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 with attenuated 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-acetylhomoserine acts as a precursor to methionine, an essential amino acid that is widely used not only as a feed and food additive, but also as a synthetic raw material for infusions and pharmaceuticals.

[0003] Methionine can be 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. 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. Patent No. 10,584,338 [Patent Document 4] U.S. Patent No. 10273491 [Patent Document 5] Korean Registration Patent No. 10-2182497 [License 6] Korean Patent Publication No. 10-2019-0003019 [License 7] Korean Registration Patent No. 10-0905381 [License 8] Korean Patent Publication No. 10-2020-0136813 [License 9] U.S. Patent No. 5981235 [License 10] Korean Registration Patent No. 10-0057684 [Non-licensed literature]

[0006] [Non-licensed Document 1] Megumi Nagano-Shoji et.al.,Molecular Microbiology.,104(4):677-689,2017 [Non-licensed Document 2] Pearson et al(1988)[Proc.Natl.Acad.Sci.USA 85]:2444 [Non-licensed Document 3] Rice et al.,2000,Trends Genet.16:276-277 [Non-licensed Document 4] Needleman and Wunsch,1970,J.Mol.Biol.48:443-453 [Non-licensed Document 5] Devereux,J.,et al,Nucleic Acids Research 12:387(1984) [Non-licensed Document 6] Atschul,[S.][F.,][ET AL,J MOLEC BIOL 215]:403(1990) [Non-licensed Document 7] Guide to Huge Computers,Martin J.Bishop,[ED.,]Academic Press,San Diego,1994 [Non-licensed document 8] [CARILLO ETA / .](1988)SIAM J Applied Math 48:1073

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

[0007] The present inventors have discovered a Corynebacterium microorganism capable of producing O-acetylhomoserine 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 that has an ability to produce O-acetylhomoserine and has attenuated SIRT-type deacetylase protein activity. Another object of the present application is to provide a method for producing O-acetylhomoserine, which includes a step of culturing the microorganism in a medium.

[0009] Furthermore, the present application aims to provide a method for producing L-methionine, which comprises 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, which comprises the microorganism.

[0010] A further object of the present application is to provide use of the microorganism of the present application for producing O-acetylhomoserine or L-methionine. [Effects of the Invention]

[0011] The microorganism capable of producing O-acetylhomoserine of the present invention can produce O-acetylhomoserine more efficiently and environmentally friendly than chemical synthesis. Furthermore, when the produced O-acetylhomoserine is used as a precursor for the synthesis of methionine and acetic acid by O-acetylhomoserine sulfhydrylase, L-methionine can be bioconverted with high efficiency. The converted L-methionine can be widely used not only in animal feed and animal feed additives but also in the production of human foods and food additives. DETAILED DESCRIPTION OF THE INVENTION

[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. Furthermore, this application is not limited to the following specific descriptions.

[0013] Furthermore, throughout this specification, many papers and patent documents are referenced and citations are provided, the disclosures of which are incorporated herein by reference in their entirety 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, "a sirtuin-type deacetylase" refers to a sirtuin (SIRT)-type deacetylase. Specifically, the SIRT-type deacetylase is known in the art, and the protein and gene sequences of the SIRT-type deacetylase can be obtained from publicly known databases, such as, but not limited to, GenBank of NCBI. More specifically, the SIRT-type deacetylase may have the amino acid sequence shown in 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 refers to 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 refers to a SIRT-type deacetylase consisting of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 that is endogenously present in Corynebacterium microorganisms, more specifically, it refers to two 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 to SEQ ID NO: 1 or SEQ ID NO: 2. Furthermore, it goes without saying that the present application also includes amino acid sequences in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added, so long as the amino acid sequence has such homology or identity and has the same or corresponding biological activity as the SIRT-type deacetylase of the present application.

[0017] Although 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 portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added is also used in the present application, as long as it has the same or equivalent activity as the polypeptide consisting of the amino acid sequence of the SEQ ID NO. For example, 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 are included.

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

[0019] As used herein, the term "conservative substitution" refers to the substitution of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made on the basis of 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 charged amino acids including aspartic acid, glutamic acid, lysine, arginine, and histidine, and uncharged amino acids are further classified into nonpolar and polar amino acids, with nonpolar amino acids including glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, and 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 amino acid deletions or additions that minimally affect 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- or post-translational 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 can be determined by standard sequence algorithms, optionally with default gap penalties established by the program used. Substantially homologous or identical sequences will generally hybridize to all or part of the sequence under moderately or highly stringent conditions. Hybridization, of course, also includes hybridization to polynucleotides with codons commonly used in polynucleotides or codons that take into account codon degeneracy.

[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 described in, for example, Non-Patent Document 2. Alternatively, the Needleman-Wunsch algorithm (Non-Patent Document 4) can be used, as implemented in the Needle program (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 3) (version 5.0.0 or later) in the EMBOSS package (which includes the GCG program package (Non-Patent Document 5), BLASTP, BLASTN, and 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 Non-Patent Document 4, as disclosed in, for example, Non-Patent Document 9. 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, non-identity a value of 0) and a weighted comparison matrix (or EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 10; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

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

[0026] For example, when any amino acid sequence is aligned with SEQ ID NO: 1, each amino acid residue in the amino acid sequence can be numbered based on the number and position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, the sequence alignment algorithm in the present application can identify the amino acid positions or positions where modifications such as substitutions, insertions, deletions, etc. occur when compared with 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 the present invention is not limited to these. Sequence alignment programs, pairwise sequence comparison algorithms, etc. known in the art can also be used as appropriate.

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

[0029] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and naturally or artificially genetically modified microorganisms, and is a microorganism in which a specific mechanism has been weakened or strengthened by inserting an exogenous gene or by 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 have the ability to 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 a strain derived therefrom, but is not limited to these.

[0031] For the purposes of this application, the O-acetylhomoserine-producing microorganism is characterized by having an improved ability to produce the target O-acetylhomoserine by weakening the activity of the SIRT-type deacetylase protein compared to the endogenous activity, 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 is 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% homologous or identical to the sequence of SEQ ID NO:29 or SEQ ID NO:30.

[0034] As an example, the microorganism with improved production ability has an O-acetylhomoserine production ability 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 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 deacetylase protein. However, any microorganism that shows an increase in + 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 that is about 1.1-fold or more, about 1.2-fold or more, about 1.21-fold or more, about 1.22-fold or more, about 1.23-fold or more, about 1.24-fold or more, about 1.25-fold or more, about 1.26-fold or more, about 1.27-fold or more, about 1.28-fold or more, about 1.29-fold or more, about 1.30-fold or more, about 1.31-fold or more, about 1.32-fold or more, about 1.33-fold or more, about 1.34-fold or more, or about 1.35-fold or more (there is no particular upper limit, and for example, about 10-fold or less, about 5-fold or less, about 3-fold or less, about 2-fold or less, about 1.5-fold or less, or about 1.4-fold or less) improved compared to the parent strain or unmodified microorganism before 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 naturally occurring strain itself, or a strain before its traits are changed due to genetic mutation caused by natural or artificial factors. For example, the unmodified microorganism refers to a strain in which the activity of the SIRT-type deacetylase protein described herein is not weakened compared to its endogenous activity, or before it is weakened. The term "unmodified microorganism" is also 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 yeasts. Specifically, the microorganisms include strains of the genera Escherichia, Corynebacterium, and Leptospira, and yeasts, and more specifically, strains of the genus Corynebacterium.

[0037] In the present application, the term "microorganisms of the genus Corynebacterium" includes all microorganisms of the genus Corynebacterium. Specifically, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium 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 of SEQ ID NO: 1 or SEQ ID NO: 2, or the polynucleotide of SEQ ID NO: 29 or SEQ ID NO: 30. On the other hand, although it is already known that microorganisms of the genus Corynebacterium produce O-acetylhomoserine, their 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 involved in the O-acetylhomoserine production mechanism, and Corynebacterium microorganisms whose O-acetylhomoserine-producing ability has been improved by introducing or strengthening the activity of exogenous genes.

[0039] In the present application, "attenuation" of a polypeptide activity is a concept that encompasses 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 terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation.

[0040] Specifically, the attenuation is, but is not limited to, inactivation, which means that the protein is not expressed at all, or that even if it is expressed, its activity is absent or reduced, 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 within the cell is reduced compared to that of a native 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 term "endogenous activity" refers to the activity of a specific polypeptide that was originally possessed by a parent strain, wild-type, or unmodified microorganism before the trait change, when the trait is changed due to genetic mutation caused by natural or artificial factors. This term is used interchangeably with "activity before modification." When the activity of a polypeptide is "inactivated," "deficient," "reduced," "down-regulated," "decreased," or "attenuated" compared to the endogenous activity, it means that the activity of the specific polypeptide is reduced compared to the activity of the specific polypeptide that was originally possessed by the parent strain or unmodified microorganism before the trait change.

[0043] The activity of such polypeptides can be attenuated 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 activity of a polypeptide of the present application can be attenuated by 1) deleting all or part of a gene encoding the polypeptide, 2) modifying an expression regulatory region (or 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 attenuate the activity of the polypeptide (for example, by 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 attenuate the activity of the polypeptide (for example, by modifying the gene sequence encoding the polypeptide so as to encode a polypeptide modified so as to delete or attenuate the activity of the polypeptide). a deletion / substitution / addition of one or more nucleic acid bases in the nucleic acid base sequence of (1) above); 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 prevents ribosome attachment 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 can be reverse-transcribed (reverse transcription engineering, RTE); or 9) a combination of two or more selected from the above 1) to 8), but this is not limited to these.

[0045] For example, 1) deleting a part or all of the gene encoding the polypeptide may be carried out by deleting the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, or by substituting a polynucleotide with a partial deletion of nucleotides or a marker gene.

[0046] Furthermore, the modification of the expression regulatory region (or expression regulatory sequence) described above in 2) may be carried out 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, modifying 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 attenuate the activity of the polypeptide, or by substituting an amino acid sequence or polynucleotide sequence that has been improved to have lower activity or to eliminate activity. For example, gene expression can be inhibited or attenuated by, but is not limited to, introducing a mutation into a polynucleotide sequence to form a stop codon.

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

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

[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 that prevents ribosome attachment is formed may be achieved by disabling or slowing down mRNA translation.

[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 it (reverse transcription engineering, RTE) may be carried out by creating an antisense nucleotide complementary to the gene transcript encoding the polypeptide to attenuate its activity.

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

[0053] Specifically, inactivation of the SIRT deacetylase protein activity may be achieved by deleting part or all of the gene encoding the protein. More specifically, this may be achieved by replacing a polynucleotide encoding an endogenous target protein in a chromosome of a microorganism with a polynucleotide lacking a partial nucleotide sequence or a marker gene using a vector for chromosomal introduction into the microorganism. One example of a method for deleting part or all of such a polynucleotide is a method for deleting a polynucleotide by homologous recombination, but this is not limited to this. Another example of a method for deleting part or all of the gene may involve inducing mutation using light such as ultraviolet light or chemicals, and then selecting a strain in which the target gene has been deleted from the resulting 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 homologous to the target gene into the microorganism to cause homologous recombination. The introduced polynucleotide sequence or vector may contain 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 refers to a DNA or RNA chain longer than a predetermined length, and more specifically refers to a polynucleotide fragment encoding the above-mentioned variant.

[0056] In the present application, the SIRT-type deacetylase is encoded by a gene comprising 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 comprising the polynucleotide sequence, a gene essentially consisting of the polynucleotide sequence, and / or a gene consisting of the polynucleotide sequence, but is not limited thereto. The nucleotide sequence of SEQ ID NO: 29 or SEQ ID NO: 30 can be obtained from a publicly known database, for example, but is not limited to, GenBank of NCBI.

[0057] In the present 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, and 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 have various modifications in the coding region to the extent that the amino acid sequence of the variants of the present application is not changed, taking into account codon degeneracy or codons preferred in the organism in which the variants of the present application are expressed. Specifically, the polynucleotide of the present application is included in the present application as long as it is a polynucleotide sequence that encodes a protein comprising an amino acid sequence having at least 80% homology to SEQ ID NO: 1 or SEQ ID NO: 2, and includes or has the nucleotide sequence of SEQ ID NO: 29 or SEQ ID NO: 30, or a nucleotide sequence that is 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% homologous or identical to the sequence of SEQ ID NO: 29 or SEQ ID NO: 30; or comprises said nucleotide sequence; or consists of or is essentially composed of the nucleotide sequence of SEQ ID NO: 29 or SEQ ID NO: 30, or a nucleotide sequence that is 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% homologous or identical to the sequence of SEQ ID NO: 29 or SEQ ID NO: 30; 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 sequence complementary to all or 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, conditions include those under which polynucleotides with high homology or identity, such as polynucleotides with 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 with lower homology or identity do not hybridize with each other; or conditions for washing once, specifically two to three times, at a salt concentration and temperature equivalent to those used in conventional Southern hybridization, namely, 60°C, 1×SSC, 0.1% SDS, specifically, 60°C, 0.1×SSC, 0.1% SDS, more specifically, 68°C, 0.1×SSC, 0.1% SDS.

[0060] Hybridization requires that two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize to 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 not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[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 can 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-type deacetylase protein in a host cell, thereby inactivating the activity of the SIRT-type 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 as to enable the target polypeptide to be expressed in a suitable host. The expression control region includes a promoter that initiates transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence that regulates 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 naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage or cosmid vectors. Examples of plasmid vectors that may be used include pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pSK, pSKH, and pET. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pSK, pSKH130, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors may be used.

[0068] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using a vector for intracellular chromosomal introduction. The polynucleotide can be inserted into a chromosome by any method known in the art, including, but not limited to, homologous recombination. A selection marker for determining whether or not the polynucleotide has been inserted into the chromosome may be further included. The selection marker is used to select cells transformed with the vector, i.e., to determine whether or not the target nucleic acid molecule has been inserted. Markers that confer selectable phenotypes, such as drug resistance, auxotrophy, resistance to cytotoxic agents, and expression of surface polypeptides, are used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit a different phenotype, allowing the selection of transformed cells.

[0069] In the present application, "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism, thereby expressing the polypeptide encoded by the polynucleotide in the host cell. The transformed polynucleotide may be any polynucleotide that can be expressed in the host cell, regardless of whether it is located intrachromosomally or extrachromosomally. Furthermore, the polynucleotide may comprise DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form that can be expressed in the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct 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 also be introduced into the host cell in its own form and operably linked to sequences necessary for expression in the host cell, but this is not limited thereto.

[0070] Furthermore, 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 microorganisms of the present application, partial or complete modification of a polynucleotide can be induced by, but is not limited to, (a) homologous recombination using a vector for chromosomal introduction into the microorganism, or genome editing using engineered nucleases (e.g., CRISPR-Cas9), and / or (b) treatment with light such as ultraviolet light or radiation and / or chemicals. Methods for partially or completely modifying the gene include methods using DNA recombination techniques. For example, partial or complete deletion of a gene can be achieved by introducing a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism and causing homologous recombination. The introduced nucleotide sequence or vector contains, but is not limited to, a dominant selection marker.

[0072] In the present application, "enhancing" a polypeptide activity means improving the activity of the polypeptide compared to its endogenous activity. The term "enhancing" is used interchangeably with "activation," "up-regulation," "overexpression," and "increase." Here, activation, enhancement, up-regulation, overexpression, and enhancement all encompass the development of an activity not originally present, as well as an improvement in activity compared to endogenous activity or activity prior to modification. The term "endogenous activity" refers to the activity of a specific polypeptide inherently possessed by a parent strain or unmodified microorganism prior to phenotypic change, when the trait is altered through genetic mutation due to natural or artificial factors. This term is also used interchangeably with "activity prior to modification." "Enhancing," "up-regulating," "overexpressing," or "improving" a polypeptide activity compared to its endogenous activity means an improvement in the activity and / or concentration (expression level) of a specific polypeptide inherently possessed by a parent strain or unmodified microorganism prior to phenotypic 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] Various methods well known in the art can be applied to enhance the activity of the polypeptide, and any method can be used as long as it can enhance the activity of the target polypeptide compared to the unmodified microorganism. Specifically, methods such as, but not limited to, conventional methods in molecular biology using genetic engineering and / or protein engineering well known to those skilled in the art (e.g., Non-Patent Documents 13 and 17).

[0075] Specifically, the enhancement of a polypeptide of the present application can be achieved 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 that encodes the polypeptide (e.g., by generating a mutation in the expression regulatory region, substituting a sequence with a higher activity, or inserting a sequence with a higher activity); 3) modifying the nucleotide sequence encoding the start codon or 5'UTR region of a gene transcript that encodes the polypeptide; 4) modifying the amino acid sequence of the polypeptide so as to enhance polypeptide activity; 5) modifying the polynucleotide sequence encoding the polypeptide so as to enhance polypeptide activity (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance polypeptide activity); 6) introducing a foreign polypeptide that exhibits polypeptide activity or a foreign polynucleotide encoding it; 7) optimizing the codons of a polynucleotide encoding the polypeptide; 8) analyzing the tertiary structure of the polypeptide and selectively modifying or chemically modifying exposed portions; or 9) a combination of two or more selected from 1) to 8) above, but is not limited thereto.

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

[0077] 2) Replacing the expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide with a sequence with stronger activity can be achieved, for example, by generating a mutation in the sequence through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by replacing the expression regulatory region with a sequence with higher activity, so as to further enhance the activity of the expression regulatory region. Examples of expression regulatory regions include, but are not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, and sequences regulating the termination of transcription and translation. For example, this can be achieved by replacing the original promoter 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 transcript encoding the polypeptide (3) can be modified, for example, by substituting it with a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate than 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) can be carried out by, but is not limited to, generating a sequence mutation through 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 to enhance the activity of the polypeptide, or by substituting an amino acid sequence or polynucleotide sequence improved to have higher activity or improved activity. Specifically, the substitution can be carried out by, but is not limited to, inserting the polynucleotide into a chromosome by homologous recombination. The vector used here may further contain a selection marker for confirming whether or not it has been inserted into the chromosome. The selection marker is as described above.

[0081] 6) Introduction of an exogenous polynucleotide exhibiting the activity of a polypeptide may be carried out by introducing into a host cell an exogenous polynucleotide encoding a polypeptide exhibiting the same or similar activity as the polypeptide. The exogenous polynucleotide may be of any origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The introduction can be carried out by a known transformation method appropriately selected by those skilled in the art, and the introduced polynucleotide is expressed in the host cell as described above, thereby producing the 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 within a host cell, or by optimizing the codons of an exogenous polynucleotide so as to achieve optimized transcription and translation within a host cell.

[0083] 8) Analyzing the tertiary structure of a polypeptide and selecting and altering or chemically modifying exposed portions may be carried out, for example, by comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, determining candidate template proteins based on the degree of sequence similarity, confirming the structure based on the candidate template proteins, and selecting and altering or modifying exposed portions 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 product produced from the polypeptide.

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

[0086] In this application, "L-methionine / branched-chain amino acid exporter YjeH" refers to a type of amino acid efflux protein (AAE) within 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, a characteristic of the APC superfamily. The L-methionine / branched-chain amino acid exporter YjeH protein and gene sequence can be obtained from publicly known databases, such as, but not limited to, GenBank at NCBI. Examples of such sequences 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 with O-acetylhomoserine export ability, protein with 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 introduced thereinto, thereby enhancing 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 composed 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 to SEQ ID NO: 10. Furthermore, it goes without saying that the present application also includes amino acid sequences in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added, so long as the amino acid sequence has such homology or identity and has the same or corresponding biological activity as the mutant of the L-methionine / branched-chain amino acid exporter YjeH of the present application.

[0090] For example, the microorganism of the present application having the ability to produce O-acetylhomoserine 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, a microorganism into which the protein has been introduced and whose ability to produce O-acetylhomoserine has been further improved.

[0091] Specifically, "protein introduction" means expressing a gene that the microorganism does not originally have in the microorganism, thereby causing the activity of a specific protein to be manifested, or causing the activity of the protein to be increased or improved compared to the endogenous activity or the activity before modification. For example, this may mean introducing a polynucleotide encoding a specific protein into a chromosome in the microorganism, or introducing a vector containing a polynucleotide encoding a specific protein into the microorganism, causing the activity of the protein to be manifested.

[0092] For the purposes of this application, a microorganism containing a mutant of the L-methionine / branched-chain amino acid exporter YjeH refers to a microorganism in which the mutant of the L-methionine / branched-chain amino acid exporter YjeH has been introduced into a microorganism that naturally has weak O-acetylhomoserine exporting ability, thereby improving the O-acetylhomoserine exporting ability. Specifically, the microorganism is characterized by the introduction of the mutant of the L-methionine / branched-chain amino acid exporter YjeH, which increases the extracellular export of O-acetylhomoserine compared to wild-type or unmodified microorganisms, thereby improving the O-acetylhomoserine producing ability. Therefore, while wild-type or unmodified microorganisms are unable to export O-acetylhomoserine or can only produce very small amounts of O-acetylhomoserine, the introduction of the mutant of the L-methionine / branched-chain amino acid exporter YjeH increases the O-acetylhomoserine export of the microorganism, thereby improving the O-acetylhomoserine producing ability.

[0093] Examples of the inner membrane protein YjeH and / or its mutants used in the present application are disclosed in Patent Document 5, 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 acetyl transferase (MetX) activity, but is not limited to this.

[0094] In the present application, "MetX" refers to homoserine acetyltransferase. Specifically, most microorganisms present in nature use O-succinylhomoserine or O-acetylhomoserine as intermediates for methionine biosynthesis. Generally, MetA produces O-succinylhomoserine, while homoserine O-acetyltransferase produces O-acetylhomoserine. Unlike MetA, MetX is not subject to feedback inhibition and has high enzymatic stability. The protein and gene sequences of MetX can be obtained from publicly known databases, such as, but not limited to, NCBI's GenBank. In the present application, "homoserine acetyltransferase" is interchangeably referred to as "MetX" and "homoserine O-acetyltransferase."

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

[0096] The nucleotide sequence of the MetX gene can be obtained from the publicly known database, GenBank, of NCBI. The polynucleotide encoding MetX is included in the present application as long as it encodes a protein comprising an amino acid sequence having at least 80% identity to MetX. Examples of the polynucleotide include, but are not limited to, a polynucleotide having 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% identity to the nucleotide sequence of SEQ ID NO: 26, a polynucleotide comprising the nucleotide sequence, or a polynucleotide consisting essentially 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% identity to the nucleotide sequence of SEQ ID NO: 26.

[0097] An example of the MetX gene used in the present application is disclosed in US Pat. No. 5,629,499, 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 to this.

[0098] Specifically, to increase O-acetyl-homoserine biosynthesis, the aspartokinase activity is further enhanced compared to that of a non-mutated microorganism, and in particular, a mutation (L377K) (Patent Document 6) has been introduced into the gene (lysC) encoding aspartokinase to remove feedback inhibition of L-lysine and L-threonine, but the present invention is not limited to this. The nucleotide sequence of the lysC can be obtained from a publicly known database and may be any nucleotide sequence encoding a protein having aspartokinase activity, such as the nucleotide 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 to this.

[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 encoding cystathionine synthase (metB) is deleted, but this is not limited to this. In the present application, the term "cystathionine γ-synthase" is used interchangeably with "cystathionine synthase." The nucleotide sequence of metB can be obtained from a publicly known database and may be any nucleotide sequence encoding a protein having cystathionine synthase activity, such as the nucleotide 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 to this.

[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 the activity of the non-mutated microorganism, and in particular, the gene (metY) encoding O-acetylhomoserine thiol lyase is deleted, but this is not limited thereto. The nucleotide sequence of the metY gene can be obtained from a publicly known database and may be any nucleotide sequence encoding a protein having O-acetylhomoserine thiol lyase activity, such as the nucleotide sequence of SEQ ID NO: 16.

[0103] Another aspect of the present application provides a method for producing O-acetylhomoserine, comprising culturing the 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 to culture the microorganisms, and those skilled in the art can easily adjust such culture processes depending on the selected strain.

[0105] The term "culturing" in the present application means growing the microorganism of the present application under appropriately controlled environmental conditions. The culturing process of the present application can be carried out using a suitable medium and culture conditions known in the art. Those skilled in the art can easily adjust such a culturing process depending on the selected strain. Specifically, the culturing may be batch, continuous, or fed-batch culture, but is not limited thereto.

[0106] The term "culture medium" as used herein refers to a mixture of nutrients necessary for culturing the microorganism of the present application as its main components, and supplies nutrients such as water essential for survival and growth, growth factors, etc. Specifically, the medium and other culture conditions used to culture the microorganism of the present application may be any medium used for culturing ordinary microorganisms, and the microorganism of the present application can be cultured in an ordinary medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins under aerobic conditions by adjusting the temperature, pH, etc.

[0107] In the present application, examples of carbon sources that can be used include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid can also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) can be used. Any other carbon source can also be used in an appropriate amount. These carbon sources can be used alone or in combination of two or more, but are not limited to these.

[0108] Examples of the nitrogen source that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate, and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its degradation products, and defatted soybean cake or its degradation products. These nitrogen sources can be used alone or in combination of two or more, but are not limited to these.

[0109] Examples of the phosphorus source that can be used include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and sodium-containing salts corresponding thereto. Examples of inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate. Other examples include amino acids, vitamins, and / or suitable precursors. These components or precursors can be added to the medium in a batch or continuous manner. However, the present invention is not limited to these.

[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. Furthermore, foam formation can be suppressed during cultivation using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, oxygen or an oxygen-containing gas may be injected into the culture to maintain an aerobic state, and nitrogen, hydrogen, or carbon dioxide gas may be injected, or no gas may be injected, to maintain anaerobic and microaerobic states, but these are not limiting.

[0111] The temperature of the culture is, but is not limited to, 25° C. to 40° C., more specifically, 28° C. to 37° C. The culture period is continued until a desired amount of useful substance is produced, and is, but is 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 the microorganism of the present application, a step of preparing a medium for culturing the microorganism, or a combination thereof (in any order), for example, before 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 culture medium (culture medium) or the microorganism. The recovery step may be further included after the culture step.

[0114] The recovery may involve 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), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography 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] The method for producing O-acetylhomoserine of the present application may further include a purification step. The purification can 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 a single step, but is not limited thereto.

[0116] The O-acetylhomoserine recovered in this manner 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 using the O-acetylhomoserine accumulated by the above method as a substrate via an enzymatic reaction using O-acetylhomoserine sulfhydrylase or the like.

[0119] When O-acetylhomoserine is used as an L-methionine precursor in the two-step process, O-acetylhomoserine sulfhydrylase derived from a microbial strain belonging to the genus Leptospira, Chromobacterium, or Hyphomonas, more specifically, 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. 5,629,499, 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 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. [Example]

[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 well understood and easily implemented by skilled artisans in the technical field of the present application or a similar technical field. [Example]

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

[0126] Specifically, to prepare the NCgl0616 deletion vector, a pair of primers (SEQ ID NOs: 3 and 4) was designed to amplify the upstream 5' end and a pair of primers (SEQ ID NOs: 5 and 6) was designed to amplify the downstream 3' end, centered 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 ATCC13032 wild-type (WT) chromosome as a template and primers represented by SEQ ID NOs: 3 and 4, and SEQ ID NOs: 5 and 6. The PCR conditions were denaturation at 95°C for 5 minutes, followed by 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 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 around the deletion site of the NCgl0616 gene.

[0129] Using the two amplified DNA fragments as templates, PCR was performed with primers of SEQ ID NOs: 3 and 6. 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 90 seconds, followed by polymerization at 72°C for 7 minutes. As a result, a 1,410-bp DNA fragment containing the site for deletion of the NCgl0616 gene was amplified.

[0130] The pDCM2 vector (SEQ ID NO: 7, Patent Document 8) was digested with SmaI, and the resulting PCR product (2912 bp DNA fragment) was fusion cloned with the pDCM2 vector digested with SmaI restriction enzyme using the In-Fusion® HD Cloning Kit (Clontech). The cloned vector was transformed into Escherichia coli DH5α, and the transformed E. coli was plated on 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 isolated by the plasmid extraction method (Patent Document 9). Finally, the pDCM2-ΔNCgl0616 recombinant vector into which the NCgl0616 deletion cassette had been cloned was constructed.

[0131] The constructed pDCM2-ΔNCgl0616 was transformed into ATCC13032 by electroporation, and a secondary crossover process was performed to obtain ATCC13032ΔNCgl0616, in which the NCgl0616 gene was deleted on the chromosome. Whether the NCgl0616 gene had been inactivated was confirmed by PCR using primers represented by SEQ ID NOs: 8 and 9, followed by comparison with ATCC13032 in which the NCgl0616 gene had not been inactivated.

[0132] [Table 2]

[0133] Example 1-2. Evaluation of O-acetylhomoserine productivity in wild-type strains 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 as follows, and the O-acetylhomoserine in the culture medium was analyzed.

[0134] One platinum loopful of the strain was inoculated into a 250 ml conical baffle flask containing 25 ml of the following medium and cultured with shaking at 33 °C and 200 rpm for 20 hours. The concentration of O-acetylhomoserine was analyzed using HPLC. The analyzed concentrations are shown in Table 3.

[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. 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 was finally confirmed by performing PCR using the primers of SEQ ID NO: 8 and 9 and then 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 Erlenmeyer flask containing 25 ml of the following medium and cultured with shaking at 33 °C and 200 rpm for 20 hours. The O-acetylhomoserine concentration 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, deletion of NCgl0616 in the KCCM12634P strain resulted in O-acetylhomoserine of 1.39 g / L, which was approximately 130% of the productivity of KCCM12634P. Therefore, the above results confirmed that in strains with improved O-acetylhomoserine productivity, deletion of NCgl0616 resulted in a greater improvement in O-acetylhomoserine productivity. Therefore, the results of Examples 1-2 and 2-2 confirmed that deletion and inactivation of the NCgl0616 gene, which is an endogenous gene of ATCC13032 of the present application, improved the productivity of the target amino acid. [Example]

[0144] Construction of NCgl0616 deletion strains in strains with improved O-acetylhomoserine production and evaluation of O-acetylhomoserine and homoserine production - 2 Example 3-1. Strain Construction-1 The metB gene, encoding cystathionine gamma-synthase in the O-acetylhomoserine degradation pathway, was isolated by PCR using Corynebacterium glutamicum ATCC13032 chromosomal DNA as a template. The metB gene sequence (NCBI accession number Ncgl2360, SEQ ID NO: 11) was obtained from the National Institutes of Health GenBank. Based on this information, primers containing the N-terminal portion of the metB gene and a linker (SEQ ID NOs: 12 and 13) and the C-terminal portion of the metB gene and a linker (SEQ ID NOs: 14 and 15) were synthesized. 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 primers of SEQ ID NOs: 12 and 13, and SEQ ID NOs: 14 and 15. PfuUltra was used as the polymerase. 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 of the metB gene and the linker region, and a 527-bp amplified gene containing the C-terminal portion of the metB gene and the linker region were obtained.

[0147] PCR was performed using the two amplified genes obtained as described above as templates. The PCR conditions were 10 cycles of denaturation at 96°C for 60 seconds, annealing at 50°C for 60 seconds, and polymerization at 72°C for 1 minute, followed by addition of SEQ ID NOs: 12 and 15 and further polymerization reaction for 20 cycles. As a result, a 1064-bp inactivation cassette containing the N-terminus, linker, and C-terminus of the metB gene was obtained.

[0148] The pDCM2 vector (SEQ ID NO: 7, Patent Document 8) was digested with SmaI, and the resulting PCR product (1064 bp) was fusion cloned with the pDCM2 vector digested with SmaI restriction enzyme using the In-Fusion® HD Cloning Kit (Clontech). The cloned vector was transformed into Escherichia coli DH5α, and the transformed E. coli was plated on LB solid medium containing 25 mg / L kanamycin. Colonies transformed with the plasmid were selected from the LB medium, and the plasmid was then isolated by the plasmid extraction method (Patent Document 9). Finally, the pDCM2-ΔmetB recombinant vector, into which the metB gene deletion cassette had been cloned, was constructed.

[0149] The constructed pDCM2-ΔmetB vector was transformed into the KCCM12634P strain by electroporation, and a secondary crossover process was performed to obtain KCCM12634PΔmetB, in which the metB gene was inactivated on the chromosome. The inactivated metB gene was identified by PCR using primers represented by SEQ ID NOs: 12 and 15, and then compared with ATCC13032, in which the metB gene was not inactivated.

[0150] Example 3-2. Strain Construction-2 The metY gene, encoding O-acetylhomoserine(thiol)-lyase in the O-acetylhomoserine degradation pathway, was isolated by PCR using chromosomal DNA from 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. Based on this information, primers containing the N-terminal portion of the metY gene and a linker portion (SEQ ID NOs: 17 and 18) and the C-terminal portion of the metY gene and a linker portion (SEQ ID NOs: 19 and 20) were synthesized. 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 primers of SEQ ID NOs: 17 and 18, and SEQ ID NOs: 19 and 20. PfuUltra was used as the polymerase. TMHigh-fidelity DNA polymerase (Stratagene) was used. PCR conditions included 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. This resulted in a 548-bp amplified gene containing the N-terminal portion of the metY gene and a linker, and a 550-bp amplified gene containing the C-terminal portion of the metY gene and a linker. PCR was performed using the two amplified genes obtained as described above as templates. PCR conditions included 10 cycles of denaturation at 96°C for 60 seconds, annealing at 50°C for 60 seconds, and polymerization at 72°C for 1 minute. SEQ ID NOs: 17 and 20 were then added, and the polymerization reaction was carried out for an additional 20 cycles. This resulted in the production of a 1,077-bp inactivation cassette containing the N-terminus, linker, and C-terminus of the metY gene.

[0153] The pDCM2 vector (SEQ ID NO: 7, Patent Document 8) was digested with SmaI, and the resulting PCR product (1077 bp) was fusion cloned with the pDCM2 vector digested with SmaI restriction enzyme using the In-Fusion® HD Cloning Kit (Clontech). The cloned vector was transformed into Escherichia coli DH5α, and the transformed E. coli was plated on LB solid medium containing 25 mg / L kanamycin. Colonies transformed with the plasmid were selected from the LB medium, and the plasmid was then isolated by the plasmid extraction method (Patent Document 9). Finally, the pDCM2-ΔmetY recombinant vector, into which the metY gene deletion cassette had been cloned, was constructed.

[0154] The constructed pDCM2-ΔmetY vector was transformed into the KCCM12634PΔmetB strain constructed in Example 3-1 by electroporation, and a second crossover process was carried out to obtain KCCM12634PΔmetBΔmetY, in which the metY gene was further inactivated on the chromosome. The inactivated metY gene was subjected to PCR using primers represented by SEQ ID NOs: 17 and 20, and then finally confirmed by comparison with ATCC13032, in which the metY gene was not inactivated.

[0155] Example 3-3. Strain Construction-3 A mutation (L377K) (Patent Document 6) was introduced into the lysC gene (SEQ ID NO: 21) encoding aspartokinase from Corynebacterium glutamicum ATCC13032 to enhance lysC gene expression and eliminate feedback inhibition by L-lysine and L-threonine. To construct a vector containing the mutant lysC gene, a pair of primers (SEQ ID NOs: 22 and 23) was designed to amplify the upstream 5' end and a pair of primers (SEQ ID NOs: 24 and 25) was designed to amplify the downstream 3' end, centered around the mutation site. The primer sequences are shown in Table 7.

[0156] [Table 7]

[0157] PCR was performed using the ATCC13032 chromosome as a template and primers represented by 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 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, centered around the mutation in the lysC gene.

[0158] Using the two amplified DNA fragments as templates, PCR was performed with primers of SEQ ID NOs: 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 at 72°C for 7 minutes. As a result, a 1011-bp DNA fragment containing the 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 digested with SmaI, and the resulting PCR product (1011 bp) was fusion cloned with the pDCM2 vector digested with SmaI restriction enzyme using the In-Fusion® HD Cloning Kit (Clontech). The cloned vector was transformed into Escherichia coli DH5α, and the transformed E. coli was plated on LB solid medium containing 25 mg / L kanamycin. Colonies transformed with the plasmid were selected from the LB medium, and the plasmid was then isolated by the plasmid extraction method (Patent Document 9). Finally, the pDCM2-lysC(L377K) recombinant vector was constructed, into which a cassette with the lysC(L377K) gene replaced the lysC(L377K) gene was cloned.

[0160] The resulting pDCM2-lysC(L377K) vector was transformed into the strain KCCM12634PΔmetBΔmetY prepared in Example 3-2 by electroporation, and a secondary crossover process was performed to obtain Corynebacterium glutamicum KCCM12634PΔmetBΔmetY lysC(L377K), which had a nucleotide mutation introduced into the lysC gene on the chromosome. The gene containing the nucleotide mutation was subjected to PCR using primers set forth in SEQ ID NOs: 22 and 25, and then finally confirmed by sequencing by comparing it with the sequence of the wild-type lysC gene.

[0161] Example 3-4. Construction of a strain lacking the endogenous gene NCgl0616-1 To maximize O-acetylhomoserine production, the pDCM2-ΔNCgl0616 vector prepared in Example 1-1 was transformed into the strain KCCM12634PΔmetBΔmetY lysC(L377K) prepared in Example 3-3 by electroporation, and a second crossover process was performed to obtain KCCM12634PΔmetBΔmetY lysC(L377K)ΔNCgl0616, in which the NCgl0616 gene was deleted on the chromosome. Whether the NCgl0616 gene had been inactivated was confirmed by PCR using primers represented by SEQ ID NOs: 8 and 9, followed by comparison with ATCC13032 in which the NCgl0616 gene had not been inactivated.

[0162] Example 3-5. Construction of a strain lacking the endogenous gene NCgl0616 - 2 To amplify the gene encoding homoserine acetyltransferase (MetX), the nucleotide sequence of the MetX gene (NCBI accession number NCgl0624, SEQ ID NO: 26) was obtained from the National Institutes of Health (NIH) GenBank. Based on this sequence, primers (SEQ ID NOs: 27 and 28) were designed with BamHI restriction enzyme sites at both ends to amplify the region from the promoter region (approximately 300 bp upstream of the start codon) to the terminator region (approximately 100 bp downstream of the stop codon). 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 at 72°C for 7 minutes. A 1546-bp DNA fragment encoding 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, which 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 with one inoculation loop 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 9.

[0167] <00006​​​​​​​​​​​​​​As a result, as shown in Table 9, when the control strain KCCM12634PΔmetBΔmetY lysC(L377K) was cultivated, 1.3 g / L of O-acetylhomoserine accumulated, and when KCCM12634PΔmetBΔmetY lysC(L377K)ΔNCgl0616 was cultivated, 1.73 g / L of O-acetylhomoserine accumulated, demonstrating 130% productivity compared to the control. Furthermore, when KCCM12634PΔmetBΔmetY lysC(L377K) / pECCG117-MetX WT was cultivated, 2.10 g / L of O-acetylhomoserine accumulated. When KCCM12634PΔmetBΔmetY lysC(L377K)ΔNCgl0616 / pECCG117-MetX WT was cultivated, 2.83 g / L of O-acetylhomoserine accumulated. This confirmed that the productivity was 135% of that of KCCM12634PΔmetBΔmetY lysC(L377K) / pECCG117-MetX WT.

[0170] Therefore, it was confirmed that the ability to produce the target amino acid was further improved by deleting and inactivating the endogenous gene NCgl0616 in a strain with improved O-acetyl-L-homoserine production ability. [Example]

[0171] Construction of an endogenous gene (NCgl0078) deletion strain and evaluation of its ability to produce O-acetylhomoserine and homoserine Example 4-1. Construction of a deletion vector for deleting NCgl0078 The NCgl0616 and NCgl0078 genes in the Corynebacterium glutamicum ATCC13032 strain are known to be homologs of two SIRT-type deacetylases in Corynebacterium glutamicum ATCC13032 (Non-Patent Document 1). Therefore, we obtained the nucleotide sequence of the NCgl0078 gene, a homolog of the NCgl0616 gene, from the National Institutes of Health GenBank (NCBI accession number NCgl0078, SEQ ID NO: 30). Based on this information, we synthesized primers containing the N-terminal portion and linker portion of the NCgl0078 gene (SEQ ID NOs: 31 and 32) and the C-terminal portion and linker portion of the NCgl0078 gene (SEQ ID NOs: 33 and 34). The primer sequences are shown in Table 10.

[0172] [Table 10]

[0173] PCR was carried out using ATCC13032 wild-type (WT) chromosomal DNA as a template and primers of SEQ ID NOs: 31 and 32, and SEQ ID NOs: 33 and 34. PfuUltra was used as the polymerase. TM High-fidelity DNA polymerase (Stratagene) was used. PCR conditions included 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. This resulted in a 1,223-bp amplified gene containing the N-terminal portion and linker of the NCgl0078 gene, and a 2,576-bp amplified gene containing the C-terminal portion and linker of the NCgl0078 gene. PCR was performed using the two amplified genes obtained as described above as templates. PCR conditions included 10 cycles of denaturation at 96°C for 60 seconds, annealing at 50°C for 60 seconds, and polymerization at 72°C for 1 minute. SEQ ID NOs: 31 and 34 were then added, and the polymerization reaction was carried out for an additional 20 cycles. This resulted in a 3,806-bp inactivation cassette containing the N-terminus, linker, and C-terminus of the NCgl0078 gene.

[0174] The pDCM2 vector (SEQ ID NO: 7, Patent Document 8) was digested with SmaI, and the resulting PCR product (1077 bp) was fusion cloned with the pDCM2 vector digested with SmaI restriction enzyme using the In-Fusion® HD Cloning Kit (Clontech). The cloned vector was transformed into Escherichia coli DH5α, and the transformed E. coli was plated on LB solid medium containing 25 mg / L kanamycin. Colonies transformed with the plasmid were selected from the LB medium, and the plasmid was then extracted. Finally, the pDCM2-ΔNCgl0078 recombinant vector, into which the NCgl0078 gene deletion cassette had been cloned, was constructed.

[0175] The prepared pDCM2-ΔNCgl0078 vector was transformed into the KCCM12634P strain prepared in Example 3-1 by electroporation, 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 represented by SEQ ID NOs: 31 and 34, and then finally confirmed by comparison with ATCC13032, in which the NCgl0078 gene was not inactivated.

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

[0177] One loopful of the strain was inoculated into a 250 ml corner-baffled 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% of that of KCCM12634P. Therefore, from the above results, it was confirmed that in the strain with improved O-acetylhomoserine production ability, the improvement effect of 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 improvement effect of 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 improvement effect of 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 description.

Claims

1. A microorganism of the genus Corynebacterium having an ability to produce O-acetylhomoserine and in which SIRT-type deacetylase protein activity is attenuated, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.

2. 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.

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

4. The Corynebacterium microorganism according to claim 1, wherein the microorganism lacks the nucleic acid base sequence of SEQ ID NO: 29 or SEQ ID NO:

30.

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

6. The Corynebacterium microorganism according to claim 5 , wherein the amino acid sequence of SEQ ID NO: 10 has been introduced into the microorganism.

7. 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 having attenuated SIRT-type deacetylase protein activity.

8. Cultivating a Corynebacterium microorganism capable of producing 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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