Isopropylmalate synthase variant and a method of producing L-leucine using the same

Abstract

A novel modified polypeptide having an isopropylmalate synthase activity, a polynucleotide encoding the same, a microorganism comprising the polypeptide, and a method of producing L-leucine by culturing the microorganism.

Claims

1. A modified polypeptide having an isopropylmalate synthase activity, wherein arginine at position 558 from a N-terminus of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with histidine, alanine or glutamine; and/or glycine at position 561 from a N-terminus of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with arginine or tyrosine.

2. The modified polypeptide according to claim 1, wherein the modified polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOS: 21, 22, 23, 25, 26, 28, 29, 31, 32, 34 and 35.

3. A modified polypeptide having an isopropylmalate synthase activity, wherein arginine at position 558 from an N-terminus of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with histidine, alanine, or glutamine, and glycine at position 561 from an N-terminus of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with aspartic acid, arginine, or tyrosine.

4. The modified polypeptide according to claim 3, wherein the modified polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOS: 27, 30, and 33.

5. A polynucleotide encoding the modified polypeptide of claim 1.

6. The polynucleotide according to claim 5, wherein the polynucleotide consists of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 36, 37, 38, 40, 41, 43, 44, 46, 47, 49 and 50.

7. A polynucleotide encoding the modified polypeptide of claim 2.

8. A polynucleotide encoding the modified polypeptide of claim 3.

9. The polynucleotide according to claim 8, wherein the polynucleotide consists of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 42, 45, and 48.

10. A microorganism of the genus Corynebacterium producing L-leucine, which is transformed with a vector comprising a polynucleotide encoding a modified polypeptide having an isopropylmalate synthase activity, wherein arginine at position 558 from a N-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid residue other than arginine; and/or glycine at position 561 from a N-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid residue other than glycine.

11. A microorganism of the genus Corynebacterium producing L-leucine, comprising the modified polypeptide of claim 1.

12. The microorganism according to claim 11, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.

13. A microorganism of the genus Corynebacterium producing L-leucine, comprising the modified polypeptide of claim 2.

14. A microorganism of the genus Corynebacterium producing L-leucine, comprising the modified polypeptide of claim 3.

15. A microorganism of the genus Corynebacterium producing L-leucine, comprising the modified polypeptide of claim 4.

16. A method of producing L-leucine, comprising: (a) culturing the microorganism of the genus Corynebacterium producing L-leucine according to claim 11 in a medium to produce L-leucine; and (b) recovering L-leucine from the cultured microorganism or the cultured medium.

17. A method of producing L-leucine, comprising: (a) culturing the microorganism of the genus Corynebacterium producing L-leucine according to claim 13 in a medium to produce L-leucine; and (b) recovering L-leucine from the cultured microorganism or the cultured medium.

18. A method of producing L-leucine, comprising: (a) culturing the microorganism of the genus Corynebacterium producing L-leucine according to claim 14 in a medium to produce L-leucine; and (b) recovering L-leucine from the cultured microorganism or the cultured medium.

19. A method of producing L-leucine, comprising: (a) culturing the microorganism of the genus Corynebacterium producing L-leucine according to claim 15 in a medium to produce L-leucine; and (b) recovering L-leucine from the cultured microorganism or the cultured medium.

Description

EXAMPLE 1

Confirmation of leuA Nucleotide Sequence of KCCM11661P, Microorganism Producing Leucine

(1) Corynebacterium glutamicum ATCC14067 was inoculated into a seed medium having the ingredients described below at 121° C. for 15 minutes, cultured for 13 hours, and then 25 mL of the culture medium was recovered. The recovered culture medium was washed with a 100 mM citrate buffer and treated with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) for 30 minutes to a final concentration of 400 μg/mL. Thereafter, the resultant was washed with a 100 mM phosphate buffer. The mortality rate of the strains treated with NTG was determined to be 99.6% as a result of smearing the strains on a minimal medium having the ingredients described below. In order to achieve variants resistant to norleucine (NL), the NTG-treated strains were smeared on the minimal media with final concentrations of 20 mM, 40 mM, and 50 mM, cultured at 30° C. for 5 days, and then variants resistant to NL were obtained.

Seed Medium

(2) Glucose (20 g), peptone (10 g), yeast extract (5 g), carbamide (1.5 g), KH2PO4 (4 g), K2HPO4 (8 g), MgSO.sub.4.7H.sub.2O (0.5 g), biotin (100 μg), thiamine hydrochloride (1,000 μg), calcium-pantothenic acid (2,000 μg), nicotinamide (2,000 μg; based on 1 liter of distilled water), pH 7.0 <Production medium >

(3) Glucose (100 g), (NH.sub.4).sub.2SO.sub.4 (40 g), soy protein (2.5 g), corn steep solid (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 hydrochloride (1,000 μg), calcium-pantothenic acid (2000 μg), nicotinamide (3,000 μg), CaCO.sub.3 (30 g; based on 1 liter of distilled water), pH 7.0

(4) The variants obtained by the method above were designated as Corynebacterium glutamicum KCJ-24 and Corynebacterium glutamicum KCJ-28 and deposited to the Korean Culture Center of Microorganisms, an international depositary authority, on Jan. 22, 2015, under the Budapest Treaty, and as a result, Corynebacterium glutamicum KCJ-24 and Corynebacterium glutamicum KCJ-28 were deposited under Accession Nos. KCCM11661P and KCCM11662P, respectively. Corynebacterium glutamicum KCJ-24 and Corynebacterium glutamicum KCJ-28 produced L-leucine at a concentration of 2.7 g/L and 3.1 g/L, respectively. Therefore, it was confirmed that the productivity of L-leucine produced from the variants was 10-fold higher than that of the wild-type.

(5) Additionally, an attempt was made to confirm whether the variation of leuA encoding isopropylmalate synthase (IPMS) occurred in the variant KCCM11661P. The amino acid sequence (SEQ ID NO: 1) of wild-type leuA was confirmed by referring to WP_003863358.1 of Genebank. The chromosomal DNA of the variant was amplified using a polymerase chain reaction (hereinafter referred to as ‘PCR’) method. Although it is known that the leuA gene consists of 616 amino acids, in some references, it is published that the translation initiation codon is indicated 35 amino acids downstream of the sequence of the leuA gene, and thereby the leuA gene consists of 581 amino acids. In such a case, the position number indicating the variation of the corresponding amino acid can vary. Therefore, in cases where the leuA gene is considered to consist of 581 amino acids, the variation position is additionally indicated in parenthesis.

(6) Specifically, PCR was performed using the chromosomal DNA of the variant as a template and using primers of SEQ ID NOs: 3 and 4 under the following conditions: denaturation at 94° C. for 1 minute; annealing at 58° C. for 30 seconds; and polymerization at 72° C. for 2 minutes using Taq DNA polymerase. Such PCR was repeated a total of 28 times to amplify a fragment of about 2,700 base pairs. The nucleotide sequence of the fragment was analyzed using the same primer, and as a result, it was confirmed that G, which is the 1673nd nucleotide of leuA in KCCM11661P, was substituted with A. This result implies that arginine, which is the 558.sup.th (or 523.sup.rd; hereinafter only indicated as 558.sup.th) amino acid, is substituted with histidine. In addition, it was also confirmed that GC, which are the 1682.sup.nd and 1683.sup.rd nucleotides, were substituted with AT. This result also implies that glycine, which is the 561.sup.st (or 526.sup.th, hereinafter only indicated as 561.sup.st) amino acid, is substituted with aspartic acid.

EXAMPLE 2

Production of Substitution Vector of IPMS Variant

(7) In order to produce a vector containing the modified nucleotide sequence confirmed in Example 1, PCR was performed using the chromosomal DNA of the variant above as a template and using primers of SEQ ID NOs: 5 and 6 under the following conditions: denaturation at 94° C. for 1 minute; annealing at 58° C. for 30 seconds; and polymerization at 72° C. for 1 minute using Pfu DNA polymerase. Such PCR was repeated a total of 25 times to amplify a fragment of about 1,460 base pairs with SalI and XbaI restriction enzyme sites. The amplified fragment was treated with restriction enzymes, SalI and XbaI, and then pDZ-leuA (R558H, G561D) was prepared by ligation with the vector pDZ (Korean Patent No: 10-0924065 and International Patent Publication No. 2008-033001) treated with the same enzymes. Additionally, in order to prepare a vector with each variation, ATCC14067 was used as a template, and then 2 fragments were amplified using primers 5 and 7, and primers 8 and 6, respectively. PCR was performed using the two prepared fragments as templates under the following conditions: denaturation at 94° C. for 1 minute; annealing at 58° C. for 30 seconds; and polymerization at 72° C. for 1 minute using Pfu DNA polymerase. Such PCR was repeated a total of 25 times to amplify a fragment of about 1,460 base pairs with SalI and XbaI restriction enzyme sites. The amplified fragment was treated with restriction enzymes SalI and XbaI, and then pDZ-leuA (R558H) was prepared by ligation with pDZ treated with the same enzymes. pDZ-leuA (G561D) was prepared using primers 5 and 9, and primers 10 and 6 by the same method above.

EXAMPLE 3

Production of Substitution Strain of IPMS Variant

(8) Corynebacterium glutamicum ATCC14067 was used as a parent strain in order to prepare a strain containing the leuA-modified nucleotide sequence which was found in the modified strain above.

(9) Corynebacterium glutamicum ATCC14067 was transformed with the vectors pDZ-leuA (R558H), pDZ-leuA (G561D), and pDZ-leuA (R558H, G561D), which were prepared in Example 2 by electroporation. Each of the strains prepared through the secondary crossover was designated as 14067::leuA (R558H), 14067::leuA (G561D), and 14067::leuA (R558H, G561D). In order to confirm whether the nucleotide of leuA was substituted, PCR was performed using primers of SEQ ID NOs: 3 and 4 under the following conditions: denaturation at 94° C. for 1 minute; annealing at 58° C. for 30 seconds; and polymerization at 72° C. for 2 minutes using Taq DNA polymerase. Such PCR was repeated a total of 28 times to amplify a fragment of about 2,700 base pairs. Thereafter, the substitution of the nucleotide of leuA was confirmed by analyzing the nucleotide sequence with the same primer.

(10) The strain, 14067::leuA (R558H, G561D) which was transformed with the vector pDZ-leuA (R558H, G561D), was designated as KCJ-0148, and deposited to the Korean Culture Center of Microorganisms on Jan. 25, 2016, and as a result, the strain was deposited under Accession No. KCCM11811P.

EXAMPLE 4

Production of L-Leucine in Substitution Strain of IPMS Variant

(11) In order to produce L-leucine from Corynebacterium glutamicum 14067::leuA (R558H), 14067::leuA (G561D), and 14067::leuA (R558H, G561D), which were prepared in Example 3, cultivation was carried out in the following manner.

(12) A platinum loop of each of the parent strain, Corynebacterium glutamicum ATCC14067, and the prepared Corynebacterium glutamicum 14067::leuA (R558H), 14067::leuA (G561D), and 14067::leuA (R558H, G561D) strains was inoculated into a corner-baffled flask (250 mL) containing a production medium (25 mL). Thereafter, L-leucine was produced by incubating in a shaking water bath at 30° C. at a rate of 200 rpm for 60 hours.

(13) After completion of the incubation, the amount of L-leucine produced was measured by high performance liquid chromatography. The concentration of L-leucine in the culture medium for each experimental strain is shown in Table 1 below.

(14) TABLE-US-00001 TABLE 1 Production of L-leucine in substitution strain of IPMS variant L-Leucine Strain concentration (g/L) ATCC14067 0.1 14067::leuA (R558H) 1.2 14067::leuA (G561D) 1.6 14067::leuA (R558H, G561D) 2.5

(15) As shown in Table 1 above, it was confirmed that the L-leucine productivity of the L-leucine-producing strains, Corynebacterium glutamicum 14067::leuA (R558H), 14067::leuA (G561D), and 14067::leuA (R558H, G561D), which have the R558H, G561D, or R558H/G561D variation in the leuA gene, was enhanced about 12- to 25-fold compared to that of the parent strain, Corynebacterium glutamicum ATCC14067.

EXAMPLE 5

Production of IPMS Variant-Overexpressing Vector

(16) In order to produce an expression vector containing the modified nucleotide sequence confirmed in Example 1, PCR was carried out using ATCC14067 and the chromosomal DNA of the 3 variants prepared in Example 3 as templates and using primers of SEQ ID NOs: 11 and 12 under the following conditions: denaturation at 94° C. for 1 minute; annealing at 58° C. for 30 seconds; and polymerization at 72° C. for 1 minute using Pfu DNA polymerase. Such PCR was repeated a total of 25 times to amplify a fragment of about 2,050 base pairs with NdeI and XbaI restriction enzyme sites. The amplified fragment was treated with restriction enzymes, NdeI and XbaI, and then expression vectors p117_PCJ7-leuA (WT), p117_PCJ7-leuA (R558H), p117_PCJ7-leuA (G561D), and p117_PCJ7-leuA (R558H, G561D) were prepared by ligation using p117_PCJ7 in which a PCJ7 promoter was inserted in the vector pECCG117 (Biotechnology letters Vol. 13, No. 10, p. 721-726 (1991)) treated with the same enzymes. The PCJ7 promoter is a promoter that enhances gene expression, and is publicly known in Korean Patent No. 10-0620092 and International Patent Publication No. 2006-065095.

EXAMPLE 6

Production of Strain Transformed With IPMS Variant-Overexpressing Vector

(17) In order to produce a strain transformed with an overexpression vector containing the leuA modified nucleotide sequence prepared in Example 5, the parent strain, which is wild-type Corynebacterium glutamicum ATCC14067, and the leucine-producing strains KCCM11661P and KCCM11662P were used.

(18) Each of the vectors p117_PCJ7-leuA (WT), p117_PCJ7-leuA (R558H), p117_PCJ7-leuA (G561D), and p117_PCJ7-leuA (R558H, G561D), prepared in Example 5, was transformed with Corynebacterium glutamicum ATCC14067, KCCM11661P, and KCCM11662P by electroporation. As a result, 14067::p117_PCJ7-leuA (WT), 14067::p117_PCJ7-leuA (R558H), 14067::p117_PCJ7-leuA (G561D), 14067::p117_PCJ7-leuA (R558H,G561D); KCCM11661P::p117_PCJ7-leuA (WT), KCCM11661P::p117_PCJ7-leuA (R558H), KCCM11661P::p117_PCJ7-leuA (G561D), KCCM11661P::p117_PCJ7-leuA (R558H, G561D); and KCCM11662P::p117_PCJ7-leuA (WT), KCCM11662P::p117_PCJ7-leuA (R558H), KCCM11662P::p117_PCJ7-leuA (G561D), KCCM11662P::p117_PCJ7-leuA (R558H, G561D) were produced.

EXAMPLE 7

Production of L-Leucine in Strain Transformed With IPMS Variant-Overexpressing Vector

(19) In order to produce L-leucine from the L-leucine-producing strains, Corynebacterium glutamicum 14067::p117_PCJ7-leuA (WT), 14067::p117_PCJ7-leuA (R558H), 14067::p117_PCJ7-leuA (G561D), 14067::p117_PCJ7-leuA (R558H, G561D); KCCM11661P:: p117_PCJ7-leuA (WT), KCCM11661P::117PCJ7-leuA (R558H), KCCM11661P::p117_PCJ7-leuA (G561D), KCCM11661P::p117_PCJ7-leuA (R558H, G561D); and KCCM11662P::p117_PCJ7-leuA (WT), KCCM11662P::p117_PCJ7-leuA (R558H), KCCM11662P::p117_PCJ7-leuA (G561D), KCCM11662P::p117_PCJ7-leuA (R558H, G561D), which were produced in Example 6, cultivation was carried out in the following manner.

(20) A platinum loop of each of the parent strains, Corynebacterium glutamicum ATCC14067, KCCM11661P, and KCCM11662P, and the strains produced in Example 6 was inoculated into a corner-baffled flask (250 mL) containing a production medium (25 mL). Thereafter, L-leucine was produced by incubating in a shaking water bath at 30° C. at a rate of 200 rpm for 60 hours.

(21) After completion of the incubation, the amount of L-leucine produced was measured by high performance liquid chromatography. The concentration of L-leucine in the culture medium for each experimental strain is shown in Table 2 below.

(22) TABLE-US-00002 TABLE 2 Production of L-leucine in strain overexpressing IPMS variant L-Leucine Strain concentration (g/L) ATCC14067 0.1 14067:: p117_PCJ7-leuA (WT) 0.3 14067:: p117_PCJ7-leuA (R558H) 4.5 14067::p117_PCJ7-leuA (G561D) 5.1 14067::p117_PCJ7-leuA (R558H, G561D) 9.8 KCCM11661P 2.7 KCCM11661P:: p117_PCJ7-leuA (WT) 3.0 KCCM11661P:: p117_PCJ7-leuA (R558H) 6.1 KCCM11661P::p117_PCJ7-leuA (G561D) 6.8 KCCM11661P::p117_PCJ7-leuA 12.3 (R558H,G561D) KCCM11662P 3.1 KCCM11662P:: p117_PCJ7-leuA (WT) 3.3 KCCM11662P:: p117_PJ7-leuA (R558H) 6.3 , KCCM11662P::p117_PCJ7-leuA (G561D) 6.9 KCCM11662P::p117_PCJ7-leuA 13.1 (R558H, G561D)

(23) As shown in Table 2 above, it was confirmed that the L-leucine production of the L-leucine-producing strains, 14067::p117_PCJ7-leuA (R558H), 14067::p117_PCJ7-leuA (G561D), and 14067::p117_PCJ7-leuA (R558H, G561D), which were transformed with the overexpression vector containing variation of the leuA gene in the strain ATCC14067, was enhanced 45- to 98-fold compared to that of the parent strain ATCC14067; the L-leucine production of the L-leucine-producing strains, KCCM11661P::p117_PCJ7-leuA (R558H), KCCM11661P::p117_PCJ7-leuA (G561D), and KCCM11661P::p117_PCJ7-leuA (R558H, G561D), which were transformed with the overexpression vector containing variation of the leuA gene in the strain KCCM11661P, was enhanced 2.3- to 4.5-fold compared to that of the parent strain KCCM11661P; and that the L-leucine production of the L-leucine-producing strains, KCCM11662P::p117_PCJ7-leuA (R558H), KCCM11662P::p117_PCJ7-leuA (G561D), and KCCM11662P::p117_PCJ7-leuA (R558H,G561D), which were transformed with the overexpression vector containing variation of the leuA gene in the strain KCKCM11662P, was enhanced 2- to 4.2-fold compared to that of the parent strain KCCM11662P.

EXAMPLE 8

Measurement of Isopropylmalate Synthase Activity in Strain Transformed With leua-Overexpressing Vector

(24) In order to measure an isopropylmalate synthase activity in the L-leucine-producing strains, Corynebacterium glutamicum 14067::p117_PCJ7-leuA (WT), 14067::p117_PCJ7-leuA (R558H), 14067::p117_PCJ7-leuA (G561D), and 14067::p117_PCJ7-leuA (R558H, G561D), produced in Example 6, experiments were carried out in the following manner.

(25) A platinum loop of each of the 4 strains above was inoculated into a corner-baffled flask (250 mL) containing the seed medium (25 mL). Thereafter, the resultants were incubated in a shaking water bath at 30° C. at a rate of 200 rpm for 16 hours. After completion of the incubation, the culture medium was centrifuged to discard the supernatant, the pellet was washed and mixed with a lysis buffer, and the cells were pulverized with a bead homogenizer. The proteins present in the lysate were quantitated according to the Bradford assay, and the activity of isopropylmalate synthase was measured by measuring the CoA produced when the lysate containing proteins (100 μg/mL) was used. The measurement results of the isopropylmalate synthase activity in each strain are shown in Table 3 below.

(26) TABLE-US-00003 TABLE 3 Strain Relative IPMS activity (%) 14067::p117_PCJ7-leuA (WT) 100 14067::p117_PCJ7-leuA (R558H) 105 14067::p117_PCJ7-leuA (G561D) 130 14067::p117_PCJ7-leuA (R558H, G561D) 328

(27) In order to confirm the degree of release of feedback inhibition by leucine in the enzyme, the isopropylmalate synthase activity was measured by measuring the CoA produced when the lysate containing proteins (100 μg/mL) was used under the condition where leucine (3 g/L) was added. The measurement results of the isopropylmalate synthase activity in each strain are shown in Table 4 below.

(28) TABLE-US-00004 TABLE 4 Leucine Leucine 0 g/L 2 g/L Strain Relative IPMS activity (%) 14067::p117_PCJ7-leuA (WT) 100 24 14067::p117_PCJ7-leuA (R558H) 100 61 14067::p117_PCJ7-leuA (G561D) 100 70 14067::p117_PCJ7-leuA (R558H, G561D) 100 89

(29) As shown in Tables 3 and 4 above, it was confirmed that the isopropylmalate synthase activity of the L-leucine-producing strains, Corynebacterium glutamicum 14067::p117_PCJ7-leuA (R558H), 14067::p117_PCJ7-leuA (G561D), and 14067::p117_PCJ7-leuA (R558H, G561D), which were transformed with the vector expressing the IPMS variant, were enhanced 1.05-fold, 1.3-fold, and 3.2-fold, respectively, compared to that of the control, Corynebacterium glutamicum 14067::p117_PCJ7-leuA (WT). In addition, the L-leucine-producing strains maintained their IPMS activity at 61%, 70%, and 89%, respectively, even when leucine (2 g/L) was added, confirming that feedback inhibition by leucine was released.

EXAMPLE 9

Production of Vector for Improving Isopropylmalate Synthase (IPMS) Variant

(30) In Examples 4, 7, and 8, since it was confirmed that the 558t.sup.h and 561st amino acids in the amino acid sequence (SEQ ID NO: 1) of isopropylmalate synthase were important sites for the activity of the IPMS variant enzyme, the attempt was made to confirm whether the enzyme activity was enhanced or whether feedback inhibition was further released when substituted with an amino acid other than the amino acids in the variant. Therefore, an attempt was made to prepare a variant substituted with an amino acid of other amino acid groups capable of causing structural variations.

(31) A variant in which the 558.sup.th amino acid, arginine, was substituted with alanine (Ala) or glutamine (Gln) was prepared. The vector p117_PCJ7-leuA (R558A), in which the 558.sup.th amino acid is substituted with alanine (Ala), and the vector p117_PCJ7-leuA (R558Q), in which the 558.sup.th amino acid is substituted with glutamine (Gln), were prepared using a site-directed mutagenesis method and by using the vector p117_PCJ7-leuA (R558H) as a template, the primer of SEQ ID NOs: 13 and 14, and the primer pair of SEQ ID NOs: 15 and 16.

(32) A variant in which the 561.sup.st amino acid, glycine, was substituted with arginine (Arg) or tyrosine (Tyr) was prepared. The vector p117_PCJ7-leuA (G561R), in which the 561.sup.st amino acid is substituted with arginine (Arg), and the vector p117_PCJ7-leuA (G561Y), in which the 561.sup.st amino acid is substituted with tyrosine (Tyr), were obtained using a site-directed mutagenesis method and by using p117_PCJ7-leuA (G561D) as a template, the primer of SEQ ID NOs: 17 and 18, and the primer pair of SEQ ID NOs: 19 and 20.

EXAMPLE 10

Production of Strain in Which Isopropylmalate-Modified Variant is Introduced

(33) In order to prepare a strain transformed with an expression vector containing the leuA-modified nucleotide sequence prepared in Example 9, wild-type Corynebacterium glutamicum ATCC14067 was used as a parent strain.

(34) Each of the vectors, p117_PCJ7-leuA (R558A), p117_PCJ7-leuA (R558Q), p117_PCJ7-leuA (G561R), and p117_PCJ7-leuA (G561Y), which were prepared in Example 9, was transformed in Corynebacterium glutamicum ATCC14067 by electroporation to prepare 14067::p117_PCJ7-leuA (R558A), 14067::p117_PCJ7-leuA (R558Q), 14067::p117_PCJ7-leuA (G561R), and 14067::p117_PCJ7-leuA (G561Y).

EXAMPLE 11

Production of L-Leucine in Strain in Which Isopropylmalate Synthase-Modified Variant is Introduced

(35) In order to produce L-leucine from the L-leucine-producing strains, Corynebacterium glutamicum 14067::p117_PCJ7-leuA (R558A), 14067::p117_PCJ7-leuA (R558Q), 14067::p117_PCJ7-leuA (G561R), and 14067::p117_PCJ7-leuA (G561Y), which were prepared in Example 10, cultivation was carried out in the following manner.

(36) A platinum loop of each of the parent strain, Corynebacterium glutamicum ATCC14067, and the 4 strains above was inoculated into a corner-baffled flask (250 mL) containing a production medium (25 mL). Thereafter, L-leucine was produced by incubating in a shaking water bath at 30° C. at a rate of 200 rpm for 60 hours.

(37) After completion of the incubation, the amount of L-leucine produced was measured by high performance liquid chromatography. The concentration of L-leucine in the culture medium for each experimental strain is shown in Table 5 below.

(38) TABLE-US-00005 TABLE 5 Production of L-leucine in strain overexpressing IPMS variant L-Leucine Strain concentration (g/L) ATCC14067 0.1 14067::p117_PCJ7-leuA (WT) 0.3 14067::p117_PCJ7-leuA (R558H) 4.5 (Example 7) 14067::p117_PCJ7-leuA (R558A) 3.8 14067::p117_PCJ7-leuA (R558Q) 3.2 14067::p117_PCJ7-leuA (G561D) 5.1 (Example 7) 14067::p117_PCJ7-leuA (G561R) 4.0 14067::p117_PCJ7-leuA (G561Y) 3.6

(39) As shown in Table 5 above, it was confirmed that the L-leucine productivity of the L-leucine-producing strains, Corynebacterium glutamicum 14067::p117_PCJ7-leuA (R558A) and 14067::p117_PCJ7-leuA (R558Q), was improved 32- to 38-fold compared to the parent strain, Corynebacterium glutamicum ATCC14067.

(40) Additionally, it was confirmed that the L-leucine productivity of the L-leucine-producing strains, Corynebacterium glutamicum 14067::p117_PCJ7-leuA (G561R) and 14067::p117_PCJ7-leuA (G561Y), was improved about 36- to 40-fold compared to that of the parent strain, Corynebacterium glutamicum ATCC14067.

(41) Based on the results above, it was confirmed the 558th and 561st amino acids in the amino acid sequence (SEQ ID NO: 1) of isopropylmalate synthase were important sites for the activity of the IPMS variant enzyme, and that even when each of the 558.sup.th and 561.sup.st amino acids of the wild type IPMS protein was substituted with histidine and aspartic acid, respectively, the L-leucine productivity was remarkably increased in the strain having such modification.

(42) While the present disclosure has been described with reference to the particular illustrative embodiments, it will be understood by those skilled in the art to which the present disclosure pertains that the present disclosure may be embodied in other specific forms without departing from the technical spirit or essential characteristics of the present disclosure. Therefore, the embodiments described above are considered to be illustrative in all respects and not restrictive. Furthermore, the scope of the present disclosure is defined by the appended claims rather than the detailed description, and it should be understood that all modifications or variations derived from the meanings and scope of the present disclosure and equivalents thereof are included in the scope of the appended claims.