Microorganisms in which the activity of pyruvate dehydrogenase complex subunit E1 is increased, and a method for producing O-acetylhomoserine or its derivatives using the same.

Abstract

This application relates to a microorganism in which the activity of pyruvate dehydrogenase complex subunit E1 is increased, and to a method for producing O-acetylhomoserine or its derivatives using the same.

Description

Technical Field

[0001] This application relates to a microorganism with increased activity of pyruvate dehydrogenase complex subunit E1 and a method for producing O-acetylhomoserine or a derivative thereof using the same.

Background Art

[0002] Microorganisms belonging to the genus Corynebacterium are Gram-positive microorganisms widely used in the production of L-amino acids. They are widely used for producing not only proteinaceous amino acids but also non-proteinaceous amino acids such as O-acetylhomoserine, L-homoserine, L-methionine, etc., which are converted from O-acetylhomoserine.

[0003] In order to produce L-amino acids and other useful substances, various studies have been conducted for the development of highly efficient production microorganisms. For example, for the production of L-amino acids, in Corynebacterium strains, approaches specific to the target substance, such as increasing the expression of genes encoding enzymes mainly involved in the biosynthesis of L-amino acids or removing genes unnecessary for the biosynthesis of L-amino acids, are mainly used (US 9644009 B2).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

[0005] [Non-licensed Document 1] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444 [Non-licensed Document 2] Rice et al., 2000, Trends Genet. 16:276-277 [Non-licensed Document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453 [Non-licensed Document 4] Devereux,J.,et al,Nucleic Acids Research 12:387 (1984) [Non-licensed Document 5] Atschul,[S.] [F.,] [ET AL,J MOLEC BIOL 215]:403 (1990) [Non-licensed Document 6] Guide to Huge Computers,Martin J. Bishop,[ED.,] Academic Press,San Diego, 1994 [Non-licensed Document 7] [CARILLO et al.](1988) SIAM J Applied Math 48:1073 [Non-licensed Document 8] Smith and Waterman,Adv. Appl. Math (1981) 2:482 [Non-licensed Document 9] Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation (1978)

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

Non-licensed Document 17

Non-Patent Document 18

Non-Patent Document 19

Summary of the Invention

Problems to be Solved by the Invention

[0006] There is an emerging need for research on methods capable of producing L-amino acids efficiently and in high yields.

Means for Solving the Problems

[0007] One aspect of the present application provides a microorganism with increased activity of pyruvate dehydrogenase complex subunit E1 (AceE).

[0008] In one specific example, the microorganism is a microorganism that produces O-acetylhomoserine or a derivative thereof.

[0009] In another specific example, the microorganism has increased activity of pyruvate dehydrogenase complex subunit E1 (AceE) compared to its intrinsic activity.

[0010] As the microorganism according to any one of the above-described specific examples, the microorganism may be a microorganism belonging to the genus Corynebacterium.

[0011] As the microorganism according to any one of the above-described specific examples, the Corynebacterium genus microorganism may be Corynebacterium glutamicum.

[0012] As a microorganism derived from any of the specific examples mentioned above, the pyruvate dehydrogenase complex subunit E1 may be derived from Corynebacterium glutamicum.

[0013] As a microorganism based on any one of the specific examples mentioned above, the pyruvate dehydrogenase complex subunit E1 may contain the amino acid sequence of Sequence ID No. 1.

[0014] As a microorganism representing any one of the specific examples mentioned above, the pyruvate dehydrogenase complex subunit E1 may be encoded by the polynucleotide of SEQ ID NO: 2 or SEQ ID NO: 3.

[0015] As a microorganism based on any one of the above-mentioned specific examples, the microorganism may have increased production capacity for O-acetylhomoserine or its derivatives compared to a non-myxoid microorganism.

[0016] As a microorganism comprising any one of the specific examples described above, the O-acetylhomoserine or its derivative may be one or more selected from O-acetylhomoserine, L-methionine, and L-homoserine.

[0017] Another aspect of this application provides a method for producing O-acetylhomoserine or a derivative thereof, comprising the step of culturing a microorganism in which the activity of pyruvate dehydrogenase complex subunit E1 is increased in a culture medium.

[0018] In one specific example, the method may further include the step of recovering O-acetylhomoserine or a derivative thereof from the cultured microorganism, the culture product of the microorganism, the fermented product of the microorganism, or the culture medium.

[0019] Another aspect of this application provides a composition for producing O-acetylhomoserine or a derivative thereof, comprising a microorganism in which pyruvate dehydrogenase complex subunit E1 activity is increased compared to its endogenous activity, a culture of the microorganism, a ferment of the microorganism, or a combination of two or more of the above.

[0020] Another aspect of this application provides the use of microorganisms in which pyruvate dehydrogenase complex subunit E1 activity is increased compared to endogenous activity for the production of O-acetylhomoserine or derivatives thereof.

[0021] Another aspect of this application provides a method for producing O-acetylhomoserine or derivatives of the microorganism, comprising modifying the microorganism to increase the pyruvate dehydrogenase complex subunit E1 activity of the present application compared to its endogenous activity. [Effects of the Invention]

[0022] The microorganism described in this application can be used to produce O-acetylhomoserine and its derivatives in high yield. [Modes for carrying out the invention]

[0023] This can be explained in more detail as follows: On the other hand, each description and embodiment disclosed in this application also applies to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the categories of this application are not limited by the specific descriptions described below. In addition, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated as a whole into this specification by reference to more clearly explain the level of the technical field to which this application belongs and the content of this application.

[0024] definition As used in the specification and claims of this application, singular articles ("a," "an," and "the") may include plural subjects unless otherwise specified. Also, plural terms may include singular forms unless otherwise specified. Furthermore, in the specification and claims of this application, unless otherwise specified, the use of "or" is used to include "and / or."

[0025] In this application, the term "about" is presented before a specific number. As used in this application, "about" includes not only the exact number that follows, but also a range that is approximately that number or close to it. The context in which the number is presented can be considered to determine whether it is close to or approximately that specific number. For example, the term "about" could refer to a range of 10% to +10% of a number. Another example is that the term "about" could refer to a range of -5% to +5% of a given number. However, it is not limited to these examples.

[0026] In this application, terms such as “first, second, third…”, “i), ii), iii)…”, or “(a), (b), (c), (d)…” are used to distinguish between different components, and when such terms are used in relation to steps of a method, use, or analysis, these terms are not limited to being performed sequentially or in order. For example, there may be no time interval between these steps, they may be performed simultaneously, or there may be intervals of a few seconds, minutes, hours, days, or months.

[0027] In this application, the term "consisting essentially of" means that the presence of the unspecified components is substantially unaffected by the presence of the unspecified components in the case that the features of the subject matter claimed in this application are not substantially affected by the presence of the unspecified components.

[0028] In this application, the term "consisting of" means that the proportion of the specific components(e) listed below the term totals 100%. The components or features following the term "consisting of" may be essential or mandatory. In some specific examples, any other optional or non-essential components or features other than those following "consisting of" are excluded.

[0029] In this application, the term “comprising” means the presence of the features, stages, or components described below in the term, and does not exclude the presence of one or more additional features, stages, or components. In this application, the components or features described below “comprising” may be essential or mandatory, but some specific examples may further include other optional or non-essential components or features.

[0030] Proteins, polypeptides In this application, the terms "protein" or "polypeptide" mean a polymer or oligomer of continuous amino acid residues. In this application, "polypeptide," "protein," and "peptide" are used interchangeably with "amino acid sequence."

[0031] Depending on the context, an active protein, polypeptide, or peptide is referred to as an "enzyme." In this application, unless otherwise specified, amino acid sequences are described as N-terminal → C-terminal orientation.

[0032] In this application, the term "mature polypeptide" means a polypeptide in a form that lacks a signal sequence or propeptide sequence. A mature protein / polypeptide / peptide may also be a functional form of a protein / polypeptide / peptide. A mature polypeptide may be the final post-translational form or the final form after post-translational modification. Examples of post-translational modification include, but are not limited to, modification of the N or C terminus, glycosylation, phosphorylation, and removal of a leader sequence.

[0033] In this application, the terms "contains" an amino acid sequence described by a specific sequence number, "consists of" an amino acid sequence described by a specific sequence number, or "has" an amino acid sequence described by a specific sequence number include polypeptides or proteins in which some amino acids are deleted, altered, substituted, conservatively substituted, or added, provided that they have the same or corresponding activity as the polypeptide or protein consisting of the amino acid sequence of the sequence number. For example, the polypeptide or protein may also include polypeptides or proteins having additions or deletions of sequences within or before / after (N-terminus or C-terminus) the polypeptide or protein sequence that do not alter the function of the protein, naturally occurring mutations, silent mutations, or conservative substitutions, provided that they have the same or corresponding activity as the polypeptide or protein.

[0034] As a specific example, polypeptides conjugated with an N-terminal signal (or leader) sequence involved in the translocation of polypeptides (proteins) co-translationally or post-translationally, or polypeptides conjugated with other sequences or linkers to enable the identification, purification, or synthesis of polypeptides (proteins), may also be included in the range of polypeptides of the amino acid sequences described by the particular sequence number.

[0035] In this application, the term "conservative substitution" means replacing one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; amino acids with nonpolar side chains (nonpolar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. As another example, amino acids can be classified into electrically charged amino acids (arginine, lysine, histidine, glutamic acid, aspartic acid) and uncharged amino acids (also called neutral amino acids) (glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine). As yet another example, phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. As yet another example, valine, leucine, and isoleucine can be classified as branched amino acids.As another example, the 20 amino acids can be classified by size into five groups, starting with the smallest amino acid group: glycine, alanine, serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, tyrosine. However, this is not necessarily the only classification. Typically, conservative substitutions have little to no effect on polypeptide activity.

[0036] Polynucleotides In this application, the term "gene" means a polynucleotide comprising a polynucleotide encoding a polypeptide and the regions before and after the coding region. In some specific examples, a gene may have sequences (introns) inserted between each coding region (exon).

[0037] In this application, the terms "polynucleotide, nucleic acid, or nucleic acid molecule" mean a polymer of nucleotides in which nucleotide monomers are covalently linked together in a long chain, and which is a DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA) chain of a certain length or longer.

[0038] homology, identity In this application, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid or nucleotide sequences that correspond to each other, and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0039] The homology or identity of sequences of conserved polynucleotides or polypeptides can be determined by standard sequencing algorithms, and a default gap penalty established by the program used may be used in conjunction with them. Substantially homologous or identical sequences can generally be hybridized under moderate to high stringent conditions by at least 50%, 60%, 70%, 80%, or 90% of the entire sequence or its full length. It is obvious that hybridization also includes polynucleotides containing codons in general or codon degeneracy in polynucleotides.

[0040] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using known computer algorithms such as the "FASTA" program with default parameters, for example, as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.] [ET AL, J MOLEC BIOL 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.] Academic Press, San (Including Diego, 1994, and [CARILLO et al.] (1988) SIAM J Applied Math 48:1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information Databases.

[0041] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as Needleman et al. (1970), J Mol Biol. 48:443, as is publicly known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In summary, the GAP program can be defined as the total number of symbols in the shorter of two sequences divided by the number of similarly sequenced symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program may include: (1) unitary matrices (with values ​​of 1 for identity and 0 for non-identity), PAM Matrix (see Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation (1978)), Gribskov et al (1986) Nucl. Acids Res. 14:6745 weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a 3.0 penalty for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0042] Furthermore, whether any two polynucleotide sequences are homologous, similar, or identical can be determined by comparing the sequences in a Southern hybridization experiment under defined stringent conditions. The defined appropriate hybridization conditions may be, but are not limited to, those within the scope of the art and well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

[0043] In this application, the term "stringent condition" means conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8). For example, the conditions may be such that polynucleotides with high homology or identity hybridize with each other, with homology or identity levels of 60% or more, 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, but do not hybridize with polynucleotides with lower homology or identity levels. Alternatively, the conditions may be such that the polynucleotides are washed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of a normal Southern hybridization: 60°C, 1×SSC, 0.1% SDS, more specifically 60°C, 0.1×SSC, 0.1% SDS, or more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0044] The aforementioned hybridization requires that the two nucleotides have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. The term “complementary” is used to describe the relationship between nucleotide bases that can hybridize with one another. For example, in DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of this application may also include isolated nucleic acid fragments that are complementary to the overall sequence, as well as substantially similar base sequences.

[0045] For example, polynucleotides homologous or identical to the polynucleotides of this application can be detected using hybridization conditions that include a hybridization step at a Tm value of 55°C, and under the conditions described above. The Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0046] The appropriate stringency for hybridizing the aforementioned polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are well known in the art (e.g., J. Sambrook et al., ibid.).

[0047] nucleic acid structures, vectors, transformations In this application, the term "nucleic acid construct" means a single or double-stranded nucleic acid molecule containing one or more regulatory sequences, which is artificially synthesized, manipulated to contain specific sequences in a manner not found in nature, or isolated from nature.

[0048] As used in this application, the term “vector” means a DNA product for delivering a target polynucleotide into a suitable host or host cell. For example, it may include a base sequence of a polynucleotide encoding the target polypeptide, operably linked to a suitable regulatory region (or regulatory sequence) so that the target polypeptide can be expressed in a suitable host. The regulatory region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences regulating the termination of transcription and decoding. After being transformed into a suitable host cell, the vector can replicate or function independently of the host genome and integrate into the genome itself.

[0049] The vectors used in this application are not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ, pDC, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. As an example, pDZ, pDC, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, and pDCM2 (WO2021-187781 A1) vectors can be used.

[0050] As an example, a target polynucleotide can be inserted into a chromosome via a chromosome insertion vector within a cell. The insertion of the polynucleotide into the chromosome can be carried out by any method known in the art, such as homologous recombination, but is not limited to these methods. A selection marker may further be included to confirm the presence or absence of the chromosome insertion. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of the target nucleic acid molecule insertion, and may be a marker that confers a selectable phenotype, such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of a surface polypeptide. Transformed cells can be selected because, in an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes.

[0051] In this application, the term "transformation" means altering the genetic traits of a host cell by introducing a vector containing a target polynucleotide into the host cell. The transformed polynucleotide may be located either inserted into or outside the chromosome of the host cell. The polynucleotide may also contain DNA or RNA. The polynucleotide may be introduced in a form appropriate to the purpose of introduction. For example, a polynucleotide for expressing a target polypeptide may be introduced into the host cell in the form of an expression cassette, which is a gene structure containing all the elements necessary for its expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, all operably linked to the polynucleotide. The expression cassette may also be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form and operably linked to the sequence necessary for expression in the host cell, but is not limited to this.

[0052] In this application, the term “operatably linked” means a configuration in which a regulatory sequence is positioned appropriately so that the regulatory sequence controls the expression of a coding sequence. Accordingly, “operatably linked” includes regulatory regions of known functional domains, such as promoters, terminators, signal sequences, or enhancer regions, that have a desired activity, and that are attached to or linked to a target (gene or polypeptide) so as to regulate its expression, secretion, or function, or to that target, by the desired activity. For example, it means a promoter sequence that initiates and mediates the transcription of a polynucleotide encoding a polypeptide, and that polynucleotide sequence is functionally linked to the polynucleotide sequence.

[0053] In this application, the term "expression" includes, but is not limited to, any step involved in the production of a polypeptide, such as transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0054] In this application, the term "expression vector" means a linear or cyclic nucleic acid molecule comprising a target polynucleotide sequence and a regulatory sequence operably linked for its expression.

[0055] In this application, the term "regulatory sequence" means a polynucleotide sequence necessary for regulating the expression of a target polynucleotide sequence. Each regulatory sequence may be native (of the same origin) or foreign (derived from another gene) sequence, a variant thereof, or another artificial sequence with respect to the coding sequence. Examples of such regulatory sequences include leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, operator sequences, sequences encoding ribosome binding sites, and sequences that regulate transcription and translation termination. The smallest unit of such regulatory sequence may include a promoter and transcription and translation termination sequences.

[0056] In relation to cells, polynucleotides, polypeptides, or vectors, the term “recombinant” in this application means that a cell, polynucleotide, polypeptide, or vector has been modified by the introduction of a heterologous nucleic acid or polypeptide or by alteration of a native polynucleotide or polypeptide, or that a cell has been derived from such a modified cell. For example, a recombinant cell may express genes not found in the cell’s native (non-recombinant) form, or native genes that are expressed, not expressed at all, or expressed abnormally.

[0057] microorganisms In this application, the terms "microorganism (or strain)" include all wild-type microorganisms and prokaryotic or eukaryotic microorganisms that have undergone natural or artificial genetic modification, and which are microorganisms in which a particular mechanism has been weakened or increased due to causes such as the insertion of external genes or the increase or inactivation of endogenous gene activity, and may include microorganisms that undergo genetic modification for the production of a target polypeptide, protein, or product. In this application, "microorganism" and "strain" may be used interchangeably without limitation as they have the same meaning.

[0058] For example, the microorganism of this application may be, but is not limited to, a microorganism in which the pyruvate dehydrogenase complex subunit E1 activity is increased compared to its endogenous activity (e.g., a recombinant strain).

[0059] In this application, the term "microorganism capable of producing O-acetylhomoserine or its derivatives" refers to a microorganism capable of producing O-acetylhomoserine or its derivatives within a living organism, and may include all microorganisms that do not inherently possess the ability to produce O-acetylhomoserine or its derivatives but have been conferred the ability to produce O-acetylhomoserine or its derivatives, or microorganisms that inherently possess the ability to produce O-acetylhomoserine or its derivatives. The ability to produce O-acetylhomoserine or its derivatives can be conferred or enhanced through selective breeding.

[0060] In this application, the term "non-myxoid microorganism (strain)" does not exclude microorganisms (strains) that include naturally occurring mutations, but rather means a wild-type microorganism (strain) or a naturally occurring microorganism (strain) itself, or a microorganism (strain) before its characteristics are altered by genetic mutation due to natural or artificial factors. The aforementioned "non-myxoid microorganism (strain)" may be used interchangeably with "pre-deformation microorganism (strain)," "non-mutant microorganism (strain)," "parent microorganism," "parent strain," "wild-type microorganism (strain)," "reference microorganism (strain)," or "standard microorganism (strain)." In this application, a non-myxoid microorganism means, but is not limited to, a microorganism in which the pyruvate dehydrogenase complex subunit E1 activity of this application has not been increased compared to the endogenous activity, or before it has been increased.

[0061] Furthermore, in this application, the non-mutated microorganism may be, but is not limited to, a microorganism containing an amino acid sequence consisting of SEQ ID NO: 1, or a polynucleotide consisting of SEQ ID NO: 2 or SEQ ID NO: 3.

[0062] Increased polypeptide activity In this application, the term "increase" of polypeptide activity means that the activity of a polypeptide in a host cell increases compared to its endogenous activity. This increase may be used interchangeably with terms such as activation, upregulation, overexpression, and enhancement. The host may be a prokaryotic or eukaryotic microorganism.

[0063] The increase in polypeptide activity may include exhibiting polypeptide activity that the host microorganism did not inherently possess, or exhibiting polypeptide activity that is improved compared to the intrinsic activity or the activity before deformation.

[0064] For example, the phrase "exhibiting activity that was not originally present" may, but is not limited to, the "introduction of polypeptides (proteins)." The introduction of polypeptides (proteins) means that a gene that was not originally present in the microorganism is expressed within the microorganism, thereby exhibiting activity of a specific polypeptide (protein), or exhibiting increased or improved activity compared to the intrinsic activity or pre-modification activity of the polypeptide (protein). For example, a polynucleotide encoding a specific polypeptide (protein) may be introduced into the chromosome of the microorganism, or a vector containing a polynucleotide encoding a specific polypeptide (protein) may be introduced into the microorganism, and its activity may be exhibited.

[0065] The aforementioned "intrinsic activity" refers to the activity of a specific polypeptide that was originally present in the microorganism or non-myxomycete before the trait change due to genetic mutation caused by natural or artificial factors. This can be used interchangeably with "activity before the change."

[0066] An increase in polypeptide activity compared to its endogenous activity means that the activity and / or concentration (expression level) of a particular polypeptide has improved compared to what was originally present in the microorganism before the trait change or in the non-myxogenic microorganism.

[0067] For example, the increase may be such that the activity of the corresponding protein / polypeptide was absent, or the activity or concentration was increased by approximately 1%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, or 500%, up to approximately 1000% or 2000% or more, relative to the activity or concentration in the initial microbial strain.

[0068] The increase in the polypeptide activity can be achieved by introducing an exogenous polypeptide or by increasing the activity of an endogenous polypeptide. The presence or absence of an increase in the polypeptide activity can be confirmed by an increase in the polypeptide's activity level, expression level, or the amount of product resulting from the polypeptide activity.

[0069] The increase in the activity of the polypeptide can be achieved by applying various methods well known in the field, and is not limited as long as it can increase the activity of the target polypeptide compared to the microorganism before deformation. Specifically, this may involve, but is not limited to, the use of gene engineering and / or protein engineering, which are routine methods in molecular biology and are well known to ordinary technicians in the field (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0070] Specifically, the increase in the activity of the polypeptide in this application is, 1) Increase in the intracellular copy number of polynucleotides encoding polypeptides; 2) Modification of gene expression regulatory regions on chromosomes that encode polypeptides (e.g., mutation within the regulatory region, replacement with a more active sequence, or insertion of a more active sequence); 3) Modifications of the nucleotide sequence encoding the start codon or 5'-UTR region of a polypeptide-encoding gene transcript; 4) Modification of the amino acid sequence of the polypeptide so as to increase polypeptide activity; 5) Modification of the polynucleotide sequence encoding the polypeptide so as to increase polypeptide activity (for example, modification of the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified to increase polypeptide activity); 6) Introduction of a foreign polypeptide exhibiting polypeptide activity or a foreign polynucleotide encoding it; 7) Codon optimization of polynucleotides encoding polypeptides; 8) Analyze the tertiary structure of the polypeptide, select exposed areas, and deform or chemically modify them; 9) Regulation of the intracellular localization of proteins (polypeptides); or 10) A combination of two or more selected from items 1) to 9) above is also acceptable, but is not particularly limited thereto.

[0071] for example, The increase in the intracellular copy number of the polynucleotide encoding the polypeptide described in 1) above may be achieved by introducing a vector containing the polynucleotide encoding the polypeptide operably linked to an appropriate regulatory sequence into a host cell. The vector may be a vector capable of replicating and functioning independently of the host. Alternatively, one or more copies of the polynucleotide encoding the polypeptide operably linked to an appropriate regulatory sequence may be introduced into the chromosomes of a host cell. The introduction into the chromosomes can be achieved by introducing a vector into the host cell that can insert the polynucleotide into the chromosomes of the host cell, but is not limited to this. The vector is as described above. The regulatory sequence may be native (of the same origin) or foreign (derived from another gene) sequence, or a variant thereof, or another artificial sequence, and can induce the expression of the polynucleotide in the host cell.

[0072] The replacement of a gene expression regulatory region (or expression regulatory sequence) on a chromosome encoding a polypeptide with a more potent sequence may, for example, involve generating a sequence mutation by deletion, insertion, substitution, or a combination thereof, to further increase the activity of the expression regulatory region, or by replacing it with a sequence having stronger activity. The expression regulatory region may include, but is not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, and sequences that regulate transcription and decoding termination. For example, the original promoter may be replaced with a potent promoter, but is not limited to this.

[0073] Examples of well-known strong promoters include, but are not limited to, the cj1-cj7 promoter (US Registered Patent US 7662943 B2), the lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Registered Patent US 10584338 B2), O2 promoter (US Registered Patent US 10273491 B2), tkt promoter, and yccA promoter.

[0074] The modification of the base sequence of the start codon or 5'-UTR region of the polypeptide-encoding gene described in 3) above may, for example, be by substitution with another start codon that has a higher polypeptide expression rate compared to the endogenous start codon, but is not limited thereto.

[0075] The modification of the amino acid sequence or polynucleotide sequence of the polypeptide described in 4) and 5) above may be, but not limited to, the deletion, insertion, non-conservative or conservative substitution, or combination thereof of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, thereby increasing the activity of the polypeptide, or by replacing it with an improved amino acid sequence or polynucleotide sequence that has stronger activity or an improved amino acid sequence or polynucleotide sequence that has increased activity. Specifically, the replacement can be carried out by inserting a polynucleotide into the chromosome by homologous recombination, but is not limited to this. The vector used in this case may further include a selection marker for confirming the presence or absence of chromosomal insertion. The selection marker is as described above.

[0076] The introduction of a foreign polynucleotide exhibiting polypeptide activity (6) above may be the introduction of a foreign polynucleotide encoding a polypeptide exhibiting the same or similar activity as the polypeptide into the host cell. The foreign polynucleotide is not restricted in its origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The method used for the introduction can be appropriately selected by those skilled in the art from known transformation methods, and the introduction of the polynucleotide into the host cell generates the polypeptide, thereby increasing its activity.

[0077] The codon optimization of the polynucleotide encoding the polypeptide described in 7) above may be codon optimization of the endogenous polynucleotide so that transcription and translation are increased in the host cell, or the codon of the exogenous polynucleotide may be optimized so that optimized transcription and translation occur in the host cell.

[0078] 8) Analyzing the tertiary structure of the polypeptide and selecting exposed sites to deform or chemically modify may, for example, involve comparing the sequence information of the polypeptide to be analyzed with a database containing sequence information of known proteins to determine candidate template proteins according to the degree of sequence similarity, confirming the structure based on that, and selecting exposed sites to deform or chemically modify.

[0079] The intracellular positional regulation of a protein (polypeptide) described in 9) above may involve targeting the protein (polypeptide) to a specific organelle or specific intracellular space within the cell. For example, it may involve targeting the periplasm or cytoplasm through the addition or removal of a leader sequence that functions to target the protein (polypeptide), but is not limited thereto.

[0080] Such an increase in polypeptide activity may be, but is not limited to, an increase in the activity or concentration of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild-type or pre-mutation microorganism, or an increase in the amount of product produced from the polypeptide.

[0081] Weakening of polypeptide activity In this application, the term “weakening” of the activity of a polypeptide (including, for example, the protein specified in the name of each enzyme) encompasses all concepts of reduced or absent activity compared to its endogenous activity. Such weakening may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decline, reduce, and attenuation.

[0082] The aforementioned weakening may include cases where the activity of the polypeptide itself is reduced or eliminated compared to the polypeptide activity originally possessed by the microorganism due to mutations in the polynucleotide encoding the polypeptide, cases where the overall polypeptide activity and / or concentration (expression level) in the cell is lower than that of the natural strain due to inhibition of the expression of the gene encoding the polynucleotide or inhibition of translation into the polypeptide, cases where the polynucleotide is not expressed at all, and / or cases where there is no polypeptide activity despite the expression of the polynucleotide. The "inactivation, deficiency, reduction, downregulation, decrease, and attenuation" of polypeptide activity compared to its endogenous activity means that it has decreased compared to the activity of the specific polypeptide originally possessed by the parent strain or non-mutant microorganism before the trait change.

[0083] The weakening of the activity of such polypeptides can be achieved by any method known in the art, but is not limited to these methods, and can be achieved by applying a variety of well-known methods in the field (e.g., Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).

[0084] Specifically, the weakening of the polypeptide activity of this application is 1) Deletion of all or part of the gene encoding the polypeptide; 2) Modification of the gene expression regulatory region (or gene expression regulatory sequence) so that the expression of the gene encoding the polypeptide is reduced; 3) Modification of the amino acid sequence constituting the polypeptide (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence) so as to remove or weaken the activity of the polypeptide; 4) Modification of the gene sequence encoding the polypeptide so that the polypeptide activity is removed or weakened (for example, deletion / substitution / addition of one or more nucleic acid bases in the nucleic acid sequence of the polypeptide gene so that it encodes a polypeptide that has been modified so that the polypeptide activity is removed or weakened); 5) Modifications of the nucleotide sequence encoding the start codon or 5'-UTR region of a polypeptide-encoding gene transcript; 6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide; 7) Addition of a sequence complementary to the Shine-Dalgarno sequence to the Shine-Dalgarno sequence of the polypeptide-encoding gene in order to form a secondary structure that cannot be attached to ribosomes; 8) Addition of a promoter that is transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the polypeptide-coding gene sequence (Reverse transcription engineering, RTE); 9) Regulation of the intracellular localization of proteins (polypeptides); or 10) A combination of two or more selected from items 1) to 9) above is also acceptable, but is not particularly limited thereto.

[0085] for example, The deletion of part or all of the gene encoding the polypeptide described in 1) above may be the removal of the entire polynucleotide encoding the endogenous target polypeptide within the chromosome, replacement with a polynucleotide in which some nucleotides are deleted, or replacement with a marker gene.

[0086] Furthermore, the modification of the expression regulatory region (or expression regulatory sequence) described in 2) above may involve deletion, insertion, non-conservative or conservative substitution, or a combination thereof, resulting in the occurrence of a mutation on the expression regulatory region (or expression regulatory sequence) or replacement with a sequence having 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 that regulates the termination of transcription and decoding.

[0087] The modifications of the amino acid sequence or polynucleotide sequence described in 3) and 4) above may include, but are not limited to, the deletion, insertion, non-conservative or conservative substitution, or combination thereof of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, thereby causing a sequence mutation, or replacement with an amino acid sequence or polynucleotide sequence modified to have weaker activity or an amino acid sequence or polynucleotide sequence modified to have no activity, in order to weaken the activity of the polypeptide. For example, gene expression can be inhibited or weakened by introducing a mutation in the polynucleotide sequence to form a stop codon, but is not limited to this.

[0088] The modification of the start codon or the nucleotide sequence encoding the 5'-UTR region of the gene transcript encoding the polypeptide (5) above may, but is not limited to, substitution with, for example, a nucleotide sequence encoding another start codon with a lower polypeptide expression rate compared to the endogenous start codon.

[0089] The introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide (6) can be done by referring to, for example, the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews-Trends in Genetics, Vol. 1(1) 1986].

[0090] 7) In order to form a secondary structure that ribosomes cannot attach to, the addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence in the polypeptide-encoding gene may make mRNA translation impossible or reduce its rate.

[0091] Furthermore, the addition of a promoter that is transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE) may weaken the activity by creating complementary antisense nucleotides in the transcript of the gene encoding the polypeptide.

[0092] The intracellular positional regulation of a protein (polypeptide) described in 9) above may involve targeting the protein (polypeptide) to a specific organelle or specific intracellular space within the cell. For example, it may involve targeting the periplasm or cytoplasm through the addition or removal of a leader sequence that functions to target the protein (polypeptide), but is not limited thereto.

[0093] Such weakening of polypeptide activity may be, but is not limited to, a reduction in the activity or concentration expression level of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild-type or pre-deformation microbial strain, or a reduction in the amount of product produced from the polypeptide.

[0094] Modification of some or all of the polynucleotides in the microorganisms of this application may be induced by (a) homologous recombination using a chromosome insertion vector within the microorganism, or by genome editing using an engineered nuclease (e.g., CRISPR-Cas9), and / or (b) by treatment with light such as ultraviolet light and radiation and / or chemical substances.

[0095] culture In this application, the term "culture" means growing microorganisms under appropriately controlled environmental conditions. The culture process can be carried out according to suitable culture media and culture conditions known in the art. Such a culture process can be easily adapted and used by those skilled in the art depending on the selected microorganisms. Specifically, the culture may be batch, continuous, and / or fed-batch.

[0096] In this application, the term "culture medium" refers to a substance mixed primarily with nutrients necessary for culturing microorganisms, supplying essential nutrients and growth factors, including water, which are indispensable for survival and growth. Specifically, the culture medium and other culture conditions used for culturing microorganisms in this application are not particularly limited and any culture medium used for culturing ordinary microorganisms can be used. However, the microorganisms in this application can be cultured under aerobic conditions in an ordinary culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, with temperature, pH, etc., adjusted. For example, culture media for Corynebacterium microorganisms can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0097] In this application, the carbon source may include carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn maceration can be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and other appropriate amounts of carbon sources can be used in a variety of ways without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited thereto.

[0098] The nitrogen sources may 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 like glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn maceration, casein hydrolysates, fish or their decomposition products, defatted soy cake or its decomposition products. These nitrogen sources may be used individually or in combination of two or more, and are not limited to these uses.

[0099] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or their corresponding sodium-containing salts. Inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate, and may also include amino acids, vitamins, and / or suitable precursors. These components or precursors may be added to the culture medium in batches or continuously, but are not limited to these methods.

[0100] Furthermore, during the cultivation of the microorganisms of this application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the culture medium in an appropriate manner to adjust the pH of the culture medium. In addition, during cultivation, antifoaming agents such as fatty acid polyglycol esters can be used to suppress the formation of bubbles. Furthermore, in order to maintain an aerobic state in the culture medium, oxygen or oxygen-containing gas can be injected into the culture medium, or in order to maintain an anaerobic and microaerobic state, no gas can be injected, or nitrogen, hydrogen, or carbon dioxide gas can be injected, but this is not limited to these methods.

[0101] In the culture described in this application, the culture temperature can be maintained at 20-45°C, specifically 25-40°C, and the culture can be performed for approximately 10-160 hours, but is not limited to this.

[0102] In this application, the term "culture" means a culture solution, concentrated culture solution, dried culture solution, culture filtrate, concentrated culture filtrate, or dried culture filtrate obtained by culturing a specific microorganism in a culture medium, wherein the culture solution contains the specific microorganism, and the culture filtrate substantially does not contain the specific microorganism (where substantially means removing the specific microorganism separated by filtration or the like, and does not mean that the microorganism is completely removed from the filtrate). The dosage form of the culture is not limited, and may be, for example, a liquid, emulsion, or solid.

[0103] In this application, the term "fermentation" refers to a process in which microorganisms use their enzymes to decompose organic matter, but which is not a putrefaction reaction. Fermentation and putrefaction reactions proceed through similar processes, but if the decomposition results in the production of useful substances, it is called fermentation, while if it produces foul odors or harmful substances, it is called putrefaction.

[0104] In this application, the method for obtaining the fermented product from the microorganism is not particularly limited and can be obtained by methods commonly used in the art or similar fields.

[0105] In this application, the term "fermented product" includes not only the fermented substance itself, but also all kinds of substances including fermented products generated from microorganisms, such as a microbial culture medium in which microorganisms and cultures coexist, a fermented product obtained by filtering microorganisms from the culture medium, a fermented product obtained by sterilizing the culture medium and filtering it, an extract obtained by extracting the fermented product or a culture medium containing it, a diluted solution or concentrate obtained by diluting the fermented product or its extract, a dried product obtained by drying the fermented product or its extract, and a lysate obtained by collecting and crushing the microbial cells of the microorganisms.

[0106] Specific description of this application The specific example of this application will be explained in more detail below.

[0107] One aspect of this application provides a microorganism in which the activity of pyruvate dehydrogenase complex subunit E1 is increased compared to its endogenous activity.

[0108] As an example of this application, the microorganism of this application may have the ability to produce O-acetylhomoserine or its derivatives.

[0109] The increased pyruvate dehydrogenase complex subunit E1 activity is defined as the increased production capacity of the microorganisms of this application for O-acetylhomoserine or its derivatives compared to the production capacity of natural wild-type microorganisms or non-mutant microorganisms (e.g., microorganisms expressing polypeptides having wild-type pyruvate dehydrogenase complex subunit E1 activity (e.g., the polypeptide of SEQ ID NO: 1) or microorganisms in which the pyruvate dehydrogenase complex subunit E1 activity of this application has not been increased compared to its endogenous activity, or before it was increased).

[0110] For example, the pyruvate dehydrogenase complex subunit E1 activity can be measured by measuring the production capacity or yield of O-acetylhomoserine or its derivatives, but is not limited to this.

[0111] In this application, "Pyruvate Dehydrogenase Complex subunit E1" is a component of the pyruvate dehydrogenase complex and may be a polypeptide containing an E1 active site that converts pyruvate to acetyl-CoA and CO2 in pyruvate dehydrogenase.

[0112] The pyruvate dehydrogenase complex subunit E1 of this application may be used interchangeably with "AceE". The pyruvate dehydrogenase complex subunit E1 is publicly known in the art and may, but is not limited to, be encoded by aceE. The amino acid and polynucleotide sequences of the pyruvate dehydrogenase complex subunit E1 can be obtained from known databases, such as NCBI's GenBank, but is not limited to these.

[0113] For example, the pyruvate dehydrogenase complex subunit E1 may include the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 60% or more homology or identity thereto. However, it is not limited to this, as long as it has the same activity as pyruvate dehydrogenase complex subunit E1. Specifically, a protein that exhibits the same efficacy as pyruvate dehydrogenase complex subunit E1, even if it includes a sequence in which some of the amino acid sequences of SEQ ID NO: 1 are deleted, modified, substituted, or added, may be included in the pyruvate dehydrogenase complex subunit E1. Furthermore, any protein having, containing, consisting of, or substantially consisting of the amino acid sequence of Sequence ID No. 1, or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with the aforementioned sequence, and exhibiting efficacy corresponding to the pyruvate dehydrogenase complex subunit E1, may be included in the pyruvate dehydrogenase complex subunit E1. For example, the pyruvate dehydrogenase complex subunit E1 may be an exogenous protein or a protein endogenously present in Corynebacterium microorganisms or Corynebacterium glutamicum, but is not limited thereto. Specifically, it may be a pyruvate dehydrogenase complex subunit E1 consisting of the amino acid sequence of Sequence ID No. 1 endogenously present in Corynebacterium microorganisms or Corynebacterium glutamicum, but is not limited thereto. An example of this is NCBI Reference No. WP_011014985.1.

[0114] Furthermore, the polynucleotide encoding pyruvate dehydrogenase complex subunit E1 having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 60% or more homology or identity thereto can be manufactured based on codon information known to the art. For example, the protein may be encoded by a polynucleotide having, containing, or consisting of, or substantially composed of, the sequence of SEQ ID NO: 2 or 3, or a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the sequence of SEQ ID NO: 2 or 3.

[0115] In this application, a gene containing the base sequence described by a specific sequence number may be mixed with a polynucleotide containing the base sequence described by a specific sequence number.

[0116] The polynucleotides of this application may undergo various modifications to their coding region, either due to codon degeneracy or considering the preferred codons in organisms expressing the pyruvate dehydrogenase complex subunit E1 of this application, as long as these modifications do not alter the amino acid sequence of the pyruvate dehydrogenase complex subunit E1 of this application. Therefore, it is obvious that polynucleotides translated by codon degeneracy into polypeptides consisting of the amino acid sequence of the pyruvate dehydrogenase complex subunit E1 of this application, or polypeptides homologous or identical thereto, may also be included in the polynucleotides of this application. For example, the polynucleotides of this application may be SEQ ID NO: 2, SEQ ID NO: 3, or their degenerated sequences.

[0117] Other examples include, but are not limited to, a polynucleotide having or containing a nucleotide sequence that is 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with SEQ ID NO: 2 or SEQ ID NO: 3, or consisting of or substantially comprising a nucleotide sequence that is 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with SEQ ID NO: 2 or SEQ ID NO: 3.

[0118] Furthermore, the polynucleotides of this application may include, without limitation, probes produced from known gene sequences, such as sequences that hybridize under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence of this application and encode the pyruvate dehydrogenase complex subunit E1 of this application.

[0119] In this application, the term "O-acetylhomoserine or its derivatives" includes O-acetylhomoserine and products produced using O-acetylhomoserine as a precursor. As one specific example, the O-acetylhomoserine or its derivatives may be one or more selected from O-acetylhomoserine, L-methionine, and L-homoserine.

[0120] For the purposes of this application, the microorganisms of this application may include all microorganisms in which the activity of pyruvate dehydrogenase complex subunit E1 is increased compared to its endogenous activity and which are capable of producing the target O-acetylhomoserine or its derivatives. For example, the microorganisms of this application are characterized by increased production capacity of O-acetylhomoserine or its derivatives due to increased pyruvate dehydrogenase complex subunit E1 activity compared to its endogenous activity, and may be, but are not limited to, genetically modified or recombinant microorganisms. Specifically, the recombinant microorganisms with increased production capacity of O-acetylhomoserine or its derivatives may be, but are not limited to, natural wild-type microorganisms or non-mutated microorganisms with endogenous activity of pyruvate dehydrogenase complex subunit E1, and may have increased O-acetylhomoserine production capacity compared to these.

[0121] As an example, microorganisms capable of producing O-acetylhomoserine or its derivatives are prokaryotic or eukaryotic microorganisms that can produce O-acetylhomoserine or its derivatives within their bodies, and can include all microorganisms that inherently possess the ability to produce O-acetylhomoserine or its derivatives, or microorganisms that do not inherently possess the ability to produce O-acetylhomoserine or its derivatives but have been conferred the ability to produce O-acetylhomoserine or its derivatives. The ability to produce O-acetylhomoserine or its derivatives is conferred or enhanced by the increased pyruvate dehydrogenase complex subunit E1 activity or species improvement described in this application.

[0122] As an example, recombinant microorganisms capable of producing O-acetylhomoserine or its derivatives according to this application may include all microorganisms that can be transformed through a vector to increase the pyruvate dehydrogenase complex subunit E1 activity according to this application and produce O-acetylhomoserine or its derivatives.

[0123] For example, a microorganism that produces O-acetylhomoserine or its derivatives may intrinsically contain a protein consisting of the amino acid sequence of SEQ ID NO: 1, or a protein consisting of an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with SEQ ID NO: 1.

[0124] For example, the microorganism producing O-acetylhomoserine or its derivatives may be a microorganism that intrinsically contains a polynucleotide sequence encoding a protein having at least 60% homology to the SEQ ID NO: 1, the base sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or a base sequence having 60% or more homology or identity with the base sequence of SEQ ID NO: 2 or SEQ ID NO: 3, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more.

[0125] The microorganisms described in this application can include all microorganisms in which the pyruvate dehydrogenase complex subunit E1 activity has been increased compared to its endogenous activity by various known methods.

[0126] As one specific example, a microorganism in which the activity of pyruvate dehydrogenase complex subunit E1 of this application has increased compared to its endogenous activity may be, but is not limited to, a microorganism in which the protein expression and activity have increased due to the substitution of a different sequence in the sequence that regulates the expression of the sequence encoding pyruvate dehydrogenase complex subunit E1, or due to a deformation of the base sequence encoding pyruvate dehydrogenase complex subunit E1, or due to a substitution of the start codon. As another specific example, a microorganism in which the activity of pyruvate dehydrogenase complex subunit E1 of this application has increased compared to its endogenous activity may be a microorganism in which the protein expression and activity have increased due to the substitution of a sequence that regulates the expression of the sequence encoding pyruvate dehydrogenase complex subunit E1 with a sequence that is more active than the endogenous expression regulatory sequence. However, is not limited to this.

[0127] For example, the microorganisms in which the production capacity of O-acetylhomoserine or its derivatives has increased in this application may be, but are not limited to, microorganisms in which the production capacity of O-acetylhomoserine or its derivatives has increased compared to non-myxoid microorganisms. For example, the non-myxoid microorganisms in which the increase in the production capacity of O-acetylhomoserine or its derivatives is compared may be, but are not limited to, the ATCC13032 strain.

[0128] As an example, a microorganism with increased production capacity for O-acetylhomoserine or its derivatives may have an increased production capacity of approximately 1% or more compared to the parent microorganism (parent strain) or non-myxoid microorganism before mutation. Specifically, this could be approximately 1% or more, approximately 2.5% or more, or approximately 5% or more. However, it is not limited to these values ​​as long as it has a positive increase compared to the production capacity of the parent microorganism (parent strain) or non-myxoid microorganism before mutation. As another example, a recombinant microorganism with increased production capacity for O-acetylhomoserine or its derivatives may have an increased production capacity of approximately 1.01 times or more, 1.02 times or more, 1.03 times or more, 1.04 times or more, 1.05 times or more, 1.06 times or more, 1.07 times or more, 1.08 times or more, 1.09 times or more, or approximately 1.1 times or more compared to the parent microorganism (parent strain) or non-myxoid microorganism before mutation.

[0129] For example, the microorganism capable of producing O-acetylhomoserine or its derivatives may be either a prokaryotic cell or a eukaryotic cell, but specifically, it may be a prokaryotic cell. Examples of such prokaryotic cells may include microbial strains belonging to the genera Escherichia, Erwinia, Serratia, Providencia, Corynebacterium, Pseudomonas, Leptospira, Salmonella, Brevibacteria, Hypomonas, Chromobacterium, and Nocardia, or fungi or yeasts. Specifically, these are microbial strains and yeasts belonging to the genera Escherichia, Corynebacterium, and Leptospira. More specifically, they may also be microbial strains of the genus Corynebacterium.

[0130] As a microorganism based on any one of the specific examples mentioned above, the microorganism of this application may be a microorganism of the genus Corynebacterium.

[0131] As an example of this application, the microorganisms of this application are Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, and Corynebacterium striatum. The microorganisms of this application may be Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens. Specifically, the microorganisms of this application may be, but are not limited to, microorganisms of the genus Corynebacterium, and more specifically, Corynebacterium glutamicum.

[0132] The Corynebacterium microorganisms having the ability to produce O-acetylhomoserine or its derivatives according to this application may include the naturally occurring wild-type microorganisms themselves, Corynebacterium microorganisms having improved O-acetylhomoserine or its derivative production ability by increasing or decreasing the activity of genes related to the production mechanism of O-acetylhomoserine or its derivatives, and Corynebacterium microorganisms having improved O-acetylhomoserine or its derivative production ability by introducing or increasing the activity of external genes.

[0133] The microorganism having the ability to produce O-acetylhomoserine or its derivatives according to this application may further include modifications that increase the ability to produce the target O-acetylhomoserine or its derivatives.

[0134] As an example, the microorganism of this application may be a microorganism in which the activity of O-acetylhomoserine transferase (MetX) is increased compared to its endogenous activity.

[0135] As an example, the microorganisms of this application may include a modification of the inner membrane protein YjeH. This can be referenced in the contents of WO2021-125896 A1.

[0136] As an example, the microorganisms of this application may include variants in which the expression of aspart kinase (LysC) is enhanced and / or feedback inhibition is removed. This may be referenced to the contents of US 10662450 B2.

[0137] For example, the microorganism of this application may be a microorganism in which the activity of cystathionine gamma-synthase is attenuated. For example, the microorganism of this application may be a microorganism in which the activity of the metB gene encoding cystathionine gamma-synthase is attenuated. For example, the microorganism may be a microorganism in which all or part of the metB gene is missing.

[0138] For example, the microorganism of this application may be a microorganism in which the activity of O-acetylhomoserine(thiol)-lyase is attenuated. For example, the microorganism of this application may be a microorganism in which the activity of the metY gene encoding O-acetylhomoserine(thiol)-lyase is attenuated. For example, the microorganism may be a microorganism in which all or part of the metY gene is missing.

[0139] For example, the microorganism of this application may be a microorganism in which the activity of the endogenous protein Ncgl0616 is weakened. For example, it may be a microorganism in which part or all of the gene encoding Ncgl0616 is missing.

[0140] Another aspect of this application provides a method for producing O-acetylhomoserine or a derivative thereof, comprising the step of culturing a microorganism in which the pyruvate dehydrogenase complex subunit E1 activity of this application is increased compared to its endogenous activity, in a culture medium.

[0141] In the method of this application, the culture of microorganisms is carried out using any culture conditions and methods known in the art. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected strain.

[0142] O-acetylhomoserine or its derivatives produced by the culture described in this application are either secreted into the culture medium or remain within the cells.

[0143] In one specific example, the method for producing O-acetylhomoserine or a derivative thereof according to the present application may further include, for example, a step of preparing the microorganism of the present application, a step of preparing a culture medium for culturing the strain, or a combination thereof (in any order), before the culturing step.

[0144] The method for producing O-acetylhomoserine or its derivatives according to this application may further include a step of recovering the target substance, specifically O-acetylhomoserine or its derivatives, from the cultured microorganism, the culture of the microorganism, the fermented product of the microorganism, or the culture medium. The recovery step may further include a step after the culture step.

[0145] The aforementioned recovery may involve collecting the target O-acetylhomoserine or its derivatives using appropriate methods known in the art, such as the microbial culture methods of this application, for example, batch, continuous, or fed-batch culture methods. For example, various chromatography methods such as centrifugation, filtration, treatment with a crystallizing protein precipitant (salting-out method), extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof can be used, and the target substance, specifically O-acetylhomoserine or its derivatives, can be recovered from the culture medium or microorganism using appropriate methods known in the art.

[0146] Furthermore, the method for producing O-acetylhomoserine or its derivatives according to this application may further include a purification step. The purification can be carried out using appropriate methods known in the art. For example, if the method for producing O-acetylhomoserine or its derivatives according to this application includes both a recovery step and a purification step, the recovery step and the purification step can be carried out sequentially or discontinuously, regardless of order, or simultaneously or integrated into a single step, but are not limited thereto.

[0147] In the method of this application, the increase in pyruvate dehydrogenase complex subunit E1 activity and O-acetylhomoserine or its derivatives are as described in the other aspects above.

[0148] Another aspect of this application provides a composition for producing O-acetylhomoserine or a derivative thereof, comprising a microorganism in which the pyruvate dehydrogenase complex subunit E1 activity of this application is increased compared to its endogenous activity, a culture of the microorganism, a fermented product of the microorganism, or a combination of two or more of the above.

[0149] The compositions of this application may further contain any suitable excipients commonly used in compositions for the production of O-acetylhomoserine or its derivatives, such excipients may include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents.

[0150] As one specific example, each component present in the composition of this application may be included in a microbiologically effective amount or in an amount that is suitable for use as a production composition.

[0151] In the composition of this application, the increase in pyruvate dehydrogenase complex subunit E1 activity and O-acetylhomoserine or its derivatives are as described in the other aspects above.

[0152] Another aspect of this application provides the use of the pyruvate dehydrogenase complex subunit E1 activity of this application in microorganisms with increased activity compared to endogenous activity for the production of O-acetylhomoserine or derivatives thereof.

[0153] In the use of this application, the increase in pyruvate dehydrogenase complex subunit E1 activity and O-acetylhomoserine or its derivatives are as described in the other aspects above.

[0154] Another aspect of this application provides a method for producing O-acetylhomoserine or derivatives of the microorganism, comprising modifying the microorganism to increase the pyruvate dehydrogenase complex subunit E1 activity of the present application compared to its endogenous activity.

[0155] In the microbial production method of this application, the increase in pyruvate dehydrogenase complex subunit E1 activity and O-acetylhomoserine or its derivatives are as described in the other aspects above. [Examples]

[0156] Example 1. Preparation of O-acetylhomoserine and homoserine-producing bacterial strains We prepared bacterial strains for use in evaluating O-acetylhomoserine and homoserine production capacity.

[0157] Example 1-1. metB deficiency The metB gene, which encodes cystathionine gamma-synthase in the O-acetylhomoserine degradation pathway, was isolated via PCR using chromosomal DNA from Corynebacterium glutamicum ATCC13032 as a template. The metB gene sequence information (NCBI registration number Ncgl2360, SEQ ID NO: 4) was obtained from the National Institutes of Health's Gene Bank (NIH GenBank), and primers containing the N-terminal and linker portions of the metB gene (SEQ ID NOs: 6 and 7), and primers containing the C-terminal and linker portions (SEQ ID NOs: 8 and 9) were synthesized based on this information. The primer sequences are listed in Table 1 below.

[0158] [Table 1]

[0159] PCR was performed using chromosomal DNA from ATCC13032 as a template and primers corresponding to SEQ ID NOs. 6 and 7, and SEQ ID NOs. 8 and 9. The polymerase used was PfuUltra. TMUsing high-fidelity DNA polymerase (Stratagene), PCR was performed under the following conditions: denaturation at 96°C for 30 seconds, annealing at 53°C for 30 seconds, and polymerization at 72°C for 1 minute, repeated 30 times. As a result, a 558 bp amplified gene containing the N-terminus and linker of the metB gene and a 527 bp amplified gene containing the C-terminus and linker of the metB gene were obtained. Using the two amplified genes obtained above as templates, PCR was performed under the following conditions: denaturation at 96°C for 60 seconds, annealing at 50°C for 60 seconds, and polymerization at 72°C for 1 minute, repeated 10 times. Then, sequence numbers 6 and 9 were added, and the polymerization reaction was repeated 20 more times. As a result, an inactivated cassette of 1064 bp containing the N-terminus, linker, and C-terminus of the metB gene was obtained.

[0160] The pDCM2 vector was treated with SmaI, and the PCR product (1064 bp) obtained above was fusion cloned with the pDCM2 vector obtained by SmaI restriction enzyme treatment using the In-Fusion(R) HD Cloning Kit (Clontech). The cloned vector was transformed into E. coli DH5α, and the transformed E. coli were streaked onto LB solid medium containing 25 mg / l kanamycin. After selecting the plasmid-transformed colonies in the LB medium, plasmids were obtained using plasmid extraction (US registration publication US 5981235 A), and finally, a pDCM2-ΔmetB recombinant vector with a cloned metB gene-deficient cassette was constructed.

[0161] The fabricated pDCM2-ΔmetB vector was transformed into the ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)(KCCM12634P;WO2021-125896 A1) strain using electropulse technology. After a secondary crossover, ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetB was obtained in which the metB gene was inactivated on the chromosome. The presence or absence of metB gene inactivation was finally confirmed by comparing it with ATCC13032, in which the metB gene was not inactivated after PCR using primers SEQ ID NOs. 6 and 9.

[0162] Examples 1-2. metY deficiency The metY gene, which encodes O-acetylhomoserine(thiol)-lyase in the O-acetylhomoserine degradation pathway, was obtained via PCR using chromosomal DNA from Corynebacterium glutamicum ATCC13032 as a template. The nucleotide sequence information for the metY gene (NCBI registration number Ncgl0625, SEQ ID NO: 19) was obtained from the National Institutes of Health's Gene Bank (NIH GenBank), and primers containing the N-terminal and linker portions of the metY gene (SEQ ID NOs: 20 and 21) and primers containing the C-terminal and linker portions (SEQ ID NOs: 22 and 23) were synthesized based on this information. The primer sequences are shown in Table 2 below.

[0163] [Table 2]

[0164] PCR was performed using chromosomal DNA from ATCC13032 as a template and primers corresponding to SEQ ID NOs. 20 and 21, and SEQ ID NOs. 22 and 23. The polymerase used was PfuUltra. TMUsing high-fidelity DNA polymerase (Stratagene), the PCR conditions were denaturation at 96°C for 30 seconds, annealing at 53°C for 30 seconds, and polymerization at 72°C for 1 minute, repeated 30 times. As a result, a 548 bp amplified gene containing the N-terminus and linker of the metY gene and a 550 bp amplified gene containing the C-terminus and linker of the metY gene were obtained. Using the two amplified genes obtained above as templates, PCR was performed, with the PCR conditions being denaturation at 96°C for 60 seconds, annealing at 50°C for 60 seconds, and polymerization at 72°C for 1 minute, repeated 10 times. Then, sequence numbers 20 and 23 were added, and the polymerization reaction was repeated 20 more times. As a result, an inactivated cassette of 1077 bp containing the N-terminus, linker, and C-terminus of the metY gene was obtained.

[0165] The pDCM2 vector was treated with SmaI, and the PCR product (1077 bp) obtained above was fused with the pDCM2 vector obtained by SmaI restriction enzyme treatment using the In-Fusion(R) HD Cloning Kit (Clontech). The cloned vector was transformed into E. coli DH5α, and the transformed E. coli were streaked onto LB solid medium containing 25 mg / l kanamycin. After selecting the plasmid-transformed colonies in the LB medium, plasmids were obtained using plasmid extraction (US registration publication US 5981235 A), and finally, a pDCM2-ΔmetY recombinant vector with a cloned meTY gene-deficient cassette was constructed.

[0166] The fabricated pDCM2-ΔmetY vector was transformed into ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetB using the electro-pulse method. Through a secondary crossover process, the metY gene was further inactivated on the chromosome, yielding ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetY.

[0167] The presence or absence of metY gene inactivation was finally confirmed by comparing the metY gene in ATCC13032, which was not inactivated after PCR using primers SEQ ID NOs. 20 and 23, with that inactivated metY gene.

[0168] Example 1-3. Introduction of lysC (L377K) A mutation (L377K) (US 10662450 B2) was introduced into the lysC gene (SEQ ID NO: 24), which encodes aspart kinase derived from Corynebacterium glutamicum ATCC13032, to enhance lysC gene expression and remove feedback inhibition to L-lysine and L-threonine. To construct a vector containing the mutant lysC gene, a pair of primers (SEQ ID NOs: 25 and 26) for amplifying the 5' upstream region around the mutation site and a pair of primers (SEQ ID NOs: 27 and 28) for amplifying the 3' downstream region were devised. The primer sequences are shown in Table 3 below.

[0169] [Table 3]

[0170] PCR was performed using the chromosome of ATCC13032 as a template, with primers SEQ ID NOs. 25 and 26, and SEQ ID NOs. 27 and 28. The PCR conditions were: denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 30 seconds, repeated 30 times, followed by polymerization at 72°C for 7 minutes. As a result, a 512 bp DNA fragment from the 5' upstream region and a 522 bp DNA fragment from the 3' downstream region were obtained, primarily due to mutations in the lysC gene. Using the two amplified DNA sections as templates, PCR was performed with primers SEQ ID NOs. 25 and 28. The PCR conditions were: denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 60 seconds, repeated 30 times, followed by polymerization at 72°C for 7 minutes. As a result, a 1011 bp DNA fragment containing the mutant lysC(L377K) gene, which encodes an aspart kinase variant in which the 377th leucine position is replaced with lysine, was amplified.

[0171] The pDCM2 vector was treated with SmaI, and the PCR product (1011 bp) obtained above was fused with the pDCM2 vector obtained by SmaI restriction enzyme treatment using the In-Fusion(R) HD Cloning Kit (Clontech). The cloned vector was transformed into Escherichia coli DH5α, and the transformed Escherichia coli was streaked onto LB solid medium containing 25 mg / l kanamycin. After selecting the plasmid-transformed colonies in the LB medium, plasmids were obtained using plasmid extraction (US registration publication US 5981235 A), and finally, a pDCM2-lysC(L377K) recombinant vector was constructed in which a cassette with the lysC(L377K) gene substituted was cloned.

[0172] The constructed pDCM2-lysC(L377K) vector was transformed into the bacterial strain ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetY using electrotherapy. Through a secondary crossover process, a nucleotide mutation was introduced into the lysC gene on the chromosome, yielding Corynebacterium glutamicum ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetY lysC(L377K). The presence or absence of the nucleotide mutation was confirmed by PCR using primers 25 and 28, followed by sequencing and comparison with the wild-type lysC gene sequence.

[0173] Examples 1-4. NCgl0616 Defect We created a vector to delete the endogenous gene NCgl0616 (SEQ ID NO: 29) in Corynebacterium glutamicum ATCC13032.

[0174] Specifically, we devised primer pairs (sequences 30 and 31) to amplify the 5' upstream region centered around the location of the NCgl0616 gene in sequence number 29, and primer pairs (sequences 32 and 33) to amplify the 3' downstream region.

[0175] [Table 4]

[0176] PCR was performed using ATCC13032 wild-type (WT) chromosomes as templates and primers SEQ ID NOs. 30, 31, 32, and 33. The PCR conditions were as follows: denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 30 seconds, repeated 30 times, followed by polymerization at 72°C for 7 minutes. As a result, a 701 bp DNA fragment from the 5' upstream region and a 699 bp DNA fragment from the 3' downstream region were obtained, centered around the deletion site of the NCgl0616 gene.

[0177] PCR was performed using two types of amplified DNA sections as templates with primers SEQ ID NO: 30 and SEQ ID NO: 33. The PCR conditions were as follows: denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 90 seconds, repeated 30 times, followed by polymerization at 72°C for 7 minutes. As a result, a 1410 bp DNA fragment containing a site that may delete the NCgl0616 gene was amplified.

[0178] The pDCM2 vector was treated with SmaI, and the PCR product (2912 bp DNA fragment) obtained above was fused with the pDCM2 vector obtained by SmaI restriction enzyme treatment using the In-Fusion(R) HD Cloning Kit (Clontech). The cloned vector was transformed into Escherichia coli DH5α, and the transformed Escherichia coli was streaked onto LB solid medium containing 25 mg / l kanamycin. After selecting the plasmid-transformed colonies from the LB solid medium, plasmids were obtained using plasmid extraction (US registration publication US 5981235 A), and finally, a pDCM2-ΔNCgl0616 recombinant vector with a cloned NCgl0616-deficient cassette was constructed.

[0179] [Table 5]

[0180] The constructed pDCM2-ΔNCgl0616 vector was used to transform strains with increased O-acetylhomoserine production capacity using electrotherapy. Through a secondary crossover process, ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetY lysC(L377K)ΔNCgl0616 was obtained, which lacked the NCgl0616 gene on the chromosome. The presence or absence of NCgl0616 gene inactivation was finally confirmed by comparing ATCC13032, whose NCgl0616 gene was not inactivated after PCR using primers 34 and 35.

[0181] Example 2. Plasmid production for enhancing the activity of pyruvate dehydrogenase complex subunit E1. We constructed a vector to enhance the activity of the pyruvate dehydrogenase complex subunit E1 (NCgl2167, hereafter referred to as aceE). Specifically, to create an enhancement vector for pyruvate dehydrogenase complex subunit E1 (NCgl2167, SEQ ID NO: 1), we used the Pcj7 promoter (US 7662943 B2) (SEQ ID NO: 3), a promoter known to be a strong promoter, to replace the wild-type promoter of the aceE gene (SEQ ID NO: 2) with Pcj7, thereby creating a plasmid that enhances aceE activity. We obtained the upstream and downstream regions of the aceE gene. Specifically, to produce strains into which aceE possessing the Pcj7 promoter was introduced, PCR was performed using chromosomal DNA from Corynebacterium glutamicum ATCC13032 as a template. The upstream region of the aceE gene was amplified using primers SEQ ID NO. 12 and SEQ ID NO. 13, and the downstream region of the aceE gene was amplified using primers SEQ ID NO. 14 and SEQ ID NO. 15. In addition, the Pcj7 promoter fragment was obtained using SEQ ID NOs. 10 and 11 as templates for pDCM2-Pcj7. The primer sequences used for each of the above PCRs are shown in Table 6 below.

[0182] [Table 6]

[0183] For polymerases used in PCR reactions, PfuUltra TMUsing high-fidelity DNA polymerase (Stratagene), the PCR conditions were denaturation at 95°C for 30 seconds; denaturation at 55°C for 30 seconds; and polymerization at 72°C for 1 minute. This denaturation, annealing, and polymerization reaction was repeated 28 times. As a result, a 318 bp DNA fragment of the Pcj7 promoter region, a 526 bp upstream DNA fragment of Corynebacterium glutamicum ATCC13032 aceE, and a 527 bp downstream DNA fragment were obtained. PCR was performed using the amplified promoter and DNA sections as templates with primers of SEQ ID NO: 12 and SEQ ID NO: 15. The PCR conditions were denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 2 minutes, repeated 28 times, followed by polymerization at 72°C for 5 minutes. After DNA purification, the two fragments obtained above were subjected to fusion cloning using the In-Fusion(R) HD cloning kit (Clontech) with a pDCM2 vector (WO2021-187781 A1) treated with SmaI restriction enzyme, according to the provided manual, to obtain a plasmid. The resulting vector was named pDCM2-Pcj7_aceE.

[0184] Example 3. Production of an O-acetylhomoserine strain with enhanced pyruvate dehydrogenase complex subunit E1 activity. To confirm whether the enhanced activity of the Corynebacterium glutamicum strain capable of producing O-acetylhomoserine and homoserine increases the production capacity of the enhanced strain, the Corynebacterium glutamicum strain ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetY lysC(L377K)ΔNCgl0616, which was prepared in Example 1 and produces O-acetylhomoserine and homoserine, was transformed into the Corynebacterium glutamicum strain ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetY lysC(L377K)ΔNCgl0616 by homologous recombination on the chromosome using the vector pDCM2-Pcj7_aceE prepared in Example 2 (van der Rest et al., Appl Microbiol). Biotechnol 52:541-545,1999).

[0185] The aforementioned recombinant strain was named CM04-8001(ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetY lysC(L377K)ΔNCgl0616-Pcj7_aceE).

[0186] Example 4. Evaluation of the production capacity of O-acetylhomoserine and homoserine strains with enhanced pyruvate dehydrogenase complex subunit E1 activity. Flask evaluations were performed to compare the production capacity of O-acetylhomoserine and homoserine-producing strains Corynebacterium glutamicum ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetY lysC(L377K)ΔNCgl0616 and CM04-8001.

[0187] The bacterial strain was inoculated into a 250 ml corner baffle flask containing 25 ml of the culture medium described below using one platinum loop, and incubated at 33°C for 20 hours with shaking at 200 rpm. The O-acetylhomoserine concentration was analyzed using HPLC, and the analyzed concentrations are shown in Table 7.

[0188] O-acetylhomoserine production medium (pH 7.2) Glucose 30g, KH2PO4 2g, Urea 3g, (NH4)2SO4 40g, Peptone 2.5g, CSL (Sigma) 5g (10ml), MgSO4.7H2O 0.5g, CaCO3 20g (based on 1 liter of distilled water)

[0189] [Table 7]

[0190] As shown in Table 3 above, we confirmed that the concentration of O-acetylhomoserine increased in O-acetylhomoserine-producing strains with enhanced pyruvate dehydrogenase complex subunit E1 activity compared to ATCC13032 and ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetY lysC(L377K)ΔNCgl0616.

[0191] Example 5. Production of an O-acetylhomoserinetransferase (MetX) introduced plasmid. To amplify the gene encoding O-acetylhomoserinetransferase (MetX), the nucleotide sequence information for the metX gene (NCgl0624, SEQ ID NO: 16) was obtained from the National Institutes of Health Gene Bank (NIH GenBank). Based on this, primers were devised to amplify from the promoter region (approximately 300 bp upstream of the start codon) to the terminator region (approximately 100 bp downstream of the stop codon) by inserting BamHI restriction enzyme sites at both ends of SEQ ID NOs: 17 and 18. The PCR conditions were denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 90 seconds, repeated 30 times, followed by polymerization at 72°C for 7 minutes. As a result, a 1546 bp DNA fragment of the metX gene encoding region was obtained. pECCG117 (KR 10-0057684 B1) vector and metX A plasmid was obtained by treating DNA fragments with the restriction enzyme BamHI, ligating them using DNA ligase, and then cloning it. This plasmid was named pECCG117-metX WT. The primer sequences are shown in Table 8 below.

[0192] [Table 8]

[0193] The prepared pECCG117-metX WT vector was introduced into the bacterial strains ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetY lysC(L377K)ΔNCgl0616 and CM04-8001, prepared in Example 1, by electropulse. The resulting cells were then streaked onto a selection medium containing 25 mg / L of kanamycin to obtain the respective transformed strains.

[0194] To compare the O-acetylhomoserine production capacity of the strains prepared as described above, the cultures were cultured using the following method and the O-acetylhomoserine in the culture medium was analyzed.

[0195] The bacterial strain was inoculated using one platinum loop (inoculation loop) into a 250 ml corner baffle flask containing 25 ml of the culture medium described below, and incubated at 33°C for 20 hours with shaking at 200 rpm. The O-acetylhomoserine concentration was analyzed using HPLC, and the analyzed concentrations are shown in Table 9.

[0196] O-acetylhomoserine production medium (pH 7.2) Glucose 30g, KH2PO4 42g, Urea 3g, (NH4)2SO4 40g, Peptone 2.5g, CSL(Sigma) 5g (10 ml), MgSO4.7H2O 0.5g, Methionine 400mg, CaCO3 20g (based on 1 liter of distilled water)

[0197] [Table 9]

[0198] As a result, as shown in Table 9 above, when the ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetY lysC(L377K)ΔNCgl0616 / pECCG117-metX WT strain was cultured, 2.83 g / L of O-acetylhomoserine accumulated, and it was confirmed that the production volume increased by 130% to 3.68 g / L when cultured with CM04-8002.

[0199] Through this process, we confirmed that in bacterial strains with increased O-acetylhomoserine production capacity, O-acetylhomoserine production capacity increased when the activity of pyruvate dehydrogenase complex subunit E1 was enhanced.

[0200] The results above indicate that enhancement of pyruvate dehydrogenase complex subunit E1 activity is effective in the production of O-acetyl-L-homoserine and its derivatives in Corynebacterium O-acetyl-L-homoserine and homoserine-producing strains.

[0201] From the above description, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are merely illustrative and not limiting. The scope of this application should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, as described below, and their equivalent concepts, rather than from the above detailed description.

Claims

1. A method for producing O-acetylhomoserine or a derivative thereof, comprising the step of culturing a microorganism in which the activity of pyruvate dehydrogenase complex subunit E1 is increased compared to its endogenous activity in a culture medium.

2. The method according to claim 1, wherein the microorganism is a microorganism of the genus Corynebacterium.

3. The method according to claim 2, wherein the Corynebacterium genus microorganism is Corynebacterium glutamicum.

4. The method according to claim 1, wherein the pyruvate dehydrogenase complex subunit E1 is derived from Corynebacterium glutamicum.

5. The method according to claim 1, wherein the pyruvate dehydrogenase complex subunit E1 contains the amino acid sequence of SEQ ID NO:

1.

6. The method according to claim 5, wherein the pyruvate dehydrogenase complex subunit E1 is encoded by the polynucleotide of SEQ ID NO: 2 or SEQ ID NO:

3.

7. The method according to claim 1, wherein the O-acetylhomoserine or its derivative is one or more selected from O-acetylhomoserine, L-methionine, and L-homoserine.

8. The method according to claim 1, further comprising the step of recovering O-acetylhomoserine or a derivative thereof from the cultured microorganism, the culture product of the microorganism, the fermented product of the microorganism, or the culture medium.

9. A microorganism that produces O-acetylhomoserine or its derivatives, in which the activity of pyruvate dehydrogenase complex subunit E1 is increased compared to its endogenous activity.

10. A composition for producing O-acetylhomoserine or its derivatives, comprising a microorganism in which the activity of pyruvate dehydrogenase complex subunit E1 is increased compared to its endogenous activity, a culture of the microorganism, a fermented product of the microorganism, or a combination of two or more of these.

11. Use of microorganisms in which pyruvate dehydrogenase complex subunit E1 activity is increased compared to endogenous activity, for the production of O-acetylhomoserine or its derivatives.

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