O-acetylhomoserine-producing microorganisms and methods for producing O-acetylhomoserine or L-methionine using the same

Abstract

The present application relates to a microorganism having attenuated GNAT family N-acetyltransferase protein activity, a method for producing O-acetylhomoserine and L-methionine using the microorganism, a composition for producing O-acetylhomoserine containing the microorganism, and use of the microorganism for producing O-acetylhomoserine or L-methionine.

Description

Technical Field

[0001] This application relates to a microorganism with weakened GNAT family N-acetyltransferase protein activity, a method for producing O-acetyl homoserine and L-methionine using the same, a composition for producing O-acetyl homoserine containing the microorganism, and the use of the microorganism for producing O-acetyl homoserine or L-methionine.

Background Art

[0002] O-acetyl homoserine acts as a precursor of methionine, which is an essential amino acid in the living body. Methionine is an essential amino acid in the living body and is widely used not only as a feed and food additive but also as a synthetic raw material for infusion solutions and pharmaceuticals.

[0003] Methionine is produced by biological synthesis or chemical synthesis. In this regard, a two-step process (Patent Document 1) for producing L-methionine by an enzymatic conversion reaction from a precursor of L-methionine produced by fermentation is also known.

[0004] In the above two-step process, O-succinyl homoserine and O-acetyl homoserine can be used as methionine precursors. Therefore, for the economical mass production of methionine, it is very important to produce O-acetyl homoserine in a high yield.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

[0006] [Non-licensed Document 1] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 [Non-licensed Document 2] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-licensed Document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-licensed Document 4] Devereux, J., et al, Nucleic Acids Research 12: 387(1984) [Non-licensed Document 5] Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990) [Non-licensed Document 6] Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

[0007] The inventors have found a Corynebacterium microorganism that produces O - acetylhomoserine and a method for producing O - acetylhomoserine or L - methionine using the same, and have completed this application. [[Means for Solving the Problems]]

[0008] This application aims to provide a Corynebacterium microorganism having the ability to produce O - acetylhomoserine with weakened GNAT family N - acetyltransferase protein activity.

[0009] Also, this application aims to provide a method for producing O - acetylhomoserine, which includes the step of culturing the microorganism in a medium.

[0010] Furthermore, this application aims to provide a method for producing L - methionine, which includes the step of culturing the microorganism in a medium.

[0011] Furthermore, this application aims to provide a composition for producing O - acetylhomoserine containing the microorganism.

[0012] Furthermore, this application aims to provide the use of the microorganism of this application for the production of O - acetylhomoserine or L - methionine. [[Effects of the Invention]]

[0013] The microorganism that produces O-acetylhomoserine according to this application is more environmentally friendly and efficient than chemical synthesis. Furthermore, when the produced O-acetylhomoserine is used as a precursor for the synthesis of methionine and acetic acid by O-acetylhomoserine sulfhydrase, it can be bioconverted to L-methionine with high efficiency. The converted L-methionine can be widely used not only in the production of animal feed and animal feed additives, but also in the production of human food or food additives. [Modes for carrying out the invention]

[0014] These will be explained in detail below. Note that each description and embodiment disclosed in this application applies to other descriptions and embodiments. That is, any combination of the various elements disclosed in this application is included. Furthermore, this application is not limited to the following specific descriptions.

[0015] Furthermore, numerous papers and patent documents are referenced throughout this specification, and their citations are indicated. The disclosures of the cited papers and patent documents are incorporated in their entirety as references within this specification, thereby providing a clearer explanation of the level of the art to which this application pertains and the content of this application.

[0016] One aspect of this application provides a Corynebacterium microorganism having O-acetylhomoserine production ability, characterized by weakened GNAT family N-acetyltransferase protein activity.

[0017] In this application, "GNAT family N-acetyltransferase" refers to an N-acetyltransferase belonging to the GNAT (Gcn5-Related N-Acetyltransferases) family.

[0018] Specifically, the GNAT family N-acetyltransferases are known in the art, and the protein and gene sequences of the GNAT family N-acetyltransferases can be obtained from known databases, such as NCBI's GenBank, but are not limited to these. More specifically, the GNAT family N-acetyltransferase may have the amino acid sequence represented by SEQ ID NO: 1, may contain the amino acid sequence, may be substantially composed of the amino acid sequence, or may consist of the amino acid sequence. For example, the protein consisting of Sequence ID No. 1 refers to, but is not limited to, the protein endogenously present in microorganisms of the genus Corynebacterium, encoded by the known NCgl0959 gene. More specifically, it refers to, but is not limited to, the GNAT family N-acetyltransferase consisting of the amino acid sequence of Sequence ID No. 1, which is endogenously present in microorganisms of the genus Corynebacterium, and more specifically, the GNAT family N-acetyltransferase of Corynebacterium glutamicum ATCC13032, encoded by the NCgl0959 gene.

[0019] Furthermore, the GNAT family N-acetyltransferases of this application may include not only the amino acid sequence represented by SEQ ID NO: 1, but also amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with SEQ ID NO: 1. Moreover, it goes without saying that this application also includes amino acid sequences having such homology or identity and possessing the same or equivalent biological activity as the GNAT family N-acetyltransferases of this application, even if some sequences are deleted, modified, substituted, conservatively substituted, or added.

[0020] Even if this application describes a polypeptide or protein containing an amino acid sequence represented by a specific sequence number, a polypeptide or protein consisting of an amino acid sequence represented by a specific sequence number, or a polypeptide or protein having an amino acid sequence represented by a specific sequence number, it goes without saying that any protein having an amino acid sequence in which some of the sequences are deleted, modified, substituted, conserved substituted, or added may be used in this application, as long as it has the same or equivalent activity as the polypeptide consisting of the amino acid sequence of the said sequence number. For example, this includes proteins having additions of sequences that do not change the function of the protein, spontaneously occurring mutations, silent mutations, or conserved substitutions at the N-terminus and / or C-terminus of the amino acid sequence.

[0021] Examples include amino acid sequences having additions or deletions of sequences that do not alter the function of the GNAT family N-acetyltransferase of this application, spontaneous mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or within the amino acid sequence.

[0022] In this application, "conservative substitution" means that one amino acid is replaced by another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Furthermore, amino acids are classified into those with electrically charged side chains and those with uncharged side chains. Examples of amino acids with electrically charged side chains include aspartic acid, glutamic acid, lysine, arginine, and histidine. Amino acids with uncharged side chains are further classified into nonpolar amino acids and polar amino acids. Examples of nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Examples of polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Typically, conservative substitutions have little to no effect on the activity of the resulting polypeptide.

[0023] Furthermore, GNAT family N-acetyltransferases may include amino acid deletions or additions that have minimal effect on the polypeptide's properties and secondary structure. For example, the polypeptide may be bound to the N-terminal signal (or leader) sequence of a protein involved in protein transfer, either co-translationally or post-translationally. Alternatively, the polypeptide may be bound to other sequences or linkers to enable the polypeptide's identification, purification, or synthesis.

[0024] In this application, "homology" or "identity" refers to the degree to which two given amino acid sequences or base sequences are similar, and is expressed as a percentage. Homology and identity are often used interchangeably.

[0025] The sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard sequencing algorithms, which may also be used in conjunction with a default gap penalty established by the program used. Substantially, homologous or identical sequences generally hybridize with each other, in whole or in part, under moderate to high stringent conditions. Hybridization also includes hybridization with polynucleotides that have common codons or codons considering codon degeneracy.

[0026] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using default parameters, such as those in Non-Patent Document 1, and known computer algorithms such as the "FASTA" program. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 3), as performed in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later) (including the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, and FASTA (Non-Patent Documents 5, 6, and 7)). For example, homology, similarity, or identity can be determined using BLAST or Clustal W from the National Center for Biotechnology Information Databases.

[0027] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as Non-Patent Document 3, as disclosed in Non-Patent Document 8, for example. In summary, the GAP program is defined as the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include (1) a binary comparison matrix (with a value of 1 for identity and 0 for non-identity) and a weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 9, as in Non-Patent Document 10; (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extended penalty of 0.5); and (3) no penalty for terminal gaps.

[0028] In this application, "corresponding to" means an amino acid residue at a position listed in the polypeptide, or an amino acid residue that is similar, identical, or equivalent to a residue listed in the polypeptide. Identifying the amino acid at the corresponding position will determine the specific amino acid in the sequence referencing the particular sequence. In this application, "corresponding region" generally means a similar or corresponding position in the related protein or reference protein.

[0029] For example, by aligning any amino acid sequence with Sequence ID No. 1, each amino acid residue in the sequence can be numbered based on the number and position of amino acid residues corresponding to the amino acid residues in Sequence ID No. 1. For example, the sequence alignment algorithm in this application can be used to identify the positions of amino acids, or the positions where modifications such as substitutions, insertions, or deletions occur, by comparing it with a query sequence (also called a "reference sequence").

[0030] For such alignment, for example, the Needleman-Wunsch algorithm (Non-Patent Literature 3) and the Needle program from the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Literature 2) can be used, but are not limited to these. Sequence alignment programs and pairwise sequence comparison algorithms known in the art can be used as appropriate.

[0031] In this application, "O-acetylhomoserine" refers to a specific intermediate substance in the methionine biosynthesis pathway of microorganisms, and means an acetyl derivative of L-homoserine. The O-acetylhomoserine is produced by an enzymatic activity that transfers the acetyl group of acetyl-CoA to homoserine, using homoserine and acetyl-CoA as substrates.

[0032] In this application, "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have been genetically modified naturally or artificially, and is a microorganism in which a specific mechanism has been weakened or strengthened due to causes such as the insertion of an external gene or the strengthening or inactivation of the activity of an endogenous gene, and is a microorganism that has been genetically modified for the production of a target polypeptide, protein, or product.

[0033] In this application, "microorganisms having O-acetylhomoserine production ability" means prokaryotic or eukaryotic microbial strains that produce O-acetylhomoserine within their bodies, and includes microorganisms in which the ability to produce O-acetylhomoserine has been conferred to a parent strain that lacks O-acetylhomoserine production ability, and microorganisms that inherently possess O-acetylhomoserine production ability. O-acetylhomoserine production ability can be conferred or improved through selective breeding. The microorganisms having O-acetylhomoserine production ability are strains that produce L-lysine, L-threonine, L-isoleucine, or L-methionine, or from which they are derived, but are not limited to these.

[0034] For the purposes of this application, the microorganisms that produce O-acetylhomoserine are characterized by having weakened activity of the GNAT family N-acetyltransferase protein compared to its endogenous activity, thereby improving their ability to produce the desired O-acetylhomoserine, and are genetically modified or recombinant microorganisms, but are not limited to these. Specifically, the recombinant strains with improved O-acetylhomoserine production ability are microorganisms that have improved O-acetylhomoserine production ability compared to natural wild-type microorganisms or unmodified microorganisms with endogenous activity of the GNAT family N-acetyltransferase protein, but are not limited to these.

[0035] For example, the microorganism producing O-acetylhomoserine may intrinsically contain a protein consisting of the amino acid sequence of SEQ ID NO: 1, or a protein consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with SEQ ID NO: 1.

[0036] For example, the microorganism producing O-acetylhomoserine may be a microorganism that intrinsically contains a polynucleotide sequence encoding a protein having at least 80% homology to SEQ ID NO: 1, the base sequence of SEQ ID NO: 2, or a base sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the sequence of SEQ ID NO: 2.

[0037] As an example, the microorganism with improved productivity is one in which the productivity has improved by approximately 1% or more compared to the O-acetylhomoserine production capacity of the parent strain before mutation or an unmodified microorganism possessing endogenous activity of the GNAT family N-acetyltransferase protein. Specifically, this improvement can be approximately 1%, 5%, 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% or more (there is no particular limit on the upper limit, for example, approximately 100% or less, approximately 50% or less, approximately 45% or less, approximately 40% or less, or approximately 35% or less). However, any microorganism with improved productivity that has increased by a positive value compared to the productivity of the parent strain before mutation or an unmodified microorganism is acceptable. As another example, the recombinant strains with improved production capacity are those in which the O-acetylhomoserine production capacity has improved by approximately 1.1 times or more, approximately 1.2 times or more, approximately 1.21 times or more, approximately 1.22 times or more, approximately 1.23 times or more, approximately 1.24 times or more, approximately 1.25 times or more, approximately 1.26 times or more, approximately 1.27 times or more, approximately 1.28 times or more, approximately 1.29 times or more, or approximately 1.30 times or more (there is no particular limit on the upper limit, for example, approximately 10 times or less, approximately 5 times or less, approximately 3 times or less, approximately 2 times or less, approximately 1.5 times or less, or approximately 1.4 times or less), but are not limited to these.

[0038] In this application, "unmodified microorganism" does not exclude strains containing naturally occurring mutations in microorganisms, but rather refers to wild-type strains or natural strains themselves, or strains before they undergo genetic mutation and changes in phenotype due to natural or artificial factors. For example, the aforementioned unmodified microorganism refers to a strain in which the GNAT family N-acetyltransferase protein activity described herein is not weakened compared to endogenous activity, or before it is weakened. The aforementioned "unmodified microorganism" is used interchangeably with "pre-modification strain," "pre-modification microorganism," "non-mutant strain," "unmodified strain," "non-mutant microorganism," or "reference microorganism."

[0039] The microorganisms having the ability to produce O-acetylhomoserine may be either prokaryotic or eukaryotic cells, and specifically prokaryotic cells. Examples of such prokaryotic cells include microbial strains belonging to the genera Escherichia, Erwinia, Serratia, Providencia, Corynebacterium, Pseudomonas, Leptospira, Salmonella, Brevibacterium, Hyphomononas, Chromobacterium, and Norcardia, or fungi or yeast. Specifically, these are microbial strains and yeasts belonging to the genera Escherichia, Corynebacterium, and Leptospira. More specifically, they are microbial strains of the genus Corynebacterium.

[0040] In this application, "microorganisms of the genus Corynebacterium" includes all microorganisms of the genus Corynebacterium. Specifically, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes These include Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens, and more specifically, Corynebacterium glutamicum.

[0041] The unmodified microorganism may be a microorganism containing an amino acid sequence consisting of SEQ ID NO: 1, or a polynucleotide consisting of SEQ ID NO: 2.

[0042] On the other hand, while it is already known that microorganisms of the genus Corynebacterium produce O-acetylhomoserine, their production capacity is very low, and the genes and mechanisms involved in the production process have not been elucidated. Therefore, the Corynebacterium microorganisms that produce O-acetylhomoserine in this application include the naturally occurring wild-type microorganisms themselves, Corynebacterium microorganisms whose O-acetylhomoserine production capacity has been improved by strengthening or weakening the activity of genes related to the O-acetylhomoserine production mechanism, and Corynebacterium microorganisms whose O-acetylhomoserine production capacity has been improved by introducing or strengthening the activity of external genes.

[0043] In this application, "weakening" of polypeptide activity encompasses all cases where activity is reduced compared to endogenous activity or where activity is eliminated. This weakening is used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation.

[0044] Specifically, the aforementioned weakening is, but is not limited to, inactivation. Inactivation means that the protein is not expressed at all, or if expressed, its activity is absent or reduced, compared to the parent strain or strains in which the protein consisting of the amino acid sequence of Sequence ID No. 1 has not been modified.

[0045] The aforementioned weakening includes at least one of the following: the activity of the polypeptide itself is reduced or eliminated compared to the original polypeptide activity of the microorganism due to mutations in the polynucleotide encoding the polypeptide; the overall degree and / or concentration (expression level) of polypeptide activity within the cell is reduced compared to the natural strain due to inhibition of the expression of the gene encoding the polynucleotide or inhibition of translation into the polypeptide; the complete absence of polynucleotide expression; and the absence of polypeptide activity even if polynucleotide is expressed.

[0046] The aforementioned "endogenous activity" refers to the activity of a specific polypeptide that was originally present in the parent strain, wild type, or unmodified microorganism before the trait change, when a trait changes due to genetic mutation caused by natural or artificial factors. This is often used interchangeably with "activity before modification." When polypeptide activity is described as "inactivated," "deficient," "reduced," "downregulated," "decreased," or "attenuated" compared to endogenous activity, it means that it is lower than the activity of the specific polypeptide that was originally present in the parent strain or unmodified microorganism before the trait change.

[0047] Such weakening of polypeptide activity is not limited to these methods and can be achieved by applying various methods well known in the field (e.g., Non-Patent Documents 11, 12, etc.).

[0048] Specifically, the weakening of polypeptide activity in this application involves: 1) deleting all or part of the gene encoding the polypeptide; 2) modifying the expression regulatory region (or expression regulatory sequence) of the gene encoding the polypeptide so that its expression is reduced; 3) modifying the amino acid sequence constituting the polypeptide so that the polypeptide activity is deleted or weakened (e.g., deleting / substituting / adding one or more amino acids in the amino acid sequence); and 4) modifying the gene sequence encoding the polypeptide so that the polypeptide activity is deleted or weakened (e.g., modifying the polypeptide gene sequence to encode a polypeptide that has been modified so that the polypeptide activity is deleted or weakened). 1) Deleting / substituting / adding one or more nucleic acid bases in the nucleic acid base sequence; 5) Modifying the base sequence encoding the start codon or 5'UTR region of the polypeptide-encoding gene transcript; 6) Introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementaryly to the polypeptide-encoding gene transcript; 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the polypeptide-encoding gene such that a secondary structure is formed that prevents ribosome attachment; 8) Adding a promoter to the 3' end of the ORF (open reading frame) of the polypeptide-encoding gene sequence to reverse transcription (Reverse transcription engineering, RTE); or 9) A combination of two or more selected from 1) to 8) above, but not limited to these.

[0049] For example, the deletion of part or all of the gene encoding the polypeptide described in 1) above may be carried out by deleting the entire polynucleotide encoding the endogenous target polypeptide within the chromosome, or by substituting it with a polynucleotide or marker gene in which some nucleotides are deleted.

[0050] Furthermore, modifying the regulatory expression region (or regulatory expression sequence) described in 2) above may be carried out by causing a mutation on the regulatory expression region (or regulatory expression sequence) through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by substituting it with a sequence having weaker activity. The regulatory expression region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates the termination of transcription and translation.

[0051] Furthermore, modifying the amino acid sequence or polynucleotide sequence described in 3) and 4) above is carried out by introducing a sequence mutation through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, so as to weaken the polypeptide's activity, or by substituting it with an improved amino acid sequence or polynucleotide sequence that has lower activity, or an improved amino acid sequence or polynucleotide sequence that eliminates activity, but is not limited to these methods. For example, gene expression can be inhibited or weakened by introducing a mutation into a polynucleotide sequence to form a stop codon, but is not limited to these methods.

[0052] Furthermore, modifying the start codon or the nucleotide sequence encoding the 5'UTR region of the gene transcript encoding the polypeptide (5) above is performed, for example, by substituting it with a nucleotide sequence encoding another start codon that has a lower polypeptide expression rate compared to the endogenous start codon, but is not limited to this.

[0053] 6) Introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcript encoding the polypeptide can be done, for example, by referring to Non-Patent Document 13.

[0054] 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence in the polypeptide-coding gene so that a secondary structure is formed that makes ribosome attachment impossible may be done by making mRNA translation impossible or slowing down the rate of mRNA translation.

[0055] The reverse transcription engineering (RTE) described in 8) above, which involves adding a promoter to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide, may be performed by creating an antisense nucleotide complementary to the gene transcript encoding the polypeptide and weakening its activity.

[0056] Specifically, the microorganisms possessing O-acetylhomoserine production ability in this application are, but are not limited to, those in which the GNAT family N-acetyltransferase protein activity has been inactivated.

[0057] Specifically, to inactivate the GNAT family N-acetyltransferase protein activity, a method may be used in which part or all of the gene encoding the protein is deleted. More specifically, this may be done by using a chromosome transfer vector in a microorganism to replace a polynucleotide encoding an endogenous target protein within the chromosome with a polynucleotide or marker gene in which part of the nucleotide sequence is deleted. One example of such a method for deleting part or all of a polynucleotide is, but is not limited to, a method of deleting a polynucleotide by homologous recombination. As another example, the method for deleting part or all of the gene may be done by inducing a mutation using light such as ultraviolet light or a chemical substance, and then selecting a bacterial strain in which the target gene is deleted from the resulting mutant.

[0058] The gene deletion method described above includes methods using genetic recombination techniques. For example, it may be carried out by introducing a polynucleotide sequence or vector containing a polynucleotide sequence homologous to the target gene into the microorganism to induce homologous recombination. Furthermore, the introduced polynucleotide sequence or vector contains a dominant selection marker. However, it is not limited to this.

[0059] In this application, "polynucleotide" means a polymer of nucleotides in which nucleotide monomers are covalently linked together in a long chain, and refers to a DNA or RNA chain longer than a predetermined length, and more specifically, refers to a polynucleotide fragment that codes for the aforementioned variant.

[0060] In this application, the GNAT family N-acetyltransferase is encoded by a gene containing the polynucleotide sequence of SEQ ID NO: 2, but is not limited thereto. More specifically, the GNAT family N-acetyltransferase is encoded by a gene having the polynucleotide sequence of SEQ ID NO: 2, a gene containing the polynucleotide sequence, a gene essentially consisting of the polynucleotide sequence, and / or a gene consisting of the polynucleotide sequence, but is not limited thereto. The nucleotide sequence of SEQ ID NO: 2 can be obtained from a known database, such as NCBI's GenBank, but is not limited thereto.

[0061] In this application, the gene containing the nucleotide sequence of SEQ ID NO: 2 is used interchangeably with a polynucleotide containing the nucleotide sequence of SEQ ID NO: 2, a gene or polynucleotide having the nucleotide sequence of SEQ ID NO: 2, or a gene or polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2.

[0062] The polynucleotides of this application may be modified in various ways in the coding region, either by codon degeneracy or by taking into consideration the preferred codons in organisms expressing the variants of this application, as long as the amino acid sequence of the variants of this application does not change. Specifically, the polynucleotides of this application include any polynucleotide sequence encoding a protein that has at least 80% homology to the amino acid sequence of SEQ ID NO: 1, and are not limited to those having the base sequence of SEQ ID NO: 2, or a base sequence that has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the sequence of SEQ ID NO: 2, or containing the said base sequence, or consisting of the base sequence of SEQ ID NO: 2, or a base sequence that has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the sequence of SEQ ID NO: 2, or substantially consisting of the said base sequence.

[0063] Furthermore, the polynucleotide of this application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, such as a complementary sequence to all or part of the polynucleotide sequence of this application. The “stringent condition” means a condition that enables specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Documents 14 and 15). For example, this could involve hybridizing polynucleotides with high homology or identity, such as polynucleotides with 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity, while not hybridizing polynucleotides with lower homology or identity. Alternatively, it could involve washing once, specifically two to three times, at a salt concentration and temperature equivalent to the typical washing conditions for Southern hybridization: 60°C, 1×SSC, 0.1%SDS, more specifically 60°C, 0.1×SSC, 0.1%SDS, or more specifically 68°C, 0.1×SSC, 0.1%SDS.

[0064] Hybridization requires that the two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of this application may include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0065] Specifically, polynucleotides homologous or identical to the polynucleotide of this application can be detected using hybridization conditions in which the hybridization step is performed at a Tm value of 55°C and the conditions described above. The Tm value may be 60°C, 63°C, or 65°C, but is not limited to these, and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0066] The appropriate stringency for hybridizing the polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are known in the art (e.g., Non-Patent Document 14).

[0067] Specifically, the microorganisms possessing O-acetylhomoserine production ability in this application are, but are not limited to, microorganisms in which the GNAT family N-acetyltransferase protein activity has been inactivated.

[0068] Specifically, the microorganisms capable of producing O-acetylhomoserine in this application are, but are not limited to, microorganisms lacking the polynucleotide encoding the GNAT family N-acetyltransferase. More specifically, the microorganisms capable of producing O-acetylhomoserine in this application are, but are not limited to, microorganisms lacking the nucleic acid base sequence of Sequence ID No. 2.

[0069] For the purposes of this application, the microorganism is characterized by having the activity of the GNAT family N-acetyltransferase protein inactivated by including an expression vector for inactivating the GNAT family N-acetyltransferase protein in a host cell, but is not limited to this.

[0070] The vector of this application comprises a DNA product comprising a polynucleotide sequence encoding a target polypeptide operably linked to a suitable regulatory region (or regulatory sequence) so as to enable expression of the target polypeptide in a suitable host. The regulatory region comprises a promoter for initiating transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences for regulating the termination of transcription and translation. When transformed into a suitable host cell, the vector can replicate and function independently of the host genome and is integrated into the genome itself.

[0071] The vectors used in this application are not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state. For example, as phage vectors or cosmid vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used, and as plasmid vectors, pDZ series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, pSK series, pSKH series, pET series, etc. can be used. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pSK, pSKH130, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

[0072] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using an intracellular chromosome introduction vector. The insertion of the polynucleotide into the chromosome can be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. The vector may further include a selection marker to confirm whether or not the polynucleotide has been inserted into the chromosome. The selection marker is used to select cells transformed by the vector, that is, to confirm whether or not the target nucleic acid molecule has been inserted, and markers that confer selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, and expression of surface polypeptides are used. In an environment treated with a selective agent, only cells expressing the selection marker will survive or exhibit different phenotypes, thus allowing for the selection of transformed cells.

[0073] In this application, "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism, thereby causing the polypeptide encoded by the polynucleotide to be expressed in the host cell. The transformed polynucleotide may be any form, regardless of whether it is inserted into or outside the host cell's chromosomes, as long as it is expressed in the host cell. The polynucleotide also contains DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced into the host cell in any form, as long as it is introduced and expressed in the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a gene structure containing all the elements necessary for its expression. Typically, the expression cassette includes a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may also be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form and operably linked to the sequence necessary for expression in the host cell, but is not limited to this.

[0074] Furthermore, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the target variant of this application.

[0075] In the microorganisms of this application, modification of part or all of the polynucleotides can be induced by (a) homologous recombination using a chromosome introduction vector in the microorganism, or genome editing using an engineered nuclease (e.g., CRISPR-Cas9), and / or (b) light and / or chemical treatment such as ultraviolet light or radiation. The methods for modifying part or all of the genes include methods using DNA recombination technology. For example, deletion of part or all of the gene can be achieved by introducing a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism to induce homologous recombination. The introduced nucleotide sequence or vector contains, but is not limited to, a dominant selection marker.

[0076] In this application, "enhancement" of polypeptide activity means improving the polypeptide activity compared to its endogenous activity. This enhancement is used interchangeably with activation, upregulation, overexpression, and increase. Here, activation, enhancement, upregulation, overexpression, and increase all include exhibiting activity that was not originally present, or improving activity compared to endogenous activity or activity before modification. "Endogenous activity" refers to the activity of a specific polypeptide that was originally present in the parent strain or unmodified microorganism before the trait change, when a trait changes due to genetic mutation caused by natural or artificial factors. This is used interchangeably with "activity before modification." When polypeptide activity is "enhanced," "upregulated," "overexpressed," or "improved" compared to its endogenous activity, it means that the activity and / or concentration (expression level) of the specific polypeptide that was originally present in the parent strain or unmodified microorganism before the trait change is improved.

[0077] The enhancement may be carried out by introducing an exogenous polypeptide, or by enhancing the activity and / or increasing the concentration (expression level) of an endogenous polypeptide. Whether or not the activity of the polypeptide has been enhanced can be confirmed by an increase in the degree of the polypeptide's activity, its expression level, or the amount of product produced from the polypeptide.

[0078] Various methods known in the field can be applied to enhance the activity of the polypeptide, and any method that can enhance the activity of the target polypeptide compared to the microorganism before modification is acceptable. Specifically, this includes, but is not limited to, conventional methods in molecular biology, including genetic engineering and / or protein engineering known to those with ordinary skill in the field (see, for example, Non-Patent Documents 12, 16, etc.).

[0079] Specifically, the enhancement of the polypeptide in this application is carried out by 1) increasing the intracellular copy number of the polynucleotide encoding the polypeptide, 2) modifying the expression regulatory region of the gene on the chromosome encoding the polypeptide (for example, by inducing a mutation in the expression regulatory region, substituting with a sequence having higher activity, or inserting a sequence having higher activity), 3) modifying the base sequence encoding the start codon or 5'UTR region of the gene transcript encoding the polypeptide, 4) modifying the amino acid sequence of the polypeptide so as to enhance polypeptide activity, 5) modifying the polynucleotide sequence encoding the polypeptide so as to enhance polypeptide activity (for example, by modifying the polynucleotide sequence of the polypeptide gene to encode a polypeptide modified to enhance polypeptide activity), 6) introducing a foreign polypeptide exhibiting polypeptide activity or a foreign polynucleotide encoding it, 7) optimizing the codon of the polynucleotide encoding the polypeptide, 8) analyzing the tertiary structure of the polypeptide and selectively modifying or chemically modifying exposed portions, or 9) a combination of two or more selected from 1) to 8) above, but is not limited to these.

[0080] More specifically, increasing the intracellular copy number of the polynucleotide encoding the polypeptide (as described in 1) above may be carried out by introducing into a host cell a vector into which the polynucleotide encoding the polypeptide is operably linked and which replicates and functions independently of the host. Alternatively, it may be carried out by introducing one or more copies of the polynucleotide encoding the polypeptide into the chromosomes within the host cell. The introduction into the chromosomes is carried out by introducing into the host cell a vector capable of inserting the polynucleotide into the chromosomes within the host cell, but is not limited to this. The vector is as described above.

[0081] 2) Substitution of a gene expression regulatory region (or expression regulatory sequence) on a chromosome encoding a polypeptide with a more potent sequence is carried out, for example, by causing a sequence mutation through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by substituting with a sequence having higher activity, so as to further enhance the activity of the expression regulatory region. The expression regulatory region includes, but is not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, and sequences that regulate the termination of transcription and translation. For example, this is carried out by substituting the original promoter with a potent promoter, but is not limited to this.

[0082] Examples of known strong promoters include, but are not limited to, the CJ1-CJ7 promoters (Patent Document 2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (Patent Document 3), the O2 promoter (Patent Document 4), the tkt promoter, and the yccA promoter.

[0083] 3) Modifying the start codon or the nucleotide sequence encoding the 5'UTR region of a gene transcript encoding a polypeptide is performed, for example, by substituting it with a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate compared to the endogenous start codon, but is not limited to this.

[0084] Modifying the amino acid sequence or polynucleotide sequence described in 4) and 5) above is carried out by, but is not limited to, introducing a sequence mutation by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, so as to enhance the polypeptide's activity, or by substituting it with an improved amino acid sequence or polynucleotide sequence that has higher activity, or an improved amino acid sequence or polynucleotide sequence that has improved activity. Specifically, the substitution is carried out by, but is not limited to, inserting the polynucleotide into the chromosome by homologous recombination. The vector used here may further include a selection marker to confirm whether or not it has been inserted into the chromosome. The selection marker is as described above.

[0085] The introduction of a foreign polynucleotide exhibiting polypeptide activity (6) above may be carried out by introducing a foreign polynucleotide encoding a polypeptide exhibiting identical or similar activity to the polypeptide into the host cell. The foreign polynucleotide may have any origin or sequence, as long as it exhibits identical or similar activity to the polypeptide. The introduction can be carried out by a person skilled in the art using a known transformation method as appropriate, and as the polynucleotide introduced as described above is expressed in the host cell, the polypeptide is produced and its activity is improved.

[0086] The optimization of codons of polynucleotides encoding polypeptides described in 7) above may be performed by optimizing endogenous polynucleotide codons so as to increase transcription or translation within the host cell, or by optimizing exogenous polynucleotide codons so as to perform optimized transcription or translation within the host cell.

[0087] The 8) analysis of the tertiary structure of the polypeptide, and the selection and modification or chemical modification of exposed portions may be carried out, for example, by comparing the sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored, determining candidate template proteins according to the degree of sequence similarity, confirming the structure based on these, and selecting and modifying or chemically modifying exposed portions.

[0088] Such enhancement of polypeptide activity is achieved by increasing the activity, concentration, or expression level of the corresponding polypeptide compared to the activity or concentration of the polypeptide expressed in the wild-type or pre-modification microbial strain, or by increasing the amount of product produced from the polypeptide, but is not limited to these methods.

[0089] The microorganisms possessing O-acetylhomoserine production ability described in this application are further enhanced in L-methionine / branched-chain amino acid exporter YjeH protein activity, but are not limited to these.

[0090] In this application, "L-methionine / branched-chain amino acid exporter YjeH" refers to a type of amino acid efflux (AAE) protein within the amino acid-polyamine-organocation (APC) superfamily of transport proteins, which mediates the extracellular export of O-acetylhomoserine and / or homoserine. It is predicted to contain 12 transmembrane α helices, 10 of which form an inverted repeat fold, a characteristic of the APC superfamily. The L-methionine / branched-chain amino acid exporter YjeH protein and gene sequence can be obtained from known databases, such as NCBI's GenBank, but are not limited to these. For example, those derived from Escherichia coli (E. coli) can be used, but are not limited to these. In this application, the L-methionine / branched-chain amino acid exporter YjeH protein is used in combination with inner membrane proteins, O-acetylhomoserine efflux proteins, proteins having O-acetylhomoserine efflux ability, proteins having O-acetylhomoserine efflux activity, YjeH protein, or YjeH.

[0091] Specifically, the microorganisms having the ability to produce O-acetylhomoserine according to this application further include, but are not limited to, a variant of the L-methionine / branched-chain amino acid exporter YjeH, and more specifically, the microorganisms having the ability to produce O-acetylhomoserine according to this application further have the L-methionine / branched-chain amino acid exporter YjeH protein activity enhanced by the introduction of a variant of the L-methionine / branched-chain amino acid exporter YjeH, but are not limited to this.

[0092] More specifically, the variant of the L-methionine / branched-chain amino acid exporter YjeH may improve the production capacity of O-acetylhomoserine by increasing the amount of O-acetylhomoserine excreted by microorganisms, or it may have the amino acid sequence represented by SEQ ID NO: 10, or it may contain the amino acid sequence, or it may be substantially composed of the amino acid sequence, or it may consist of the amino acid sequence.

[0093] Furthermore, the variant of the L-methionine / branched-chain amino acid exporter YjeH may include not only the amino acid sequence represented by SEQ ID NO: 10, but also an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with SEQ ID NO: 10. Moreover, it goes without saying that this application also includes amino acid sequences having such homology or identity and possessing the same or equivalent biological activity as the variant of the L-methionine / branched-chain amino acid exporter YjeH of this application, even if some of the sequences are deleted, modified, substituted, conservatively substituted, or added.

[0094] For example, the microorganism having the ability to produce O-acetylhomoserine according to this application further comprises a protein consisting of the amino acid sequence of SEQ ID NO: 10, or a protein consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with SEQ ID NO: 10. Specifically, it may be a microorganism into which the protein has been introduced and whose ability to produce O-acetylhomoserine has been further improved.

[0095] Specifically, "protein introduction" means causing a gene that was not originally present in the microorganism to be expressed within that microorganism, thereby causing the activity of a specific protein to appear, or causing an increase or improvement in the activity of the protein compared to its endogenous activity or the activity before modification. For example, this may involve introducing a polynucleotide encoding a specific protein into the chromosome of the microorganism, or introducing a vector containing a polynucleotide encoding a specific protein into the microorganism, thereby causing its activity to appear.

[0096] For the purposes of this application, the microorganism containing the L-methionine / branched-chain amino acid exporter YjeH variant refers to a microorganism that naturally has weak O-acetylhomoserine efflux ability, but in which the L-methionine / branched-chain amino acid exporter YjeH variant has been introduced to improve O-acetylhomoserine efflux ability. Specifically, the microorganism is characterized in that, by introducing the L-methionine / branched-chain amino acid exporter YjeH variant, the amount of O-acetylhomoserine excreted from the extracellular space increases compared to wild-type or unmodified microorganisms, thereby improving the O-acetylhomoserine production capacity. Therefore, while wild-type or unmodified microorganisms are unable to excrete O-acetylhomoserine, or can only excrete it in trace amounts, introducing the L-methionine / branched-chain amino acid exporter YjeH variant increases the amount of O-acetylhomoserine excreted by the microorganism, thereby improving the O-acetylhomoserine production capacity.

[0097] Examples of the intermembryonic protein YjeH and / or its variants used in this application are disclosed in Patent Document 5, and the full text of the specification of the above patent is incorporated herein by reference.

[0098] The microorganisms possessing O-acetylhomoserine production ability described in this application are, but are not limited to, those with enhanced homoserine acetyltransferase (MetX) activity.

[0099] In this application, "MetX" refers to homoserine acetyltransferase. Specifically, most microorganisms in nature use O-succinyl homoserine or O-acetyl homoserine as intermediates to biosynthesize methionine, but generally MetA produces O-succinyl homoserine, and homoserine O-acetyltransferase produces O-acetyl homoserine. Unlike MetA, MetX is not subject to feedback inhibition and has high enzyme stability. The protein and gene sequences of MetX can be obtained from known databases, such as NCBI's GenBank, but are not limited to these. In this application, "homoserine acetyltransferase" is used interchangeably with "MetX" and "homoserine O-acetyltransferase".

[0100] Specifically, the microorganisms possessing O-acetylhomoserine production ability described in this application are, but are not limited to, those in which the expression of the MetX gene encoding O-acetylhomoserine transferase is further amplified.

[0101] The nucleotide sequence of the MetX gene is obtained from the NCBI's GenBank, a known database. The polynucleotide encoding MetX is included in this application as long as it is a polynucleotide sequence encoding a protein that includes an amino acid sequence having at least 80% homology to MetX, and is not limited to having a nucleotide sequence of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or less than 100% homology or identity with the nucleotide sequence of SEQ ID NO: 26, or includes the nucleotide sequence, or consists of a nucleotide sequence of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or less than 100% homology or identity with the nucleotide sequence of SEQ ID NO: 26, or substantially consists of the nucleotide sequence.

[0102] An example of the MetX gene used in this application is disclosed in Patent Document 5, and the full text of the specification of the above patent is incorporated herein by reference.

[0103] The microorganisms possessing O-acetylhomoserine production ability described in this application are, but are not limited to, those with enhanced aspartokinase activity.

[0104] Specifically, in order to increase O-acetyl-homoserine biosynthesis, the aspartokinase activity is further enhanced compared to non-mutated microorganisms, and in particular, a mutation (L377K) (Patent Document 6) for releasing feedback inhibition to L-lysine and L-threonine is introduced into the gene encoding aspartokinase (lysC), but it is not limited to this. The nucleotide sequence of lysC can be any nucleotide sequence that encodes a protein having aspartokinase activity and can be obtained from a known database, for example, the nucleotide sequence of Sequence ID No. 21.

[0105] The microorganisms possessing O-acetylhomoserine production ability described in this application are, but are not limited to, those with further weakened cystathionine gamma synthase activity.

[0106] Specifically, the weakening mentioned above is inactivation, but is not limited to this.

[0107] More specifically, the cystathionine γ-synthase activity is reduced or inactivated compared to that of non-mutant microorganisms, and in particular, the gene encoding cystathionine synthase (metB) is deleted, but is not limited to these. In this application, "cystathionine γ-synthase" is used interchangeably with "cystathionine synthase". The nucleotide sequence of metB may be any nucleotide sequence obtained from a known database that encodes a protein having cystathionine synthase activity, for example, the nucleotide sequence of Sequence ID No. 11.

[0108] The microorganisms possessing O-acetylhomoserine production ability described in this application are, but are not limited to, those with further weakened O-acetylhomoserine (thiol)-lyase activity.

[0109] Specifically, the weakening mentioned above is inactivation, but is not limited to this.

[0110] More specifically, the O-acetylhomoserine thiol lyase activity is reduced or inactivated compared to that of non-mutant microorganisms, and in particular, the gene encoding O-acetylhomoserine thiol lyase (metY) is deleted, but is not limited to these. The nucleotide sequence of metY can be any nucleotide sequence obtained from a known database that encodes a protein having O-acetylhomoserine thiol lyase activity, for example, the nucleotide sequence of Sequence ID No. 16.

[0111] Another aspect of this application provides a method for producing O-acetylhomoserine, comprising the step of culturing the microorganism in a culture medium.

[0112] The aforementioned microorganism, O-acetylhomoserine, is as described above.

[0113] In the method of this application, any culture conditions and methods known in the art are used for culturing microorganisms. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected strain.

[0114] In this application, "cultivation" means growing the microorganisms of this application under appropriately adjusted environmental conditions. The cultivation process of this application can be carried out using suitable culture media and cultivation conditions known in the art. Such a cultivation process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the cultivation is batch, continuous, and fed-batch culture, but is not limited to these.

[0115] In this application, "culture medium" refers to a substance that is a mixture mainly composed of nutrients necessary for culturing the microorganisms of this application, and supplies nutrients and growth factors, including water, which are essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the microorganisms of this application may be any that are normally used for culturing microorganisms, and the microorganisms of this application can be cultured in a normal culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, under aerobic conditions, with the temperature, pH, etc. adjusted.

[0116] In this application, the carbon source can be carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn maceration liquid can be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and any other carbon source in an appropriate amount can be used. These carbon sources can be used individually or in combination of two or more, but are not limited to these uses.

[0117] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate can be used, as well as organic nitrogen sources such as amino acids like glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn maceration liquid, casein hydrolysates, fish or their decomposition products, defatted soybean cake or its decomposition products. These nitrogen sources can be used individually or in combination of two or more, but are not limited to these uses.

[0118] As the phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or equivalent sodium-containing salts can be used. As inorganic compounds, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc., can be used, and in addition, amino acids, vitamins, and / or suitable precursors can be used. These components or precursors can be added to the culture medium in batches or continuously, but are not limited to these.

[0119] In this application, the pH of a microbial culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture in a manner suitable for the culture. Furthermore, during cultivation, the formation of bubbles can be suppressed using an antifoaming agent such as fatty acid polyglycol ester. In addition, oxygen or oxygen-containing gas may be injected into the culture to maintain an aerobic state, and to maintain an anaerobic and microaerobic state, no gas injection is necessary, but nitrogen, hydrogen, or carbon dioxide gas may be injected, although these are not limited to these.

[0120] The culture temperature is 25°C to 40°C, more specifically 28°C to 37°C, but is not limited to this range. The culture period continues until the desired amount of useful substance is produced, specifically 1 hour to 100 hours, but is not limited to this range.

[0121] O-acetylhomoserine produced by the culture described in this application is either secreted into the culture medium or remains within the cells.

[0122] The method for producing O-acetylhomoserine according to this application may further include, for example, the steps of preparing the microorganism of this application, preparing a culture medium for culturing the microorganism, or a combination thereof (in any order) before the culturing step.

[0123] The method for producing O-acetylhomoserine according to this application may further include a step of recovering O-acetylhomoserine from the culture medium (culture medium) or microorganism used in the culture. The recovery step may be further included after the culture step.

[0124] The aforementioned recovery may involve collecting the target O-acetylhomoserine using a suitable method known in the art, depending on the microorganism culture method of this application, such as batch, continuous, or fed-batch culture. For example, various chromatography methods such as centrifugation, filtration, crystallization, treatment with protein precipitants (salting-out method), extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof can be used, and the target O-acetylhomoserine can be recovered from the culture medium or microorganism using a suitable method known in the art.

[0125] Furthermore, the method for producing O-acetylhomoserine according to this application may further include a purification step. The purification can be carried out by a suitable method known in the art. For example, if the method for producing O-acetylhomoserine according to this application includes both a recovery step and a purification step, the recovery step and the purification step may be carried out sequentially or discontinuously, regardless of order, simultaneously, or integrated as a single step, but are not limited thereto.

[0126] The O-acetylhomoserine recovered in this manner can be used to produce methionine using a two-step process (Patent Document 7).

[0127] A further aspect of this application provides a method for producing L-methionine, comprising the steps of: culturing the microorganism in a culture medium; producing O-acetylhomoserine from the cultured microorganism or culture medium; and converting the O-acetylhomoserine to L-methionine.

[0128] The aforementioned microorganisms, O-acetylhomoserine, and L-methionine are as described above.

[0129] The aforementioned two-step process includes a step of producing L-methionine and an organic acid by an enzymatic reaction using an enzyme having O-acetyl homoserine sulfhydrylase activity or a strain containing the enzyme, with O-acetyl homoserine and methyl mercaptan produced by the L-methionine precursor-producing strain as substrates.

[0130] More specifically, this application provides a method for producing L-methionine using O-acetylhomoserine accumulated by the above method as a substrate, by an enzymatic reaction such as that of O-acetylhomoserine sulfhydrase.

[0131] In the aforementioned two-step process, when O-acetylhomoserine is used as an L-methionine precursor, specifically, O-acetylhomoserine sulfhydrase derived from microbial strains belonging to the genera Leptospira sp., Chromobacterium sp., and Hyphomonas sp., more specifically, from microbial strains belonging to Leptospira meyeri, Pseudomonas aeruginosa, Hyphomonas Neptunium, and Chromobacterium Violaceum, can be used.

[0132] The above response is as follows: CH3SH + O-acetylhomoserine ⇔ acetic acid + methionine

[0133] Further methionine production processes are disclosed in Patent Document 7, and the entire specification of the above patent is incorporated into this application as reference material.

[0134] A further aspect of this application provides a composition for the production of O-acetylhomoserine, comprising the microorganism.

[0135] The aforementioned microorganism, O-acetylhomoserine, is as described above.

[0136] The composition of this application may further contain any suitable excipients commonly used in compositions for the production of O-acetylhomoserine. Examples of such excipients include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0137] Further embodiments of this application provide applications for the production of O-acetylhomoserine or L-methionine from the microorganism. [Examples]

[0138] The present application will be described in more detail below with reference to examples. However, these examples are merely preferred embodiments illustrating the present application, and the application is not limited thereto. Technical matters not described herein can be fully understood and readily implemented by a skilled technician in the art of this application or a similar art. [Examples]

[0139] Creation of bacterial strains lacking the endogenous gene (NCgl0959) and evaluation of O-acetylhomoserine and homoserine production capacity. Example 1-1. Preparation of a deletion vector for NCgl0959 deletion. To determine the effectiveness of the endogenous gene NCgl0959 in Corynebacterium glutamicum ATCC13032, a deletion vector was created in which the gene NCgl0959 (SEQ ID NO: 2) was deleted.

[0140] Specifically, to construct an NCgl0959 deletion vector, we designed one pair of primers (SEQ ID NOs: 3 and 4) to amplify the upstream 5' end and one pair of primers (SEQ ID NOs: 5 and 6) to amplify the downstream 3' end, centering on the NCgl0959 gene location in SEQ ID NO: 2. The primer sequences are shown in Table 1.

[0141] [Table 1]

[0142] PCR was performed using ATCC13032 wild-type (WT) chromosomes as templates and primers SEQ ID NOs. 3 and 4, and SEQ ID NOs. 5 and 6. The PCR conditions were denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 30 seconds, for 30 cycles, followed by polymerization at 72°C for 7 minutes. As a result, a 1476 bp DNA fragment upstream of the 5' end and a 1456 bp DNA fragment downstream of the 3' end were obtained, centered around the deletion site of the NCgl0959 gene.

[0143] PCR was performed using the two amplified DNA fragments as templates with primers SEQ ID NOs: 3 and 6. The PCR conditions were denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 90 seconds, for 30 cycles, followed by polymerization at 72°C for 7 minutes. As a result, a 2912 bp DNA fragment containing the deletion site for the NCgl0959 gene was amplified.

[0144] The pDCM2 vector (SEQ ID NO: 7, Patent Document 8) was treated with SmaI, and the PCR product (2912 bp DNA fragment) obtained as described above was fusion cloned with the pDCM2 vector treated with SmaI restriction enzyme using the In-Fusion® HD Cloning Kit (Clontech). The cloned vector was transformed into E. coli DH5α, and the transformed E. coli were streaked onto LB solid medium containing 25 mg / l kanamycin. Colonies transformed with the plasmid were selected from the LB solid medium, and the plasmid was then obtained by plasmid extraction (Patent Document 9). Finally, a pDCM2-ΔNCgl0959 recombinant vector with a cloned NCgl0959 deletion cassette was prepared.

[0145] The prepared pDCM2-ΔNCgl0959 was transformed into the ATCC13032 strain using electrophoretic pulses, and after a secondary crossover process, ATCC13032ΔNCgl0959 was obtained, in which the NCgl0959 gene was deleted on the chromosome. Whether or not the NCgl0959 gene was inactivated was confirmed by PCR using primers of sequence numbers 8 and 9, and then by comparing it with ATCC13032 in which the NCgl0959 gene was not inactivated.

[0146] [Table 2]

[0147] Examples 1-2. Evaluation of O-acetylhomoserine production capacity in wild-type strains. To compare the O-acetylhomoserine (O-AH) production capacity of ATCC13032ΔNCgl0959 prepared in Example 1-1 with that of the wild-type strain ATCC13032, the two strains were cultured using the following method, and the O-acetylhomoserine in the culture medium was analyzed.

[0148] One platinum loopful of the strain was inoculated into a 250-ml conical baffle flask containing 25 ml of the following medium, and cultured with shaking at 33 °C and 200 rpm for 20 hours. The concentration of O-acetylhomoserine was analyzed using HPLC. The analyzed concentrations are shown in Table 3. <O-acetylhomoserine production medium (pH 7.2)> Glucose 30 g, KH2PO4 2 g, Urea 3 g, (NH4)2SO4 40 g, Peptone 2.5 g, CSL (Sigma) 5 g (10 ml), MgSO4·7H2O 0.5 g, CaCO3 20 g (in 1 liter of distilled water)

[0149]

Table 3

[0150] As a result, as shown in Table 3, when ATCC13032 was cultured, 0.27 g / L of O-acetylhomoserine was accumulated. When ATCC13032ΔNCgl0959, in which the endogenous gene NCgl0959 gene was deleted, was cultured, 0.30 g / L of O-acetylhomoserine was accumulated, and it was confirmed that it showed a production ability of 120% compared to the control group ATCC13032.

Example

[0151] Preparation of an NCgl0959 deletion strain in a strain with improved O-acetylhomoserine production ability and evaluation of O-acetylhomoserine and homoserine production ability - 1 Example 2-1. Preparation of strain The pDCM2-ΔNCgl0959 vector prepared in Example 1-1 was transformed into the KCCM12634P (Patent Document 5, ATCC13032ΔNCgl2335::PCJ7-YjeH(eco,F351L)) strain with improved O-acetylhomoserine production ability by the electropulse method. Through the secondary crossover process, KCCM12634PΔNCgl0959 in which the NCgl0959 gene was deleted on the chromosome was obtained. Whether the NCgl,0959 gene was inactivated or not was finally confirmed by performing PCR using the primers of SEQ ID NO: 8 and 9 and comparing it with ATCC13032 in which the NCgl0959 gene was not inactivated.

[0152] Example 2-2. Evaluation of O-acetylhomoserine production ability To compare the O-acetylhomoserine (O-AH; O-Acetyl Homoserine) production abilities of the KCCM12634P strain and the KCCM12634PΔNCgl0959 strain prepared in Example 2-1, they were cultured by the following method, and O-acetylhomoserine in the culture broth was analyzed.

[0153] One platinum loop of the strain was inoculated into a 250 ml conical baffle flask containing 25 ml of the following medium, and cultured with shaking at 33 °C and 200 rpm for 20 hours. The concentration of O-acetylhomoserine was analyzed using HPLC. The analyzed concentrations are shown in Table 4. <O-acetylhomoserine production medium (pH 7.2)> Glucose 30 g, KH2PO4 2 g, urea 3 g, (NH4)2SO4 40 g, peptone 2.5 g, CSL (Sigma) 5 g (10 ml), MgSO4·7H2O 0.5 g, CaCO3 20 g (in 1 liter of distilled water)

[0154]

Table 4

[0155] As a result, as shown in Table 4, in the KCCM12634P strain, when NCgl0959 was deleted, O-acetylhomoserine was 1.31 g / L, and it was confirmed that the production ability was about 130% that of KCCM12634P. Therefore, from the above results, it was confirmed that in the strain with improved O-acetylhomoserine production ability, the effect of improving O-acetylhomoserine production ability was greater due to the deletion of NCgl0959.

[0156] Therefore, from the results of Examples 1-2 and 2-2, it was confirmed that the deletion and inactivation of the NCgl0959 gene, which is an endogenous gene of ATCC13032 of the present application, improved the production ability of the target amino acid.

Examples

[0157] Preparation of NCgl0959-deficient strains in strains with improved O-acetylhomoserine production capacity and evaluation of O-acetylhomoserine and homoserine production capacity - 2 Example 3-1. Strain Preparation - 1 The metB gene, which encodes cystathionine gamma-synthase in the O-acetylhomoserine degradation pathway, was obtained by PCR using chromosomal DNA from Corynebacterium glutamicum ATCC13032 as a template. The nucleotide sequence information for the metB gene (NCBI registration number Ncgl2360, SEQ ID NO: 11) was obtained from the National Institutes of Health (NIH) GenBank, and primers containing the N-terminus and linker portion of the metB gene (SEQ ID NOs: 12 and 13) and primers containing the C-terminus and linker portion (SEQ ID NOs: 14 and 15) were synthesized based on this information. The primer sequences are shown in Table 5.

[0158] [Table 5]

[0159] PCR was performed using chromosomal DNA from ATCC13032 as a template, with primers numbered SEQ ID NOs. 12 and 13, and SEQ ID NOs. 14 and 15. PfuUltra polymerase was used. TM Using high-fidelity DNA polymerase (Stratagene), the PCR conditions were denaturation at 96°C for 30 seconds, annealing at 53°C for 30 seconds, and polymerization at 72°C for 1 minute, repeated for 30 cycles. As a result, two amplified genes were obtained: a 558 bp gene containing the N-terminal and linker portions of the metB gene, and a 527 bp gene containing the C-terminal and linker portions of the metB gene.

[0160] As described above, PCR was performed using the two amplified genes obtained as templates. The PCR conditions were denaturation at 96°C for 60 seconds, annealing at 50°C for 60 seconds, polymerization at 72°C for 1 minute, followed by 10 cycles of this process, after which SEQ ID NOs. 12 and 15 were added and the polymerization reaction was carried out for another 20 cycles. As a result, an inactivated cassette of 1064 bp containing the N-terminus, linker, and C-terminus of the metB gene was obtained.

[0161] The pDCM2 vector (SEQ ID NO: 7, Patent Document 8) was treated with SmaI, and the PCR product (1064 bp) obtained as described above was fusion cloned with the pDCM2 vector treated with SmaI restriction enzyme using the In-Fusion® HD Cloning Kit (Clontech). The cloned vector was transformed into E. coli DH5α, and the transformed E. coli were streaked onto LB solid medium containing 25 mg / l kanamycin. Colonies transformed with the plasmid were selected from the LB medium, and the plasmid was then obtained by plasmid extraction (Patent Document 9), and finally, a pDCM2-ΔmetB recombinant vector with a cloned metB gene deletion cassette was constructed.

[0162] The prepared pDCM2-ΔmetB vector was transformed into the KCCM12634P strain using electrotherapy, and after a secondary crossover, KCCM12634PΔmetB was obtained in which the metB gene was inactivated on the chromosome. The inactivated metB gene was then subjected to PCR using primers SEQ ID NOs. 12 and 15, and finally confirmed by comparison with ATCC13032, in which the metB gene was not inactivated.

[0163] Example 3-2. Strain Preparation - 2 The metY gene, which encodes O-acetylhomoserine (thiol)-lyase in the O-acetylhomoserine degradation pathway, was obtained by PCR using chromosomal DNA from Corynebacterium glutamicum ATCC13032 as a template. The nucleotide sequence information for the metY gene (NCBI registration number Ncgl0625, SEQ ID NO: 16) was obtained from the National Institutes of Health (NIH) GenBank, and primers containing the N-terminus and linker portion of the metY gene (SEQ ID NOs: 17 and 18) and primers containing the C-terminus and linker portion (SEQ ID NOs: 19 and 20) were synthesized based on this information. The primer sequences are shown in Table 6.

[0164] [Table 6]

[0165] PCR was performed using chromosomal DNA from ATCC13032 as a template, with primers numbered SEQ ID NOs. 17 and 18, and SEQ ID NOs. 19 and 20. PfuUltra polymerase was used. TM Using high-fidelity DNA polymerase (Stratagene), the PCR conditions were denaturation at 96°C for 30 seconds, annealing at 53°C for 30 seconds, and polymerization at 72°C for 1 minute, repeated for 30 cycles. As a result, an amplified gene of 548 bp containing the N-terminus and linker of the metY gene and an amplified gene of 550 bp containing the C-terminus and linker of the metY gene were obtained. PCR was performed using the two amplified genes obtained as described above as templates. The PCR conditions were denaturation at 96°C for 60 seconds, annealing at 50°C for 60 seconds, and polymerization at 72°C for 1 minute, repeated for 10 cycles, after which sequence numbers 17 and 20 were added and the polymerization reaction was repeated for another 20 cycles. As a result, an inactivated cassette of 1077 bp containing the N-terminus, linker, and C-terminus of the metY gene was obtained.

[0166] The pDCM2 vector (SEQ ID NO: 7, Patent Document 8) was treated with SmaI, and the PCR product (1077 bp) obtained as described above was fusion cloned with the pDCM2 vector treated with SmaI restriction enzyme using the In-Fusion® HD Cloning Kit (Clontech). The cloned vector was transformed into E. coli DH5α, and the transformed E. coli were streaked onto LB solid medium containing 25 mg / l kanamycin. Colonies transformed with the plasmid were selected from the LB medium, and the plasmid was then obtained by plasmid extraction (Patent Document 9), and finally, a pDCM2-ΔmetY recombinant vector with a cloned meTY gene deletion cassette was constructed.

[0167] The prepared pDCM2-ΔmetY vector was transformed into the strain KCCM12634PΔmetB prepared in Example 3-1 using the electropulse method. After a secondary crossover process, KCCM12634PΔmetBΔmetY was obtained in which the metY gene was further inactivated on the chromosome. The inactivated metY gene was subjected to PCR using primers SEQ ID NOs. 17 and 20, and then finally confirmed by comparison with ATCC13032, in which the metY gene was not inactivated.

[0168] Example 3-3. Strain Preparation - 3 A mutation (L377K) (Patent Document 6) was introduced into the lysC gene (SEQ ID NO: 21), which encodes aspart kinase derived from Corynebacterium glutamicum ATCC13032, to enhance lysC gene expression and remove feedback inhibition to L-lysine and L-threonine. To construct a vector containing the mutant lysC gene, a pair of primers (SEQ ID NOs: 22 and 23) for amplifying the upstream 5' end and a pair of primers (SEQ ID NOs: 24 and 25) for amplifying the downstream 3' end were designed, centered around the mutation site. The primer sequences are shown in Table 7.

[0169] [Table 7]

[0170] PCR was performed using the chromosome of ATCC13032 as a template, with primers SEQ ID NOs. 22 and 23, and SEQ ID NOs. 24 and 25. The PCR conditions were denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 30 seconds, repeated 30 cycles, and then polymerization at 72°C for 7 minutes. As a result, mainly mutations in the lysC gene were obtained, yielding a 512 bp DNA fragment upstream of the 5' end and a 522 bp DNA fragment downstream of the 3' end.

[0171] PCR was performed using the two amplified DNA fragments as templates with primers SEQ ID NOs. 22 and 25. The PCR conditions were denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 60 seconds, for 30 cycles, and then polymerization at 72°C for 7 minutes. As a result, a 1011 bp DNA fragment containing the mutant lysC(L377K) gene encoding an aspart kinase variant in which leucine at position 377 is replaced with lysine was amplified.

[0172] The pDCM2 vector (SEQ ID NO: 7, Patent Document 8) was treated with SmaI, and the PCR product (1011 bp) obtained as described above was fusion cloned with the pDCM2 vector treated with SmaI restriction enzyme using the In-Fusion® HD Cloning Kit (Clontech). The cloned vector was transformed into E. coli DH5α, and the transformed E. coli were streaked onto LB solid medium containing 25 mg / l kanamycin. Colonies transformed with the plasmid were selected from the LB medium, and the plasmid was then obtained by plasmid extraction (Patent Document 9). Finally, a pDCM2-lysC(L377K) recombinant vector was prepared, in which a cassette with the lysC(L377K) gene substituted was cloned.

[0173] The prepared pDCM2-lysC(L377K) vector was transformed into the strain KCCM12634PΔmetBΔmetY prepared in Example 3-2 using the electropulse method. After a secondary crossover, Corynebacterium glutamicum KCCM12634PΔmetBΔmetY lysC(L377K) was obtained in which a nucleotide mutation was introduced into the lysC gene on the chromosome. The gene into which the nucleotide mutation was introduced was subjected to PCR using primers SEQ ID NOs. 22 and 25, and then finally confirmed by sequencing by comparing it with the sequence of the wild-type lysC gene.

[0174] Example 3-4. Preparation of a bacterial strain lacking the endogenous gene NCgl0959 - 1 To maximize O-acetylhomoserine production, the pDCM2-ΔNCgl0959 vector prepared in Example 1-1 was transformed into the strain KCCM12634PΔmetBΔmetY lysC(L377K) prepared in Example 3-3 using electropulse technology. After a secondary crossover process, KCCM12634PΔmetBΔmetY lysC(L377K)ΔNCgl0959 was obtained, which had a deletion of the NCgl0959 gene on the chromosome. Whether or not the NCgl0959 gene was inactivated was confirmed by PCR using primers of sequence numbers 8 and 9, and then by comparison with ATCC13032, in which the NCgl0959 gene was not inactivated.

[0175] Example 3-5. Preparation of bacterial strains lacking the endogenous gene NCgl0959 - 2 To amplify the gene encoding homoserine acetyltransferase (MetX), the nucleotide sequence information of the MetX gene (NCBI registration number NCgl0624, SEQ ID NO: 26) was obtained from the National Institutes of Health (NIH) GenBank. Based on this, primer pairs (SEQ ID NOs: 27 and 28) were designed by inserting BamHI restriction enzyme sites at both ends to amplify the region from the promoter site (approximately 300 bp upstream of the start codon) to the terminator site (approximately 100 bp downstream of the stop codon). The PCR conditions consisted of denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 90 seconds, repeated 30 cycles, followed by polymerization at 72°C for 7 minutes. As a result, a 1546 bp DNA fragment of the MetX gene encoding region was obtained. The pECCG117 vector (Patent Document 10) and a MetX DNA fragment were treated with the restriction enzyme BamHI, ligated using a DNA ligation enzyme, and then cloned to obtain a plasmid. This plasmid was named pECCG117-MetX WT. The primer sequences are shown in Table 8.

[0176] [Table 8]

[0177] The prepared pECCG117-MetX WT vector was introduced into the bacterial strain KCCM12634PΔmetBΔmetY lysC(L377K) prepared in Example 3-3 and the bacterial strain KCCM12634PΔmetBΔmetY lysC(L377K)ΔNCgl0959 prepared in Example 3-4, respectively, using the electropulse method. These were then spread onto a selective medium containing 25 mg / l of kanamycin to obtain the transformed strains KCCM12634PΔmetBΔmetY lysC(L377K) / pECCG117-MetX WT and KCCM12634PΔmetBΔmetY lysC(L377K)ΔNCgl0959 / pECCG117-MetX WT, respectively.

[0178] Example 3-6. Evaluation of O-acetylhomoserine production ability In order to compare the O-acetylhomoserine production abilities of the strains prepared in Examples 3-3 to 3-5, they were cultured by the following method, and O-acetylhomoserine in the culture broth was analyzed.

[0179] One platinum loop of the strain was inoculated into a 250 ml corner baffle flask containing 25 ml of the following medium, and shake-cultured at 33 °C and 200 rpm for 20 hours. The concentration of O-acetylhomoserine was analyzed using HPLC. The analyzed concentrations are shown in Table 9. <O-acetylhomoserine production medium (pH 7.2)> Glucose 30 g, KH2PO4 2 g, urea 3 g, (NH4)2SO4 40 g, peptone 2.5 g, CSL (Sigma) 5 g (10 ml), MgSO4·7H2O 0.5 g, methionine 400 mg, CaCO3 20 g (in 1 liter of distilled water)

[0180]

Table 9

[0181] As a result, as shown in Table 9, when the control group strain KCCM12634PΔmetBΔmetY lysC (L377K) was cultured, 1.3 g / L of O-acetylhomoserine accumulated, and when KCCM12634PΔmetBΔmetY lysC (L377K)ΔNCgl0959 was cultured, 1.69 g / L of O-acetylhomoserine accumulated, and it was confirmed that the production ability was 130% compared to the control group.

[0182] Furthermore, when the KCCM12634PΔmetBΔmetY lysC(L377K) / pECCG117-MetX WT strain was cultured, 2.10 g / L of O-acetylhomoserine accumulated. When the KCCM12634PΔmetBΔmetY lysC(L377K)ΔNCgl0959 / pECCG117-MetX WT strain was cultured, 2.73 g / L of O-acetylhomoserine accumulated. It was confirmed that this strain exhibits 130% of the production capacity of the KCCM12634PΔmetBΔmetY lysC(L377K) / pECCG117-MetX WT strain.

[0183] Therefore, it was confirmed that in bacterial strains with improved O-acetyl-L homoserine production capacity, deletion and inactivation of the endogenous gene NCgl0959 further improved the production capacity of the target amino acid.

[0184] From the above explanation, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. It should be understood that the above embodiments are merely illustrative and not limiting. This application should be interpreted as including all modified or altered forms derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. A Corynebacterium microorganism having O-acetylhomoserine production ability, wherein the GNAT family N-acetyltransferase protein activity is weakened, and the Corynebacterium microorganism comprises an amino acid sequence having at least 90% identity with the amino acid sequence of Sequence ID No.

1.

2. The Corynebacterium microorganism according to claim 1, wherein the Corynebacterium microorganism is Corynebacterium glutamicum.

3. The Corynebacterium microorganism according to claim 1, wherein the GNAT family N-acetyltransferase is a protein consisting of the amino acid sequence of SEQ ID NO:

1.

4. The weakening is inactivation, as described in claim 1, for a microorganism of the genus Corynebacterium.

5. The Corynebacterium microorganism according to claim 1, wherein the microorganism is one in which the nucleic acid base sequence of Sequence ID No. 2 is deleted.

6. The Corynebacterium microorganism according to claim 1, wherein the microorganism further has enhanced L-methionine / branched-chain amino acid exporter YjeH protein activity.

7. The Corynebacterium microorganism according to claim 6, wherein the microorganism is one into which the amino acid sequence of Sequence ID No. 10 has been introduced.

8. A method for producing O-acetylhomoserine, comprising the step of culturing a Corynebacterium microorganism having O-acetylhomoserine production ability and weakened GNAT family N-acetyltransferase protein activity in a culture medium, wherein the GNAT family N-acetyltransferase protein comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

1.

9. The steps include culturing a Corynebacterium microorganism with weakened GNAT family N-acetyltransferase protein activity and O-acetylhomoserine production ability in a culture medium, The steps include producing O-acetylhomoserine from the cultured microorganism or culture medium, A method for producing L-methionine, comprising the step of converting O-acetylhomoserine to L-methionine, wherein the GNAT family N-acetyltransferase protein comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1.

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