Modified homoserine dehydrogenase and method for producing homoserine or L-amino acid derived from homoserine using the same

Abstract

The present disclosure relates to modified homoserine dehydrogenase and a method for producing homoserine or a homoserine-derived L-amino acid using the same.

Claims

1. A modified homoserine dehydrogenase comprising an amino acid sequence, wherein the amino acid sequence is substituted with histidine at a position corresponding to position 407 in the amino acid sequence of SEQ ID NO: 1.

2. A polynucleotide encoding the modified homoserine dehydrogenase of claim 1.

3. A microorganism of the genus Corynebacterium, comprising the modified homoserine dehydrogenase of claim 1.

4. The microorganism according to claim 3, wherein the microorganism of the genus Corynebacterium produces homoserine or a homoserine-derived L-amino acid.

5. The microorganism according to claim 4, wherein the homoserine-derived L-amino acid is at least one kind selected from the group consisting of L-threonine, L-isoleucine, O-acetyl homoserine, and L-methionine.

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

7. A method for producing homoserine or a homoserine-derived L-amino acid, comprising: culturing in a medium a microorganism of the genus Corynebacterium comprising the modified homoserine dehydrogenase of claim 1; and recovering homoserine or a homoserine-derived L-amino acid from the cultured microorganism or cultured medium.

8. The method according to claim 7, wherein the homoserine-derived L-amino acid is at least one kind selected from the group consisting of L-threonine, L-isoleucine, O-acetyl homoserine, and L-methionine.

9. The method according to claim 7, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.

10. A modified homoserine dehydrogenase that comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1, and has histidine at a position corresponding to position 407 in the amino acid sequence of SEQ ID NO: 1.

11. The modified homoserine dehydrogenase of claim 10, wherein the modified homoserine dehydrogenase is at least 90% identical to the amino acid sequence of SEQ ID NO: 1.

12. The modified homoserine dehydrogenase of claim 10, wherein the modified homoserine dehydrogenase is at least 95% identical to the amino acid sequence of SEQ ID NO: 1.

13. A polynucleotide encoding the modified homoserine dehydrogenase of claim 10.

14. A microorganism of the genus Corynebacterium, comprising the modified homoserine dehydrogenase of claim 10.

15. The microorganism according to claim 14, wherein the microorganism of the genus Corynebacterium produces homoserine or a homoserine-derived L-amino acid.

16. The microorganism according to claim 15, wherein the homoserine-derived L-amino acid is at least one kind selected from the group consisting of L-threonine, L-isoleucine, O-acetyl homoserine, and L-methionine.

17. The microorganism according to claim 14, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.

18. A method for producing homoserine or a homoserine-derived L-amino acid, comprising: culturing in a medium a microorganism of the genus Corynebacterium comprising the modified homoserine dehydrogenase of claim 10; and recovering homoserine or a homoserine-derived L-amino acid from the cultured microorganism or cultured medium.

19. The method according to claim 18, wherein the homoserine-derived L-amino acid is at least one kind selected from the group consisting of L-threonine, L-isoleucine, O-acetyl homoserine, and L-methionine.

20. The method according to claim 18, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Hereinafter, the present disclosure will be described in detail through exemplary embodiments. However, these exemplary embodiments are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

Example 1: Screening for AHV-Resistant Microorganisms Through Artificial Modification

(2) In this Example, an experiment of imparting resistance against 2-amino-3-hydroxy-valerate (hereinafter, “AHV”), which is an L-threonine analog, was conducted using Corynebacterium glutamicum KFCC10881 (KR Patent No. 0159812) as a parent strain, so as to release the feedback inhibition by L-threonine of homoserine dehydrogenase (hereinafter, “Hom”, EC:1.1.1.3).

(3) Modification was induced by an artificial modification method using N-methyl-N′-nitro-N-nitrosoguanidine (hereinafter, “NTG”). The KFCC10881 strain, which had been cultured in a seed medium for 18 hours, was inoculated into 4 mL of the seed medium, and then cultured until OD.sub.660 reached about 1.0. The culture medium was centrifuged to recover the cells, and then the cells were washed twice with a 50 mM Tris-malate buffer (pH 6.5) and suspended in the final 4 mL of the same buffer. An NTG solution (2 mg/mL in a 0.05 M Tris-malate buffer (pH 6.5)) was added to the cell suspension to have a final concentration of 150 mg/L, and then allowed to stand at room temperature for 20 minutes. Thereafter, the cells were recovered by centrifugation, and washed twice with the same buffer to remove the NTG. The finally washed cells were suspended in 4 mL of a 20% glycerol solution and then stored at −70° C. until use. The NTG-treated strains were plated on a minimal medium containing 3 g/L of AHV, and then 126 AHV-resistant strains derived from KFCC10881 were obtained through the above procedure.

(4) Seed Medium (pH 7.0)

(5) glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH.sub.2PO.sub.4 4 g, K.sub.2HPO.sub.4 8 g, MgSO.sub.4 7H.sub.2O 0.5 g, biotin 100 μg, thiamine HCl 1,000 μg, calcium pantothenate 2,000 μg, nicotinamide 2,000 μg (based on 1 L of distilled water)

(6) Minimal Medium (pH 7.2)

(7) glucose 5 g, KH.sub.2PO.sub.4 1 g, (NH.sub.4).sub.2SO.sub.4 5 g, MgSO.sub.4 7H.sub.2O 0.4 g, NaCl 0.5 g, biotin 200 μg, thiamine HCl 100 μg, calcium pantothenate 100 μg, nicotinamide 0.03 g, urea 2 g, Na.sub.2B.sub.4O.sub.7 10H.sub.2O 0.09 mg, (NH.sub.4).sub.6Mo.sub.7O.sub.27 4H.sub.2O 0.04 mg, ZnSO.sub.4 7H.sub.2O 0.01 mg, CuSO.sub.4 5H.sub.2O, MnCl.sub.2 4H.sub.2O 0.01 mg, FeCl.sub.3 6H.sub.2O 1 mg, CaCl.sub.2 0.01 mg (based on 1 L of distilled water)

Example 2: L-Threonine Production Test for AHV-Resistant Strain Derived from KFCC10881

(8) A test for the L-threonine-producing ability was conducted on the 126 AHV-resistant strains obtained in Example 1. The 126 strains obtained in Example 1 were inoculated into each corner-baffled flask (250 mL) containing the seed medium (25 mL), and then cultured with shaking at 30° C. at 200 rpm for 20 hours. The seed culture medium (1 mL) was inoculated into each corner-baffled flask (250 mL) containing the L-threonine production medium (24 mL) below, and then cultured with shaking at 30° C. at 200 rpm for 48 hours.

(9) L-Threonine Production Medium (pH 7.2)

(10) glucose 30 g, KH.sub.2PO.sub.4 2 g, urea 3 g, (NH.sub.4).sub.2SO.sub.4 40 g, peptone 2.5 g, CSL (Sigma) 5 g (10 mL), MgSO.sub.4 7H.sub.2O 0.5 g, leucine 400 mg, CaCO.sub.3 20 g (based on 1 L of distilled water)

(11) After the culture, the amounts of various amino acids produced were measured using HPLC. The concentrations of the amino acids in the culture media for the top 5 strains, which were shown to have excellent L-threonine-producing abilities among the 126 strains experimented on, are shown in Table 1. The 5 candidate strains confirmed through the above procedure were named KFCC10881-1 to KFCC10881-5.

(12) TABLE-US-00001 TABLE 1 Experiments on L-threonine production of excellent AHV-resistant strains Thr + Hse + OD Thr Hse Gly Ile Lys Gly + Ile KFCC10881 60.1 0.0 0.1 0.2 0.0 12.3 0.3 KFCC10881-1 53.6 4.1 1.3 1.4 1.2  2.0 8.0 KFCC10881-2 53.3 2.2 0.9 1.0 1.1  8.3 5.2 KFCC10881-3 68.5 1.5 1.2 1.1 0.2 10.8 4.0 KFCC10881-4 59.1 1.2 0.9 1.0 0.7  1.9 3.8 KFCC10881-5 49.6 2.4 1.1 1.2 0.9  5.4 5.6

(13) As shown in Table 1, the amounts of L-threonine, L-homoserine, glycine, L-alanine, and L-isoleucine, which are produced by the 5 types of strains having resistance to AHV, were increased compared to a control group, whereas the amount of L-lysine produced was decreased.

(14) The biosynthetic pathways of L-threonine and L-lysine are separated from aspartate-semialdehyde (hereinafter, “ASA”) as a branching point. That is, the amount of L-lysine produced is decreased as the amount of L-threonine produced is increased. Accordingly, the amounts of homoserine (Hse), glycine (Gly), and L-isoleucine (Ile), which can be by-products in the L-threonine biosynthetic pathway, may be increased as the amount of L-threonine produced is increased, and thus the total amount thereof produced (Thr+Hse+Gly+Ile) was also confirmed.

(15) Therefore, among the AHV-resistant strains above, the KFCC10881-1 strain, which showed a reduced amount of L-lysine production, a high amount of L-threonine production, and a high amount of total (Thr+Hse+Gly+Ile) production, was selected as the most excellent AHV-resistant strain.

Example 3: Analysis of Nucleotide Sequences of Strains Having Excellent Ability to Produce Threonine Derived from KFCC10881

(16) To analyze the nucleotide sequences of the L-threonine biosynthesis enzymes of the strain selected in Example 2 above, the following experiment was conducted. Based on the gene information provided by the Kyoto Encyclopedia of Genes and Genomes (KEGG), each of the nucleotide sequence of hom (SEQ ID NO: 2, NCg11136), which encodes homoserine dehydrogenase of Corynebacterium glutamicum ATCC13032, and the nucleotide sequence of thrB (SEQ ID NO: 3, Gene No. NCg11137), which encodes homoserine kinase, were obtained. Both hom and thrB genes are known to have an operon structure (Peoples et al., Mol. Biol. 2(1):63-72, 1988).

(17) To obtain the DNA fragment containing the hom-thrB operon of the selected strain PCR was carried out using the genomic DNA of the strain as a template and a primer set of SEQ ID NO: 4 and SEQ ID NO: 5. PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as a polymerase for the PCR reaction. PCR conditions were as follows: 30 cycles of denaturation at 96° C. for 30 seconds, annealing at 52° C. for 30 seconds, and polymerization at 72° C. for 3 minutes. As a result, it was possible to amplify a gene fragment (2,778 bp; SEQ ID NO: 6), which includes the nucleotide sequence (300 bp) containing a promoter region upstream of the initiation codon of SEQ ID NO: 2 to include the 200 bp downstream of the termination codon of SEQ ID NO: 3.

(18) The nucleotide sequence was determined using the prepared primers by an ABI PRISM 3730XL Analyzer (96 capillary type; Applied Biosystems). In the nucleotide sequence corresponding to hom of the hom-thrB operon in the KFCC10881-1 strain, guanine (i.e., the nucleotide at position 1,220 of SEQ ID NO: 2) was modified to adenine, and thus the CGT gene codon encoding an arginine residue was modified to the CAT gene codon encoding a histidine residue (hereinafter, “R407H modification”; SEQ ID NO: 7). Meanwhile, no modification was discovered in the thrB gene corresponding to SEQ ID NO: 3.

(19) From the nucleotide sequence analyses above, it was possible to conclude that the feedback inhibition by L-threonine was desensitized by the modification of arginine (i.e., the 407.sup.th amino acid residue) of the Hom (SEQ ID NO: 8) in the KFCC10881-1 strain to histidine (hereinafter, “R407H modification”).

Example 4: Preparation of Novel Strains to which Homoserine Dehydrogenase is Introduced

(20) A primer set of SEQ ID NO: 9 and SEQ ID NO: 10 was prepared so as to prepare strains in which the variant (R407H) identified in Example 2 was introduced to their wild-type strains.

(21) To prepare strains to which each of the R407H hom modification is introduced, PCR was carried out using the genomic DNA extracted from the KFCC10811-1 strain as a template and the primer set of SEQ ID NO: 9 and SEQ ID NO: 10. PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as a polymerase for the PCR reaction. PCR conditions were 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 2 minutes. As a result, a gene fragment (1,668 bp) including a promoter region (about 300 bp) of the hom gene (1,338 bp) was obtained. The amplified product was purified using a PCR purification kit (QIAGEN) and used as an insert DNA fragment for the preparation of a vector. Meanwhile, after treating with restriction enzyme SmaI, the ratio of the molar concentration (M) of the pDZ vector (KR Patent No. 10-0924065) heat-treated at 65° C. for 20 minutes to the insert DNA fragment amplified by the PCR above was set to be 1:2, and the vector was cloned using an Infusion Cloning Kit (TaKaRa) according to the manufacturer's manual, and thereby the vector for introducing the R407H modification into the chromosome, pDZ-R407H, was prepared.

(22) The pDZ-R407H vector was transformed into Corynebacterium glutamicum ATCC13032 by electroporation and subjected to secondary crossover, and thereby a strain in which a substitution of a modified nucleotide was introduced into the chromosome was obtained. Using the primer sets listed below and a Mutant Allele Specific Amplification (MASA) PCR technique (Takeda et al., Hum. Mutation, 2, 112-117 (1993)), the appropriateness of the substitution was primarily determined by selecting the strain amplified using the primer set corresponding to the modified sequence (SEQ ID NO: 11 and SEQ ID NO: 12). In addition, analysis of the hom sequence of the selected strain was conducted to secondarily confirm the appropriateness of the substitution using the primer set of SEQ ID NO: 11 and SEQ ID NO: 13 and by analyzing the modified sequence in the same manner as in Example 2. The strain substituted with the modified nucleotide was named CA09-0900.

(23) The strain CA09-0900 was deposited at the Korean Culture Center of Microorganisms (KCCM), an International Depositary Authority under the Budapest Treaty, on Dec. 14, 2018, and was assigned Accession No. KCCM12418P.

Example 5: Measurement of Activity of Homoserine Dehydrogenase

(24) The activity of the enzyme Hom was measured in the prepared strain. The wild-type strain ATCC13032 (the control group) and the CA09-0900 strain prepared in Example 4 were each inoculated into 25 mL of the seed medium and cultured until the strains reached the late log phase. The cells of each strain were recovered by centrifugation, washed twice with a 0.1 M potassium phosphate buffer (pH 7.6), and finally suspended in 2 mL of the same buffer containing glycerol at a concentration of 30%. Each cell suspension was physically disrupted by a conventional glass bead vortexing method for 10 minutes, and each supernatant was recovered through two centrifugations (13,000 rpm, 4° C., 30 minutes) and used as a crude extract for measuring the activity of Hom. For the measurement of the activity of Hom, a coenzyme solution (0.1 mL) was added to a reaction solution for measuring the enzyme activity (a potassium phosphate (pH 7.0) buffer, 25 mM NADPH, 5 mM aspartate semi-aldehyde) and reacted at 30° C. The Hom enzyme activity U was defined as the number of NADPH consumed per minute according to the presence of L-threonine (0 mM, 10 mM), and the measurement results of the enzyme activity are shown in Table 2 below.

(25) TABLE-US-00002 TABLE 2 Measurement of Hom activity (U) and desensitization by L-threonine Enzyme Activity (U) according to Amount of L-Threonine Added (mM) Strain 0 mM 10 mM ATCC13032 0.91 0.02 CA09-0900 1.37 1.23

(26) As a result of the experiment, it was confirmed that in the Hom including the R407H modification, the inhibition of the activity was reduced under the condition where 10 mM L-threonine was contained, unlike the wild-type Hom, thus confirming the occurrence of desensitization to L-threonine.

Example 6: Preparation and Evaluation of Microorganism Strains of the Genus Corynebacterium Having L-Threonine-Producing Ability

(27) Strains producing L-threonine were developed from the wild-type Corynebacterium glutamicum ATCC13032. Specifically, to resolve the feedback inhibition by aspartate kinase (lysC) (i.e., an important enzyme which is acted upon first in the threonine biosynthesis pathway), leucine (i.e., which is an amino acid at position 377 of lysC) was substituted with lysine (SEQ ID NO: 14).

(28) More specifically, to prepare strains in which the lysC (L377K) modification is introduced, PCR was carried out using the chromosome of ATCC13032 as a template and the primers set of SEQ ID NOS: 15 and 16 or SEQ ID NOS: 17 and 18. PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as a polymerase for the PCR reaction. PCR conditions were 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. As a result, a DNA fragment (515 bp) in the 5′ upstream region and a DNA fragment (538 bp) in the 3′ downstream region, with the modification site of lysC gene as the center, were each obtained. PCR was carried out with the two amplified DNA fragments as a template and the primer set of SEQ ID NO: 15 and SEQ ID NO: 18. PCR was carried out 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. As a result, the DNA fragment (1,023 bp) including the modification of lysC gene, which encodes an aspartokinase variant in which leucine at position 377 is substituted with lysine, was amplified. The amplified product was purified using a PCR purification kit (QIAGEN) and used as an insert DNA fragment for the preparation of a vector. Meanwhile, after treating with restriction enzyme SmaI, the ratio of the molar concentration (M) of the pDZ vector (KR Patent No. 10-0924065) heat-treated at 65° C. for 20 minutes to the insert DNA fragment amplified by the PCR above was set to be 1:2, and the vector was cloned using an Infusion Cloning Kit (TaKaRa) according to the manufacturer's manual, and thereby the vector for introducing the L377K modification into the chromosome, pDZ-L377K, was prepared.

(29) The prepared pDZ-L377K vector was transformed into the ATCC13032 strain and subjected to secondary crossover, and thereby a strain in which a substitution of a modified nucleotide was introduced into the chromosome was obtained. The strain was named CJP1. The CJP1 strain was named again as CA01-2307, deposited at the Korean Culture Center of Microorganisms (KCCM), an International Depositary Authority under the Budapest Treaty, on Mar. 29, 2017, and was assigned Accession No. KCCM12000P.

(30) To clearly confirm the changes in the L-threonine production of the above strain, the modification identified in Example 4 was introduced into a gene encoding homoserine dehydrogenase. Specifically, to introduce the R407H modification into the CJP1 strain, the pDZ-R407H vector prepared in Example 4 was transformed into the CJP1 strain by electroporation and subjected to a secondary crossover, and thereby a strain in which a modified nucleotide was introduced into the chromosome was obtained. The strain substituted with a modified nucleotide was named CJP1-R407H.

(31) TABLE-US-00003 TABLE 3 Confirmation of L-threonine-producing ability of prepared strains Amino acid (g/L) Strain Thr Lys CJP1 0.36 3.62 CJP1-R407H 1.50 2.47

(32) As a result, in the strain where the modification was introduced, the amount of L-lysine produced was decreased and the amount of L-threonine produced was increased by 1.14 g/L, compared to the CJP1 strain (the control group), thus confirming a significant improvement in the effect of desensitization.

Example 7: Preparation and Evaluation of Microorganism Strains of the Genus Corynebacterium Producing L-Isoleucine

(33) To prepare isoleucine-producing strains, a vector was prepared for enhancing the expression of the modified gene ilvA(V323A) (Appl. Enviro. Microbiol., December 1996, p. 4345-4351), which encodes known L-threonine dehydratase (the first enzyme in the isoleucine biosynthesis pathway), in the strains prepared in Example 6.

(34) Specifically, to prepare a vector for introducing a modification, which targets the gene ilvA, a pair of primers (SEQ ID NOS: 19 and 20) for amplifying the 5′ upstream region and a pair of primers (SEQ ID NOS: 21 and 22) for amplifying the 3′ downstream region were devised with the modification site as the center. BamHI restriction enzyme sites were inserted at each end of the primers of SEQ ID NOS: 19 and 22, and the primers of SEQ ID NOS: 20 and 21 were designed such that a nucleotide-substituted modification can be positioned at a region where a crossover is to be induced.

(35) PCR was carried out with the chromosome of the wild-type strain as a template using the primers of SEQ ID NOS: 19, 20, 21, and 22. PCR was carried out as follows: denaturation at 95° C. for 5 minutes; 30 cycles of denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 30 seconds; and polymerization at 72° C. for 7 minutes. As a result, a DNA fragment (627 bp) in the 5′ upstream region and a DNA fragment (608 bp) in the 3′ downstream region were obtained with the modification site of the gene ilvA as the center.

(36) PCR was carried out using the two amplified DNA fragments as a template and the primer set of SEQ ID NOS: 19 and 22. PCR was carried out as follows: denaturation at 95° C. for 5 minutes; 30 cycles of denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 60 seconds; and polymerization at 72° C. for 7 minutes. As a result, a DNA fragment (1,217 bp) was amplified, in which the DNA fragment included a modification of the gene ilvA that encodes an IlvA variant where valine at position 323 was substituted with alanine. The vector pECCG117 (KR Patent No. 10-0057684) and the DNA fragment (1,217 bp) were treated with restriction enzyme BamHI, ligated using DNA ligase, and then cloned to obtain a plasmid. The thus-obtained plasmid was named pECCG117-ilvA(V323 A).

(37) The pECCG117-ilvA(V323A) vector was introduced to the CJP1-R407H strain prepared in Example 6 by electroporation and plated on a selective medium containing kanamycin (25 mg/L) to obtain the transformed strains. The thus-obtained transformed strains were cultured by the same flask culture method of Example 2, and the concentrations of L-isoleucine in the culture media were analyzed. The results thereof are shown in Table 4 below.

(38) TABLE-US-00004 TABLE 4 Confirmation of L-isoleucine-producing ability of prepared strains Strain L-Isoleucine (g/L) CJP1/pECCG117-ilvA(V323A) 0.7 CJP1-R407H/pECCG117-ilvA(V323A) 1.4

(39) As a result, it was confirmed that in the strain including the hom(R407H) modification, the L-isoleucine-producing ability was improved by 0.7 g/L compared to the control strain.

Example 8: Preparation and Evaluation of O-Acetyl-Homoserine (OAH)-Producing Strain Substituted with Modified Hom

(40) 8-1. Preparation of ATCC13032 Strain Substituted with Modified Hom

(41) The R407H modification was introduced into the hom gene of the ATCC13032 strain in the same manner as in Example 4, and the thus-prepared strain was named ATCC13032::Hom.sup.FBR.

(42) 8-2. Deletion of metB Gene

(43) In this Example, the metB gene encoding cystathionine gamma-synthase in the O-acetyl-homoserine degradation pathway was obtained through PCR using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template. Based on the GenBank of the National Institutes of Health (NIH GenBank), the information of the nucleotide sequence of the metB was obtained (NCBI Registration No. Ncg12360; SEQ ID NO: 23). In addition, based on this, the primers (SEQ ID NOS: 24 and 25) containing the N-terminus and linker sequence of the metB gene and the primers (SEQ ID NOS: 26 and 27) containing the C-terminus and linker sequence of the metB gene were synthesized. PCR was carried out using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template and the oligonucleotides of the nucleotide sequences of SEQ ID NOS: 24 and 25 and SEQ ID NOS: 26 and 27 as the primer sets. PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as a polymerase. PCR was carried out as follows: 30 cycles of denaturation at 96° C. for 30 seconds, annealing at 53° C. for 30 seconds, and polymerization at 72° C. for 1 minute; and polymerization at 72° C. for 7 minutes. As a result, an amplified gene (500 bp) containing the N-terminus and linker of the metB gene and an amplified gene (500 bp) containing the C-terminus and linker of the metB gene were obtained.

(44) PCR was carried out using the two thus-obtained amplified genes as a template and the primer set of SEQ ID NOS: 24 and 27 under the following conditions: 30 cycles of denaturation at 96° C. for 60 seconds, annealing at 50° C. for 60 seconds, and polymerization at 72° C. for 1 minute; and polymerization at 72° C. for 7 minutes. As a result, an amplified ΔmetB gene (1,000 bp), which is a metB inactivation cassette containing the N-terminal-linker-C-terminal of the metB gene, was obtained. The metB gene obtained though the PCR was treated with restriction enzymes XbaI and SalI included at the termini, and then cloned into a pDZ vector, which was treated in advance with the restriction enzymes XbaI and SalI, via ligation. Thereafter, a recombinant pDZ-ΔmetB vector in which the metB inactivation cassette is finally cloned was prepared.

(45) The prepared pDZ-ΔmetB vector was transformed into the Corynebacterium glutamicum ATCC13032 and ATCC13032::Hom.sup.FBR strains. After secondary crossover, the Corynebacterium glutamicum ATCC13032 ΔmetB and ATCC13032::Hom.sup.FBR ΔmetB strains, in which the metB gene is inactivated on the chromosome, were obtained. The inactivated metB gene was finally confirmed by carrying out PCR using the primer set of SEQ ID NOS: 24 and 27, followed by comparing the sequence with the ATCC13032 strain in which the metB gene is not inactivated.

(46) 8-3. Deletion of metY Gene

(47) In this Example, the metY gene encoding O-acetylhomoserine (thiol)-lyase in the 0-acetyl-homoserine degradation pathway was obtained through PCR using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template. Based on GenBank of the National Institutes of Health (NIH GenBank), the information of the nucleotide sequence of the metY gene was obtained (NCBI Registration No. Ncg10625; SEQ ID NO: 28). In addition, based on this, the primers (SEQ ID NOS: 29 and 30) containing the N-terminus and linker sequence of the metY gene and the primers (SEQ ID NOS: 31 and 32) containing the C-terminus and linker sequence of the metY gene were synthesized.

(48) PCR was carried out with the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template using the oligonucleotides of the nucleotide sequences of SEQ ID NOS: 29 and 30 and SEQ ID NOS: 31 and 32 as the primer sets. PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as a polymerase. PCR was carried out as follows: 30 cycles of denaturation at 96° C. for 30 seconds, annealing at 53° C. for 30 seconds, and polymerization at 72° C. for 1 minute; and polymerization at 72° C. for 7 minutes. As a result, an amplified gene (500 bp) containing the N-terminus and linker of the metY gene and an amplified gene (500 bp) containing the C-terminus and linker of the metY gene were obtained. PCR was carried out using the two thus-obtained amplified genes as a template and the primer set of SEQ ID NOS: 29 and 32 under the following conditions: 10 cycles of denaturation at 96° C. for 60 seconds, annealing at 50° C. for 60 seconds, and polymerization at 72° C. for 1 minute; and polymerization at 72° C. for 7 minutes. As a result, an amplified ΔmetY gene (1,000 bp), which is a metY inactivation cassette containing the N-terminal-linker-C-terminal of the metY gene, was obtained.

(49) The metY gene obtained through the PCR was treated with restriction enzymes XbaI and SalI included at the termini, and then cloned into a pDZ vector, which was treated in advance with the restriction enzymes XbaI and SalI, via ligation. Thereafter, a recombinant pDZ-ΔmetY vector in which the metY inactivation cassette is finally cloned was prepared.

(50) The prepared pDZ-ΔmetY vector was transformed into the Corynebacterium glutamicum ATCC13032, ATCC13032::Hom.sup.FBR, ATCC13032 ΔmetB, and ATCC13032::Hom.sup.FBR ΔmetB strains. After secondary crossover, Corynebacterium glutamicum ATCC13032 ΔmetY, ATCC13032::Hom.sup.FBR ΔmetY, ATCC13032 ΔmetB ΔmetY, and ATCC13032::Hom.sup.FBR ΔmetB ΔmetY, in which the metY gene is inactivated on the chromosome, were obtained. The inactivated metY gene was finally confirmed by carrying out PCR using the primer set of SEQ ID NOS: 29 and 32, followed by comparing the sequence with ATCC13032 in which the metY gene is not inactivated.

(51) 8-4. Preparation and Evaluation of Strain Producing O-Acetyl-Homoserine

(52) Comparison was made between the O-acetyl-homoserine-producing abilities of the ATCC13032, ATCC13032 ΔmetB, ATCC13032 ΔmetY, ATCC13032 ΔmetBΔmetY, ATCC13032::Hom.sup.FBR, ATCC13032::Hom.sup.FBR ΔmetB, ATCC13032::Hom.sup.FBR ΔmetY, and ATCC13032::Hom.sup.FBR ΔmetBΔmetY strains prepared in Examples 8-1 to 8-3, in which the metB, metY, and metBY genes are deleted and the modified hom gene is substituted therein.

(53) Specifically, single colonies were cultured in a solid LB medium overnight in a 32° C. incubator, and one loopful of each of the single colonies was inoculated into O-acetyl-homoserine titer media (25 mL), and then the resultants were cultured at 32° C. at 250 rpm for 42 to 64 hours. The O-acetyl-homoserine from each culture was analyzed by HPLC, and the results thereof are shown in Table 5 below.

(54) O-Acetyl-L-Homoserine Production Medium (pH 7.2)

(55) glucose 30 g, KH.sub.2PO.sub.4 2 g, urea 3 g, (NH.sub.4).sub.2SO.sub.4 40 g, peptone 2.5 g, CSL (Sigma) 5 g (10 mL), MgSO.sub.4.7H.sub.2O 0.5 g, methionine 400 mg, leucine 400 mg, CaCO.sub.3 20 g (based on 1 L of distilled water)

(56) TABLE-US-00005 TABLE 5 Evaluation of O-acetyl-homoserine production Strains O-AH production (g/L) ATCC13032 — 0.0 metB 0.3 metY 0.3 metBY 0.5 ATCC13032::Hom.sup.FBR — 0.0 (R407H) metB 1.3 metY 1.5 metBY 3.7

(57) As a result, as shown in Table 5 above, O-acetyl-L-homoserine was not accumulated when Corynebacterium glutamicum ATCC13032, the control strain, was cultured; whereas 0-acetyl-L-homoserine was accumulated in an amount of 0.3 g/L, 0.3 g/L, and 0.5 g/L for each of the ATCC13032 ΔmetB, ATCC13032 ΔmetY, and ATCC13032 ΔmetB ΔmetY strains, respectively, in which the metB,metY, and metBY genes are inactivated.

(58) Additionally, in the case of the ATCC13032::Hom.sup.FBR strain in which the hom gene is substituted in the form of R407H, and the ATCC13032::Hom.sup.FBR ΔmetB, ATCC13032::Hom.sup.FBR ΔmetY, and ATCC13032::Hom.sup.FBR ΔmetB ΔmetY strains in which the metB, metY, and metBY genes are inactivated, respectively, it was confirmed that 0-acetyl-L-homoserine was accumulated in an amount of 1.3 g/L, 1.5 g/L, and 3.7 g/L for each of these strains.

(59) Therefore, it was confirmed from the above results that the production amount of the target amino acid, of which homoserine is a precursor, can be significantly increased using the modified hom of the present disclosure.

Example 9: Preparation and Evaluation of Strains Producing L-Methionine

Example 9-1: Preparation of Recombinant Vector for Deletion of mcbR Gene

(60) In this Example, to prepare methionine-producing strains, a vector for inactivation of the mcbR gene (J. Biotechnol. 103:51-65, 2003), which encodes known methionine and cysteine transcription regulatory proteins in the strains prepared in Example 6, was prepared.

(61) Specifically, a recombinant plasmid vector was prepared using the method below so as to delete the mcbR gene on the chromosome of Corynebacterium ATCC13032. Based on the nucleotide sequences reported in the GenBank of the National Institutes of Health (NIH GenBank), the mcbR gene and its surrounding sequence (SEQ ID NO: 33) of Corynebacterium glutamicum were obtained.

(62) For the purpose of mcbR-deletion, PCR was carried out using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template and the primer sets of SEQ ID NOS: 34 and 35 and SEQ ID NOS: 36 and 37 under the following conditions: denaturation at 95° C. for 5 minutes; 30 cycles of denaturation at 95° C. for 30 seconds; annealing at 53° C. for 30 seconds, and polymerization at 72° C. for 30 seconds; and polymerization at 72° C. for 7 minutes. As a result, DNA fragments (700 bp) were obtained.

(63) A pDZ vector, which cannot be replicated in Corynebacterium glutamicum, and the amplified mcbR gene fragments were treated with restriction enzyme SmaI for chromosomal insertion. Thereafter, they were ligated using DNA ligase, transformed into E. coli DH5a, and plated on the same solid LB medium containing kanamycin (25 mg/L). Colonies transformed with the vector, in which deleted fragments of the target genes are inserted through PCR, were selected, and a plasmid was obtained using a plasmid extraction method. The thus-obtained plasmid was named pDZ-ΔmcbR.

Example 9-2: Preparation and Evaluation of Microorganism Strains of Genus Corynebacterium Producing L-Methionine

(64) The pDZ-AmcbR vector prepared in Example 9-1 by homologous recombination on the chromosome was transformed to each of the CJP1-R407H and CJP1 strains, which had been prepared in Example 6, by electroporation (van der Rest et al., Appl. Microbiol. Biotechnol. 52:541-545, 1999). Thereafter, secondary recombination was carried out on a solid medium containing X-gal. Strains in which the mcbR gene is deleted were confirmed by a PCR method with the transformed Corynebacterium glutamicum strains, in which the secondary recombination had been completed, using the primer set of SEQ ID NOS: 38 and 39. These recombinant strains were named “CJP1-R407HAmcbR” and “CJP1ΔmcbR”, respectively.

(65) To analyze the L-methionine-producing ability of the prepared CJP1-R407HAmcbR strain, the strain was cultured together with the CJP1ΔmcbR strain in the following manner.

(66) Corynebacterium glutamicum CJP1/ΔmcbR and the inventive strain (Corynebacterium glutamicum CJP1-R407HAmcbR) were inoculated into a 250 mL corner-baffled flask containing the seed medium below (25 mL), and then cultured with shaking at 30° C. at 200 rpm for 20 hours. Thereafter, the seed culture medium (1 mL) was inoculated into a 250 mL corner-baffled flask containing the production medium below (24 mL), and then cultured with shaking at 30° C. at 200 rpm for 48 hours. The compositions of the seed medium and production medium are as follows.

(67) <Seed Medium (pH 7.0)>

(68) glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH.sub.2PO.sub.4 4 g, K.sub.2HPO.sub.4 8 g, MgSO.sub.4.7H.sub.2O 0.5 g, biotin 100 μg, thiamine HCl 1,000 μg, calcium pantothenate 2,000 μg, nicotinamide 2,000 μg (based on 1 L of distilled water)

(69) <Production Medium (pH 8.0)>

(70) glucose 50 g, (NH.sub.4).sub.2S.sub.2O.sub.3 12 g, yeast extract 5 g, KH.sub.2PO.sub.4 1 g, MgSO.sub.4.7H.sub.2O 1.2 g, biotin 100 μg, thiamine HCl 1,000 μg, calcium pantothenate 2,000 μg, nicotinamide 3,000 μg, CaCO.sub.3 30 g (based on 1 L of distilled water)

(71) After the cultivation using the above cultivation method, the concentration of L-methionine in each culture medium was analyzed, and the results are shown in Table 6.

(72) TABLE-US-00006 TABLE 6 Evaluation of L-methionine-producing abilities of prepared strains Strain L-Methionine (g/L) CJP1ΔmcbR 0.01 CJP1-R407HΔmcbR 0.19

(73) As a result, it was confirmed that in the strain including the R407H hom modification, the L-methionine-producing ability was improved by 0.18 g/L compared to the control strain.

(74) Based on the results above, it was confirmed that the amount of L-methionine produced can be significantly increased using the modified hom of the present disclosure.

(75) 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.