O-acetylhomoserine producing microorganism and method for producing O-acetylhomoserine or L-methionine using the same

Abstract

The present application relates to a microorganism having weakened SIRT-type deacetylase 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] The present application relates to a microorganism having weakened SIRT-type deacetylase 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. [Background technology]

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

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

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

[0005] [Patent Document 1] International Publication No. 2008 / 013432 [Patent Document 2] U.S. Patent No. 7,662,943 [Patent Document 3] U.S. Pat. No. 1,058,438 [Patent Document 4] U.S. Patent No. 10273491 [Patent Document 5] Korea Registration Patent No. 10-2182497 [Patent Document 6] Korean Patent Publication No. 10-2019-0003019 [Patent Document 7] Korean Patent Registration No. 10-0905381 [Patent Document 8] Korean Patent Publication No. 10-2020-0136813 [Patent Document 9] U.S. Patent No. 5981235 [Patent Document 10] Korea Registration Patent No. 10-0057684

Non-licensed documents

[0006]

Non-patent document 1

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Non-licensed literature 9

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Non-licensed Document 11

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Non-licensed Document 15

[0007] The present inventors have discovered an O-acetylhomoserine-producing Corynebacterium microorganism and a method for producing O-acetylhomoserine or L-methionine using the same, and have completed the present application. [Means for solving the problem]

[0008] An object of the present application is to provide a Corynebacterium microorganism having an ability to produce O-acetylhomoserine and attenuated SIRT-type deacetylase protein activity. Another object of the present application is to provide a method for producing O-acetylhomoserine, which comprises a step of culturing the microorganism in a medium.

[0009] Furthermore, the present application aims to provide a method for producing L-methionine, comprising the step of culturing the microorganism in a medium. A further object of the present application is to provide a composition for producing O-acetylhomoserine, comprising the microorganism.

[0010] Furthermore, the present application aims to provide use of the microorganism of the present application for producing O-acetylhomoserine or L-methionine. Effect of the Invention

[0011] The microorganism capable of producing O-acetylhomoserine of the present application can produce O-acetylhomoserine in an environmentally friendly and highly efficient manner compared to chemical synthesis. In addition, when the produced O-acetylhomoserine is used as a precursor for the synthesis of methionine and acetic acid by O-acetylhomoserine sulfhydrylase, it can bioconvert L-methionine with high efficiency, and the converted L-methionine can be widely used not only for animal feed and animal feed additives but also for the production of human food and food additives. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] These will be described in detail below. Note that each description and embodiment disclosed in this application also applies to other descriptions and embodiments. In other words, all combinations of various elements disclosed in this application are included in this application. In addition, this application is not limited to the following specific description.

[0013] Furthermore, throughout this specification, many papers and patent documents are referenced and cited, and the disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety in order to more clearly explain the state of the art to which this application pertains and the contents of this application.

[0014] One aspect of the present application provides a Corynebacterium microorganism having an ability to produce O-acetylhomoserine and attenuated SIRT-type deacetylase protein activity. In the present application, the term "SIRT-type deacetylase" refers to a sirtuin (SIRT)-type deacetylase. Specifically, the SIRT-type deacetylase is known in the art, and the protein and gene sequence of the SIRT-type deacetylase can be obtained from a known database, for example, GenBank of NCBI, but is not limited thereto. More specifically, the SIRT-type deacetylase may have an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, may include the amino acid sequence, may be essentially composed of the amino acid sequence, or may consist of the amino acid sequence.

[0015] For example, a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 means a protein that is endogenously present in Corynebacterium microorganisms and is encoded by the publicly known NCgl0616 gene or NCgl0078 gene, respectively, but is not limited to these. Specifically, it means a SIRT-type deacetylase consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 that is endogenously present in Corynebacterium microorganisms, more specifically, it means two types of SIRT-type deacetylase homologs of Corynebacterium glutamicum ATCC13032 that are encoded by the NCgl0616 gene or NCgl0078 gene, respectively (Non-Patent Document 1), but is not limited to these.

[0016] Furthermore, the SIRT-type deacetylase of the present application may include not only the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, 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: 1 or SEQ ID NO: 2. Furthermore, it goes without saying that the present application also includes an amino acid sequence having such homology or identity and having the same or corresponding biological activity as the SIRT-type deacetylase of the present application, in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added.

[0017] Even if the present application describes "a polypeptide or protein comprising an amino acid sequence represented by a specific SEQ ID NO," "a polypeptide or protein consisting of an amino acid sequence represented by a specific SEQ ID NO," or "a polypeptide or protein having an amino acid sequence represented by a specific SEQ ID NO," it goes without saying that any protein having an amino acid sequence in which a part of the sequence has been deleted, modified, substituted, conservatively substituted, or added is also used in the present application, so long as it has the same or corresponding activity as the polypeptide consisting of the amino acid sequence of the SEQ ID NO. For example, there are proteins having an addition of a sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation, or a conservative substitution at the N-terminus and / or C-terminus of the amino acid sequence.

[0018] For example, the amino acid sequence may have an addition or deletion of a sequence that does not change the function of the SIRT-type deacetylase of the present application, a naturally occurring mutation, a silent mutation, or a conservative substitution at the N-terminus, C-terminus, and / or internally.

[0019] In the present application, "conservative substitution" refers to the replacement of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally occur based on similarity in 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. Amino acids are further classified into those with electrically charged side chains and those with uncharged side chains, with the charged side chains including aspartic acid, glutamic acid, lysine, arginine, and histidine, and the uncharged side chains including nonpolar and polar amino acids, with the nonpolar amino acids including glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, and the polar amino acids including serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Typically, conservative substitutions have little or no effect on the activity of the resulting polypeptide.

[0020] The SIRT-type deacetylase may also contain deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide may be linked to an N-terminal signal (or leader) sequence of a protein involved in co-translationally or post-translationally protein transfer. The polypeptide may also be linked to other sequences or linkers that allow the polypeptide to be identified, purified, or synthesized.

[0021] In this application, "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, expressed as a percentage. Homology and identity are often used interchangeably.

[0022] Sequence homology or identity of conserved polynucleotides or polypeptides may be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences will generally hybridize to the entire sequence or to a portion thereof under moderate or high stringent conditions. Hybridization, of course, also includes hybridization to polynucleotides having common codons or codons that take into account the degeneracy of codons in the polynucleotide.

[0023] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program with default parameters as in, for example, Non-Patent Document 2. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 4) as implemented in the Needle program (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 3) (version 5.0.0 or later) of the EMBOSS package (including the GCG program package (Non-Patent Document 5), BLASTP, BLASTN, FASTA (Non-Patent Documents 6, 7 and 8)). For example, BLAST or Clustal W from the National Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0024] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as that disclosed in, for example, J. Biol. 1999, 143:1311-1354 (2001). Briefly, the GAP program defines 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 (identity takes a value of 1 and non-identity takes a value of 0) and a weighted comparison matrix (or EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in, (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 extension penalty of 0.5), and (3) no penalty for terminal gaps.

[0025] "Corresponding to" in this application means the amino acid residue at the recited position in the polypeptide, or an amino acid residue similar, identical or equivalent to the recited residue in the polypeptide. Identifying the amino acid at the corresponding position will determine the particular amino acid of the sequence to which the particular sequence is referenced. "Corresponding region" in this application generally refers to a similar or corresponding position in a related or reference protein.

[0026] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, and based thereon, each amino acid residue of the amino acid sequence can be numbered with reference to the number and position of the amino acid residues corresponding to the amino acid residues in SEQ ID NO: 1. For example, the sequence alignment algorithm in the present application can identify the positions of amino acids or positions where modifications such as substitutions, insertions, deletions, etc. occur when compared to a query sequence (also referred to as a "reference sequence").

[0027] For such alignment, for example, the Needleman-Wunsch algorithm (Non-Patent Document 4) or the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 3) can be used, but is not limited to these. Sequence alignment programs and pairwise sequence comparison algorithms known in the technical field can be used as appropriate.

[0028] In the present application, "O-acetylhomoserine" refers to a specific intermediate substance in the methionine biosynthesis pathway of microorganisms, and refers to an acetyl derivative of L-homoserine. The O-acetylhomoserine is produced by the enzyme activity of homoserine and acetyl-CoA as substrates, which transfers the acetyl group of acetyl-CoA to homoserine.

[0029] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have been genetically modified, either naturally or artificially, and is a microorganism in which a specific mechanism has been weakened or enhanced by, for example, inserting an exogenous gene or enhancing or inactivating the activity of an endogenous gene, and is a microorganism that has been genetically modified to produce a desired polypeptide, protein, or product.

[0030] In the present application, the term "microorganism capable of producing O-acetylhomoserine" refers to a prokaryotic or eukaryotic microbial strain that produces O-acetylhomoserine in the organism, and includes microorganisms in which the ability to produce O-acetylhomoserine has been imparted to a parent strain that does not produce O-acetylhomoserine, and microorganisms that have the ability to produce O-acetylhomoserine endogenously. The ability to produce O-acetylhomoserine can be imparted or improved by breeding. The microorganism capable of producing O-acetylhomoserine is a strain that produces L-lysine, L-threonine, L-isoleucine, or L-methionine, or one derived therefrom, but is not limited thereto.

[0031] For the purpose of this application, the O-acetylhomoserine-producing microorganism is characterized in that the activity of the SIRT-type deacetylase protein is weakened as compared to the endogenous activity, and the target O-acetylhomoserine-producing ability is improved, and is a genetically modified microorganism or a recombinant microorganism, but is not limited thereto. Specifically, the recombinant strain with improved O-acetylhomoserine-producing ability is a microorganism with improved O-acetylhomoserine-producing ability compared to a natural wild-type microorganism or an unmodified microorganism having the endogenous activity of the SIRT-type deacetylase protein, but is not limited thereto.

[0032] For example, the microorganism that produces O-acetylhomoserine may be a microorganism that endogenously contains a protein consisting of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, 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 to SEQ ID NO:1 or SEQ ID NO:2.

[0033] For example, the microorganism that produces O-acetylhomoserine may be a microorganism that endogenously contains a polynucleotide sequence encoding a protein comprising an amino acid sequence having at least 80% homology to SEQ ID NO:1 or SEQ ID NO:2, a nucleotide sequence of SEQ ID NO:29 or SEQ ID NO:30, or a nucleotide 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 to the sequence of SEQ ID NO:29 or SEQ ID NO:30.

[0034] As an example, the microorganism with improved production ability is improved by about 1% or more, specifically about 1% or more, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 21% or more, about 22% or more, about 23% or more, about 24% or more, about 25% or more, about 26% or more, about 27% or more, about 28% or more, about 29% or more, about 30% or more, about 31% or more, about 32% or more, about 33% or more, about 34% or more, or about 35% or more (there is no particular limit to the upper limit, for example, about 100% or less, about 50% or less, about 45% or less, or about 40% or less), compared to the O-acetylhomoserine production ability of the parent strain before mutation or an unmodified microorganism having the endogenous activity of a SIRT-type deacetylase protein. However, any microorganism that shows an increase in the + value compared to the production ability of the parent strain or unmodified microorganism before mutation may be used. In other examples, the recombinant strain with improved production ability has an O-acetylhomoserine production ability improved by about 1.1 times or more, about 1.2 times or more, about 1.21 times or more, about 1.22 times or more, about 1.23 times or more, about 1.24 times or more, about 1.25 times or more, about 1.26 times or more, about 1.27 times or more, about 1.28 times or more, about 1.29 times or more, about 1.30 times or more, about 1.31 times or more, about 1.32 times or more, about 1.33 times or more, about 1.34 times or more, or about 1.35 times or more (there is no particular limit to the upper limit, and for example, about 10 times or less, about 5 times or less, about 3 times or less, about 2 times or less, about 1.5 times or less, or about 1.4 times or less) compared to the parent strain or unmodified microorganism before the mutation, but is not limited thereto.

[0035] In the present application, the term "unmodified microorganism" does not exclude strains containing naturally occurring mutations in microorganisms, but refers to a wild-type strain or a natural strain itself, or a strain before its characteristics are changed by genetic mutation due to natural or artificial factors. For example, the unmodified microorganism refers to a strain in which the SIRT-type deacetylase protein activity described herein is not weakened compared to the endogenous activity, or before it is weakened. The term "unmodified microorganism" is used interchangeably with "strain before modification", "microorganism before modification", "non-mutated strain", "non-modified strain", "non-mutated microorganism", or "reference microorganism".

[0036] The microorganism capable of producing O-acetylhomoserine may be either a prokaryotic cell or a eukaryotic cell, specifically a prokaryotic cell. Examples of the prokaryotic cell include microbial strains belonging to the genera Escherichia, Erwinia, Serratia, Providencia, Corynebacteria, Pseudomonas, Leptospira, Salmonella, Brevibacterium, Hyphomonas, Chromobacterium, and Norcardia, or fungi or yeast. Specifically, the microorganisms include Escherichia, Corynebacterium, and Leptospira strains, and yeasts, and more specifically, Corynebacterium strains.

[0037] In the present application, the term "microorganism of the genus Corynebacterium" includes any microorganism 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, ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens, and more specifically Corynebacterium glutamicum.

[0038] The unmodified microorganism may be a microorganism comprising the amino acid sequence consisting of SEQ ID NO:1 or SEQ ID NO:2, or the polynucleotide consisting of SEQ ID NO:29 or SEQ ID NO:30. On the other hand, it is already known that microorganisms of the genus Corynebacterium produce O-acetylhomoserine, but the production ability is very low, and the genes acting on the production mechanism and the mechanism principle have not been elucidated. Therefore, the O-acetylhomoserine-producing microorganisms of the genus Corynebacterium of the present application include all of the following: natural wild-type microorganisms themselves, Corynebacterium microorganisms whose O-acetylhomoserine-producing ability has been improved by strengthening or weakening the activity of genes related to the O-acetylhomoserine production mechanism, and Corynebacterium microorganisms whose O-acetylhomoserine-producing ability has been improved by introducing or strengthening the activity of an exogenous gene.

[0039] In the present application, the term "attenuation" of a polypeptide activity is a concept that includes all of the following: a decrease in activity compared to the endogenous activity, or the absence of activity. The term "attenuation" is also used interchangeably with other terms such as inactivation, deficiency, down-regulation, decrease, reduction, and attenuation.

[0040] Specifically, the weakening is, but is not limited to, inactivation, which means that the protein is not expressed at all, or is expressed but has no or reduced activity, compared to a parent strain or a strain in which the protein consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is not modified.

[0041] The weakening includes at least one of the following: the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide originally possessed by the microorganism due to, for example, a mutation in the polynucleotide encoding the polypeptide; the overall level and / or concentration (expression amount) of polypeptide activity in the cell is reduced compared to that of a natural strain due to, for example, inhibition of expression of the gene of the polynucleotide encoding it or inhibition of translation into the polypeptide; no expression of the polynucleotide at all; and no polypeptide activity even if the polynucleotide is expressed.

[0042] The "endogenous activity" refers to the activity of a specific polypeptide that a parent strain, wild-type, or unmodified microorganism originally had before the trait change when the trait is changed due to genetic mutation caused by natural or artificial factors. This is used interchangeably with "activity before modification." When the activity of a polypeptide is "inactivated," "lacking," "reduced," "down-regulated," "decreased," or "attenuated" compared to the endogenous activity, it means that the activity of the specific polypeptide is decreased compared to the activity of the specific polypeptide that a parent strain or unmodified microorganism originally had before the trait change.

[0043] Such attenuation of the activity of a polypeptide can be achieved by applying various methods well known in the art, including, but not limited to, those described in Non-Patent Documents 12 and 13.

[0044] Specifically, the attenuation of a polypeptide activity in the present application can be achieved by 1) deleting all or a part of a gene encoding the polypeptide, 2) modifying an expression regulatory region (or an expression regulatory sequence) so as to reduce the expression of a gene encoding the polypeptide, 3) modifying an amino acid sequence constituting the polypeptide so as to delete or weaken the activity of the polypeptide (e.g., deleting / substituting / adding one or more amino acids in the amino acid sequence), or 4) modifying a gene sequence encoding the polypeptide so as to delete or weaken the activity of the polypeptide (e.g., modifying the gene sequence encoding the polypeptide so as to encode a polypeptide modified so as to delete or weaken the activity of the polypeptide). a nucleic acid sequence encoding a polypeptide; 5) modifying the base sequence encoding the start codon or 5'UTR region of the gene transcript encoding the polypeptide; 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcript encoding the polypeptide; 7) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding the polypeptide so that a secondary structure that makes ribosome attachment impossible is formed; 8) adding a promoter to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide so that it is reverse transcribed (Reverse Transcription Engineering, RTE); or 9) a combination of two or more selected from 1) to 8) above, but is not particularly limited thereto.

[0045] For example, 1) deleting a part or the whole of the gene encoding the polypeptide may be carried out by deleting the entire polynucleotide encoding the endogenous target polypeptide in a chromosome, or by replacing it with a polynucleotide lacking some nucleotides or a marker gene.

[0046] In addition, the expression regulatory region (or expression regulatory sequence) may be modified by generating a mutation in the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by substituting a sequence having a weaker activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0047] Furthermore, the modification of the amino acid sequence or polynucleotide sequence of 3) and 4) above can be performed by, but is not limited to, generating a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide so as to weaken the activity of the polypeptide, or by replacing the sequence with an amino acid sequence or polynucleotide sequence improved to have a lower activity, or an amino acid sequence or polynucleotide sequence improved to have no activity. For example, gene expression can be inhibited or weakened by, but is not limited to, introducing a mutation into a polynucleotide sequence to form a stop codon.

[0048] Furthermore, the nucleotide sequence encoding the start codon or 5'UTR region of the gene transcription product encoding the polypeptide can be modified, for example, by substituting it with a nucleotide sequence encoding another start codon that has a lower expression rate of the polypeptide compared to the endogenous start codon, but is not limited to this.

[0049] Regarding 6) the introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcription product encoding the polypeptide, Non-Patent Document 14 can be referred to, for example.

[0050] 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide so that a secondary structure is formed that makes ribosome attachment impossible may be achieved by making mRNA translation impossible or slowing down the rate.

[0051] 8) Adding a promoter to the 3' end of the ORF (open reading frame) of a gene sequence encoding a polypeptide so as to reverse transcribe the promoter (Reverse transcription engineering, RTE) may be carried out by creating an antisense nucleotide complementary to the gene transcript encoding the polypeptide to attenuate the activity.

[0052] Specifically, the microorganism capable of producing O-acetylhomoserine of the present application is one in which SIRT deacetylase protein activity has been inactivated, but is not limited thereto.

[0053] Specifically, in order to inactivate the SIRT-type deacetylase protein activity, a method of deleting a part or all of the gene encoding the protein may be used. More specifically, the method may be performed by replacing a polynucleotide encoding an endogenous target protein in a chromosome with a polynucleotide lacking a part of the nucleotide sequence or a marker gene using a vector for chromosomal introduction into a microorganism. One example of a method of deleting a part or all of such a polynucleotide is a method of deleting a polynucleotide by homologous recombination, but is not limited thereto. As another example, the method of deleting a part or all of the gene may be performed by inducing a mutation using light such as ultraviolet light or a chemical substance, and selecting a strain in which the target gene is deleted from the obtained mutants.

[0054] The gene deletion method includes a method using genetic recombination techniques. For example, the gene deletion may be performed by introducing a polynucleotide sequence or vector containing a polynucleotide sequence having homology to a target gene into the microorganism to cause homologous recombination. The introduced polynucleotide sequence or vector contains a dominant selection marker. However, the gene deletion method is not limited to this.

[0055] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and means a DNA or RNA chain longer than a certain length, and more specifically, means a polynucleotide fragment encoding the mutant.

[0056] In the present application, the SIRT-type deacetylase is encoded by a gene containing the polynucleotide sequence of SEQ ID NO: 29 or SEQ ID NO: 30, but is not limited thereto. More specifically, the SIRT-type deacetylase is encoded by a gene having the polynucleotide sequence of SEQ ID NO: 29 or SEQ ID NO: 30, 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 base sequence of SEQ ID NO: 29 or SEQ ID NO: 30 can be obtained from a known database, for example, from NCBI's GenBank, but is not limited thereto.

[0057] In this application, a gene comprising the nucleotide sequence of SEQ ID NO:29 or SEQ ID NO:30 is used interchangeably with a polynucleotide comprising the nucleotide sequence of SEQ ID NO:29 or SEQ ID NO:30, a gene or polynucleotide having the nucleotide sequence of SEQ ID NO:29 or SEQ ID NO:30, or a gene or polynucleotide consisting of the nucleotide sequence of SEQ ID NO:29 or SEQ ID NO:30.

[0058] The polynucleotides of the present application may be modified in various ways in the coding regions to accommodate codon degeneracy or preferred codons in the organism in which the variant of the present application is expressed, without altering the amino acid sequence of the variant of the present application. Specifically, the polynucleotide of the present application is included in the present application as long as it is a polynucleotide sequence encoding a protein comprising an amino acid sequence having at least 80% homology to SEQ ID NO:1 or SEQ ID NO:2, and includes the nucleotide sequence of SEQ ID NO:29 or SEQ ID NO:30, or a nucleotide 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 to the sequence of SEQ ID NO:29 or SEQ ID NO:30, or includes said nucleotide sequence, or consists of the nucleotide sequence of SEQ ID NO:29 or SEQ ID NO:30, or a nucleotide 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 to the sequence of SEQ ID NO:29 or SEQ ID NO:30, or is substantially composed of said nucleotide sequence, but is not limited thereto.

[0059] Furthermore, the polynucleotide of the present application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a complementary sequence to all or a part of the polynucleotide sequence of the present application. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Documents 15 and 16). For example, the conditions include conditions under which polynucleotides having high homology or identity, such as polynucleotides having a homology or identity 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 99% or more, hybridize with each other, but polynucleotides having lower homology or identity than that do not hybridize with each other, or conditions under which washing is performed once, specifically two to three times, at a salt concentration and temperature equivalent to normal Southern hybridization washing conditions of 60°C, 1 x SSC, 0.1% SDS, specifically 60°C, 0.1 x SSC, 0.1% SDS, more specifically 68°C, 0.1 x SSC, 0.1% SDS.

[0060] Hybridization requires that two nucleic acids have complementary sequences, even though mismatches between bases are possible depending on the stringency of 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. Thus, the polynucleotides of the present application may include isolated nucleic acid fragments that are complementary to the entire sequence, as well as substantially similar nucleic acid sequences.

[0061] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the hybridization conditions described above, in which the hybridization step is performed at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and may be appropriately adjusted by those skilled in the art depending on the purpose.

[0062] The appropriate stringency for hybridizing the polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables known in the art (eg, Non-Patent Document 15).

[0063] Specifically, the microorganism capable of producing O-acetylhomoserine of the present application is a microorganism in which SIRT type deacetylase protein activity has been inactivated, but is not limited thereto.

[0064] Specifically, the microorganism capable of producing O-acetylhomoserine of the present application is a microorganism lacking a polynucleotide encoding a SIRT-type deacetylase, but is not limited thereto. More specifically, the microorganism capable of producing O-acetylhomoserine of the present application is a microorganism lacking the nucleic acid base sequence of SEQ ID NO: 29 or SEQ ID NO: 30, but is not limited thereto.

[0065] For the purposes of this application, the microorganism comprises an expression vector for inactivating a SIRT deacetylase protein in a host cell, thereby inactivating the activity of the SIRT deacetylase protein, but is not limited thereto.

[0066] The vector of the present application includes a DNA product comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host. The expression control region includes a promoter for initiating transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence 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.

[0067] The vector used in the present application is 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 a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. may be used as phage or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pSK, pSKH, pET, etc. may be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pSK, pSKH130, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. may be used.

[0068] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome by a vector for intracellular chromosome introduction. The polynucleotide can be inserted into a chromosome by any method known in the art, such as, but not limited to, homologous recombination. A selection marker for confirming whether or not it has been inserted into the chromosome may be further included. The selection marker is for selecting cells transformed with the vector, i.e., for confirming whether or not the target nucleic acid molecule has been inserted, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide is used. In an environment treated with a selection agent, only cells expressing the selection marker survive or show a different phenotype, so that transformed cells can be selected.

[0069] In the present application, "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or a microorganism, thereby expressing the polypeptide encoded by the polynucleotide in the host cell. The transformed polynucleotide may be any polynucleotide that is expressed in the host cell, regardless of whether it is inserted into the chromosome of the host cell or located extrachromosomally. The polynucleotide may include DNA and / or RNA that encodes the target polypeptide. The polynucleotide may be introduced in any form as long as it is introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic structure containing all elements necessary for its own expression. Typically, the expression cassette contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. The polynucleotide may be introduced into the host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0070] Additionally, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target variant of the present application.

[0071] In the microorganism of the present application, modification of a part or the whole of a polynucleotide can be induced by (a) homologous recombination using a vector for chromosomal introduction in the microorganism, or genome editing using engineered nuclease (e.g., CRISPR-Cas9), and / or (b) light such as ultraviolet light or radiation and / or chemical treatment, but is not limited thereto. Methods for modifying a part or the whole of the gene include methods using DNA recombination techniques. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene is introduced into the microorganism to cause homologous recombination, thereby deleting a part or the whole of the gene. The introduced nucleotide sequence or vector contains a dominant selection marker, but is not limited thereto.

[0072] In the present application, "enhancement" of a polypeptide activity means improving the activity of the polypeptide compared to its endogenous activity. The enhancement is used interchangeably with activation, up-regulation, overexpression, increase, etc. Here, activation, enhancement, up-regulation, overexpression, and increase all include the display of an activity that was not originally present, and the improvement of activity compared to the endogenous activity or activity before modification. The "endogenous activity" means the activity of a specific polypeptide originally possessed by a parent strain or an unmodified microorganism before the trait change when the trait is changed due to genetic mutation caused by natural or artificial factors. This is used interchangeably with "activity before modification." The "enhancement," "up-regulation," "overexpression," or "improvement" of a polypeptide activity compared to its endogenous activity means the improvement of the activity and / or concentration (expression level) of a specific polypeptide originally possessed by a parent strain or an unmodified microorganism before the trait change.

[0073] The enhancement may be achieved by introducing a foreign polypeptide, or by enhancing the activity and / or increasing the concentration (expression level) of an endogenous polypeptide. Whether the activity of the polypeptide has been enhanced can be confirmed by an increase in the level of activity of the polypeptide, the expression level, or the amount of a product produced from the polypeptide.

[0074] To enhance the activity of the polypeptide, various methods well known in the art can be applied, and any method can be used as long as it can enhance the activity of the target polypeptide compared to the unmodified microorganism. Specifically, the method is a conventional method in molecular biology that uses genetic engineering and / or protein engineering well known to those skilled in the art, but is not limited to these (e.g., Non-Patent Documents 13, 17, etc.).

[0075] Specifically, the enhancement of the polypeptide of the present application is carried out by 1) increasing the intracellular copy number of a polynucleotide encoding the polypeptide, 2) modifying the expression regulatory region of a gene on a chromosome encoding the polypeptide (e.g., generating a mutation in the expression regulatory region, substituting a sequence having a higher activity, or inserting a sequence having a higher activity), 3) modifying the base sequence encoding the start codon or 5'UTR region of a gene transcript encoding the polypeptide, 4) modifying the amino acid sequence of the polypeptide so as to enhance the polypeptide activity, 5) modifying the polynucleotide sequence encoding the polypeptide so as to enhance the polypeptide activity (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the polypeptide activity), 6) introducing a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same, 7) optimizing the codons of a polynucleotide encoding the polypeptide, 8) analyzing the tertiary structure of the polypeptide and selecting and modifying or chemically modifying an exposed portion, or 9) a combination of two or more selected from 1) to 8) above, but is not particularly limited thereto.

[0076] More specifically, the above 1) increasing the intracellular copy number of the polynucleotide encoding the polypeptide may be achieved by introducing into the host cell a vector to which the polynucleotide encoding the polypeptide is operably linked, the vector replicating and functioning independently of the host. Alternatively, the above may be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide into a chromosome in the host cell. The introduction into the chromosome is achieved by introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome in the host cell, but is not limited thereto. The vector is as described above.

[0077] The expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide is replaced with a sequence having a stronger activity, for example, by generating a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so that the activity of the expression regulatory region is further enhanced, or by replacing the expression regulatory region with a sequence having a higher activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating the termination of transcription and translation, and the like. For example, the expression regulatory region is replaced with a strong promoter, but is not limited to this.

[0078] Examples of known strong promoters include, but are not limited to, the CJ1 to 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.

[0079] The nucleotide sequence encoding the start codon or 5'UTR region of the gene transcription product encoding the polypeptide (3) can be modified, for example, by substituting it with a nucleotide sequence encoding another start codon that has a higher expression rate of the polypeptide compared to the endogenous start codon, but is not limited to this.

[0080] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) above can be performed by, but is not limited to, generating a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, so as to enhance the activity of the polypeptide, or by replacing the sequence with an amino acid sequence or polynucleotide sequence improved to have a higher activity, or an amino acid sequence or polynucleotide sequence improved to improve the activity. Specifically, the replacement can be performed by, but is not limited to, inserting the polynucleotide into a chromosome by homologous recombination. Here, the vector used may further include a selection marker for checking whether or not it has been inserted into the chromosome. The selection marker is as described above.

[0081] The above 6) introduction of a foreign polynucleotide exhibiting the activity of the polypeptide may be carried out by introducing into a host cell a foreign polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide. The foreign polynucleotide may be of any origin or sequence as long as it exhibits the same / similar activity as the polypeptide. The introduction can be carried out by a person skilled in the art by appropriately selecting a known transformation method, and the introduced polynucleotide is expressed in the host cell as described above, thereby producing a polypeptide and improving its activity.

[0082] 7) Optimizing the codons of a polynucleotide encoding a polypeptide may be carried out by optimizing the codons of an endogenous polynucleotide so as to increase transcription or translation in a host cell, or by optimizing the codons of an exogenous polynucleotide so as to perform optimized transcription or translation in a host cell.

[0083] 8) Analyzing the tertiary structure of a polypeptide and selecting and altering or chemically modifying an exposed portion may be carried out, for example, by comparing sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored, determining candidates for template proteins depending on the degree of sequence similarity, confirming the structure based on the candidates, and selecting and altering or modifying the exposed portion to be altered or chemically modified.

[0084] Such enhancement of polypeptide activity can be achieved by, but is not limited to, improving the activity, concentration, or expression level of the corresponding polypeptide compared to the activity or concentration of the polypeptide expressed in a wild-type or unmodified microbial strain, or by increasing the amount of a product produced from the polypeptide.

[0085] The microorganism having O-acetylhomoserine producing ability of the present application further has enhanced activity of the L-methionine / branched-chain amino acid exporter YjeH protein, but is not limited thereto.

[0086] In the present application, the term "L-methionine / branched-chain amino acid exporter YjeH" refers to a type of amino acid export protein (AAE) in the amino acid-polyamine-organocation (APC) superfamily of transporter 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, which is a characteristic of the APC superfamily. The L-methionine / branched-chain amino acid exporter YjeH protein and gene sequence can be obtained from a known database, such as, but not limited to, GenBank of NCBI. Examples of the protein and gene sequence include, but are not limited to, those derived from Escherichia coli (E. coli). In the present application, the L-methionine / branched-chain amino acid exporter YjeH protein is referred to as inner membrane protein, O-acetylhomoserine export protein, protein having O-acetylhomoserine export ability, protein having O-acetylhomoserine export activity, YjeH protein, or YjeH.

[0087] Specifically, the microorganism capable of producing O-acetylhomoserine of the present application further includes, but is not limited to, a mutant of the L-methionine / branched-chain amino acid exporter YjeH. More specifically, the microorganism capable of producing O-acetylhomoserine of the present application further includes, but is not limited to, a mutant of the L-methionine / branched-chain amino acid exporter YjeH, into which a mutant of the L-methionine / branched-chain amino acid exporter YjeH has been introduced to enhance the activity of the L-methionine / branched-chain amino acid exporter YjeH protein.

[0088] More specifically, the mutant of the L-methionine / branched-chain amino acid exporter YjeH may improve the ability to produce O-acetylhomoserine by increasing the amount of O-acetylhomoserine excreted by the microorganism, and may have the amino acid sequence shown in SEQ ID NO: 10, may contain the amino acid sequence, may be essentially consisting of the amino acid sequence, or may consist of the amino acid sequence.

[0089] Furthermore, the mutant 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. Furthermore, it goes without saying that the present application also includes an amino acid sequence having such homology or identity and having the same or corresponding biological activity as the mutant of the L-methionine / branched-chain amino acid exporter YjeH of the present application, in which a part of the sequence has been deleted, modified, substituted, conservatively substituted, or added.

[0090] For example, the microorganism having O-acetylhomoserine producing ability of the present application may further comprise 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 to SEQ ID NO: 10, specifically, the microorganism may be a microorganism into which the protein has been introduced and in which O-acetylhomoserine producing ability has been further improved.

[0091] Specifically, "introduction of a protein" means that a gene not originally possessed by a microorganism is expressed in the microorganism to cause the activity of a specific protein to appear, or that the activity of the protein is increased or improved compared to the endogenous activity or activity before modification. For example, it may be that a polynucleotide encoding a specific protein is introduced into a chromosome in a microorganism, or that a vector containing a polynucleotide encoding a specific protein is introduced into a microorganism to cause the activity to appear.

[0092] For the purpose of this application, the microorganism containing the mutant of the L-methionine / branched-chain amino acid exporter YjeH means a microorganism having a naturally weak O-acetylhomoserine exporting ability, in which the mutant of the L-methionine / branched-chain amino acid exporter YjeH is introduced to improve the O-acetylhomoserine exporting ability. Specifically, the microorganism is characterized in that the mutant of the L-methionine / branched-chain amino acid exporter YjeH is introduced to increase the extracellular export of O-acetylhomoserine compared to wild-type or unmodified microorganisms, thereby improving the O-acetylhomoserine production ability. Thus, while wild-type or unmodified microorganisms cannot export O-acetylhomoserine, or can only produce a very small amount of O-acetylhomoserine even if they do, by introducing the mutant of the L-methionine / branched-chain amino acid exporter YjeH, the amount of O-acetylhomoserine exported by the microorganism is increased, thereby improving the O-acetylhomoserine production ability.

[0093] Examples of the inner membrane protein YjeH and / or its mutants used in the present application are disclosed in WO 2006 / 023991, the entire specification of which is incorporated herein by reference. The microorganism capable of producing O-acetylhomoserine of the present application further has enhanced homoserine acetyltransferase (MetX) activity, but is not limited thereto.

[0094] In the present application, "MetX" means homoserine acetyltransferase. Specifically, most microorganisms existing in nature use O-succinylhomoserine or O-acetylhomoserine as intermediates to synthesize methionine, but generally, MetA produces O-succinylhomoserine, and homoserine O-acetyltransferase produces O-acetylhomoserine. 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 publicly known databases, such as, but not limited to, NCBI GenBank. In the present application, "homoserine acetyltransferase" is used interchangeably with "MetX" and "homoserine O-acetyltransferase".

[0095] Specifically, the microorganism capable of producing O-acetylhomoserine of the present application is one in which expression of the MetX gene encoding O-acetylhomoserine transferase is further amplified, but is not limited thereto.

[0096] The nucleotide sequence of the MetX gene can be obtained from GenBank, a publicly known database of NCBI. The polynucleotide encoding MetX is included in the present application as long as it is a polynucleotide sequence encoding a protein containing an amino acid sequence having at least 80% homology with MetX, and may have or contain the nucleotide sequence of SEQ ID NO: 26 or a nucleotide 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 nucleotide sequence of SEQ ID NO: 26, or may consist of or be substantially composed of the nucleotide sequence of SEQ ID NO: 26 or a nucleotide 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 nucleotide sequence of SEQ ID NO: 26, but is not limited thereto.

[0097] An example of the MetX gene used in the present application is disclosed in US Pat. No. 5,399,633, the entire specification of which is incorporated herein by reference. The microorganism capable of producing O-acetylhomoserine of the present application further has enhanced aspartokinase activity, but is not limited thereto.

[0098] Specifically, in order to increase the biosynthesis of O-acetyl-homoserine, the aspartokinase activity is further enhanced compared to that of a non-mutated microorganism, and in particular, a mutation (L377K) for releasing the feedback inhibition of L-lysine and L-threonine in the gene (lysC) encoding aspartokinase (Patent Document 6) is introduced, but the present invention is not limited thereto. The base sequence of the lysC can be obtained from a known database and may be any base sequence that encodes a protein having aspartokinase activity, for example, the base sequence of SEQ ID NO: 21.

[0099] The microorganism capable of producing O-acetylhomoserine of the present application further has weakened cystathionine gamma synthase activity, but is not limited thereto.

[0100] Specifically, said weakening is, but is not limited to, inactivation. More specifically, the activity of the cystathionine γ-synthase is reduced or inactivated compared to that of a non-mutated microorganism, and in particular, the gene (metB) encoding cystathionine synthase is deleted, but this is not limited thereto. In the present application, "cystathionine γ-synthase" is used interchangeably with "cystathionine synthase." The base sequence of metB can be obtained from a publicly known database and may be any base sequence that encodes a protein having cystathionine synthase activity, for example, the base sequence of SEQ ID NO: 11.

[0101] The microorganism capable of producing O-acetylhomoserine of the present application further has weakened O-acetylhomoserine(thiol)-lyase activity, but is not limited thereto.

[0102] Specifically, said weakening is, but is not limited to, inactivation. More specifically, the activity of the O-acetylhomoserine thiol lyase is reduced or inactivated compared to that of a non-mutated microorganism, and in particular, the gene (metY) encoding O-acetylhomoserine thiol lyase is deleted, but this is not limited thereto. The base sequence of metY can be obtained from a publicly known database and may be any base sequence that encodes a protein having O-acetylhomoserine thiol lyase activity, for example, the base sequence of SEQ ID NO: 16.

[0103] Another aspect of the present application provides a method for producing O-acetylhomoserine, comprising culturing said microorganism in a medium. The microorganism and O-acetylhomoserine are as described above.

[0104] In the method of the present application, any culture conditions and culture methods known in the art can be used for culturing the microorganisms. Such culture processes can be easily adjusted by those skilled in the art depending on the strain selected.

[0105] In the present application, "culturing" means growing the microorganism of the present application under appropriately adjusted environmental conditions. The culturing process of the present application can be carried out in a suitable medium and under suitable culture conditions known in the art. Such a culturing process can be easily adjusted and used by a person skilled in the art depending on the strain selected. Specifically, the culturing can be batch, continuous, or fed-batch culture, but is not limited thereto.

[0106] In the present application, the term "culture medium" refers to a substance that is a mixture of nutrients necessary for culturing the microorganism of the present application as the main components, and that supplies nutrients such as water essential for survival and growth, growth factors, etc. Specifically, the culture medium and other culture conditions used for culturing the microorganism of the present application may be any medium that is used for culturing a normal microorganism, and the microorganism of the present 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, etc., under aerobic conditions by adjusting the temperature, pH, etc.

[0107] In the present application, the carbon source may be carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc., sugar alcohols such as mannitol, sorbitol, etc., organic acids such as pyruvic acid, lactic acid, citric acid, etc., amino acids such as glutamic acid, methionine, lysine, etc. Also, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, corn steeping liquid, etc. may be used, specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and any other suitable carbon source may be used. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0108] The nitrogen source may be an inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, or ammonium nitrate, or an organic nitrogen source such as an amino acid such as glutamic acid, methionine, or glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its decomposition product, or defatted soybean cake or its decomposition product. These nitrogen sources may be used alone or in combination of two or more kinds, but are not limited thereto.

[0109] The phosphorus source may be potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or a sodium-containing salt equivalent thereto. The inorganic compound may be sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, or other suitable compounds, including amino acids, vitamins, and / or suitable precursors. These components or precursors may be added to the medium in a batch or continuous manner. However, they are not limited thereto.

[0110] In the present application, the pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture in a suitable manner during the cultivation of the microorganism. In addition, foam generation can be suppressed during the cultivation using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, in order to maintain the aerobic state of the culture, oxygen or an oxygen-containing gas may be injected into the culture, and in order to maintain the anaerobic and microaerobic state, no gas may be injected, and nitrogen, hydrogen, or carbon dioxide gas may be injected, but is not limited thereto.

[0111] The temperature of the culture is 25° C. to 40° C., more specifically, but not limited to, 28° C. to 37° C. The culture period is continued until a desired amount of useful substance is produced, specifically, but not limited to, 1 hour to 100 hours.

[0112] The O-acetylhomoserine produced by the culture of the present application is either secreted into the medium or remains intracellularly. The method for producing O-acetylhomoserine of the present application may further include a step of preparing a microorganism of the present application, a step of preparing a medium for culturing the microorganism, or a combination thereof (in any order), for example prior to the culturing step.

[0113] The method for producing O-acetylhomoserine of the present application may further include a step of recovering O-acetylhomoserine from the medium used in the culture (the medium in which the culture was carried out) or the microorganism. The recovery step may be further included after the culture step.

[0114] The recovery may be a method of collecting the target O-acetylhomoserine using a suitable method known in the art depending on the culture method of the microorganism of the present application, such as batch, continuous, or fed-batch culture. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographies such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination thereof can be used, and the target O-acetylhomoserine can be collected from the medium or the microorganism using a suitable method known in the art.

[0115] In addition, the method for producing O-acetylhomoserine of the present application may further include a purification step. The purification may be performed by any suitable method known in the art. For example, when the method for producing O-acetylhomoserine of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the order, or may be performed simultaneously or integrated into one step, but is not limited thereto.

[0116] The O-acetylhomoserine thus recovered can be used to produce methionine by a two-stage process (Patent Document 7). Yet another aspect of the present application provides a method for producing L-methionine, comprising the steps of culturing the microorganism in a medium, producing O-acetylhomoserine from the cultured microorganism or the medium, and converting the O-acetylhomoserine to L-methionine.

[0117] The microorganism, O-acetylhomoserine and L-methionine are as described above. The two-step process includes a step of producing L-methionine and an organic acid by an enzymatic reaction using O-acetylhomoserine and methyl mercaptan produced by the L-methionine precursor-producing strain as substrates and an enzyme having O-acetylhomoserine sulfhydrylase activity or a strain containing the enzyme.

[0118] More specifically, the present application provides a method for producing L-methionine by using the O-acetylhomoserine accumulated by the above-mentioned method as a substrate through an enzymatic reaction with O-acetylhomoserine sulfhydrylase or the like.

[0119] In the two-step process, when O-acetylhomoserine is used as an L-methionine precursor, O-acetylhomoserine sulfhydrylase derived from a microbial strain belonging to the genera Leptospira sp., Chromobacterium sp., or Hyphomonas sp., more specifically, a microbial strain belonging to Leptospira meyeri, Pseudomonas aeruginosa, Hyphomonas Neptunium, or Chromobacterium Violaceum, can be used.

[0120] The above reaction is as follows: CH3SH + O-acetylhomoserine ⇔ Acetate + Methionine Such an additional process for producing methionine is disclosed in US Pat. No. 5,399,633, the entire specification of which is incorporated herein by reference.

[0121] Yet another aspect of the present application provides a composition for producing O-acetylhomoserine, comprising the microorganism. The microorganism and O-acetylhomoserine are as described above.

[0122] The composition of the present application may further contain any suitable excipient that is commonly used in compositions for producing O-acetylhomoserine, including, but not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, isotonicity agents, etc.

[0123] Yet another aspect of the present application provides use of the microorganism for producing O-acetylhomoserine or L-methionine. EXAMPLES

[0124] 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 present application is not limited thereto. Note that technical matters not described in this specification are fully understood and easily implemented by a skilled engineer in the technical field of the present application or a similar technical field. EXAMPLES

[0125] Construction of an endogenous gene (NCgl0616) deletion strain and evaluation of O-acetylhomoserine and homoserine production Example 1-1. Construction of a deletion vector for deleting NCgl0616 To assess the effectiveness of the endogenous gene NCgl0616 in Corynebacterium glutamicum ATCC13032, a deletion vector was constructed in which the gene NCgl0616 (SEQ ID NO: 29) was deleted.

[0126] Specifically, to prepare an NCgl0616 deletion vector, a pair of primers (SEQ ID NOs: 3 and 4) for amplifying the 5'-end upstream and a pair of primers (SEQ ID NOs: 5 and 6) for amplifying the 3'-end downstream were designed, centering on the NCgl0616 gene position of SEQ ID NO: 29. The primer sequences are shown in Table 1.

[0127] [Table 1]

[0128] PCR was performed using the chromosome of ATCC13032 wild type (WT) as a template and primers of SEQ ID NO: 3 and 4, and SEQ ID NO: 5 and 6. The PCR conditions were denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds, followed by polymerization reaction at 72°C for 7 minutes. As a result, a 701 bp DNA fragment upstream of the 5' end and a 699 bp DNA fragment downstream of the 3' end were obtained, centered on the deletion site of the NCgl0616 gene.

[0129] Using the two amplified DNA fragments as templates, PCR was performed with primers of SEQ ID NO: 3 and SEQ ID NO: 6. The PCR conditions were denaturation at 95° C. for 5 minutes, followed by 30 cycles of denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 90 seconds, followed by polymerization reaction at 72° C. for 7 minutes. As a result, a 1410 bp DNA fragment containing the site for deleting the NCgl0616 gene was amplified.

[0130] 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 an In-Fusion (registered trademark) HD ​​Cloning Kit (Clontech). The cloned vector was transformed into Escherichia coli DH5α, and the transformed Escherichia coli was smeared on an 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 the plasmid extraction method (Patent Document 9), and finally, the pDCM2-ΔNCgl0616 recombinant vector in which the NCgl0616 deletion cassette was cloned was prepared.

[0131] The prepared pDCM2-ΔNCgl0616 was transformed into ATCC13032 strain by electric pulse method, and a secondary crossover process was performed to obtain ATCC13032ΔNCgl0616 in which the NCgl0616 gene was deleted on the chromosome. Whether or not the NCgl0616 gene was inactivated was finally confirmed by performing PCR using primers of SEQ ID NOs: 8 and 9, and then comparing with ATCC13032 in which the NCgl0616 gene was not inactivated.

[0132] [Table 2]

[0133] Example 1-2. Evaluation of O-acetylhomoserine productivity in wild-type strains In order to compare the O-acetylhomoserine (O-AH) producing abilities of ATCC13032ΔNCgl0616 prepared in Example 1-1 and the wild-type strain ATCC13032, they were cultured in the following manner, and the O-acetylhomoserine in the culture medium was analyzed.

[0134] One platinum loopful of the strain was inoculated into a 250-ml Erlenmeyer 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.

[0135] <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)

[0136]

Table 3

[0137] As a result, as shown in Table 3, when ATCC13032 was cultured, 0.27 g / L of O-acetylhomoserine was accumulated, and when ATCC13032ΔNCgl0616 with the deletion of the endogenous gene NCgl0616 was cultured, 0.33 g / L of O-acetylhomoserine was accumulated, and it was confirmed that the production ability was 125% that of the control group ATCC13032.

Example

[0138] Preparation of an NCgl0616 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 the strain The pDCM2-ΔNCgl0616 vector prepared in Example 1-1 was transformed into the KCCM12634P strain (Patent Document 5, ATCC13032ΔNCgl2335::PCJ7-YjeH(eco,F351L)) with improved O-acetylhomoserine production ability by the electroporation method. After undergoing a secondary crossover process, KCCM12634PΔNCgl0616 with the NCgl0616 gene deleted on the chromosome was obtained. Whether the NCgl0616 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 NCgl0616 gene was not inactivated.

[0139] 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 KCCM12634PΔNCgl0616 strain prepared in Example 2-1, they were cultured by the following method, and O-acetylhomoserine in the culture broth was analyzed.

[0140] 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.

[0141] <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)

[0142]

Table 4

[0143] As a result, as shown in Table 4, it was confirmed that when NCgl0616 was deleted in the KCCM12634P strain, O-acetylhomoserine was 1.39 g / L, which is about 130% of the productivity of KCCM12634P. Therefore, it was confirmed from the above results that in a strain with improved O-acetylhomoserine productivity, the deletion of NCgl0616 provides a greater effect of improving O-acetylhomoserine productivity. Therefore, it was confirmed from the results of Examples 1-2 and 2-2 that the deletion and inactivation of the NCgl0616 gene, which is an endogenous gene of ATCC13032 of the present application, improves the productivity of the target amino acid. EXAMPLES

[0144] Construction of NCgl0616 deletion strain in strain with improved O-acetylhomoserine production and evaluation of O-acetylhomoserine and homoserine production - 2 Example 3-1. Preparation of strain-1 The metB gene, which encodes cystathionine gamma-synthase in the O-acetylhomoserine decomposition pathway, was isolated by PCR using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template. The nucleotide sequence of the metB gene (NCBI accession number Ncgl2360, SEQ ID NO: 11) was obtained from the National Institutes of Health GenBank, and primers containing the N-terminal portion and linker portion of the metB gene (SEQ ID NO: 12 and 13) and the C-terminal portion and linker portion of the metB gene (SEQ ID NO: 14 and 15) were synthesized based on the information. The primer sequences are shown in Table 5.

[0145] [Table 5]

[0146] PCR was carried out using the chromosomal DNA of ATCC13032 as a template and the primers of SEQ ID NOs: 12 and 13, and SEQ ID NOs: 14 and 15. TMHigh fidelity DNA polymerase (Stratagene) was used, and the PCR conditions were 30 cycles of denaturation at 96° C. for 30 seconds, annealing at 53° C. for 30 seconds, and polymerization at 72° C. for 1 minute. As a result, a 558 bp amplified gene containing the N-terminal portion and linker portion of the metB gene and a 527 bp amplified gene containing the C-terminal portion and linker portion of the metB gene were obtained.

[0147] 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 for 10 cycles, followed by addition of SEQ ID NOs: 12 and 15 and further polymerization reaction for 20 cycles. As a result, an inactivation cassette of 1064 bp containing the N-terminus-linker-C-terminus of the metB gene was obtained.

[0148] 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 an In-Fusion (registered trademark) HD ​​Cloning Kit (Clontech). The cloned vector was transformed into Escherichia coli DH5α, and the transformed Escherichia coli was smeared on an 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 the plasmid extraction method (Patent Document 9), and finally a pDCM2-ΔmetB recombinant vector in which a metB gene deletion cassette was cloned was prepared.

[0149] The constructed pDCM2-ΔmetB vector was transformed into the KCCM12634P strain by electric pulse method, and a second crossover process was performed to obtain KCCM12634PΔmetB in which the metB gene was inactivated on the chromosome. The inactivated metB gene was subjected to PCR using primers of SEQ ID NOs: 12 and 15, and then finally confirmed by comparing with ATCC13032 in which the metB gene was not inactivated.

[0150] Example 3-2. Preparation of strain-2 The metY gene, which encodes O-acetylhomoserine(thiol)-lyase in the O-acetylhomoserine degradation pathway, was isolated by PCR using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template. The nucleotide sequence of the metY gene (NCBI accession number Ncgl0625, SEQ ID NO: 16) was obtained from the National Institutes of Health GenBank, and primers containing the N-terminal portion and linker portion of the metY gene (SEQ ID NO: 17 and 18) and the C-terminal portion and linker portion of the metY gene (SEQ ID NO: 19 and 20) were synthesized based on the information. The primer sequences are shown in Table 6.

[0151] [Table 6]

[0152] PCR was carried out using the chromosomal DNA of ATCC13032 as a template and the primers of SEQ ID NOs: 17 and 18, and SEQ ID NOs: 19 and 20. TMHigh fidelity DNA polymerase (Stratagene) was used, and 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 for 30 cycles. As a result, a 548 bp amplified gene containing the N-terminal portion and linker portion of the metY gene and a 550 bp amplified gene containing the C-terminal portion and linker portion of the metY gene were obtained. 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, and polymerization at 72°C for 1 minute for 10 cycles, and then SEQ ID NOs: 17 and 20 were added and the polymerization reaction was performed for another 20 cycles. As a result, an inactivated cassette of 1077 bp containing the N-terminus-linker-C-terminus of the metY gene was obtained.

[0153] 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 an In-Fusion (registered trademark) HD ​​Cloning Kit (Clontech). The cloned vector was transformed into Escherichia coli DH5α, and the transformed Escherichia coli was smeared on an 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 a plasmid extraction method (Patent Document 9), and finally a pDCM2-ΔmetY recombinant vector in which a metY gene deletion cassette was cloned was prepared.

[0154] The prepared pDCM2-ΔmetY vector was transformed into the strain KCCM12634PΔmetB prepared in Example 3-1 by an electric pulse method, and a second crossover process was performed to obtain KCCM12634PΔmetBΔmetY in which the metY gene was further inactivated on the chromosome. The inactivated metY gene was subjected to PCR using primers of SEQ ID NOs: 17 and 20, and then finally confirmed by comparing with ATCC13032 in which the metY gene was not inactivated.

[0155] Example 3-3. Preparation of strain-3 A mutation (L377K) (Patent Document 6) was introduced into the lysC gene (SEQ ID NO: 21) encoding aspartokinase derived from Corynebacterium glutamicum ATCC13032 to enhance expression of the lysC gene and to release feedback inhibition against L-lysine and L-threonine, and a pair of primers (SEQ ID NO: 22 and 23) for amplifying the upstream 5' end and a pair of primers (SEQ ID NO: 24 and 25) for amplifying the downstream 3' end were designed around the mutation site to prepare a vector containing the mutant lysC gene. The primer sequences are shown in Table 7.

[0156] [Table 7]

[0157] PCR was performed using the chromosome of ATCC13032 as a template and the primers of 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 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds, followed by polymerization reaction at 72°C for 7 minutes. As a result, a 512 bp DNA fragment upstream of the 5' end and a 522 bp DNA fragment downstream of the 3' end were obtained, focusing on the mutation of the lysC gene.

[0158] Using the two amplified DNA fragments as templates, PCR was performed with primers of SEQ ID NO: 22 and 25. The PCR conditions were as follows: denaturation at 95° C. for 5 minutes, followed by 30 cycles of denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 60 seconds, followed by polymerization reaction at 72° C. for 7 minutes. As a result, a 1011 bp DNA fragment containing a mutant lysC (L377K) gene encoding an aspartokinase mutant in which leucine at position 377 was replaced with lysine was amplified.

[0159] 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 an In-Fusion (registered trademark) HD ​​Cloning Kit (Clontech). The cloned vector was transformed into Escherichia coli DH5α, and the transformed Escherichia coli was smeared on an 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 the plasmid extraction method (Patent Document 9), and finally a pDCM2-lysC(L377K) recombinant vector was prepared in which a cassette in which the lysC(L377K) gene was replaced was cloned.

[0160] The prepared pDCM2-lysC(L377K) vector was transformed into the strain KCCM12634PΔmetBΔmetY prepared in Example 3-2 by the electric pulse method, and through a secondary crossover process, Corynebacterium glutamicum KCCM12634PΔmetBΔmetY lysC(L377K) in which a nucleotide mutation was introduced into the lysC gene on the chromosome was obtained. The gene in which the nucleotide mutation was introduced was subjected to PCR using the primers of SEQ ID NO: 22 and 25, and then finally confirmed by comparing with the sequence of the wild-type lysC gene by sequencing.

[0161] Example 3-4. Preparation of a strain lacking endogenous gene NCgl0616-1 In order to maximize the production of O-acetylhomoserine, the pDCM2-ΔNCgl0616 vector prepared in Example 1-1 was transformed into the strain KCCM12634PΔmetBΔmetY lysC(L377K) prepared in Example 3-3 by electric pulse method, and a second crossover process was performed to obtain KCCM12634PΔmetBΔmetY lysC(L377K)ΔNCgl0616 in which the NCgl0616 gene was deleted on the chromosome. Whether or not the NCgl0616 gene was inactivated was finally confirmed by performing PCR using primers of SEQ ID NOs: 8 and 9, and then comparing with ATCC13032 in which the NCgl0616 gene was not inactivated.

[0162] Example 3-5. Construction of a strain lacking endogenous gene NCgl0616-2 In order to amplify the gene encoding homoserine acetyl transferase (MetX), the nucleotide sequence information of the MetX gene (NCBI accession number NCgl0624, SEQ ID NO: 26) was obtained from the National Institutes of Health GenBank (NIH GenBank), and based on that, primers (SEQ ID NOs: 27 and 28) for amplifying the promoter site (approximately 300 bp upstream of the start codon) to the terminator site (approximately 100 bp downstream of the stop codon) were designed by inserting BamHI restriction enzyme sites at both ends. The PCR conditions were denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 90 seconds, followed by polymerization reaction at 72°C for 7 minutes. As a result, a DNA fragment of 1546 bp of the coding site of the MetX gene was obtained. The pECCG117 (Patent Document 10) vector and the MetX DNA fragment were treated with the restriction enzyme BamHI, ligated using DNA ligation enzyme, and then cloned to obtain a plasmid. This was named pECCG117-MetX WT. The primer sequences are shown in Table 8.

[0163] [Table 8]

[0164] The prepared pECCG117-MetX WT vector was introduced into the strain KCCM12634PΔmetBΔmetY lysC(L377K) prepared in Example 3-3 and the strain KCCM12634PΔmetBΔmetY lysC(L377K)ΔNCgl0616 prepared in Example 3-4 by the electroporation method, and then spread on a selective medium containing 25 mg / l of kanamycin, and the transformants KCCM12634PΔmetBΔmetY lysC(L377K) / pECCG117-MetX WT and KCCM12634PΔmetBΔmetY lysC(L377K)ΔNCgl0616 / pECCG117-MetX WT were obtained, respectively.

[0165] Example 3-6. Evaluation of O-acetylhomoserine production ability 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.

[0166] The strain was inoculated into a 250 ml Erlenmeyer flask containing 25 ml of the following medium with one inoculation loop, 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 9.

[0167] <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)

[0168]

Table 9

[0169] As a result, as shown in Table 9, when the control strain KCCM12634PΔmetBΔmetY lysC(L377K) was cultivated, 1.3 g / L of O-acetylhomoserine was accumulated, and when KCCM12634PΔmetBΔmetY lysC(L377K)ΔNCgl0616 was cultivated, 1.73 g / L of O-acetylhomoserine was accumulated, demonstrating 130% productivity compared to the control. In addition, when the KCCM12634PΔmetBΔmetY lysC(L377K) / pECCG117-MetX WT strain was cultivated, 2.10 g / L of O-acetylhomoserine was accumulated, and when the KCCM12634PΔmetBΔmetY lysC(L377K)ΔNCgl0616 / pECCG117-MetX WT strain was cultivated, 2.83 g / L of O-acetylhomoserine was accumulated, which was confirmed to be 135% of the productivity of the KCCM12634PΔmetBΔmetY lysC(L377K) / pECCG117-MetX WT strain.

[0170] Therefore, it was confirmed that the productivity of the target amino acid was further improved by deleting and inactivating the endogenous gene NCgl0616 in the strain with improved O-acetyl-L-homoserine productivity. EXAMPLES

[0171] Construction of an endogenous gene (NCgl0078) deletion strain and evaluation of O-acetylhomoserine and homoserine production Example 4-1. Construction of a deletion vector for deleting NCgl0078 The NCgl0616 gene and the NCgl0078 gene in the Corynebacterium glutamicum ATCC13032 strain are known as two SIRT-type deacetylase homologues of Corynebacterium glutamicum ATCC13032 (Non-Patent Document 1). Therefore, the nucleotide sequence information of the NCgl0078 gene, which is a homologue of the NCgl0616 gene, was obtained from the National Institutes of Health GenBank (NIH GenBank) (NCBI accession number NCgl0078, SEQ ID NO: 30), and based on that, primers containing the N-terminal part and linker part of the NCgl0078 gene (SEQ ID NO: 31 and 32) and primers containing the C-terminal part and linker part of the NCgl0078 gene (SEQ ID NO: 33 and 34) were synthesized. The primer sequences are shown in Table 10.

[0172] [Table 10]

[0173] PCR was carried out using chromosomal DNA of ATCC13032 wild type (WT) as a template and primers of SEQ ID NOs: 31 and 32, and SEQ ID NOs: 33 and 34. TM High fidelity DNA polymerase (Stratagene) was used, and 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 for 30 cycles. As a result, a 1223 bp amplified gene containing the N-terminal part and linker part of the NCgl0078 gene and a 2576 bp amplified gene containing the C-terminal part and linker part of the NCgl0078 gene were obtained. 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, and polymerization at 72°C for 1 minute for 10 cycles, and then SEQ ID NOs: 31 and 34 were added and the polymerization reaction was performed for another 20 cycles. As a result, an inactivated cassette of 3806 bp containing the N-terminus-linker-C-terminus of the NCgl0078 gene was obtained.

[0174] 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 an In-Fusion (registered trademark) HD ​​Cloning Kit (Clontech). The cloned vector was transformed into Escherichia coli DH5α, and the transformed Escherichia coli was smeared on an 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 a plasmid extraction method, and finally a pDCM2-ΔNCgl0078 recombinant vector in which the NCgl0078 gene deletion cassette was cloned was prepared.

[0175] The prepared pDCM2-ΔNCgl0078 vector was transformed into the KCCM12634P strain prepared in Example 3-1 by an electric pulse method, and a secondary crossover process was performed to obtain KCCM12634PΔNCgl0078 in which the NCgl0078 gene was inactivated on the chromosome. The inactivated NCgl0078 gene was subjected to PCR using primers of SEQ ID NOs: 31 and 34, and then finally confirmed by comparing with ATCC13032 in which the NCgl0078 gene was not inactivated.

[0176] Example 4-2. Evaluation of O-acetylhomoserine productivity In order to compare the O-acetyl homoserine (O-AH) producing abilities of the KCCM12634P strain and the KCCM12634PΔNCgl0616 strain prepared in Example 2-1 and the KCCM12634PΔNCg0078 strain prepared in Example 4-1, they were cultured by the following method, and the O-acetyl homoserine in the culture medium was analyzed.

[0177] A loopful of the strain was inoculated into a 250 ml corner baffle flask containing 25 ml of the following medium, and cultured at 33°C and 200 rpm for 20 hours with shaking. The O-acetylhomoserine concentration was analyzed using HPLC. The analyzed concentrations are shown in Table 11.

[0178] <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)

[0179]

Table 11

[0180] As a result, as shown in Table 11, when NCgl0078 was deleted in the KCCM12634P strain, O-acetylhomoserine was 1.27 g / L, and it was confirmed that the production ability was about 120% 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 NCgl0078. In addition, when NCgl0078 was deleted in the KCCM12634P strain, it was confirmed that the effect of improving O-acetylhomoserine production ability equivalent to that when NCgl0616, a homolog, was deleted (about 130%) was obtained.

[0181] Summarizing these, it was confirmed that in the strain with improved O-acetyl-homoserine production ability, the effect of improving O-acetylhomoserine production ability was greater due to the deletion and / or inactivation of the endogenous gene NCgl0616 and its homolog NCgl0078.

[0182] From the above description, those skilled in the art to which the present application pertains will understand that the present application can be implemented in other specific forms without changing its technical idea and essential features. It should be understood that the above examples are merely illustrative and not restrictive. The present application should be construed as including all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. A microorganism of the genus Corynebacterium having an ability to produce O-acetylhomoserine and having weakened activity of a SIRT-type deacetylase protein.

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 SIRT deacetylase is a protein consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

4. The Corynebacterium microorganism according to claim 1 , wherein the attenuation is inactivation.

5. The microorganism of claim 1 , wherein the nucleic acid base sequence of SEQ ID NO: 29 or SEQ ID NO: 30 is deleted.

6. The microorganism of claim 1, further comprising enhanced activity of an L-methionine / branched-chain amino acid exporter YjeH protein.

7. The microorganism of claim 6 , wherein the amino acid sequence of SEQ ID NO: 10 has been introduced.

8. A method for producing O-acetylhomoserine, comprising the step of culturing in a medium a Corynebacterium microorganism having an ability to produce O-acetylhomoserine and attenuated SIRT deacetylase protein activity.

9. Cultivating a Corynebacterium microorganism having an ability to produce O-acetylhomoserine and having attenuated SIRT deacetylase protein activity in a medium; Producing O-acetylhomoserine from the cultured microorganism or medium; and converting the O-acetylhomoserine into L-methionine.

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