MODIFIED POLYPEPTIDE OF MESO-DIAMINOPIMELATE DEHYDROGENASE AND METHOD FOR PRODUCING L-THREONINE USING THE SAME

Abstract

The present disclosure relates to a modified polypeptide, in which the activity of meso-diaminopimelate is weakened, and a method for producing L-threonine using the same.

Claims

1. A modified polypeptide, in which the amino acid corresponding to the 169.sup.th amino acid of SEQ ID NO: 1 is substituted with leucine, phenylalanine, glutamate, or cysteine, and which has a sequence homology to the amino acid sequence of SEQ ID NO: 1 of 80% or higher and less than 100%, and has an activity of meso-diaminopimelate dehydrogenase.

2. The modified polypeptide according to claim 1, wherein the activity of the meso-diaminopimelate dehydrogenase of the modified polypeptide is weaker than that of wild-type meso-diaminopimelate dehydrogenase having the amino acid sequence of SEQ ID NO: 1.

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

4. The polynucleotide according to claim 3, wherein the polynucleotide consists of a nucleotide sequence of SEQ ID NO: 4.

5. A microorganism of the genus Corynebacterium, which comprises: a modified polypeptide, in which the amino acid corresponding to the 169.sup.th amino acid of SEQ ID NO: 1 is substituted with leucine, phenylalanine, glutamate, or cysteine, and which has a sequence homology to the amino acid sequence of SEQ ID NO: 1 of 80% or higher and less than 100%, and has an activity of meso-diaminopimelate dehydrogenase; or a polynucleotide comprising the same.

6. The microorganism according to claim 5, wherein the microorganism of the genus Corynebacterium further comprises one or more selected from the modified polypeptides of (1) to (3) shown below: (1) a modified polypeptide, wherein the activity of dihydrodipicolinate reductase (dapB) is weakened; (2) a modified polypeptide, wherein the activity of diaminopimelate decarboxylase (lysA) is weakened; and (3) a modified polypeptide, wherein the activity of dihydrodipicolinate synthase (dapA) is weakened.

7. The microorganism according to claim 6, wherein the modified polypeptide comprises one or more selected from the modified polypeptides of (1) to (3) shown below: (1) a modified polypeptide of dihydrodipicolinate reductase (dapB), wherein the 13.sup.th amino acid in the amino acid sequence of SEQ ID NO: 81, arginine (R), is substituted with asparagine (N); (2) a modified polypeptide of diaminopimelate decarboxylase (lysA), wherein the 408.sup.th amino acid in the amino acid sequence of SEQ ID NO: 82, methionine (M), is substituted with alanine (A); and (3) a modified polypeptide of dihydrodipicolinate synthase (dapA), wherein the 119.sup.th amino acid in the amino acid sequence of SEQ ID NO: 83, tyrosine (T), is substituted with phenylalanine (F).

8. The microorganism according to claim 5, wherein the microorganism has an enhanced ability of producing L-threonine compared to a non-modified strain.

9. The microorganism according to claim 5, wherein the microorganism is Corynebacterium glutamicum.

10. A method for preparing L-threonine, comprising a step of culturing in a medium a microorganism of the genus Corynebacterium comprising a modified polypeptide, in which the amino acid corresponding to the 169.sup.th amino acid of SEQ ID NO: 1 is substituted with leucine, phenylalanine, glutamate, or cysteine, and which has a sequence homology to the amino acid sequence of SEQ ID NO: 1 of 80% or higher and less than 100%, and which has an activity of meso-diaminopimelate dehydrogenase.

11. The method according to claim 10, wherein the step of culturing the microorganism further comprises a step of recovering L-threonine from the cultured medium and the microorganism.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0095] 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 scope of the invention is not limited by these Examples.

Example 1: Preparation of Vector Library for Introduction of Modification within ORF of Ddh Gene

[0096] In order to discover variants in which the expression level of the ddh gene of Corynebacterium glutamicum or an activity thereof is reduced, a library was prepared by the method shown below.

[0097] First, in order to introduce 0 to 4.5 modifications per 1 kb of a DNA fragment (963 bp) consisting of the ddh gene (963 bp), a GenemorphII Random Mutagenesis kit (Stratagene) was used. Error-prone PCR was performed using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 (WT) as a template along with primers of SEQ ID NOS: 5 and 6. Specifically, the reaction solution, which contained the chromosomal DNA of the WT strain (500 ng), primers of SEQ ID NOS: 5 and 6 (125 ng each), Mutazyme II reaction buffer (1×), dNTP mix (40 mM), and Mutazyme II DNA polymerase (2.5 U), was subjected to the following conditions: denaturation at 94° C. for 2 minutes; 25 cycles of denaturation at 94° C. for 1 minute, annealing at 56° C. for 1 minute, and polymerization at 72° C. for 3 minutes; and polymerization at 72° C. for 10 minutes.

[0098] The amplified gene fragment was ligated to the pCRII vector using a TOPO TA Cloning Kit (Invitrogen), and the resulting vector was transformed into E. coli DH5a, and the transformants were plated on a LB solid medium containing kanamycin (25 mg/L). After selecting 20 kinds of transformed colonies, a plasmid was obtained from each of the transformed colonies. As a result of analysis of the nucleotide sequences, it was found that modifications were introduced on mutually-different locations at a frequency of 0.5 mutations/kb. Finally, about 10,000 transformed E. coli colonies were collected and the plasmid was extracted therefrom. The resultant was named as pTOPO-ddh(mt) library.

Example 2: Preparation of Ddh-Deleted Strain and Screening of Random Mutagenesis Library

[0099] In order to confirm the effect of ddh deletion on L-lysine production, the Corynebacterium glutamicum KCCM11016P strain (Korean Patent No. 10-0159812) was used. To prepare the Corynebacterium glutamicum KCCM11016P strain (in which the ddh gene is deleted) a pDZ-Δddh vector (in which the ddh gene is deleted) was prepared as follows.

[0100] Specifically, the vector was prepared in such a form that DNA fragments (600 bp each) located at 5′ and 3′ ends of the ddh gene were each ligated to the pDZ vector (Korean Patent Application Publication No. 2009-0094433). Based on the nucleotide sequence of the ddh gene reported (SEQ ID NO: 2), primers of SEQ ID NOS: 7 and 8 (into which the recognition site of the restriction enzyme XbaI was inserted at the 5′ fragment and the 3′ fragment, respectively) and primers of SEQ ID NOS: 9 and 10 (which are separated from the SEQ ID NOS: 7 and 8 by 663 bp, respectively) were synthesized (Table 1). The 5′ end gene fragment was prepared by PCR using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template along with primers of SEQ ID NOS: 7 and 9. In the same manner, the gene fragment located at the 3′ end of the ddh gene was prepared by PCR using the primers of SEQ ID NOS: 8 and 10. The PCR was performed as follows: denaturation at 94° C. for 2 minutes; 30 cycles of denaturation at 94° C. for 1 minute, annealing at 56° C. for 1 minute, and polymerization at 72° C. for 40 seconds; and polymerization at 72° C. for 10 minutes.

[0101] Meanwhile, the pDZ vector (which was digested with a restriction enzyme XbaI and then subjected to heat treatment at 65° C. for 20 minutes) was ligated to the insertion DNA fragment amplified through PCR using the Infusion Cloning kit, and the resultant was transformed into E. coli DH5a, and the transformants were plated on a LB solid medium containing kanamycin (25 mg/L). After selecting the colonies transformed with the vector, into which the desired gene was inserted through PCR using the primers of SEQ ID NOS: 7 and 8, the plasmid was obtained by a plasmid extraction method commonly known in the art, and the obtained plasmid was named as pDZ-Δddh.

TABLE-US-00001 TABLE 1 SEQ ID NO Sequence (5′->3′) SEQ ID NO: 7 CGGGGATCCTCTAGATGACCAACATCCGCG SEQ ID NO: 8 CAGGTCGACTCTAGATTAGACGTCGCGTGCG SEQ ID NO: 9 CGGTGAAATCGGCGACATCAAAGACTG SEQ ID NO: 10 GATGTCGCCGATTTCACCGCTTCCTC

[0102] The prepared vector pDZ-Δddh was transformed into the Corynebacterium glutamicum KCCM11016P strain by electroporation (Van der Rest et al., Appl. Microbiol. Biotecnol. 52:541-545, 1999), and then a strain in which the ddh gene is deleted was prepared by homologous chromosome recombination. The prepared strain in which the ddh gene is deleted was named as Corynebacterium glutamicum WT:Δddh.

[0103] Additionally, the pTOPO-ddh(mt) library, which was prepared in Example 1 above, was transformed into the KCCM11016P:Δddh strain by electroporation, and the transformants were plated on a complex plate medium containing kanamycin (25 mg/L), and about 20,000 colonies were obtained therefrom. Each colony was inoculated into the following selection medium (300 μL) and then cultured in a 96-deep well plate at 1,000 rpm at 32° C. for about 24 hours.

[0104] <Selection Medium (pH 8.0)>

[0105] 10 g glucose, 5.5 g ammonium sulfate, 1.2 g MgSO.sub.4.7H.sub.2O, 0.8 g KH.sub.2PO.sub.4, 16.4 g K.sub.2HPO.sub.4, 100 μg biotin, 1 mg thiamine HCl, 2 mg calcium-pantothenate, 2 mg nicotinamide (per 1 L distilled water)

[0106] The amount of L-lysine produced in the culture solution was analyzed using the ninhydrin method (Moore, S., Stein, W. H., Photometric ninhydrin method for use in the chromatography of amino acids. J. Biol. Chem. 1948, 176, 367-388).

[0107] After completion of the culture, the culture supernatant (10 μL) and the ninhydrin reaction solution (190 μL) were reacted at 65° C. for 30 minutes, and the absorbance was measured at the wavelength of 570 nm using a spectrophotometer. The WT strain and the WT::Δddh strain were used as control groups. Sixty kinds of strains, which showed a lower absorbance compared to the WT strain (i.e., the wild-type) while showing a higher absorbance compared to the WT::Δddh strain, were selected.

[0108] The selected 60 kinds of strains were cultured again in the same manner as described above, and the ninhydrin reaction was performed repeatedly. As a result, top 5 kinds of mutant strains, which showed an enhanced ability of producing L-lysine compared to the KCCM11016P::Δddh strain but a reduced ability of producing L-lysine compared to the KCCM11016P strain, were selected. The selected 5 kinds of strains were named as KCCM11016P::ddh(mt)-1 to KCCM11016P::ddh(mt)-5 (Table 2), respectively.

TABLE-US-00002 TABLE 2 Concentration of L-lysine production by 5 kinds of selected random mutant strains Absorbance (572 nm) Batch Batch Batch Aver- Strain 1 2 3 age Control KCCM11016P 0.228 0.205 0.216 0.215 Group 1 KCCM11016P::ddh(mt)-1 0.214 0.193 0.205 0.204 2 KCCM11016P::ddh(mt)-2 0.185 0.181 0.179 0.182 3 KCCM11016P::ddh(mt)-3 0.164 0.163 0.145 0.157 4 KCCM11016P::ddh(mt)-4 0.135 0.141 0.128 0.135 5 KCCM11016P::ddh(mt)-5 0.198 0.201 0.189 0.196 Control KCCM11016P::Δddh 0.106 0.112 0.098 0.105 Group

Example 3: Confirmation of Nucleotide Sequences of 5 Kinds of Modified Strains of ddh

[0109] In order to confirm the nucleotide sequences of the ddh gene of the 5 kinds of selected strains (i.e., KCCM11016P::ddh(mt)-1 to KCCM11016P::ddh(mt)-5), the DNA fragments including the ddh gene in the chromosome were amplified by PCR using the primers shown in Example 1 (SEQ ID NOS: 5 and 6). The PCR was performed as follows: denaturation at 94° C. for 2 minutes; 30 cycles of denaturation at 94° C. for 1 minute, annealing at 56° C. for 1 minute, and polymerization at 72° C. for 40 seconds; and polymerization at 72° C. for 10 minutes.

TABLE-US-00003 TABLE 3 SEQ ID NO Sequence (5′->3′) SEQ ID NO: 5 ATGACCAACATCCGCGTAGC SEQ ID NO: 6 TTAGACGTCGCGTGCGATCAG

[0110] As a result of the analysis of the nucleotide sequences of the amplified gene, it was found that the 5 kinds of strains were: 1) a variant, in which a modification is introduced into the nucleotide sequence located at the 37.sup.th position downstream of the ORF start codon of the ddh gene, and thus, the original sequence ‘AAC’ is converted to ‘GAC’ (i.e., the 13.sup.th amino acid from the N-terminus (i.e., asparagine) is substituted with aspartic acid); ii) a variant, in which three modifications are introduced into the nucleotide sequence including the 106.sup.th to the 108.sup.th nucleotides downstream of the ORF start codon of the ddh gene, and thus, the original sequence ‘CGC’ is converted to ‘ATG’ (i.e., the 36.sup.th amino acid from the N-terminus (i.e., arginine) is substituted with methionine); iii) a variant, in which two modifications are introduced into the nucleotide sequence including the 448.sup.th to the 449.sup.th nucleotides downstream of the ORF start codon of the ddh gene, and thus, the original sequence ‘CAG’ is converted to ‘ATG’ (i.e., the 150.sup.th amino acid from the N-terminus (i.e., glutamine) is substituted with methionine); iv) a variant, in which two modifications are introduced into the nucleotide sequence including the 505.sup.th to the 506.sup.th nucleotides downstream of the ORF start codon of the ddh gene, and thus, the original sequence ‘ACC’ is converted to ‘CTC’ (i.e., the 169.sup.th amino acid from the N-terminus (i.e., threonine) is substituted with leucine); and v) a variant, in which two modifications are introduced into the nucleotide sequence including the 584.sup.th to the 585.sup.th nucleotides downstream of the ORF start codon of the ddh gene, and thus, the original sequence ‘CGC’ is converted to ‘CAA’ (i.e., the 195.sup.th amino acid from the N-terminus (i.e., arginine) is substituted with glutamine).

Example 4: Preparation of ATCC13032 Strains into which 5 Kinds of ddh Modifications are Introduced, and Evaluation of their Abilities of Producing Threonine and Lysine

[0111] With respect to the 5 kinds of modifications confirmed in Example 3 above, in order to finally select the strains where the ability of producing L-lysine is reproducibly reduced while the ability of producing L-threonine is increased, wild-type-derived strains into which a modification is introduced were prepared.

[0112] In order to prepare strains into which a modified ddh gene is introduced in the Corynebacterium glutamicum ATCC13032 strain, 5 kinds of vectors, into which the modified ddh gene can be introduced (i.e., pDZ::ddh ml to pDZ::ddh m5), were prepared as follows.

[0113] Specifically, the vector was prepared in such a form that DNA fragments (963 bp each) located at 5′ and 3′ ends of the ddh gene were each ligated to the pDZ vector (Korean Patent No. 2009-0094433). Based on the nucleotide sequence of the ddh gene reported (SEQ ID NO: 2), a primer of SEQ ID NO: 11 (into which the recognition site of the restriction enzyme XbaI was inserted at the 5′ fragment and the 3′ fragment, respectively) and a primer of SEQ ID NO: 12 (which is separated from the SEQ ID NO: 11 by 931 bp, respectively) were synthesized.

[0114] Modified DNA fragments were prepared by PCR using the chromosomal DNA of KCCM11016P::ddh(mt)-1 to KCCM11016P::ddh(mt)-5 confirmed in Example 3 above along with the primers of SEQ ID NOS: 11 and 12. The PCR was performed as follows: denaturation at 94° C. for 2 minutes; 30 cycles of denaturation at 94° C. for 1 minute, annealing at 56° C. for 1 minute, and polymerization at 72° C. for 40 seconds; and polymerization at 72° C. for 10 minutes.

[0115] Meanwhile, the pDZ vector (which was digested with a restriction enzyme XbaI and then subjected to heat treatment at 65° C. for 20 minutes) was ligated to the modified DNA fragments amplified through PCR using the Infusion Cloning kit, and the resultants were each transformed into E. coli DH5a, and the transformants were plated on a LB solid medium containing kanamycin (25 mg/L). After selecting the colonies transformed with the vector, into which the desired gene was inserted through PCR using the primers of SEQ ID NOS: 11 and 12, the plasmids were obtained by a plasmid extraction method commonly known in the art, and the obtained plasmids were named as pDZ::ddh(mt)1 to pDZ::ddh(mt)5, respectively.

[0116] The prepared vectors (i.e., pDZ::ddh(mt)1 to pDZ::ddh(mt)5) were each transformed into the Corynebacterium glutamicum ATCC13032 strain by electroporation, and were then subjected to a second cross-over process, and thereby strains, in each of which part of the nucleotide sequence of the ddh gene is substituted with a modified nucleotide(s) on the chromosome, were obtained. Whether the substitution was appropriate was determined by the mutant allele specific amplification (MASA) PCR technology (Takeda et al., Hum. Mutation, 2, 112-117 (1993)) using the following primer pairs, where in the primer pair of SEQ ID NO: 13 and SEQ ID NO: 14, which agrees with the modified sequences, the appropriateness of the substitution was first determined by selecting the strain to be amplified, and the sequence analysis of the ddh gene of the selected strain was confirmed secondarily by analyzing the modified sequences using the primer pair of SEQ ID NO: 13 and SEQ ID NO: 15. The prepared strains, into each of which a modified ddh gene is introduced, were named as Corynebacterium glutamicum ATCC13032::ddh (mt)1 to Corynebacterium glutamicum ATCC13032::ddh(mt)5, respectively.

TABLE-US-00004 TABLE 4 SEQ ID NO Sequence (5′->3′) SEQ ID NO: 11 CGGGGATCCTCTAGATGACCAACATCCGCG SEQ ID NO: 12 CAGGTCGACTCTAGATTAGACGTCGCGTGCG SEQ ID NO: 13 CACAATTTTGGAGGATTAC SEQ ID NO: 14 TGGGTGACCACGATCAGAT SEQ ID NO: 15 GGAAACCACACTGTTTCC

[0117] With respect to the 5 kinds of strains into which 5 kinds of modifications are introduced, in order to finally select the strains where the ability of producing L-lysine is reproducibly reduced while the ability of producing L-threonine is increased, flask culture was performed using the following media. After completion of the culture, the concentrations of L-lysine and threonine in the culture solution were analyzed using HPLC, and the concentrations of L-lysine and threonine produced in each mutant strain are shown in Tables 5 and 6 below.

[0118] <Seed Medium (pH 7.0)>

[0119] 20 g glucose, 10 g peptone, 5 g yeast extract, 1.5 g urea, 4 g KH.sub.2PO.sub.4, 8 g K.sub.2HPO.sub.4, 0.5 g MgSO.sub.4.7H.sub.2O, 100 μg biotin, 1 mg thiamine HCl, 2 mg calcium-pantothenate, 2 mg nicotinamide (per 1 L distilled water)

[0120] <Production Medium (pH 7.0)>

[0121] 100 g glucose, 40 g (NH.sub.4).sub.2SO.sub.4, 2.5 g soybean protein, 5 g corn steep solids, 3 g urea, 1 g KH.sub.2PO.sub.4, 0.5 g MgSO.sub.4.7H.sub.2O, 100 μg biotin, 1 mg thiamine HCl, 2 mg calcium-pantothenate, 3 mg nicotinamide, 30 g CaCO.sub.3 (per 1 L distilled water)

TABLE-US-00005 TABLE 5 Concentrations of L-lysine produced by 5 kinds of selected random mutant strains Glucose L-lysine (g/L) Consumption Strain Batch 1 Batch 2 Batch 3 Average Rate (g/hr) Control ATCC13032 1.25 1.20 1.19 1.21 4.33 Group 1 ATCC13032::ddh (mt)1 1.20 1.15 1.19 1.18 4.30 2 ATCC13032::ddh (mt)2 1.05 1.10 1.02 1.06 4.21 3 ATCC13032::ddh (mt)3 0.85 0.88 0.90 0.88 3.79 4 ATCC13032::ddh (mt)4 0.75 0.79 0.76 0.77 3.71 5 ATCC13032::ddh (mt)5 1.11 1.08 1.13 1.11 4.12 Control ATCC13032::Δddh 0.70 0.68 0.71 0.70 3.56 Group

TABLE-US-00006 TABLE 6 Concentrations of L-threonine produced by 5 kinds of selected random mutant strains L-threonine (g/L) Batch Batch Batch Aver- Strain 1 2 3 age Control ATCC13032 0.35 0.37 0.36 0.36 Group 1 ATCC13032::ddh (mt)1 0.38 0.37 0.35 0.37 2 ATCC13032::ddh (mt)2 0.39 0.39 0.37 0.38 3 ATCC13032::ddh (mt)3 0.40 0.39 0.41 0.40 4 ATCC13032::ddh (mt)4 0.42 0.43 0.42 0.42 5 ATCC13032::ddh (mt)5 0.37 0.38 0.37 0.37 Control ATCC13032::Δddh 0.45 0.41 0.42 0.43 Group

[0122] Among the selected 5 kinds of mutant strains, as a strain in which the ability of producing L-lysine is significantly reduced while the ability of producing L-threonine is enhanced, the ATCC13032::ddh (mt)4 strain was selected.

Example 5: Preparation of ATCC13869 Strains into which 5 Kinds of Ddh Modifications are Introduced, and Evaluation of their Abilities of Producing Threonine and Lysine

[0123] With respect to the 5 kinds of modifications confirmed in Example 3 above, in order to finally select the strains where the ability of producing L-lysine is reproducibly reduced while the ability of producing L-threonine is increased, wild-type-derived strains into which a modification is introduced were prepared.

[0124] In order to prepare strains into each of which a modified ddh gene is introduced in the Corynebacterium glutamicum ATCC13869 strain, the vectors prepared in Example 4 (i.e., pDZ::ddh(mt)1 to pDZ::ddh(mt)5) were transformed into the Corynebacterium glutamicum ATCC13869 strain by electroporation, and the transformants were subjected to a second cross-over, and thereby strains, in each of which part of the nucleotide sequence of the ddh gene is substituted with a modified nucleotide(s) on the chromosome, were obtained. Whether the substitution was appropriate was determined by the mutant allele specific amplification (MASA) PCR technology (Takeda et al., Hum. Mutation, 2, 112-117 (1993)) using the following primer pairs, where in the primer pair of SEQ ID NO: 13 and SEQ ID NO: 14, which agrees with the modified sequences, the appropriateness of the substitution was first determined by selecting the strain to be amplified, and the sequence analysis of the ddh gene of the selected strain was confirmed secondarily by analyzing the modified sequences using the primer pair of SEQ ID NO: 13 and SEQ ID NO: 15. The prepared strains, into each of which a modified ddh gene is introduced, were named as Corynebacterium glutamicum ATCC13869::ddh (mt)1 to Corynebacterium glutamicum ATCC13869::ddh (mt)5, respectively.

[0125] With respect to the 5 kinds of strains into which 5 kinds of modifications are introduced, in order to finally select the strains where the ability of producing L-lysine is reproducibly reduced while the ability of producing L-threonine is increased, flask culture was performed using the following media. After completion of the culture, the concentrations of L-lysine and threonine in the culture solution were analyzed using HPLC, and the concentrations of L-lysine and threonine produced in mutant strains are shown in Tables 7 and 8 below.

[0126] <Seed Medium (pH 7.0)>

[0127] 20 g glucose, 10 g peptone, 5 g yeast extract, 1.5 g urea, 4 g KH.sub.2PO.sub.4, 8 g K.sub.2HPO.sub.4, 0.5 g MgSO.sub.4.7H.sub.2O, 100 μg biotin, 1 mg thiamine HCl, 2 mg calcium-pantothenate, 2 mg nicotinamide (per 1 L distilled water)

[0128] <Production Medium (pH 7.0)>

[0129] 100 g glucose, 40 g (NH.sub.4).sub.2SO.sub.4, 2.5 g soybean protein, 5 g corn steep solids, 3 g urea, 1 g KH.sub.2PO.sub.4, 0.5 g MgSO.sub.4.7H.sub.2O, 100 μg biotin, 1 mg thiamine HCl, 2 mg calcium-pantothenate, 3 mg nicotinamide, 30 g CaCO.sub.3 (per 1 L distilled water)

TABLE-US-00007 TABLE 7 Concentrations of L-lysine produced by 5 kinds of selected random mutant strains Glucose L-lysine (g/L) Consumption Strain Batch 1 Batch 2 Batch 3 Average Rate (g/hr) Control ATCC13869 1.21 1.22 1.22 1.22 4.03 Group 1 ATCC13869::ddh (mt)1 1.19 1.19 1.20 1.19 3.98 2 ATCC13869::ddh (mt)2 1.08 1.07 1.10 1.08 3.89 3 ATCC13869::ddh (mt)3 0.88 0.87 0.85 0.87 3.75 4 ATCC13869::ddh (mt)4 0.73 0.77 0.76 0.75 3.68 5 ATCC13869::ddh (mt)5 1.09 1.11 1.12 1.11 3.89 Control ATCC13032::Δddh 0.71 0.69 0.71 0.70 3.47 Group

TABLE-US-00008 TABLE 8 Concentrations of L-threonine produced by 5 kinds of selected random mutant strains L-threonine (g/L) Batch Batch Batch Aver- Strain 1 2 3 age Control ATCC13869 0.25 0.27 0.28 0.27 Group 1 ATCC13869::ddh (mt)1 0.27 0.29 0.27 0.28 2 ATCC13869::ddh (mf)2 0.30 0.31 0.31 0.31 3 ATCC13869::ddh (mf)3 0.35 0.33 0.36 0.35 4 ATCC13869::ddh (mf)4 0.38 0.39 0.38 0.38 5 ATCC13869::ddh (mf)5 0.31 0.29 0.32 0.31 Control ATCC13869::Δddh 0.40 0.41 0.39 0.40 Group

[0130] With respect to the ATCC13869::Δddh strain, in which ddh is deleted compared to the ATCC13869 strain (i.e., a wild-type strain), it was confirmed that the glucose consumption rate was significantly reduced and thus inhibiting the growth of the strain. In contrast, with respect to the selected 5 kinds of strains, it was confirmed that the amount of L-lysine production was reduced but the amount of L-threonine production was increased, while the glucose consumption rate was maintained at a level equivalent to that of the wild-type strain.

[0131] Among the selected 5 kinds of mutant strains, as a strain in which the ability of producing L-lysine is significantly reduced while the ability of producing L-threonine is enhanced, the ATCC13032::ddh (mt)4 strain was selected as in Example 4.

Example 6: Preparation of Strains into which Modified Ddh is Introduced in Microorganism of the Genus Corynebacterium Having Ability of Producing L-Threonine and Evaluation of the Ability of Producing L-Threonine

[0132] A strain producing L-threonine was developed from the wild-type Corynebacterium glutamicum ATCC13032 strain. Specifically, in order to release the feedback inhibition of aspartate kinase (lysC), which acts as the first important enzyme in the L-threonine biosynthesis pathway, the 377.sup.th amino acid of lysC (i.e., leucine) was substituted with lysine (SEQ ID NO: 16).

[0133] More specifically, in order to prepare strains into each of which a lysC (L377K) modification is introduced, PCR was performed using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template along with a primer pair of SEQ ID NOS: 17 and 18 or a primer pair of SEQ ID NOS: 19 and 20, respectively. PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as polymerase for a PCR reaction. The PCR was performed as follows: 28 cycles of denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 1 minute.

[0134] As a result, with respect to the modification of the lysC gene, a 515 bp DNA fragment in the 5′ upstream region and a 538 bp DNA fragment in the 3′ downstream region were obtained, respectively. PCR was performed using the two amplified DNA fragments as templates along with the primers of SEQ ID NO: 17 and SEQ ID NO: 20. The PCR was performed as follows: denaturation at 95° C. for 5 minutes; 28 cycles of denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 2 minutes; and polymerization at 72° C. for 5 minutes.

TABLE-US-00009 TABLE 9 SEQ ID NO Sequence (5′->3′) SEQ ID NO: 17 TCGAGCTCGGTACCCGCTGCGCAGTGTTGAATAC SEQ ID NO: 18 TGGAAATCTTTTCGATGTTCACGTTGACAT SEQ ID NO: 19 ATGTCAACGTGAACATCGAAAAGATTTCCA SEQ ID NO: 20 CTCTAGAGGATCCCCGTTCACCTCAGAGACGATT

[0135] As a result, a 1,023 bp DNA fragment, which includes a modification of the lysC gene that encodes an aspartokinase variant where the 377.sup.th amino acid (i.e., leucine) is substituted with lysine, was amplified. The amplified product was purified using a PCR purification kit (QIAGEN) and used as an insertion DNA fragment for the preparation of a vector. Meanwhile, a pDZ-L377K vector for the introduction of an L377K modification into the chromosome was prepared as follows: the pDZ vector (which was digested with a restriction enzyme SmaI and then subjected to heat treatment at 65° C. for 20 minutes) and the insertion DNA fragment (which was amplified by PCR above) were combined in a molar concentration ratio (M) of 1:2, and cloning was performed using an Infusion Cloning kit (TaKaRa) according to the manual provided.

[0136] The prepared vector was transformed into the ATCC13032 strain by electroporation, and the transformed strain was subjected to a second cross-over, and thereby, a strain in which each nucleotide is substituted with a modified nucleotide on the chromosome was obtained. The strain was named as CJP1. The CJP1 was named as CA01-2307 and deposited at the Korean Culture Center of Microorganisms (KCCM), which is an international depositary authority under the Budapest Treaty, on Mar. 29, 2017, and was assigned Accession No. KCCM12000P.

[0137] In order to release the feedback inhibition of homoserine dehydrogenase (hom), which acts as the second important enzyme in the L-threonine production, the 407.sup.th amino acid of hom (i.e., arginine) was substituted with histidine (SEQ ID NO: 21).

[0138] More specifically, in order to prepare strains into each of which a hom (R407H) modification is introduced, PCR was performed using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template along with a primer pair of SEQ ID NOS: 22 and 23 or a primer pair of SEQ ID NOS: 24 and 25, respectively. PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as polymerase for the PCR reaction. The PCR was performed as follows: 28 cycles of denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 1 minute.

TABLE-US-00010 TABLE 10 SEQ ID NO Sequence (5′->3′) SEQ ID NO: 22 TCGAGCTCGGTACCCCGGATGATGTGTACTGCG SEQ ID NO: 23 GACCACGATCAGATGTGCATCATCATCGCGC SEQ ID NO: 24 GATGATGATGCACATCTGATCGTGGTCACCC SEQ ID NO: 25 CTCTAGAGGATCCCCGAGTCAGCGGGAAATCCG

[0139] As a result, with respect to the modification of the horn gene, a 533 bp DNA fragment in the 5′ upstream region and a 512 bp DNA fragment in the 3′ downstream region were obtained, respectively. PCR was performed using the two amplified DNA fragments as templates along with the primers of SEQ ID NO: 22 and SEQ ID NO: 25. The PCR was performed as follows: denaturation at 95° C. for 5 minutes; 28 cycles of denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 2 minutes; and polymerization at 72° C. for 5 minutes.

[0140] As a result, a 1,018 bp DNA fragment, which includes a modification of the horn gene that encodes an aspartokinase variant where the 407.sup.th amino acid (i.e., arginine) is substituted with histidine, was amplified. The amplified product was purified using a PCR purification kit (QIAGEN) and used as an insertion DNA fragment for the preparation of a vector. Meanwhile, a pDZ-R407H vector for the introduction of an R407H modification into the chromosome was prepared as follows: the pDZ vector (which was digested with a restriction enzyme SmaI and then subjected to heat treatment at 65° C. for 20 minutes) and the insertion DNA fragment (which was amplified by PCR above) were combined in a molar concentration ratio (M) of 1:2, and cloning was performed using an Infusion Cloning kit (TaKaRa) according to the manual provided.

[0141] The prepared vector was transformed into the CJP1 strain by electroporation, and the transformed strain was subjected to a second cross-over, and thereby, a strain in which each nucleotide is substituted with a modified nucleotide on the chromosome was obtained. The strain was named as CA09-0900 (Accession NO. KCCM12418P).

[0142] In order to clearly confirm the changes in the production of L-threonine and L-lysine of the above strain, a T169L modification, which showed the highest L-threonine production and the highest reduction in L-lysine production in Examples 5 and 6 with respect to the gene encoding meso-diaminopimelate dehydrogenase (DDH), was introduced thereinto. Specifically, in order to introduce the T169L modification into the CA09-0900 strain, the pDZ::ddh(mt)4 vector prepared in Example 5 was transformed into the CA09-0900 strain by electroporation, and the transformed strain was subjected to a second cross-over in the same manner as in Example 4, and thereby, a strain in which a nucleotide is substituted with a modified nucleotide on the chromosome was obtained. The resulting strain was named as CA09-0904.

[0143] The CA09-0904 strain was deposited at the Korean Culture Center of Microorganisms (KCCM), which is an international depositary authority under the Budapest Treaty, on Apr. 25, 2019, and was assigned Accession No. KCCM12503P.

TABLE-US-00011 TABLE 11 Confirmation of abilities of prepared strains for producing L-threonine and L-lysine Amino acid (g/L) Strain Thr Lys CA09-0900 1.50 2.67 CA09-0904 2.35 1.58

[0144] As a result, the strain introduced with the modification showed a decrease of L-lysine production by 1.09 g/L and an increase of L-threonine production by 0.85 g/L compared to the CA09-0900 strain (control group) (Table 11). Therefore, it was confirmed that the activity of Ddh was significantly reduced and that the weakening of the L-lysine production pathway was positive for L-threonine production.

Example 7: Preparation of Various Strains in which 169.SUP.th .Amino Acid (i.e., Asparagine) of Ddh Gene is Substituted with Different Amino Acid

[0145] Through the CA09-0904 strain prepared in Example 6, it was confirmed that the strain which reduced L-lysine production has a positive effect on L-threonine production. An attempt was made to confirm whether any substitution of the 169.sup.th amino acid (i.e., threonine) in the ddh gene with a proteogenic amino acid other than threonine of the wild-type may increase the threonine production.

[0146] In order to introduce 19 kinds of modifications of heterogeneous nucleotide substitution including the T169L modification confirmed in Example 6, each recombinant vector was prepared as follows.

[0147] First, primers (SEQ ID NOS: 26 and 27), into which a recognition site of the restriction enzyme (XbaI) was inserted into the 5′ fragment and the 3′ fragment, about 600 bp apart downstream and upstream from the positions of the 505.sup.th to the 506.sup.th nucleotides of the ddh gene, respectively, were synthesized using the genomic DNA extracted from the WT strain as a template. In order to introduce the 19 kinds of heterogeneous nucleotide-substituted modifications, primers (SEQ ID NOS: 28 to 65) for substituting the 505.sup.th to the 506.sup.th nucleotides in the nucleotide sequences of the ddh gene were synthesized (Table 12).

[0148] Specifically, the pDZ-ddh(T169A) plasmid was prepared in such a form that the DNA fragments (600 bp each) located at the 5′ and 3′ ends of the ddh gene were ligated to the pDZ vector (Korean Patent No. 2009-0094433). The 5′ end gene fragment of the ddh gene was prepared by PCR using the chromosomal DNA of the WT strain as a template along with primers of SEQ ID NOS: 26 and 28. The PCR was performed as follows: denaturation at 94° C. for 2 minutes; 30 cycles of denaturation at 94° C. for 1 minute, annealing at 56° C. for 1 minute, and polymerization at 72° C. for 40 seconds; and polymerization at 72° C. for 10 minutes. Likewise, the 3′ end gene fragment of the ddh gene was prepared by PCR using primers of SEQ ID NOS: 27 and 29. The amplified DNA fragments were purified using a PCR Purification kit (Qiagen) and used as insertion DNA fragments for the preparation of vectors.

[0149] Meanwhile, the insertion DNA fragments amplified by PCR and the pDZ vector, which was digested with a restriction enzyme (XbaI) and then heat treated at 65° C. for 20 minutes, were ligated using the Infusion Cloning Kit and then transformed into E. coli DH5a. The resulting strain was plated on a solid LB medium containing kanamycin (25 mg/L). The transformed colonies in which the target gene was inserted into the vector by PCR using the primers of SEQ ID NOS: 26 and 27 were selected, and the plasmid was obtained using a conventionally known plasmid extraction method and named as pDZ-ddh(T169A).

[0150] Likewise, the plasmids were prepared as follows: the pDZ-ddh(T169V) using primers (SEQ ID NOS: 26 and 30 and SEQ ID NOS: 27 and 31); the pDZ-ddh(T169Q) using primers (SEQ ID NOS: 26 and 32 and SEQ ID NOS: 27 and 33); the pDZ-ddh(T169H) using primers (SEQ ID NOS: 26 and 34 and SEQ ID NOS: 27 and 35); the pDZ-ddh(T169R) using primers (SEQ ID NOS: 26 and 36 and SEQ ID NOS: 27 and 37); the pDZ-ddh(T169P) using primers (SEQ ID NOS: 26 and 38 and SEQ ID NOS: 27 and 39); the pDZ-ddh(T169L) using primers (SEQ ID NOS: 26 and 40 and SEQ ID NOS: 27 and 41); the pDZ-ddh(T169Y) using primers (SEQ ID NOS: 26 and 42 and SEQ ID NOS: 27 and 43); the pDZ-ddh(T169S) using primers (SEQ ID NOS: 26 and 44 and SEQ ID NOS: 27 and 45); the pDZ-ddh(T169K) using primers (SEQ ID NOS: 26 and 46 and SEQ ID NOS: 27 and 47); the pDZ-ddh(T169M) using primers (SEQ ID NOS: 26 and 48 and SEQ ID NOS: 27 and 49); the pDZ-ddh(T169I) using primers (SEQ ID NOS: 26 and 50 and SEQ ID NOS: 27 and 51); the pDZ-ddh(T169E) using primers (SEQ ID NOS: 26 and 52 and SEQ ID NOS: 27 and 53); the pDZ-ddh(T169D) using primers (SEQ ID NOS: 26 and 54 and SEQ ID NOS: 27 and 55); the pDZ-ddh(T169G) using primers (SEQ ID NOS: 26 and 56 and SEQ ID NOS: 27 and 57); the pDZ-ddh(T169W) using primers (SEQ ID NOS: 26 and 58 and SEQ ID NOS: 27 and 59); the pDZ-ddh(T169C) using primers (SEQ ID NOS: 26 and 60 and SEQ ID NOS: 27 and 61); the pDZ-ddh(T169F) using primers (SEQ ID NOS: 26 and 62 and SEQ ID NOS: 27 and 63); and the pDZ-ddh(T169N) using primers (SEQ ID NOS: 26 and 64 and SEQ ID NOS: 27 and 65).

TABLE-US-00012 TABLE 12 SEQ ID NO Sequence (5′->3′) SEQ ID NO: 26 CGGGGATCCTCTAGAATGACCAACATCCGC GTAG SEQ ID NO: 27 CAGGTCGACTCTAGATTAGACGTCGCGTGC GATC SEQ ID NO: 28 TCCAGTACGCTCTCCCATCCGAAGACGCCC SEQ ID NO: 29 GGATGGGAGAGCGTACTGGACTGCCTTTTG SEQ ID NO: 30 TCCAGTACGTCCTCCCATCCGAAGACGCCC SEQ ID NO: 31 GGATGGGAGGACGTACTGGACTGCCTTTTG SEQ ID NO: 32 TCCAGTACCAGCTCCCATCCGAAGACGCCC SEQ ID NO: 33 GGATGGGAGCTGGTACTGGACTGCCTTTTG SEQ ID NO: 34 TCCAGTACCAGCTCCCATCCGAAGACGCCC SEQ ID NO: 35 GGATGGGAGCTGGTACTGGACTGCCTTTTG SEQ ID NO: 36 TCCAGTACCGACTCCCATCCGAAGACGCCC SEQ ID NO: 37 GGATGGGAGTCGGTACTGGACTGCCTTTTG SEQ ID NO: 38 TCCAGTACCCTCTCCCATCCGAAGACGCCC SEQ ID NO: 39 GGATGGGAGAGGGTACTGGACTGCCTTTTG SEQ ID NO: 40 TCCAGTACTTACTCCCATCCGAAGACGCCC SEQ ID NO: 41 GGATGGGAGTAAGTACTGGACTGCCTTTTG SEQ ID NO: 42 TCCAGTACTACCTCCCATCCGAAGACGCCC SEQ ID NO: 43 GGATGGGAGGTAGTACTGGACTGCCTTTTG SEQ ID NO: 44 TCCAGTACTCCCTCCCATCCGAAGACGCCC SEQ ID NO: 45 GGATGGGAGGGAGTACTGGACTGCCTTTTG SEQ ID NO: 46 TCCAGTACAAGCTCCCATCCGAAGACGCCC SEQ ID NO: 47 GGATGGGAGCTTGTACTGGACTGCCTTTTG SEQ ID NO: 48 TCCAGTACATGCTCCCATCCGAAGACGCCC SEQ ID NO: 49 GGATGGGAGCATGTACTGGACTGCCTTTTG SEQ ID NO: 50 TCCAGTACATGCTCCCATCCGAAGACGCCC SEQ ID NO: 51 GGATGGGAGCATGTACTGGACTGCCTTTTG SEQ ID NO: 52 TCCAGTACGAACTCCCATCCGAAGACGCCC SEQ ID NO: 53 GGATGGGAGTTCGTACTGGACTGCCTTTTG SEQ ID NO: 54 TCCAGTACGATCTCCCATCCGAAGACGCCC SEQ ID NO: 55 GGATGGGAGATCGTACTGGACTGCCTTTTG SEQ ID NO: 56 TCCAGTACGGTCTCCCATCCGAAGACGCCC SEQ ID NO: 57 GGATGGGAGACCGTACTGGACTGCCTTTTG SEQ ID NO: 58 TCCAGTACTGGCTCCCATCCGAAGACGCCC SEQ ID NO: 59 GGATGGGAGCCAGTACTGGACTGCCTTTTG SEQ ID NO: 60 TCCAGTACTCCCTCCCATCCGAAGACGCCC SEQ ID NO: 61 GGATGGGAGGGAGTACTGGACTGCCTTTTG SEQ ID NO: 62 TCCAGTACTTCCTCCCATCCGAAGACGCCC SEQ ID NO: 63 GGATGGGAGGAAGTACTGGACTGCCTTTTG SEQ ID NO: 64 TCCAGTACAAGCTCCCATCCGAAGACGCCC SEQ ID NO: 65 GGATGGGAGGTTGTACTGGACTGCCTTTTG

[0151] Each of the prepared vectors was transformed into the CA09-0901 strain by electroporation. The 19 strains into each of which a modification of heterogeneous nucleotide substitution is introduced to the ddh gene were named as follows: CA09-0900::ddh(T169A), CA09-0900::ddh(T169V), CA09-0900::ddh(T169Q), CA09-0900::ddh(T169H), CA09-0900::ddh(T169R), CA09-0900::ddh(T169P), CA09-0900::ddh(T169L), CA09-0900::ddh(T169Y), CA09-0900::ddh(T169S), CA09-0900::ddh(T169K), CA09-0900::ddh(T169M), CA09-0900::ddh(T1690, CA09-0900::ddh(T169E), CA09-0900::ddh(T169D), CA09-0900::ddh(T169G), CA09-0900::ddh(T169W), CA09-0900::ddh(T169C), CA09-0900::ddh(T169F), and CA09-0900::ddh(T169N).

[0152] The ddh gene in the CA09-0900 strain was deleted by the method used in Example 2, and the resulting strain was named as CA09-0900:Δddh. The CA09-0900 and CA09-0900Δddh strains were used as control groups, and the selected 19 kinds of strains were cultured by the method shown below, and the concentrations of lysine and threonine and their glucose consumption rates were measured.

TABLE-US-00013 TABLE 13 Measurements of lysine-producing ability, threonine- producing ability, and glucose consumption rates Glucose Thr Conc. Lys Conc. Consumption Strain (g/L) (g/L) Rate (g/hr) CA09-0901 1.43 2.75 4.53 CA09-0900::Δddh 2.67 1.38 2.41 CA09-0900::ddh(T169A) 1.32 2.73 3.98 CA09-0900::ddh(T169V) 1.43 2.58 3.89 CA09-0900::ddh(T169Q) 1.38 2.62 3.91 CA09-0900::ddh(T169H) 1.67 2.63 4.23 CA09-0900::ddh(T169R) 1.72 2.41 2.44 CA09-0900::ddh(T169P) 1.81 2.25 3.16 CA09-0900::ddh(T169L) 2.48 1.52 3.97 CA09-0900::ddh(T169Y) 1.50 2.66 4.51 CA09-0900::ddh(T169S) 1.62 2.33 4.28 CA09-0900::ddh(T169K) 1.91 1.50 2.22 CA09-0900::ddh(T169M) 1.02 1.75 2.38 CA09-0900::ddh(T169I) 1.97 1.68 3.08 CA09-0900::ddh(T169E) 1.54 1.66 2.59 CA09-0900::ddh(T169D) 1.99 1.87 3.65 CA09-0900::ddh(T169G) 1.42 2.61 4.07 CA09-0900::ddh(T169W) 1.53 2.58 3.99 CA09-0900::ddh(T169C) 1.91 1.74 3.78 CA09-0900::ddh(T169F) 1.80 1.18 4.03 CA09-0900::ddh(T169N) 1.44 2.77 4.35

[0153] In the strain where the ddh gene is deleted, the threonine concentration was increased by 1.24 g/L and the lysine concentration was decreased by 1.37 g/L compared to its parent stain. Considering that the glucose was decreased by 46.1% P, in a case where no DDH activity is present due to the deletion of the ddh gene, the growth of the strain is inhibited although the THR production is increased and the LYS production is decreased, thus making it difficult to use the strain industrially. In the cases of strains including a modified polypeptide, in each of which the 169.sup.th amino acid of SEQ ID NO: 1 is substituted with a different amino acid, the LYS production was decreased and the THR production was increased while the growth of the strain was maintained at a level to be applicable in the industry. That is, it was confirmed that when the ddh gene is weakened, it helps to increase the THR production while LYS production is decreased, and the ddh gene is weakened due to the change in the 169.sup.th amino acid of SEQ ID NO: 1 (Table 13). Additionally, with respect to the modification of the 169.sup.th amino acid, the modification where threonine is substituted with lysine results in a significant increase in the reduction of lysine production and an increase of THR production and a glucose consumption rate in a commercially available level, and was thus determined to be most effective.

Example 8: Preparation and Evaluation of Strains into which Modified Ddh and Modified dapB are Introduced in Microorganism Strain of Genus Corynebacterium Having Ability of Producing L-Threonine

[0154] From the CA09-0904 strain prepared in Example 6, it was confirmed that the strain in which L-lysine production is reduced has a positive effect on the production of L-threonine. In order to confirm whether the ability of producing L-threonine can be further enhanced by further weakening the L-lysine biosynthesis pathway in the above strain, strains were developed.

[0155] Specifically, in order to weaken the activity of the enzyme involved in the second reaction of the L-lysine biosynthesis pathway (i.e., 4-hydroxy-tetrahydrodipicolinate reductase (dapB)), the 13.sup.th amino acid of dapB (i.e., arginine) was substituted with asparagine (SEQ ID NO: 66).

[0156] More specifically, in order to prepare strains into which the dapB(R13N) modification is introduced, PCR was performed using the chromosomal DNA of the ATCC13032 strain as a template along with a primer pair of SEQ ID NOS: 67 and 68 or a primer pair of SEQ ID NOS: 69 and 70, respectively. PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as polymerase for a PCR reaction. The PCR was performed as follows: 28 cycles of denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 1 minute.

[0157] As a result, with respect to the modification of the dapB gene, a 512 bp DNA fragment in the 5′ upstream region and a 514 bp DNA fragment in the 3′ downstream region were obtained, respectively. PCR was performed using the two amplified DNA fragments as templates along with the primers of SEQ ID NO: 67 and SEQ ID NO: 70. The PCR was performed as follows: denaturation at 95° C. for 5 minutes; 28 cycles of denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 2 minutes; and polymerization at 72° C. for 5 minutes.

[0158] As a result, a 1,001 bp DNA fragment, which includes a modification of the dapB gene that encodes a 4-hydroxy-tetrahydrodipicolinate reductase variant where the 13.sup.th amino acid (i.e., arginine) is substituted with asparagine, was amplified. The amplified product was purified using a PCR purification kit (QIAGEN) and used as an insertion DNA fragment for the preparation of a vector. Meanwhile, a pDZ-R13N vector for the introduction of an R13N modification into the chromosome was prepared as follows: the pDZ vector (which was digested with a restriction enzyme SmaI and then subjected to heat treatment at 65° C. for 20 minutes) and the insertion DNA fragment (which was amplified by PCR above) were combined in a molar concentration ratio (M) of 1:2, and cloning was performed using an Infusion Cloning kit (TaKaRa) according to the manual provided.

[0159] The prepared vector was transformed into the CA09-0904 strain by electroporation, and the transformed strain was subjected to a second cross-over, and thereby, a strain in which each nucleotide is substituted with a modified nucleotide on the chromosome was obtained. The strain was named as CA09-0904-R13N.

TABLE-US-00014 TABLE 14 Confirmation of abilities of prepared strains for producing L-threonine and L-lysine Amino acid (g/L) Strain Thr Lys CA09-0900 1.52 2.70 CA09-0904 2.41 1.53 CA09-0904-R13N 3.03 1.08

[0160] As a result, the strain introduced with the modification showed a decrease of L-lysine production by 1.62 g/L and an increase of L-threonine production by 1.51 g/L compared to the CA09-0900 strain (control group), while showing a decrease of L-lysine production by 0.48 g/L and an increase of L-threonine production by 0.62 g/L compared to the CA09-0904 strain (Table 14). Therefore, it was confirmed that the weakening of the L-lysine production pathway was positive for L-threonine production.

Example 9: Preparation and Evaluation of Strains into which Modified ddh and Modified lysA are Introduced in Microorganism Strain of Genus Corynebacterium Having Ability of Producing L-Threonine

[0161] From the CA09-0904 strain prepared in Example 6, it was confirmed that the strain in which L-lysine production is reduced has a positive effect on the production of L-threonine. In order to confirm whether the ability of producing L-threonine can be further enhanced by further weakening the L-lysine biosynthesis pathway in the above strain, strains were developed.

[0162] Specifically, in order to weaken the activity of the enzyme involved in the final reaction of the L-lysine biosynthesis pathway (i.e., diaminopimelate decarboxylase (lysA)), the 408.sup.th amino acid of lysA (i.e., methionine) was substituted with alanine (Biochemical and Biophysical Research Communications, Volume 495, Issue 2, 8 Jan. 2018) (SEQ ID NO: 71).

[0163] More specifically, in order to prepare strains into which the lysA(M408A) modification is introduced, PCR was performed using the chromosomal DNA of the ATCC13032 strain as a template along with a primer pair of SEQ ID NOS: 72 and 73 or a primer pair of SEQ ID NOS: 74 and 75, respectively. PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as polymerase for a PCR reaction. The PCR was performed as follows: 28 cycles of denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 1 minute.

[0164] As a result, with respect to the modification of the lysA gene, a 534 bp DNA fragment in the 5′ upstream region and a 527 bp DNA fragment in the 3′ downstream region were obtained, respectively. PCR was performed using the two amplified DNA fragments as templates along with the primers of SEQ ID NO: 72 and SEQ ID NO: 75. The PCR was performed as follows: denaturation at 95° C. for 5 minutes; 28 cycles of denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 2 minutes; and polymerization at 72° C. for 5 minutes.

TABLE-US-00015 TABLE 15 SEQ ID NO Sequence (5′->3′) SEQ ID NO: 72 TCGAGCTCGGTACCCGTTGGGCCTGTACTCACAG SEQ ID NO: 73 TAGCGGGAGCTCGCGGCGTAGCAGTATGCGCC SEQ ID NO: 74 TACTGCTACGCCGCGAGCTCCCGCTACAACGC SEQ ID NO: 75 CTCTAGAGGATCCCGTGCAAGGTGAACCAACTG

[0165] As a result, a 1,035 bp DNA fragment, which includes a modification of the lysA gene that encodes a diaminopimelate decarboxylase variant where the 408.sup.th amino acid (i.e., methionine) is substituted with alanine, was amplified. The amplified product was purified using a PCR purification kit (QIAGEN) and used as an insertion DNA fragment for the preparation of a vector. Meanwhile, a pDZ-M408A vector for the introduction of an M408A modification into the chromosome was prepared as follows: the pDZ vector (which was digested with a restriction enzyme SmaI and then subjected to heat treatment at 65° C. for 20 minutes) and the insertion DNA fragment (which was amplified by PCR above) were combined in a molar concentration ratio (M) of 1:2, and cloning was performed using an Infusion Cloning kit (TaKaRa) according to the manual provided.

[0166] The prepared vector was transformed into the CA09-0904 strain by electroporation, and the transformed strain was subjected to a second cross-over, and thereby, a strain in which each nucleotide is substituted with a modified nucleotide on the chromosome was obtained. The strain was named as CA09-0904-M408A.

TABLE-US-00016 TABLE 16 Confirmation of abilities of prepared strains for producing L-threonine and L-lysine Amino acid (g/L) Strain Thr Lys CA09-0900 1.61 2.51 CA09-0904 2.63 1.52 CA09-0904-M408A 3.08 1.10

[0167] As a result, the strain introduced with the modification showed a decrease of L-lysine production by 1.41 g/L and an increase of L-threonine production by 1.33 g/L compared to the CA09-0900 strain (control group), while showing a decrease of L-lysine production by 0.42 g/L and an increase of L-threonine production by 0.35 g/L compared to the CA09-0904 strain (Table 16). Therefore, it was confirmed that the weakening of the L-lysine production pathway was positive for L-threonine production.

Example 10: Preparation and Evaluation of Strains into which Modified ddh and Modified dapA are Introduced in Microorganism Strain of Genus Corynebacterium Having Ability of Producing L-Threonine

[0168] From the CA09-0904 strain prepared in Example 6, it was confirmed that the strain in which L-lysine production is reduced has a positive effect on the production of L-threonine. In order to confirm whether the ability of producing L-threonine can be further enhanced by further weakening the L-lysine biosynthesis pathway in the above strain, strains were developed.

[0169] Specifically, in order to weaken the activity of the enzyme involved in the second reaction of the L-lysine biosynthesis pathway (i.e., 4-hydroxy-tetrahydrodipicolinate synthase (dapA)), the 119.sup.th amino acid of dapA (i.e., tyrosine) was substituted with phenylalanine (Journal of Molecular biology, Volume 338, Issue 2, 23 Apr. 2004)) (SEQ ID NO: 76).

[0170] More specifically, in order to prepare strains into which the dapA(Y119F) modification is introduced, PCR was performed using the chromosomal DNA of the ATCC13032 strain as a template along with a primer pair of SEQ ID NOS: 77 and 78 or a primer pair of SEQ ID NOS: 79 and 80, respectively. PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as polymerase for a PCR reaction. The PCR was performed as follows: 28 cycles of denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 1 minute.

[0171] As a result, with respect to the modification of the dapA gene, a 538 bp DNA fragment in the 5′ upstream region and a 528 bp DNA fragment in the 3′ downstream region were obtained, respectively. PCR was performed using the two amplified DNA fragments as templates along with the primers of SEQ ID NO: 77 and SEQ ID NO: 80. The PCR was performed as follows: denaturation at 95° C. for 5 minutes; 28 cycles of denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 2 minutes; and polymerization at 72° C. for 5 minutes.

TABLE-US-00017 TABLE 17 SEQ ID NO Sequence (5′->3′) SEQ ID NO: 77 TCGAGCTCGGTACCCTTCATATAGTTAAGACAAC SEQ ID NO: 78 CGGCTTGGAGAAATAAGGAGTTACAACTAAAAG SEQ ID NO: 79 TAACTCCTTATTTCTCCAAGCCGAGCCAAGAG SEQ ID NO: 80 CTCTAGAGGATCCCGAGCCTCAAGTTCCTGCTC

[0172] As a result, a 1,000 bp DNA fragment, which includes a modification of the dapA gene that encodes a 4-hydroxy-tetrahydrodipicolinate synthase variant where the 119.sup.th amino acid (i.e., tyrosine) is substituted with phenylalanine, was amplified. The amplified product was purified using a PCR purification kit (QIAGEN) and used as an insertion DNA fragment for the preparation of a vector. Meanwhile, a pDZ-Y119F vector for the introduction of a dapA(Y119F) modification into the chromosome was prepared as follows: the pDZ vector (which was digested with a restriction enzyme SmaI and then subjected to heat treatment at 65° C. for 20 minutes) and the insertion DNA fragment (which was amplified by PCR above) were combined in a molar concentration ratio of 1:2, and cloning was performed using an Infusion Cloning kit (TaKaRa) according to the manual provided.

[0173] The prepared vector was transformed into the CA09-0904 strain by electroporation, and the transformed strain was subjected to a second cross-over, and thereby, a strain in which each nucleotide is substituted with a modified nucleotide on the chromosome was obtained. The strain was named as CA09-0904-Y119F.

TABLE-US-00018 TABLE 18 Confirmation of abilities of prepared strains for producing L-threonine and L-lysine Amino acid (g/L) Strain Thr Lys CA09-0900 1.48 2.68 CA09-0904 2.52 1.57 CA09-0904-Y119F 3.31 0.82

[0174] As a result, the strain introduced with the modification showed a decrease of L-lysine production by 1.86 g/L and an increase of L-threonine production by 1.83 g/L compared to the CA09-0900 strain (control group), while showing a decrease of L-lysine production by 0.75 g/L and an increase of L-threonine production by 0.79 g/L compared to the CA09-0904 strain (Table 18). Therefore, it was confirmed that the weakening of the L-lysine production pathway was positive for L-threonine production.

[0175] The above results suggest that a strain which includes a modified polypeptide of meso-diaminopimelate dehydrogenase, in which the 169.sup.th amino acid in the amino acid sequence of SEQ ID NO: 1 of the present disclosure is substituted with leucine, phenylalanine, glutamate, or cysteine, eventually has an enhanced ability of producing L-threonine through the decrease of the amount of L-lysine production and an increase of the amount of L-threonine production, compared to non-modified strains.

[0176] From the foregoing, one of ordinary skill 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.