KR20010010940A - metB gene isolated from Corynebacterium glutamicum and production of cystathionine γ-synthase - Google Patents

Abstract

PURPOSE: metB gene separated from Corynebacterium glutamicum and cystationine gamma-synthase are provided to produce methionine-producing bacteria or improve the productivity by increasing amount of cystationine gamma-synthase expressed by metB gene or artificially mutating with metB gene. CONSTITUTION: metB gene is cloned. Then, metB gene is sub-cloned. Base sequence of metB gene is determined, and amino acid sequence is analyzed from the base sequence. Cell extract of Corynebacterium glutamicum is prepared. Cystationine gamma-synthase expression-induced by metB gene is assayed. Chromosomal metB gene is disrupted and the mutation characteristics are determined.

Description

{MetB gene isolated from Corynebacterium glutamicum and production of cystathionine γ-synthase} isolated from Corynebacterium glutamicum

The present invention relates to a metB gene isolated from Corynebacterium glutamicum and a method for expressing cystathionine γ-synthase using the gene. More specifically, the present invention is to determine the nucleotide sequence by separating the Corynebacterium glutamicum metB gene and to express the metB gene to mass-produce cystathionine γ-synthase enzyme and to artificially mutate the mutation using the metB gene. It is intended to be used to produce methionine producing bacteria or to increase the efficiency of the producing bacteria.

Corynebacterium glutamicum is a gram-positive spore-forming organism that is widely used for the industrial production of amino acids. Processing strains to optimize and increase the final yield of metabolites has long been a subject of concern in the food and feed industry. Using genetic and molecular tools, it is possible to design and regulate new pathways at the molecular level to develop Corynebacterium and related bacteria. In addition, using isolated genes makes it easier to precisely regulate target pathways at the gene and protein levels. These genetic or biochemical treatments require genes related to the biosynthetic pathway of interest. Biosynthetic pathways that induce methionine from many strains have been studied to reveal similarities and differences. Acylation of homoserine, the first step, is common in all strains, even if the transferred acyl groups differ. Escherichia coli and its related bacteria use Succinyl-CoA, whereas Saccharomyces cerevisiae, Brevibacterium flavum, Corynebacterium glutamicum, and Leptospira meeri Leptospira meyeri uses acyl-CoA as an acyl donor. Formation of homocysteine from acyl homoserine can be produced in two ways (FIG. 1). 1 shows an overview of the methionine biosynthetic pathway. In FIG. 1, 5-methylTHF represents 5-methyltetrahydrofolate, 5,10-methyleneTHF represents 5,10-methylenetetrahydrofolate, and OAH OH-sulfhydrylase stands for o-acetylhomoserine sulfhydrylase. E. coli expresses homoserine succinyltransferase from metA and forms Ο-succinyl homoserine as a product. In contrast, Brevibacterium plaboom and Corynebacterium glutamicum express homoserine acetyltransferase and produce Ο-acetylhomoserine as a product. Depending on the strain, the formation of homocysteine from O-acetylhomoserine can be produced by two different pathways. Unlike enterobacteria, which first use the cystathionine pathway (yellowing) of the two pathways, Breviflavum, which is closely related to Corynebacterium, only uses the O-acetylhomoserine sulfhydrylase pathway to produce homocysteine. Only (direct sulfhydrylation) is used.

Escherichia coli utilizes a yellowing pathway, which is promoted by cystathionine γ-synthase (product of metB) and β-cystionase (product of metC). Brewer's yeast, Brevibacterium plaboom, Pseudomonas aeruginosa, and Leptospira meriri use a direct sulfhydrylation pathway that is promoted by acylhomoserine sulfhydridase. The formation of methionine from homocysteine, a similar last step among several species, is facilitated by homocysteine methylase. Unlike the Brevibacterium flaboom, which is closely associated with Corynebacterium using only a direct sulfhydrylation pathway, the enzymatic activity of the yellowing pathway was found in Corynebacterium cell extracts and the pathway was the methionine biosynthetic pathway in the cells. (Kase and Nakayama, Methionine biosynthesis in Corynebacterium glutamicum, Agr. Biol. Chem., 38, 2021-2030, 1974). Despite the industrial importance of Corynebacterium glutamicum as an amino acid producing strain, information on the biosynthetic pathway leading to methionine is still very limited and the biosynthetic pathway is still unclear. We have recently isolated metA genes from cells and demonstrated the functionality of the yellowing pathway at the genetic stage (Park et al., Isolation and analysis of metA, a methionine biosynthetic gene encoding homoserine acetyltransferase in Corynebacterium glutamicum, Molecules and Cells 8, 286-294, 1998). As a next step in understanding the biosynthetic pathway leading to methionine from Corynebacterium glutamicum at the molecular level, we isolated and characterized the metB gene from the organism. Here we completed the present invention by cloning metB, determining the sequence and investigating the biosynthetic pathway in Corynebacterium glutamicum.

An object of the present invention is to provide a metB gene and its base sequence isolated from Corynebacterium glutamicum. Another object of the present invention is to provide a Corynebacterium glutamicum strain (Accession Number: KCCM-10156) recombined with the metB gene. Another object of the present invention is to cultivate the recombinant strain to mass expression of cystathionine γ-synthase.

The object of the present invention is to confirm that the metB gene isolated from Corynebacterium glutamicum is complementary to metB mutant Escherichia coli DNA, subcloning the identified metB gene to determine the base sequence and accordingly estimated amino acid sequence By analyzing the amino acid sequence and similarity to cystathionine γ-synthase of other strains, the expression of cystathionine γ-synthase enzyme was assayed from Corynebacterium glutamicum recombined with metB gene. This was accomplished by disrupting the chromosome metB with the cloned DNA fragment to assess the identity of the cloned DNA and to investigate the methionine biosynthetic pathway and to identify mutational characteristics.

Hereinafter, the configuration and operation of the present invention.

1 is a schematic diagram illustrating a methionine biosynthetic pathway.

2 shows the complementarity of E. coli metB mutants with Corynebacterium glutamicum metB clones and subclones.

3 shows the nucleotide sequence of the Corynebacterium glutamicum metB gene and its surrounding region and the amino acid sequence thus estimated.

4 is a diagram showing multiple alignments of cystathionine γ-synthase of Corynebacterium glutamicum with Mycobacterium tuberculosis, Helicobacter pylori, influenza, and cystathionine γ-synthase of E. coli. .

5 is a schematic diagram showing induction of metB gene inactivation.

The present invention comprises the steps of cloning the metB gene; subcloning the metB gene; determining the nucleotide sequence of the metB gene and analyzing the amino acid sequence thus estimated; Preparing a Corynebacterium glutamicum cell extract; assaying for cystathionine γ-synthase enzyme derived from metB gene; The chromosome metB gene is disrupted and the mutation is characterized.

Corynebacterium glutamicum and Escherichia coli used in the present invention were incubated in MB and LB liquid medium, respectively. The minimal medium of Escherichia coli and Corynebacterium was modified with M9 and MCGC (Gubler et al., Effects of phosphoenolpyruvate carboxylase, respectively). deficiency on metabolism and lysine production in Corynebacterium glutamicum, Appl. Microbiol. Biotechnol. 40, 409-416, 1993). Glucose was added to a final concentration of 1% and antibiotic addition was as follows (μg / mL): ampicillin, 50; Kanamycin, 25; Nalidixic acid (NA), 25; Addition of amino acids, vitamins, and other additives is as follows: methionine, 9.3 mM; Arginine, 9.3 mM; Histidine, 9.3 mM; Thiamine, 0.05 mM.

Corynebacterium glutamicum of the present invention was deposited on March 18, 1999 with the accession number KCCM 10156 to the Yonsei University Korean spawn association.

Corynebacterium glutamicum and E. coli cells were cultured at 30 ° C. and 37 ° C., respectively, and the bacterial strains and plasmids used in the present invention are shown in Tables 1a and 1b.

Bacteria Strains and Plasmids Used in the Present Invention Strain or plasmid Related genotypes or phenotypes Reference Escherichia coli CGSC4896 argF58, relA1, spoT1, metB1 CGSC ^b S17-1 tra from RP4-2 integrated in the chromosome Schwartzer et al., 1991 DH5α ^{F - Φ 80dlacZDM15 Δ (lacZYA-} argF) U169 deoR endA1 hsdR17 supE44 thi-1 recA1 gyrA96 relA1 λ - Bethesda ResearchLaboratories Corynebacterium glutamicum ASO19 Spontaneous rifampin resistant mutant of ATCC 13059 ^d Yoshihama et al., 1985 ASO19E12 Restriction-deficient varient of ASO19 Follettie at al., 1993 HL345 metB mutant The present invention Plasmid pUC19 Ap ^r Yanish-Peron et al., 1985 pSL109 Shuttle vector; Ap ^r (E. coli), Km ^r (Corynebacterium glutamicum) Kim at al., 1997 pSUP301 pACYC177 derivative, Ap ^r , Km ^r , mob Schwartzer et al., 1991 pSL18 Gene disruption vectors, Ap ^r , Km ^r , mob, no replication in Corynebacterium, Corynebacterium gene-disruption vector Kim and Lee, 1996 pSL68 PSL109 with 4.5kb metB clone The present invention NOTE The ^a r superscript indicates resistance. Ap, Ampicillin, Km, kanamycin ^b E. coli Genetic Stock Center, Yale University, New Haven, Conn., USA ^c National Institute of Genetics Stock Research Center, Mishima, 411 Japan ^d ATCC, American Type Culture Collection, Rockville, MD ., USA

Bacteria Strains and Plasmids Used in the Present Invention Strain or plasmid Related genotypes or phenotypes Reference Plasmid pSL69 PSL109 with 4.6kb metB clone The present invention pSL121 pSL109 with 3.5kb KpnI-SalI fragment of pSL68 The present invention pSL122 pSL109 with the 4kb XhoI-SalI fragment of pSL68 The present invention pSL123 pSL109 with the 3kb KpnI-SalI fragment of pSL68 The present invention pSL124 pSL109 with 1 kb XhoI-SalI fragment of pSL123 The present invention pSL132 pSL109 with the 2kb KpnI-XhoI fragment of pSL123 The present invention pSL139 pUC19 with 2 kb KpnI-XhoI fragment of pSL123 The present invention pSL140 pUC19 with a 1.5kb PstI-SalI fragment of pSL123 The present invention pSL141 pUC19 with 1 kb XhoI-SalI fragment of pSL140 The present invention pSL147 pUC19 with a 1.5kb KpnI-PstI fragment of pSL139 The present invention pSL157 pUC19 with 2.2kb EcoRI-SalI fragment of pSL123 The present invention pSL161 pUC19 with 1 kb EcoRV-PstI fragment of pSL147 The present invention pSL162 pUC19 with 0.8 kb KpnI-ClaI fragment of pSL123 The present invention pSL158 pSL18 with 0.5kb PstI-XhoI fragment of pSL157 The present invention NOTE The ^a r superscript indicates resistance. Ap, Ampicillin, Km, kanamycin ^b E. coli Genetic Stock Center, Yale University, New Haven, Conn., USA ^c National Institute of Genetics Stock Research Center, Mishima, 411 Japan ^d ATCC, American Type Culture Collection, Rockville, MD ., USA

Hereinafter, the present invention will be described in detail with reference to the following examples, but the scope of the present invention is not limited to these examples.

Example 1 Cloning of the metB Gene

Molecular cloning, transformation, and electrophoresis used standard procedures. Cloning was carried out using E. coli DH5α as a host. Mini plasmid preparation of Corynebacterium glutamicum cells was carried out according to the method of Yoshihama (Yoshihama et al., Cloning vector system for Corynebacterium glutamicum, J. Bacteriol., 162, 591-507). , 1985). Chromosomal DNA of Corynebacterium glutamicum ASO19 was prepared by Gubler (Gubler et al., Effects of phosphoenolpyruvate carboxylase deficiency on metabolism and lysine production in Corynebacterium glutamicum, Appl.Microbiol. Biotechnol., 40, 409- 416, 1993). The Corynebacterium glutamicum genome library was constructed from 4 kb to 13 kb MboI fragments cloned into E. coli-Corynebacterium shuttle vector pSL109. Corynebacterium glutamicum ASO19 chromosomal DNA partially digested with restriction enzyme MboI was sized by 10-40% sucrose gradient centrifugation, linked to a BamHI cleaved vector, transformed into E. coli DH5α and approximately 5,000 Recombinants were obtained. Recombinants were pooled and plasmids isolated. Escherichia coli CGSC4896 cells were transformed with isolated plasmid DNA and plated in M9 minimal medium containing ampicillin and an appropriate additive (arginine) to corynebacterium glutamicum ASO19 constructed in Corynebacterium-E. Coli shuttle vector pSL109. Genomic libraries were screened for complementation with E. coli metB mutant CGSC4896. Plate medium was incubated at 37 ° C. for 5 days to separate colonies and screened for plasmid components to analyze several positive clones that allowed E. coli metB mutant CGSC4896 to grow in minimal medium. Approximately one positive clone was found per 2,500 screened conjugates and the plasmids pSL68 and pSL69 of the positive clones had inserted DNA fragments of 4.5 kb and 4.6 kb, respectively. Analysis was selected plasmid pSL68 and examined for the presence of the metB gene.

Example 2: Subcloning of the metB Gene

Several subclones were prepared from pSL68 to locate the metB gene in the isolated plasmid pSL68, inserted into the E. coli metB mutant strain, and tested for growth in M9 minimal medium lacking additional methionine (FIG. 2). Plasmids pSL122 and pSL123 prepared from plasmid pSL68 were complementary to E. coli metB mutant strain CGSC4896, but plasmids pSL157 and pSL139 did not support the growth of E. coli metB mutant strain CGSC4896. These experimental results indicate that metB gene is present in close proximity to EcoRI and XhoI (FIG. 2).

Figure 2 shows the complementarity of Corynebacterium glutamicum metB clone with E. coli metB with subclones. Here, the vector DNA is not shown and the arrow indicates the region coding for metB identified by DNA-base sequencing and for the complementarity test, the plasmid was placed in the metB mutant Escherichia coli CGSC4896 and supported the growth of the mutant in the M9 minimum medium. The ability to do this was tested. Restriction enzymes located at the ends of pSL68 come from a vector. The restriction site of Pst I did not determine the location for pSL68 and pSL122. In Figure 2, + indicates growth in M9 medium, and "-" indicates no growth in M9 medium.

Plasmids pSL121 and pSL122 were prepared by linking the 3.5kb KpnI-SalI fragment of pSL68 and the 4kb XhoI-SalI fragment in pSL109, respectively. Plasmid pSL123 was constructed by linking overlapping fragments (3kb KpnI-SalI fragments) of pSL121 and pSL122 in pSL109. Plasmids pSL124 and pSL132 were prepared by linking 1kb XhoI-SalI fragments and 2kb KpnI-XhoI fragments of pSL123 in pSL109, respectively. Plasmids pSL139 and pSL141 were constructed by moving the insertion fragments of pSL132 and pSL124 in pUC19, respectively. Plasmid pSL140 was constructed by linking 1.5kb PstI-SalI of pSL123 in pUC19. Plasmid pSL147 was constructed by linking 1.5 kb KpnI-PstI fragments of pSL123 in pUC19. Plasmid pSL157 was constructed by inserting a 2.2 kb EcoRI-SalI fragment of pSL123 in pUC19. Plasmids pSL161 and pSL162 were constructed by transferring 0.8kb KpnI-ClaI fragments and 1kb EcoRI-PstI fragments of pSL147 in pUC19, respectively (Table 1).

Example 3 Sequencing of MetB Gene and Presumed Amino Acid Sequencing

The complete nucleotide sequence for the metB gene was determined by dideoxynucleotide chain termination method (Sanger et al., 1977) using a commercially available sequencing kit (Pharmacia Biotech). The reaction was analyzed by an ALF-express automatic sequencer (ALF-express automatic sequencer; Pharmacia Biotech). A series of subclones were constructed from pSL68 and used as template for sequencing. Cloning vector pUC19 was used for this purpose. If necessary, nucleotide sequences were determined using synthetic oligonucleotide primers. As a result, sequencing of the inserted DNA of the plasmid pSL123 revealed one open reading frame (ORF) consisting of 1,161 nucleotides. As expected, the ORF of the metB gene was located 22 nucleotides downstream from the EcoRI site and the position of the ORF was consistent with the results obtained by the deletion assay in FIG. The base sequence of the complete metB gene and its surrounding area are shown in FIG. 3. Figure 3 shows the gene sequence of the Corynebacterium glutamicum metB gene and its surrounding area and the amino acid sequence estimated accordingly. The ribosome-binding site AGGAG was designated RBS and the sequence was assigned GenBank accession number AF126953. TTG initiation codons were selected based on amino acid sequence similarity between inferred metB and other cystathionine γ-synthase. Based on the length of the ORF, one could no longer expect the role of ATG as the start codon in the upward direction. AGGAG, a potential ribosomal binding site (Shine and Dalgarno, 1975), was located 10 bp upwards from TTG, the speculative transcription initiation codon. The GC component of metB was 55.5%, which is typical of the Corynebacterium glutamicum gene. The selected codons were very similar to the previously published Corynebacterium gene, which also indicates that the ORF encodes a protein expressed at low levels. ORF encodes a polypeptide of molecular weight 41,655 consisting of 386 amino acids and the expected isoelectric point of a complete peptide is 4.8. The encoded sequences are fairly evenly distributed throughout the sequence and contain charged amino acids. The amino acid sequence of the transcribed metB gene was compared to a protein database using BLAST software. Among the known metB genes, cystathionine γ-synthase (388 amino acids, Cole et al., 1998) of Mycobacterium tuberculosis most closely associated with Corynebacterium glutamicum showed the highest similarity score and compared. Overall similarity in the region was 63%. The same amino acids were evenly distributed throughout the sequence. The amino acid sequence similarity between the transcribed metB gene and the E. coli metB gene product (Duchange et al., Structure of the metJBLF cluster in Escherichia coli K12, J. Biol. Chem. 258, 14868-14871, 1998) was relatively low. Figure 4 shows corynebacterium glutamicum, cole et al., Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence, Nature 393, 537-544, 1998, and Helicobacter pylori (Tomb et al., The complete). genome sequence of the gastric pathogen Helicobacter pylori, Nature 388, 538-547, 1997), influenza bacteria (Fleischmann et al., Whole-genome random sequencing and assembly of Haemophilus influenzae Rd, Science 269, 496-512, 1995), and Multiple alignments of several cystathionine γ-synthase of E. coli (Blattner et al., The complete genome sequence of Escherichia coli K-12, Science 277, 1453-1462, 1997) are shown. In FIG. 4, an asterisk (*) indicates a perfect match, two dots (:) indicate high similarity, and one dot (·) indicates low similarity.

Example 4 Preparation of Cell Extract of Corynebacterium glutamicum

Cell extracts were prepared by the method of Jetten and Sinski (Jetten and Sinskey, Characterization of phosphoenolpyruvate carboxykinase from Corynebacterium glutamicum, FEMS Microbiol. Lett. 111, 183-188, 1993). Cells were collected by centrifugation, washed with 100 mM Tris-HCl (pH 7.5) containing 20 mM KCl, 33 mM MgCl ₂ , 10 mM MnCl ₂ , and 5% glycerol and resuspended with 5 mL of the same buffer. Cells were shaken vigorously with 212-300 microns glass beads (Sigma, USA) in a mini-bead beater (Biospec Products, USA) to crush cell debris at 15,000 × g. It was removed by centrifugation for 30 minutes. Supernatant was used as crude extracts.

Example 5: Cystathionine γ-Synthase Enzyme Assay

Cystathionine γ-syntheses were assayed using Rabanel's method (Ravanel et al., Purification and characterization of cystathionine γ-synthase from spinach chloroplasts, Arch. Biochem. Biophys. 316, 572-584, 1995). 0.1 mL assay mixture containing 20 mM Mops-NaOH (pH 7.5), 1 mM cysteine, 10 mM Ο-acetylhomoserine, 1 mM dithiothreitol, and an appropriate amount of enzyme for 10 minutes. Incubated at 30 ° C. The reaction was stopped by adding 50 μl of 20% trichloroacetic acid (hereinafter, referred to as TCA). Precipitated proteins were removed by centrifugation at 15,000 × g for 5 minutes. The activity of cystathionine γ-synthase present in the supernatant was assayed by measuring the loss of cysteine. 50 μl of TCA treated reaction mixture was mixed with 100 μl of concentrated acetic acid and 200 μl of ninhydrin solution (2.5 g dissolved in a 100 mL mixture containing 60 mL of concentrated acetic acid and 40 mL of concentrated hydrochloric acid). It was added to the contained glass tube. The mixture was boiled for 10 minutes and 650 μl of cold 95% ethanol was added. The amount of cysteine was determined by measuring absorbance at 560 nm (coefficient of extinction of cysteine = 25,000 M ⁻¹ cm ⁻¹ ). O-acetylhomoserine was synthesized by Nagai and Pravin's method (Nagai and Flavin., Synthesis of O-acetylhomoserine, Methods Enzymol. 17B, 423-424, 1971). The ability of the metB gene to express cystathionine γ-synthase was determined by enzymatic assay. Crude extracts were prepared and assayed from Corynebacterium glutamicum ASO19E12 (Accession No .: KCCM-10156) in which plasmid pSL123 was incubated. Insertion of plasmid pSL123 into Corynebacterium glutamicum ASO19E12 increased cystathionine γ-synthase activity by approximately 10 fold, apparently due to gene-dose effects (Table 2).

Kista thionine γ- synthase expression of the ^a Strain Plasmid Characteristics Specific activity, nmol min ^-1 mg ^-1 Growth at MM ^b Corynebacterium glutamicum / pSL109 pSL109 - 3.7 + Corynebacterium glutamicum / pSL123 (Accession Number: KCCM-10156) pSL123 metB clone 37.4 ND ^c Corynebacterium glutamicum / HL345 - metB mutant ^d ND + Escherichia coli CGSC4896 - metB mutant ND - Note: ^a enzyme was induced to stop phase growth in a minimal medium containing 1% glucose. The cells were pooled and disrupted to assay for activity. ^b MCGC minimal media ^c Not determined ^d Mutants produced in the present invention

Example 6: Crushing and Mutant Properties of Chromosome metB Gene

Electroporation of Corynebacterium glutamicum strain was carried out as follows. Corynebacterium glutamicum cells were cultured in 100 mL of MB liquid medium. When the absorbance reached 0.6 at 600 nm, ampicillin was added at a final concentration of 1.5 μg / mL. After incubating the culture for 1 and a half hours, the cells were collected by centrifugation at 5,000 g for 10 minutes. The pellets were washed three times with 10 mL of EPB1 (20 mM Hepes pH7.2, 5% glycerol) and resuspended with 1.5 mL of EPB2 (5 mM Hepes pH7.2, 15% glycerol). Cell suspension (150 μl) was mixed with plasmid DNA (1-2 μl), incubated on ice for 5 minutes and subjected to electroporation at 2.5 kV in a 2-mm cuvette. Immediately after the pulse, 1 mL of recovery liquid medium (per L, 40 g Brain Haat Infuzon, 30 g Sorbitol, 10 g sucrose) was added and the mixture was incubated at 30 ° C. for 1 and a half hours. After incubation, aliquots were plated on plate medium (40 liters of Brain Haat Efuzone, 40 grams of sorbitol, 10 g of sucrose, 15 mg of kanamycin, 16 g of agar). Plasmid mobilization, or transconjugation, from E. coli to Corynebacterium was performed according to Gubler's method (Gubler et al., Effects of phosphoenolpyruvate carboxylase deficiency on metabolism and lysine production in Corynebacterium glutamicum, Appl. Microbiol.Biotechnol. 40, 409-416, 1993). Corynebacterium transconjugants were selected from MB agar media containing nalidixic acid (NA) and kanamycin.

Molecular cloning, transformation, and electrophoresis used standard treatments. Genomic DNA was isolated from Corynebacterium strains according to Gubler's method (Gubler et al., 1993). For Southern blot analysis, chromosomal DNA digested with appropriate restriction enzymes were electrophoretically separated on agarose gels, denatured and transferred to nitrocellulose filters. A probe was used to isolate 0.5 kb PStI-XhoI fragments from plasmid pSL140. Probes in random primed DNA-labeling reactions as described by the manufacturer were labeled with Digoxygenin-11-dUTP (Boehringer Mannheim, Germany). Hybridization, washing and colorimetric screening were performed using Genius-System (Boehringer Mannheim, Germany) according to the manufacturer's instructions for filter hybridization.

An attempt was made to disrupt the chromosome metB with the cloned DNA fragment to assess the identity of the cloned DNA and to investigate the methionine biosynthetic pathway. To accomplish this goal, a 0.5 kb PstI-XhoI fragment of pSL157 present in the region coding for metB was selected (FIG. 2). The fragment was cloned into pSL18 (FIG. 5), a gene disruption vector, to prepare plasmid pSL158, which was transferred by conjugation from E. coli S17-1 to Corynebacterium glutamicum ASO19E12. Plasmid pSL158 provides resistance to kanamycin and cannot be replicated in Corynebacterium, so making a plate of kanamycin causes pSL158 to enter the genome by a single cross between the plasmid metB fragment and the genomic metB locus. Integrated clones can be selected. This disrupts chromosomal replication of metB due to the fusion of the plasmids. Single site integration of the plasmid into the chromosome was verified using Southern blot (FIG. 5). In FIG. 5, A was used to disrupt the chromosome metB of 0.5 kb PstI-XhoI fragment, and inserted into PstI and SalI sites located at the multiple cloning site (MCS) of pSL18 (4.8 kb) to prepare pSL158. B crosses once in the homologous region labeled chromosomes metB and metB 'resulting in the fusion of the plasmid into the metB locus and producing a clone resistant to kanamycin. This results in the disruption of the chromosome metB, which is designated metB "and metB. The gene disruption vector pSL158 is not replicated in Corynebacterium. C is the southern blot analysis of the constructed mutant and is the parent strain Coryne. The genomic DNA of bacterium glutamicum ASO19E12 (lane 2) and the genomic DNA of metB mutant HL345 (lane 3) were digested with PstI and XhoI, electrophoresed on agarose gel and blotted onto nitrocellulose filter. (blotting) and hybridization with a labeled probe The probe used a 0.5 kb Pst-Xho I fragment of plasmid pSL157.The upper band, indicated by the arrow, was about 3 kb in size and the lower band was 0.5 in size. kb The size marker (kb) of λ DNA cut with HindIII is shown in lane 1. In Fig. 5, E is EcoR I, X is Xho I, P is Pst I, S is Sal I, and MCS (multiple cloning sites) As a result of the experiment, the obtained mutants did not lose growth in the minimal medium lacking additional methionine No significant difference in growth rate was found between the parent strain and the mutant strain. It suggests that another methionine biosynthetic pathway that bypasses the step promoted by γ-synthase may exist in Corynebacterium glutamicum.

Identification of the cloned DNA as metB firstly complementarity of the E. coli metB strain, secondly expression of cystathionine γ-synthase activity in Corynebacterium glutamicum, and finally other cystathionine γ-synthase Showed amino acid sequence similarity with. The isoelectric point of the protein was very similar to the values of other known cystathionine γ-synthase. The molecular weight predicted to be 41,655 Dalton was also similar to that of known influenza bacteria, Bacillus subtilis, and tuberculosis cystathionine γ-synthase. These overall similarities indicate that the TTG initiation codon selected in the present invention was very accurate.

E. coli and related species are known to express homoserine succinyltransferase from the metA gene, while corynebacterium glutamicum expresses homoserine acetyltransferase, as was previously reported. It is interesting to note that the metB of Corynebacterium may be complementary to the E. coli metB mutation. This means that the cystathionine γ-synthase of Corynebacterium glutamicum used Ο-succinyl homoserine as Ο-acetylhomoserine, which is clearly the substrate of a plate medium with natural substrate in the medium. However, it is possible to predict that E. coli may have O-acetylhomoserine as an intracellular product. Cystathionine γ-synthase from Corynebacterium glutamicum appears to be different from its counterparts in other microorganisms, including E. coli. The similarity of the predicted amino acid sequence of cystathionine γ-synthase between Corynebacterium and Escherichia coli is low. As mentioned above, low similarity may reflect functional and catalytic differences between the two proteins. The biosynthetic pathway leading to methionine has not been clearly identified in Corynebacterium glutamicum. Since it is cell-dependent, the formation of homoserine from o-acetylhomoserine can occur in two different pathways (FIG. 1). Enterobacteriaceae and Corynebacterium glutamicum preferentially use the yellowing pathway, whereas Brevibacterium flabme, closely related to Corynebacterium glutamicum, only directly sulfhydryl to produce homoserine. Use the traversal path. As demonstrated in the present invention by the Corynebacterium metB mutation, Corynebacterium glutamicum apparently has another route for synthesizing methionine. In the present invention, it was speculated that another pathway may be a direct sulfhydrylation pathway promoted by o-acetylhomoserine sulfhydridase, but separation of genes and enzymes is required to demonstrate the existence of the pathway. In eukaryotes, fungi and yeasts have been reported to have both yellowing and direct sulfhydrylation pathways (Marzluf, Molecular genetics of sulfur assimilation in filamentous fungi and yeast, Annu. Rev. Microbiol. 51, 73- 96, 1997). To date, nothing has been found to have two pathways in prokaryotes.

As described above, the present invention increases the amount of cystathionine γ-synthase enzyme expressed by the metB gene or artificially forms a mutation using the metB gene to produce methionine producing bacteria or Since there is an excellent effect to increase the efficiency, it is a very useful invention in the food industry and feed industry that needs to increase the production of methionine using cystathionine γ-synthase.

〈110〉 BASF Aktiengesellschaft

〈120〉 metB gene isolated from Corynebacterium glutamicum and expression

of cystathionine gamma-synthase using its gene

〈160〉 1

〈170〉 KOPATIN 1.5

〈210〉 1

<211> 1638

<212> DNA

213 Corynebacterium glutamicum

〈220〉

<221> RBS

222 (234) .. (238)

223 ribosome binding site

〈220〉

<221> CDS

222 (249) .. (1409)

〈223〉 metB gene open reading frame

<400> 1

atcctcatga tcaaggacgc ccgcatactg gcttcgggga ctgtttcaga agtgatgact 60

cctgaaaatt tgggcgcgct gtatgacatg tcggtgtcgt tggaaactgt gcgcagccgg 120

tggttcgcgt tcgatgctct gcattaaaag gggctagttt tacacaaaag tggacagctt 180

ggtctatcat tgccagaaga ccggtccttt tagggccata gaattctgat tacaggagtt 240

gatctacc ttg tct ttt gac cca aac acc cag ggt ttc tcc act gca tcg 290

Leu Ser Phe Asp Pro Asn Thr Gln Gly Phe Ser Thr Ala Ser

1 5 10

att cac gct ggg tat gag cca gac gac tac tac ggt tcg att aac acc 338

Ile His Ala Gly Tyr Glu Pro Asp Asp Tyr Tyr Gly Ser Ile Asn Thr

15 20 25 30

cca atc tat gcc tcc acc acc ttc gcg cag aac gct cca aac gaa ctg 386

Pro Ile Tyr Ala Ser Thr Thr Phe Ala Gln Asn Ala Pro Asn Glu Leu

35 40 45

cgc aaa ggc tac gag tac acc cgt gtg ggc aac ccc acc atc gtg gca 434

Arg Lys Gly Tyr Glu Tyr Thr Arg Val Gly Asn Pro Thr Ile Val Ala

50 55 60

tta gag cag acc gtc gca gca ctc gaa ggc gca aag tat ggc cgc gca 482

Leu Glu Gln Thr Val Ala Ala Leu Glu Gly Ala Lys Tyr Gly Arg Ala

65 70 75

ttc tcc tcc ggc atg gct gca acc gac atc ctg ttc cgc atc atc ctc 530

Phe Ser Ser Gly Met Ala Ala Thr Asp Ile Leu Phe Arg Ile Ile Leu

80 85 90

aag ccg ggc gat cac atc gtc ctc ggc aac gat gct tac ggc gga acc 578

Lys Pro Gly Asp His Ile Val Leu Gly Asn Asp Ala Tyr Gly Gly Thr

95 100 105 110

tac cgc ctg atc gac acc gta ttc acc gca tgg ggc gtc gaa tac acc 626

Tyr Arg Leu Ile Asp Thr Val Phe Thr Ala Trp Gly Val Glu Tyr Thr

115 120 125

gtt gtt gat acc tcc gtc gtg gaa gag gtc aag gca gcg atc aag gac 674

Val Val Asp Thr Ser Val Val Glu Glu Val Lys Ala Ala Ile Lys Asp

130 135 140

aac acc aag ctg atc tgg gtg gaa acc cca acc aac cca gca ctt ggc 722

Asn Thr Lys Leu Ile Trp Val Glu Thr Pro Thr Asn Pro Ala Leu Gly

145 150 155

atc acc gac atc gaa gca gta gca aag ctc acc gaa ggc acc aac gcc 770

Ile Thr Asp Ile Glu Ala Val Ala Lys Leu Thr Glu Gly Thr Asn Ala

160 165 170

aag ctg gtt gtt gac aac acc ttc gca tcc cca tac ctg cag cag cca 818

Lys Leu Val Val Asp Asn Thr Phe Ala Ser Pro Tyr Leu Gln Gln Pro

175 180 185 190

cta aaa ctc ggc gca cac gca gtc ctg cac tcc acc acc aag tac atc 866

Leu Lys Leu Gly Ala His Ala Val Leu His Ser Thr Thr Lys Tyr Ile

195 200 205

gga gga cac tcc gac gtt gtt ggc ggc ctt gtg gtt acc aac gac cag 914

Gly Gly His Ser Asp Val Val Gly Gly Leu Val Val Thr Asn Asp Gln

210 215 220

gaa atg gac gaa gaa ctg ctg ttc atg cag ggc ggc atc gga ccg atc 962

Glu Met Asp Glu Glu Leu Leu Phe Met Gln Gly Gly Ile Gly Pro Ile

225 230 235

cca tca gtt ttc gat gca tac ctg acc gcc cgt ggc ctc aag acc ctt 1010

Pro Ser Val Phe Asp Ala Tyr Leu Thr Ala Arg Gly Leu Lys Thr Leu

240 245 250

gca gtg cgc atg gat cgc cac tgc gac aac gca gaa aag atc gcg gaa 1058

Ala Val Arg Met Asp Arg His Cys Asp Asn Ala Glu Lys Ile Ala Glu

255 260 265 270

ttc ctg gac tcc cgc cca gag gtc tcc acc gtg ctc tac cca ggt ctg 1106

Phe Leu Asp Ser Arg Pro Glu Val Ser Thr Val Leu Tyr Pro Gly Leu

275 280 285

aag aac cac cca ggc cac gaa gtc gca gcg aag cag atg aag cgc ttc 1154

Lys Asn His Pro Gly His Glu Val Ala Ala Lys Gln Met Lys Arg Phe

290 295 300

ggc ggc atg atc tcc gtc cgt ttc gca ggc ggc gaa gaa gca gct aag 1202

Gly Gly Met Ile Ser Val Arg Phe Ala Gly Gly Glu Glu Ala Ala Lys

305 310 315

aag ttc tgt acc tcc acc aaa ctg atc tgt ctg gcc gag tcc ctc ggt 1250

Lys Phe Cys Thr Ser Thr Lys Leu Ile Cys Leu Ala Glu Ser Leu Gly

320 325 330

ggc gtg gaa tcc ctc ctg gag cac cca gca acc atg acc cac cag tca 1298

Gly Val Glu Ser Leu Leu Glu His Pro Ala Thr Met Thr His Gln Ser

335 340 345 350

gct gcc ggc tct cag ctc gag gtt ccc cgc gac ctc gtg cgc atc tcc 1346

Ala Ala Gly Ser Gln Leu Glu Val Pro Arg Asp Leu Val Arg Ile Ser

355 360 365

att ggt att gaa gac att gaa gac ctg ctc gca gat gtc gag cag gcc 1394

Ile Gly Ile Glu Asp Ile Glu Asp Leu Leu Ala Asp Val Glu Gln Ala

370 375 380

ctc aat aac ctt tag a aactatttgg cggcaagcag cttttcaata taagcaatgc 1450

Leu Asn Asn Leu ***

385

gagcctccac catgtagccg aagagttcgt cagaagttga gacggactct tcgactgctt 1510

tacgggtcag tggcgcttcc acatctgggt tctcatcaag ccatggctta ggaaccggag 1570

caaacacatc cggcttttcg ccctctggac gattgtcaaa ggtgtagtca gaagtcaggg 1630

tgaagctg 1638

〈210〉 2

<211> 386

<212> PRT

213 Corynebacterium glutamicum

<400> 2

Leu Ser Phe Asp Pro Asn Thr Gln Gly Phe Ser Thr Ala Ser Ile His

1 5 10 15

Ala Gly Tyr Glu Pro Asp Asp Tyr Tyr Gly Ser Ile Asn Thr Pro Ile

20 25 30

Tyr Ala Ser Thr Thr Phe Ala Gln Asn Ala Pro Asn Glu Leu Arg Lys

35 40 45

Gly Tyr Glu Tyr Thr Arg Val Gly Asn Pro Thr Ile Val Ala Leu Glu

50 55 60

Gln Thr Val Ala Ala Leu Glu Gly Ala Lys Tyr Gly Arg Ala Phe Ser

65 70 75 80

Ser Gly Met Ala Ala Thr Asp Ile Leu Phe Arg Ile Ile Leu Lys Pro

85 90 95

Gly Asp His Ile Val Leu Gly Asn Asp Ala Tyr Gly Gly Thr Tyr Arg

100 105 110

Leu Ile Asp Thr Val Phe Thr Ala Trp Gly Val Glu Tyr Thr Val Val

115 120 125

Asp Thr Ser Val Val Glu Glu Val Lys Ala Ala Ile Lys Asp Asn Thr

130 135 140

Lys Leu Ile Trp Val Glu Thr Pro Thr Asn Pro Ala Leu Gly Ile Thr

145 150 155 160

Asp Ile Glu Ala Val Ala Lys Leu Thr Glu Gly Thr Asn Ala Lys Leu

165 170 175

Val Val Asp Asn Thr Phe Ala Ser Pro Tyr Leu Gln Gln Pro Leu Lys

180 185 190

Leu Gly Ala His Ala Val Leu His Ser Thr Thr Lys Tyr Ile Gly Gly

195 200 205

His Ser Asp Val Val Gly Gly Leu Val Val Thr Asn Asp Gln Glu Met

210 215 220

Asp Glu Glu Leu Leu Phe Met Gln Gly Gly Ile Gly Pro Ile Pro Ser

225 230 235 240

Val Phe Asp Ala Tyr Leu Thr Ala Arg Gly Leu Lys Thr Leu Ala Val

245 250 255

Arg Met Asp Arg His Cys Asp Asn Ala Glu Lys Ile Ala Glu Phe Leu

260 265 270

Asp Ser Arg Pro Glu Val Ser Thr Val Leu Tyr Pro Gly Leu Lys Asn

275 280 285

His Pro Gly His Glu Val Ala Ala Lys Gln Met Lys Arg Phe Gly Gly

290 295 300

Met Ile Ser Val Arg Phe Ala Gly Gly Glu Glu Ala Ala Lys Lys Phe

305 310 315 320

Cys Thr Ser Thr Lys Leu Ile Cys Leu Ala Glu Ser Leu Gly Gly Val

325 330 335

Glu Ser Leu Leu Glu His Pro Ala Thr Met Thr His Gln Ser Ala Ala

340 345 350

Gly Ser Gln Leu Glu Val Pro Arg Asp Leu Val Arg Ile Ser Ile Gly

355 360 365

Ile Glu Asp Ile Glu Asp Leu Leu Ala Asp Val Glu Gln Ala Leu Asn

370 375 380

Asn Leu ***

385

Claims (6)

A metB gene having the nucleotide sequence set forth in SEQ ID NO: 1, which encodes cystathionine γ-synthase and is isolated from Corynebacterium glutamicum. The amino acid sequence of claim 1 derived from the metB gene of claim 1. A recombinant shuttle vector, pSL123, which has a nucleotide sequence of metB gene isolated from Corynebacterium glutamicum and increases the expression activity of cystathionine γ-synthase. Recombinant Corynebacterium glutamicum strain transformed with pSL123, a recombinant vector containing metB gene fragment isolated from Corynebacterium glutamicum (Accession Number: KCCM-10156). Cystathionine γ-synthase obtained by culturing the recombinant Corynebacterium glutamicum strain according to claim 4 (Accession No .: KCCM-10156) in a minimal medium containing glucose. Characterized by using a direct sulfhydrylation pathway promoted by acetylhomoserine sulfhydridase in addition to the yellowing pathway in the synthesis pathway for forming homoserine from o-acetylhomoserine present in Corynebacterium glutamicum. Methionine production method.