Modified Acetohydroxy Acid Synthase is Introduced and Comparison of L-Valine-Producing Abilities

(71) The overexpression vectors for L-valine biosynthesis prepared in Examples 8 and 9 (i.e., pECCG117-ilvBN, pECCG117-ilvB(W503Q)N, pECCG117-ilvB(T96S)N and pECCG117-ilvB(W503N)N, pECCG117-ilvB(W503L)N, pECCG117-ilvB(T96A)N, and pECCG117-ilvB(T96C)N) were each inserted into the wild-type Corynebacterium glutamicum strain (ATCC13032) by electroporation. The prepared strains were each named Corynebacterium glutamicum ATCC13032::pECCG117-ilvBN, Corynebacterium glutamicum ATCC13032::pECCG117-ilvB (W503 Q)N, Corynebacterium glutamicum ATCC13032::pECCG117-ilvB(T96S)N, Corynebacterium glutamicum ATCC13032::pECCG117-ilvB(W503N)N, Corynebacterium glutamicum ATCC13032::pECCG117-ilvB(W503L)N, Corynebacterium glutamicum ATCC13032::pECCG117-ilvB(T96A)N, and Corynebacterium glutamicum ATCC13032::pECCG117-ilvB(T96C)N.

(72) Since those strains which are transformed with these vectors will be provided with kanamycin resistance, the presence of transformation was confirmed by checking the growth of these strains in a medium containing kanamycin at a concentration of 25 mg/L.

(73) Each of the strains was inoculated into a 250 mL corner-baffle flask containing 25 mL of the production medium and cultured with shaking at 200 rpm at 30° C. for 72 hours. The concentration of L-valine in each culture was analyzed by HPLC (Table 4).

(74) TABLE-US-00013 TABLE 4 Concentration of L-valine Production by Strains in Which Wild-Type-Derived Modified Acetohydroxy Acid Synthase is Introduced L-Valine (g/L) Batch Batch Batch Strain 1 2 3 Mean Control ATCC13032::pECCG117-ilvBN 0.1 0.1 0   0.1 1 ATCC13032::pECCG117- 0.8 0.8 0.7 0.8 ilvB(W503Q)N 2 ATCC13032::pECCG117-ilvB(T96S)N 0.4 0.5 0.5 0.5 3 ATCC13032::pECCG117- 0.7 0.6 0.5 0.6 ilvB(W503N)N 4 ATCC13032::pECCG117- 0.7 0.7 0.5 0.5 ilvB(W503L)N 5 ATCC13032::pECCG117-ilvB(T96A)N 0.2 0.3 0.2 0.2 6 ATCC13032::pECCG117-ilvB(T96C)N 0.4 0.3 0.5 0.4

(75) As a result, it was confirmed that the novel modifications in which the 96.sup.th or 503.sup.rd amino acid of acetohydroxy acid synthase is substituted with another amino acid showed a maximum increase of 700% in the L-valine-producing ability compared to the control group. This result confirmed the importance of the 96.sup.th and 503.sup.rd amino acid positions of acetohydroxy acid synthase, and these amino acid positions are expected to affect the ability of producing other branched-chain amino acids as well as L-valine.

Example 11: Preparation of Strains in which Modified Acetohydroxy Acid Synthase is Introduced and Comparison of L-Valine-Producing Abilities

(76) To confirm whether the acetohydroxy acid synthase large subunit variants of the present disclosure have an influence on the increase in the ability of producing other L-branched-chain amino acids, as another embodiment of the L-branched-chain amino acids, the ability of producing L-leucine was examined.

(77) Specifically, the two vectors in which novel modifications were introduced prepared in Example 6 were each transformed by a two-step homologous recombination into the Corynebacterium glutamicum KCCM11661P (Korean Patent Application No. 10-2015-0119785 and Korean Patent Application Publication No. 10-2017-0024653), which is an L-leucine-producing strain. Then, the strains in which the ilvB gene modification is introduced on the chromosome thereof were selected by nucleotide sequence analysis, and the strains in which the ilvB gene modification is introduced were named KCCM11661P::ilvB(W503Q) and KCCM11661P::ilvB(T96S).

(78) The Corynebacterium glutamicum KCCM11661P having resistance to norleucine (NL) is a mutant strain derived from Corynebacterium glutamicum ATCC 14067 and was obtained as follows.

(79) Specifically, the Corynebacterium glutamicum ATCC 14067 was cultured in an activation medium for 16 hours, and the activated strain was inoculated into a seed medium, which was sterilized at 121° C. for 5 minutes, and cultured for 14 hours, and 5 mL of the culture was recovered. The recovered culture was washed with 100 mM citric acid buffer and N-methyl-N′-nitro-N-nitrosoguanidine (NTG) was added thereto to a final concentration of 200 mg/L and treated for 20 minutes, and washed with 100 mM phosphate buffer. The strains treated with NTG were plated on a minimal medium and the death rate was calculated, and as a result, the death rate was shown to be 85%.

(80) To obtain a mutant strain having resistance to norleucine (NL), the NTG-treated strains were plated on a minimal medium containing NL at a final concentration of 20 mM, 30 mM, 40 mM, and 50 mM, respectively. Then, the strains were cultured at 30° C. for 5 days, and thereby an NL-resistant mutant strain was obtained.

(81) <Activation Medium>

(82) Meat Juice (1%), Polypeptone (1%), NaCl (0.5%), Yeast Extract (1%), Agar (2%), pH 7.2

(83) <Seed Medium>

(84) Glucose (5%), Bactopeptone (1%), NaCl (0.25%), Yeast Extract (1%), Urea (0.4%), pH 7.2

(85) <Minimal Medium>

(86) Glucose (1%), Ammonium Sulfate (0.4%), Magnesium Sulfate (0.04%), Monopotassium Phosphate (0.1%), Urea (0.1%), Thiamine (0.001%), Biotin (200 μg/L), Agar (2%), pH 7.0

(87) The thus-obtained mutant strain was named Corynebacterium glutamicum KCJ-24 and deposited at the Korean Culture Center of Microorganisms (KCCM), which is recognized as an international depositary authority under the Budapest Treaty, on Jan. 22, 2015, under the Accession Number KCCM11661P.

(88) The KCCM11661P::ilvB(W503Q) and KCCM11661P::ilvB(T96S) were cultured in the same manner as in Example 4, and the L-leucine concentration in each culture therefrom was analyzed (Table 5).

(89) TABLE-US-00014 TABLE 5 Concentration of L-Leucine Production by Strains in Which KCCM11661P-Dervied Modified Acetohydroxy Acid Synthase is Introduced (g/L) Strain Batch 1 Batch 2 Batch 3 Mean Control KCCM11661P 2.7 2.6 2.9 2.7 1 KCCM11661P:: 3.1 3.3 3.3 3.2 ilvB(W503Q) 2 KCCM11661PP:: 3.0 3.2 3.1 3.1 ilvB(T96S)

(90) The two strains in which novel modifications were introduced (i.e., KCCM11661P::ilvB(W503Q) and KCCM11661P::ilvB(T96S)) showed a maximum increase of 26.9% in the L-leucine-producing ability compared to their parent strain.

Example 12: Preparation of Strains in which KCCM11662P-Derived Modified Acetohydroxy Acid Synthase is Introduced and Comparison of L-Leucine-Producing Abilities

(91) The two vectors in which novel modifications were introduced prepared in Example 6 were each transformed by a two-step homologous recombination into the Corynebacterium glutamicum KCCM11662P (Korean Patent Application No. 10-2015-0119785 and Korean Patent Application Publication No. 10-2017-0024653), which is an L-leucine-producing strain. Then, the strains in which the ilvB gene modification is introduced on the chromosome thereof were selected by nucleotide sequence analysis, and the strains in which the ilvB gene modification is introduced were named KCCM11662P::ilvB(W503Q) and KCCM11662P::ilvB(T96S).

(92) The Corynebacterium glutamicum KCCM11662P having resistance to norleucine (NL) is a mutant strain derived from Corynebacterium glutamicum ATCC 13869 and was obtained as follows.

(93) Specifically, using Corynebacterium glutamicum ATCC 13869 as the parent strain, the strain was cultured in the same manner for obtaining the KCCM11662P of Example 11 and finally an NL-resistant mutant strain was obtained.

(94) The thus-obtained mutant strain was named Corynebacterium glutamicum KCJ-28 and deposited at the Korean Culture Center of Microorganisms (KCCM), which is recognized as an international depositary authority under the Budapest Treaty, on Jan. 22, 2015, under the Accession Number KCCM11662P.

(95) The KCCM11662P::ilvB(W503Q) and KCCM11662P::ilvB(T96S) were cultured in the same manner as in Example 4, and the L-leucine concentration in each culture therefrom was analyzed (Table 6).

(96) TABLE-US-00015 TABLE 6 Concentration of L-Leucine Production by Strains in Which KCCM11662P-Dervied Modified Acetohydroxy Acid Synthase is Introduced (g/L) Strain Batch 1 Batch 2 Batch 3 Mean Control KCCM11662P 3.1 3.0 3.1 3.1 1 KCCM11662P:: 3.5 3.4 3.3 3.4 ilvB(W503Q) 2 KCCM11662PP:: 3.3 3.3 3.2 3.3 ilvB(T96S)

(97) The two strains in which novel modifications were introduced (i.e., KCCM11662P::ilvB(W503Q) and KCCM11662P::ilvB(T96S)) showed a maximum increase of 13.3% in the L-leucine-producing ability compared to their parent strain.

(98) From the foregoing, a skilled person in the art to which the present disclosure pertains will be able to understand that the present disclosure may be embodied in other specific forms without modifying the technical concepts or essential characteristics of the present disclosure. In this regard, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present disclosure. On the contrary, the present disclosure is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalents, and other embodiments that may be included within the spirit and scope of the present disclosure as defined by the appended claims.