Microorganism having increased pyruvate dehydrogenase complex subunit E1 activity and method for producing o-acetylhomoserine or derivative thereof using same

Abstract

The present disclosure relates to a microorganism having increased pyruvate dehydrogenase complex subunit E1 activity, and a method for producing O-acetyl homoserine or a derivative thereof using the same.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a microorganism having increased activity of pyruvate dehydrogenase complex subunit E1, and a method for producing O-acetylhomoserine or a derivative thereof using the same. BACKGROUND

[0002] Corynebacterium microorganisms are gram-positive microorganisms widely used for the production of L-amino acids, and in addition to O-acetylhomoserine classified as a non-protein amino acid, and protein amino acids, they are widely used for the production of L-homoserine, L-methionine, etc., which can be converted from O-acetylhomoserine.

[0003] In order to produce L-amino acids and other useful substances, various studies are being conducted to develop microorganisms that efficiently produce them. For example, a substance-specific method (e.g., mainly increasing the expression of a gene encoding an enzyme involved in L-amino acid biosynthesis or removing an unnecessary gene for L-amino acid biosynthesis in a Corynebacterium strain) is mainly used for the production of L-amino acids (US 9644009 B2). SUMMARY

[0004] TECHNICAL PROBLEM

[0005] There is still a need for research into a method for efficiently producing L-amino acids in high yield.

[0006] TECHNICAL SOLUTION

[0007] One object of the present disclosure is to provide a microorganism having increased activity of pyruvate dehydrogenase complex subunit E1 (AceE).

[0008] In one embodiment, the microorganism is a microorganism for producing O-acetylhomoserine or a derivative thereof.

[0009] In another embodiment, the microorganism has increased activity of pyruvate dehydrogenase complex subunit E1 (AceE) compared to endogenous activity.

[0010] As the microorganism according to any one of the above embodiments, the microorganism can be a Corynebacterium microorganism.

[0011] As the microorganism according to any one of the above embodiments, the Corynebacterium microorganism can be Corynebacterium glutamicum.

[0012] As the microorganism according to any one of the above embodiments, the pyruvate dehydrogenase complex subunit El can be derived from Corynebacterium glutamicum.

[0013] As the microorganism according to any one of the above embodiments, the pyruvate dehydrogenase complex subunit El can include the amino acid sequence of SEQ ID NO: 1.

[0014] As the microorganism according to any one of the above embodiments, the pyruvate dehydrogenase complex subunit El can be encoded by the polynucleotide of SEQ ID NO: 2 or SEQ ID NO: 3.

[0015] As the microorganism according to any one of the above embodiments, the microorganism can be a microorganism having an increased ability to produce O-acetylhomoserine or a derivative thereof compared to an unmodified microorganism.

[0016] As the microorganism according to any one of the above embodiments, the O-acetylhomoserine or a derivative thereof can be any one or more selected from the group consisting of O-acetylhomoserine, L-methionine, and L-homoserine.

[0017] Another object of the present disclosure is to provide a method of producing O-acetylhomoserine or a derivative thereof, comprising: culturing a microorganism having increased activity of pyruvate dehydrogenase complex subunit El in a culture medium.

[0018] In one embodiment, the method can further include the step of recovering O-acetylhomoserine or a derivative thereof from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism, or the culture medium.

[0019] Another object of the present disclosure is to provide a composition for producing O-acetylhomoserine or a derivative thereof, comprising: a microorganism in which the activity of pyruvate dehydrogenase complex subunit El is increased compared to its endogenous activity; a culture of the microorganism; a fermentation product of the microorganism; or a combination of two or more thereof.

[0020] Another object of the present disclosure is to provide use of a microorganism in which the activity of pyruvate dehydrogenase complex subunit El is increased compared to its endogenous activity for producing O-acetylhomoserine or a derivative thereof.

[0021] Another object of the present disclosure is to provide a method of preparing a microorganism for producing O-acetylhomoserine or a derivative thereof, comprising: modifying a microorganism to have increased activity of pyruvate dehydrogenase complex subunit El compared to its endogenous activity.

[0022] Advantages

[0023] O-acetylhomoserine or a derivative thereof can be produced at a high yield using the microorganism of the present disclosure. DETAILED DESCRIPTION

[0024] The present disclosure will be described in detail below. At the same time, each description and embodiment disclosed in the present disclosure can also be applied to other descriptions and embodiments. That is, all combinations of various elements disclosed in the present disclosure fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the specific descriptions described below. In addition, many papers and patent documents are referred to and cited throughout the specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to further clarify the level and scope of the subject matter of the present disclosure.

[0025] DEFINITIONS

[0026] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Singular terms will include the plural and plural terms will include the singular, unless they are otherwise expressly stated herein. As used in the specification and the appended claims, the use of “or” can be used to mean “and / or,” unless otherwise indicated.

[0027] As used herein, the term “about” can precede a particular numerical value. The term “about” as used herein, includes not only the precise number listed after the term, but also a range that is close or approximate to the number. Whether a number is close or approximate to a particular number presented can be determined in view of the context in which the number is presented. In one example, the term “about” can refer to a range of -10% to +10% of an index value. In another example, the term “about” can refer to a range of -5% to +5% of a given numerical value, but is not limited thereto.

[0028] As used herein, descriptions such as the terms “first, second, third, …” “i), ii), iii), …” or “(a), (b), (c), (d), …” can be used to distinguish respective constituents. When these terms are used to refer to steps of a method, use, or assay, these terms are not limited to being performed consecutively or sequentially. For example, there can be no time interval between these steps, or they can be performed simultaneously, or can be performed with an interval of several seconds, minutes, hours, days, or months.

[0029] As used herein, the term “consisting essentially of’ can mean that when the features of the subject matter claimed herein are substantially unaffected by the presence of unspecified features, unspecified features can be present.

[0030] As used herein, the term "consisting of refers to a total percentage of 100% of the specific components listed after this term. Components or features listed after the term "consisting of can be necessary or mandatory. In some embodiments, any other components or features, or non-essential components or features, can be excluded in addition to the components or features listed after the term "consisting of.

[0031] As used herein, the term "comprising" means that the features, steps, or components listed after this term are present, but does not exclude the presence of one or more additional features, steps, or components. Components or features listed after the term "comprising" in this document can be necessary or mandatory. However, in some embodiments, this term can also include any other or non-essential components or features.

[0032] Protein, polypeptide

[0033] As used herein, the term "protein" or "polypeptide" refers to a polymer or oligomer of consecutive amino acid residues. In the present disclosure, "polypeptide", "protein", and "peptide" can be used interchangeably with each other.

[0034] In some cases, a protein, polypeptide, or peptide that exhibits activity can be referred to as an "enzyme". In the present disclosure, an amino acid sequence is described in the direction of N-terminus to C-terminus, unless otherwise specified.

[0035] As used herein, the term "mature polypeptide" refers to a polypeptide in the form without a signal sequence or pro-peptide sequence. The mature protein / polypeptide / peptide can be a functional form of the protein / polypeptide / peptide. The mature polypeptide can refer to a final form of the polypeptide after translation, or after post-translational modification. Examples of post-translational modification include N-terminal or C-terminal modification, glycosylation, phosphorylation, leader sequence removal, etc., but are not limited thereto.

[0036] With respect to the amino acid sequences in the present disclosure, although it is described as a polypeptide or protein "comprising / including" the amino acid sequence described by a particular sequence number, a polypeptide or protein "consisting of" the amino acid sequence described by a particular sequence number, or a polypeptide or protein "having" the amino acid sequence described by a particular sequence number, it is obvious that any polypeptide or protein having an amino acid sequence in which part of the sequence is deleted, modified, substituted, conservatively substituted, or added, if it has the same or corresponding activity as the polypeptide or protein consisting of the amino acid sequence of the corresponding sequence number, can fall within the scope of the present application. For example, polypeptides or proteins having sequence additions or deletions that do not change the function of the protein, naturally occurring mutations, silent mutations thereof, or conservative substitutions within or upstream or downstream (N-terminal or C-terminal) of the polypeptide or protein sequence can be included, as long as they have the same or corresponding activity as the activity of the polypeptide or protein.

[0037] In one specific example, a polypeptide (protein) that can be conjugated to an N-terminal signal (or leader) sequence involved in translocation of the polypeptide (protein) in a co-translational or post-translational manner, or a polypeptide (protein) that can be conjugated to another sequence or linker to identify, purify, or synthesize the polypeptide (protein), can also fall within the scope of the polypeptide of the amino acid sequence described by a particular number.

[0038] As used herein, the term "conservative substitution" refers to the replacement of an amino acid by another amino acid having similar structure and / or chemical properties. Such amino acid substitutions can typically occur based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of residues. Amino acids can be grouped as follows: In one 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; amino acids with polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In another example, amino acids with charged side chains (charged amino acids) include arginine, lysine, histidine, glutamic acid, and aspartic acid; and amino acids with uncharged side chains (uncharged amino acids; also known as neutral amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In another example, aromatic amino acids include phenylalanine, tryptophan, and tyrosine. In another example, branched-chain amino acids include valine, leucine, and isoleucine. In another example, the 20 amino acids can be grouped into 5 groups according to their size, starting with the relatively small amino acid group, i.e., glycine, alanine, serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, and tyrosine, although the classification of amino acids is not limited thereto. Generally, conservative substitutions have little or no effect on the activity of a polypeptide.

[0039] polynucleotide

[0040] As used herein, the term "gene" refers to a polynucleotide that encodes a polypeptide and includes regions upstream and downstream of the coding region. In some embodiments, a gene can have sequences (introns) inserted between individual coding regions (exons).

[0041] As used herein, the terms "polynucleotide," "nucleic acid," or "nucleic acid molecule" refer to a nucleotide polymer composed of nucleotide monomers joined by covalent bonds into a long chain, which is a DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA) chain of at least some length.

[0042] homology, identity

[0043] As used herein, the terms "homology" or "identity" refer to the relatedness of two given amino acid sequences or nucleotide sequences and can be expressed in percentage. The terms "homology" and "identity" can generally be used interchangeably with each other.

[0044] Conserved polynucleotide or polypeptide sequence homology or identity can be determined by standard alignment algorithms and can use the default gap penalties established by the program used. Essentially, homologous or identical sequences are generally expected to hybridize under moderately or highly stringent conditions to all or at least about 50%, 50%, 60%, 70%, 80%, or 90% of the length of the sequence. Polynucleotides containing degenerate codons rather than codons can also be considered.

[0045] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, by known computer algorithms, such as the "FASTA" program using default parameters (Pearson et al., (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444). Alternatively, it can be determined by the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) using implementation of the Needleman program (preferably version 5.0.0 or later) in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (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 Diego, 1994 and [CARILLO ET AL. (1988) SIAM J Applied Math 48: 1073). Homology, similarity or identity can be determined, for example, by using the BLAST or ClustalW of the National Center for Biotechnology Information (NCBI).

[0046] Homology, similarity or identity of polynucleotides or polypeptides can be determined, for example, by comparing sequence information using, for example, the GAP computer program, as disclosed in Needleman et al. (1970), J Mol Biol. 48: 443, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In sum, the GAP program defines homology, similarity or identity as a value obtained by dividing the number of similar aligned symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include (1) unitary matrices (containing values of 1 for identical and 0 for non-identical), PAM matrices, as disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation (1978), and the weighted comparison matrix of Gribskov et al., (1986), Nucl. Acids Res. 14:6745 (or the EDNAFULL substitution matrix (EMBOSS version of NCBI NUC4.4)) ; (2) a per residue score of 3.0, and a per residue indel penalty of 0.10 (or a gap open penalty of 10, and a gap extend penalty of 0.5); and (3) no penalty for end gaps.

[0047] Further, whether any two polynucleotide sequences have homology, similarity or identity can be determined by comparing sequence using a Southern hybridization experiment under defined stringent conditions, and suitable hybridization conditions to be defined can be determined by methods within the scope of the present disclosure, as known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York), but not limited thereto.

[0048] "Stringent conditions" refer to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (Sambrook et al., supra, 9.50-9.51, 11.7-11.8). For example, stringent conditions can include conditions in which polynucleotides with high homology or identity, i.e., polynucleotides with 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 homology or identity, hybridize to each other, and polynucleotides with less homology or identity than the above do not hybridize to each other, or can include washing conditions of ordinary Southern hybridization, i.e., one, in particular two or three, washes at a salt concentration and temperature corresponding to 60°C, 1xSSC, 0.1% SDS, specifically 60°C, 0.1xSSC, 0.1% SDS, more specifically 68°C, 0.1xSSC, 0.1% SDS.

[0049] Hybridization requires that the two nucleic acids have complementary sequences, although 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 to each other. For example, for DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present disclosure can also include isolated nucleic acid fragments that are complementary to the entire sequence, as well as nucleic acid sequences that are substantially similar thereto.

[0050] For example, a hybridization condition including a hybridization step of T m The hybridization condition of T m The value can be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by a person skilled in the art according to the purpose thereof.

[0051] The appropriate stringency of the hybridized polynucleotide depends on the length and complementarity of the polynucleotide, and these variables are well known in the art (e.g., Sambrook et al.).

[0052] Nucleic acid construct, vector, transformation

[0053] As used herein, the term "nucleic acid construct" refers to a single- or double-stranded nucleic acid molecule that includes one or more regulatory sequences, and is either artificially synthesized, or engineered to contain specific sequences in a manner that does not exist in nature, or isolated from nature.

[0054] As used herein, the term "vector" refers to a DNA construct used to deliver a target polynucleotide into a suitable host or host cell. In one example, the vector can contain a nucleotide sequence of a polynucleotide encoding a target polypeptide, which is operably linked to a suitable expression regulatory region (expression regulatory sequence) so that the target polypeptide can be expressed in a suitable host cell, but is not limited thereto.

[0055] The expression regulatory sequence can include a promoter capable of initiating transcription, any operator sequence that controls transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence that regulates transcription and translation termination. Once transformed into a suitable host cell, the vector can replicate independently of the host genome or function, or can be integrated into its genome.

[0056] The vector used in the present disclosure is not particularly limited, and any vector known in the art can be used. Examples of the vector commonly used can include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc. can be used as a bacteriophage vector or a cosmid vector; those based on pDZ, pDC, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET, etc. can be used as a plasmid vector. Specifically, pDZ, pDC, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pDCM2 (WO2021-187781 A1) vector, etc. can be used.

[0057] In one example, the target polynucleotide can be inserted into a chromosome by a vector for intracellular chromosomal insertion. Inserting the polynucleotide into a chromosome can be performed by any method known in the art, for example, homologous recombination, but is not limited thereto. The vector can further include a selection marker to confirm insertion into the chromosome. The selection marker is used to select cells transformed by the vector, i.e., to confirm whether the target nucleic acid molecule is inserted, and a marker that provides a selectable phenotype (such as drug resistance, auxotrophy, cytotoxic agent resistance, or surface polypeptide expression) can be used. Only cells expressing the selection marker can survive in an environment treated with a selection agent or show a different phenotype, and thus transformed cells can be selected.

[0058] As used herein, the term "transformation" refers to introduction of a vector containing a target polynucleotide into a host cell to alter the genetic properties of the host cell. The transformed polynucleotide can be integrated into the chromosome of the host cell and located therein or located extrachromosomally. In addition, the polynucleotide can include DNA and / or RNA. Depending on the purpose of introduction, the polynucleotide can be introduced in a suitable form. For example, the polynucleotide for expression of a target polypeptide can be introduced into a host cell in the form of an expression cassette, which is a genetic construct comprising all elements necessary for autonomous expression thereof. The expression cassette can generally comprise a promoter operably linked to the polynucleotide, a transcription terminator, a ribosome binding site, or a translation terminator. The expression cassette can be in the form of a self-replicating expression vector. In addition, the polynucleotide can be introduced into a host cell as it is, and can be operably linked to a sequence required for expression in the host cell, but is not limited thereto.

[0059] As used herein, the term "operably linked" refers to the configuration in which a regulatory sequence is placed in the appropriate position relative to the coding sequence so as to control the expression of the coding sequence. Thus, the term "operably linked" includes attachment or ligation between a regulatory region (such as a promoter, a stop codon, a signal sequence, or an enhancer) having a known or desired activity and a target (gene or polypeptide) such that the expression, secretion, or function of the target can be regulated according to the known or desired activity. For example, it can refer to the functional linkage of a polynucleotide sequence with a promoter sequence that initiates and mediates transcription of the polynucleotide encoding a polypeptide.

[0060] As used herein, the term "expression" includes any steps involved in the production of a polypeptide, such as transcription, post-transcriptional modification, translation, post-translational modification, and secretion, etc., but is not limited thereto.

[0061] As used herein, the term "expression vector" refers to a linear or circular nucleic acid molecule comprising a target polynucleotide sequence and an operably linked regulatory sequence for expression thereof.

[0062] As used herein, the term "regulatory sequence" refers to a polynucleotide sequence necessary for the expression of a target polynucleotide sequence. Each regulatory sequence can be native (derived from the same source) or foreign (derived from a different gene) to the coding sequence, or can be a mutant sequence or other artificial sequence thereof. Examples of regulatory sequences can include a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating transcription and translation termination. The minimum unit of the regulatory sequence can include a promoter and a sequence terminating transcription and translation.

[0063] As used herein, the term "recombinant" with respect to a cell, polynucleotide, polypeptide, or vector means that the cell, polynucleotide, polypeptide, or vector has been modified by the introduction of a heterologous nucleic acid or polypeptide or alteration of a native polynucleotide or polypeptide, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell can express genes that it does not normally express, or can express a native gene abnormally, underexpress, or not express at all.

[0064] Microorganism

[0065] As used herein, the term "microorganism (or strain)" includes all wild-type microorganisms or prokaryotic or eukaryotic microorganisms that have been naturally or artificially genetically modified, and it can be a microorganism in which a particular mechanism is weakened or enhanced due to the insertion of an exogenous gene, or the enhancement or inactivation of an endogenous gene activity, etc., and can be a genetically modified microorganism including the production of a desired polypeptide, protein, or product. In the present disclosure, "microorganism" and "strain" can be used interchangeably with each other.

[0066] For example, the microorganism of the present disclosure can be a microorganism (e.g., a recombinant strain) in which the activity of pyruvate dehydrogenase complex subunit E1 is increased compared to its endogenous activity, but is not limited thereto.

[0067] As used herein, the term "microorganism having the ability to produce O-acetylhomoserine or a derivative thereof" is a microorganism capable of producing O-acetylhomoserine or a derivative thereof in an organism, and can include all of a non-endogenous microorganism having the ability to produce O-acetylhomoserine or a derivative thereof that is conferred the ability to produce O-acetylhomoserine or a derivative thereof, or an endogenous microorganism having the ability to produce O-acetylhomoserine or a derivative thereof. The ability to produce O-acetylhomoserine or a derivative thereof can be conferred or enhanced by species improvement.

[0068] As used herein, the term "unmodified microorganism (strain)" does not exclude a microorganism (strain) containing a mutation that can occur naturally, and can refer to a wild-type microorganism (strain) or a native microorganism (strain) itself, or a microorganism (strain) before its properties are changed due to genetic modification by natural or artificial factors. As used herein, "unmodified microorganism (strain)" can be used interchangeably with "microorganism (strain) before modification", "unmutated microorganism (strain)", "parent microorganism", "parent strain", "wild-type microorganism (strain)", "reference microorganism (strain)", or "standard microorganism (strain)". In the present disclosure, an unmodified microorganism can be a microorganism in which the activity of pyruvate dehydrogenase complex subunit E1 is not increased compared to its endogenous activity, or a microorganism before it is increased.

[0069] Also, in the present disclosure, the unmodified microorganism can be a microorganism including a polynucleotide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3, but is not limited thereto.

[0070] Increase in polypeptide activity

[0071] As used herein, the term "increase" in polypeptide activity refers to an increase in the activity of a polypeptide in a host cell compared to its endogenous activity. Increase can be used interchangeably with terms such as activation, upregulation, overexpression, enhancement, etc. The host cell can be a prokaryotic or eukaryotic microorganism.

[0072] The increase in polypeptide activity can include two cases in which the polypeptide activity is exhibited non-endogenously possessed by the host cell, or the polypeptide activity is enhanced compared to the endogenous activity or the activity before modification.

[0073] For example, the description "exhibiting non-endogenously possessed polypeptide activity" can be caused by "introduction of a polypeptide", but is not limited thereto. As used herein, the term "introduction of a polypeptide" refers to expression of a gene not originally possessed by a microorganism in the microorganism, so that the microorganism exhibits activity of a specific polypeptide, or the activity of the polypeptide is increased or enhanced compared to the endogenous activity or the activity before modification of the corresponding polypeptide. For example, it can be the case that a polynucleotide encoding a specific polypeptide is introduced into the chromosome of a microorganism, or a vector containing a polynucleotide encoding a specific polypeptide is introduced into a microorganism, thereby exhibiting its activity.

[0074] "Endogenous activity" refers to the activity of a specific polypeptide originally possessed by a microorganism before transformation or an unmodified microorganism when the trait is changed due to genetic variation caused by natural or artificial factors. Endogenous activity can also be used interchangeably with "activity before modification".

[0075] The increase in polypeptide activity compared to the endogenous activity means that the activity and / or concentration (expression level) of the polypeptide of the microorganism is improved compared to the activity and / or concentration (expression level) of the polypeptide originally possessed by the microorganism before transformation or the unmodified microorganism.

[0076] In one example, the increase can mean that the activity of the corresponding protein / polypeptide that was not originally exhibited is exhibited, or its activity or concentration is generally increased by about 1% or more, about 10% or more, about 25% or more, about 50% or more, about 75% or more, about 100% or more, about 150% or more, about 200% or more, about 300% or more, about 400% or more, or about 500% or more, up to about 1000% or about 2000% or more, but is not limited thereto.

[0077] The increase in the activity of the polypeptide can be achieved by introducing an exogenous polypeptide or increasing the activity of an endogenous polypeptide. Whether the activity of the polypeptide is increased can be confirmed by the level of activity of the corresponding polypeptide, an increase in the expression level thereof, or an increase in the amount of product produced by the corresponding polypeptide.

[0078] The increase in the activity of the polypeptide can be achieved by various methods well known in the art, and is not limited as long as it can increase the activity of the target polypeptide compared to the microorganism before modification. Specifically, genetic engineering and / or protein engineering well known to those skilled in the art, which are conventional methods of molecular biology, can be used, but the method is not limited thereto (for example, Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al., Molecular Cloning 2012, etc.).

[0079] Specifically, the increase in the activity of the polypeptide of the present disclosure can be achieved by:

[0080] 1) increasing the copy number of the polynucleotide encoding the polypeptide within the cell;

[0081] 2) modifying the expression regulatory region of the gene encoding the polypeptide on the chromosome (for example, modifying within the expression regulatory region, replacing with a sequence having stronger activity, or inserting a sequence having stronger activity);

[0082] 3) modifying the nucleotide sequence of the coding start codon or the 5'-UTR of the transcript of the gene encoding the polypeptide;

[0083] 4) modifying the amino acid sequence of the polypeptide such that the activity of the polypeptide is increased;

[0084] 5) modifying the polynucleotide sequence encoding the polypeptide such that the activity of the polypeptide is increased (for example, modifying the polynucleotide sequence of the polypeptide gene to encode a polypeptide that has been modified to increase the activity of the polypeptide);

[0085] 6) introducing an exogenous polypeptide exhibiting the activity of the polypeptide or an exogenous polynucleotide encoding the same;

[0086] 7) codon optimization of the polynucleotide encoding the polypeptide;

[0087] 8) analyzing the tertiary structure of the polypeptide and selecting and modifying or chemically modifying the exposed site therefrom;

[0088] 9) regulating the cellular localization of the protein (polypeptide); or

[0089] 10) a combination of two or more selected from the above 1) to 9), but is not particularly limited thereto.

[0090] For example,

[0091] 1) A method of increasing the copy number of a polynucleotide encoding a polypeptide in a cell can be achieved by introducing a vector comprising a polynucleotide encoding a polypeptide into a host cell, the polynucleotide being operably linked to a suitable regulatory sequence. The vector can be a vector capable of functioning independently of the host cell. Alternatively, the method can be achieved by introducing one copy or two or more copies of a polynucleotide encoding a polypeptide into the chromosome of a host cell, the polynucleotide being operably linked to a suitable regulatory sequence. Introduction into the chromosome can be performed by introducing a vector capable of inserting a polynucleotide into the chromosome of a host cell, but is not limited thereto. The vector is as described above. The regulatory sequence can be native (derived from the same source) or foreign (derived from a different gene) to the encoding polynucleotide sequence, or can be a mutated sequence or other artificial sequence thereof, and can induce expression of the polynucleotide in the host cell.

[0092] 2) A method of replacing the expression regulatory region (or expression regulatory sequence) of a gene encoding a polypeptide on a chromosome with a sequence having strong activity can be achieved by, for example, introducing a modification in the sequence by deletion, insertion, substitution, or a combination thereof to further enhance the activity of the expression regulatory region, or by replacing the sequence with a sequence having stronger activity. The expression regulatory region can include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating transcription and translation termination, etc. In one example, the method can include replacing the original promoter with a strong promoter, but is not limited thereto.

[0093] Examples of known strong promoters include cj1 to cj7 promoters (US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US 10584338 B2), O2 promoter (US 10273491 B2), tkt promoter, yccA promoter, etc., but the strong promoter is not limited thereto.

[0094] 3) A method of modifying the nucleotide sequence encoding the initiation codon of a gene encoding a polypeptide or the 5'-UTR can be achieved by, for example, replacing the initiation codon with another initiation codon having a higher polypeptide expression rate compared to the endogenous initiation codon, but is not limited thereto.

[0095] 4) and 5) The method of modifying the amino acid sequence of the polypeptide or the polynucleotide sequence can be achieved by inducing a modification on the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to increase the activity of the polypeptide, or by replacing the sequence with an amino acid sequence or a polynucleotide sequence modified to have stronger activity, or an amino acid sequence or a polynucleotide sequence modified to increase the activity, but is not limited thereto. Specifically, the replacement can be performed by inserting the polynucleotide into the chromosome through homologous recombination, but is not limited thereto. The vector used herein can also include a selection marker to confirm insertion into the chromosome. The selection marker is as described above.

[0096] 6) The method of introducing an exogenous polynucleotide exhibiting the activity of the polypeptide can be achieved by introducing an exogenous polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide into a host cell. The exogenous polynucleotide can be used without limitation regardless of its origin or sequence, as long as it exhibits the same / similar activity as the polypeptide. The introduction can be performed by appropriately selecting a transformation method known in the art by those of ordinary skill in the art, and the expression of the introduced polynucleotide in the host cell can produce the polypeptide, thereby increasing its activity.

[0097] 7) The method of codon optimization of the polynucleotide encoding the polypeptide can be achieved by codon optimization of the endogenous polynucleotide to increase transcription or translation in the host cell, or by optimizing the codon of the exogenous polynucleotide so that optimized transcription and translation of the polynucleotide can be achieved in the host cell.

[0098] 8) The method of analyzing the tertiary structure of the polypeptide and thereby selecting and modifying or chemically modifying the exposed site can be achieved by, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing known protein sequence information to determine template protein candidates according to the degree of sequence similarity, and thereby confirming the structure based on the information to select and transform or modify the exposed site to be modified or chemically modified.

[0099] 9) The method of regulating the cellular localization of the protein (polypeptide) can be achieved by targeting the protein (polypeptide) to a specific organelle or a specific space in the cell. For example, the protein (polypeptide) can be targeted to the periplasm or cytoplasm by adding or removing a leader sequence that functions in targeting the protein (polypeptide), but is not limited thereto.

[0100] Such enhancement of the activity of the polypeptide can mean 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 microorganism or the microorganism before modification, or an increase in the amount of the product produced by the corresponding polypeptide, but is not limited thereto.

[0101] Weakening of the activity of the polypeptide

[0102] As used herein, the term "attenuation" of the activity of a polypeptide (e.g., including the proteins designated by the name of each enzyme) is a comprehensive concept including a decrease in activity or no activity as compared to its endogenous activity. Attenuation can be used interchangeably with terms such as inactivation, lack, down-regulation, reduction, decrease, weakening, etc.

[0103] Attenuation can also include cases where the activity of the polypeptide itself is reduced or eliminated as compared to the polypeptide activity originally possessed by the microorganism due to mutation, etc. of the polynucleotide encoding the polypeptide; cases where the overall level of polypeptide activity and / or concentration (expression level) in the cell is reduced as compared to the natural strain due to inhibition of gene expression of the polynucleotide encoding the polypeptide or inhibition of translation into the polypeptide, etc.; cases where the polynucleotide is not expressed at all; and / or cases where no polypeptide activity is observed even when the polynucleotide is expressed. The expression that the activity of the polypeptide is "inactivated, lacks, reduced, down-regulated, decreased, or weakened" as compared to the endogenous activity means that the activity of the polypeptide is reduced as compared to the activity of the specific polypeptide originally possessed by the parent strain before transformation or the unmodified microorganism.

[0104] Attenuation of the activity of the polypeptide can be performed by any method known in the art, but the method is not limited thereto, and can be achieved by applying various methods well known in the art (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.).

[0105] Specifically, attenuation of the activity of the polypeptide of the present disclosure can be achieved by:

[0106] 1) deletion of part or all of the gene encoding the polypeptide;

[0107] 2) modification of the expression regulatory region (expression regulatory sequence) so that the expression of the gene encoding the polypeptide is reduced;

[0108] 3) modification of the amino acid sequence constituting the polypeptide so that the activity of the polypeptide is eliminated or attenuated (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence);

[0109] 4) modification of the gene sequence encoding the polypeptide so that the activity of the polypeptide is eliminated or attenuated (e.g., deletion / substitution / addition of one or more nucleotides in the nucleotide sequence of the polypeptide gene to encode a modified polypeptide to eliminate or attenuate the activity of the polypeptide);

[0110] 5) modifying the nucleotide sequence of the coding start codon or 5'-UTR of the transcript of the gene encoding the polypeptide;

[0111] 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide;

[0112] 7) adding a sequence complementary to the Shine-Dalgarno (SD) sequence of the gene encoding the polypeptide to the front of the SD sequence to form a secondary structure, thereby inhibiting ribosome attachment;

[0113] 8) reverse transcription engineering (RTE) that adds a promoter to be reverse-transcribed to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide;

[0114] 9) modulating the cellular localization of the protein (polypeptide); or

[0115] 10) a combination of two or more selected from the above 1) to 9), but not particularly limited thereto.

[0116] For example,

[0117] 1) The method of deleting part or all of the gene encoding the polypeptide can be achieved by deleting all of the polynucleotides encoding the endogenous target polypeptide within the chromosome, or by replacing the polynucleotides with polynucleotides in which nucleotides are partially deleted or with marker genes.

[0118] 2) The method of modifying the expression regulatory region (expression regulatory sequence) can be achieved by inducing modification of the expression regulatory region (expression regulatory sequence) by deletion, insertion, non-conservative substitution, or conservative substitution, or a combination thereof; or by replacing the sequence with a sequence having weaker activity. The expression regulatory region can include a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating transcription and translation termination, but is not limited thereto.

[0119] 3) and 4) The method of modifying the amino acid sequence or the polynucleotide sequence can be achieved by inducing modification of the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to weaken the activity of the polypeptide, or by replacing the sequence with an amino acid sequence or a polynucleotide sequence modified to have weaker activity or an amino acid sequence or a polynucleotide sequence modified to have no activity, but is not limited thereto. For example, the expression of the gene can be inhibited or weakened by introducing a mutation in the polynucleotide sequence to form a stop codon, but is not limited thereto.

[0120] 5) The method of modifying the nucleotide sequence of the coding start codon or 5'-UTR of the transcript of the gene encoding the polypeptide can be achieved by, for example, replacing the nucleotide sequence with a nucleotide sequence encoding another start codon having a lower polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0121] 6) The method of introducing an antisense oligonucleotide (e.g., antisense RNA) complementary to the transcript of the gene encoding the polypeptide can be found in the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

[0122] 7) The method of adding a sequence complementary to the Shine-Dalgarno (SD) sequence of the gene encoding the polypeptide to the front of the SD sequence to form a secondary structure, thereby inhibiting ribosome attachment, can be achieved by inhibiting mRNA translation or reducing its speed.

[0123] 8) Reverse transcription engineering (RTE), which adds a promoter to be reverse-transcribed to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide, can be achieved by forming an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide to weaken activity.

[0124] In addition, 9) the method of regulating the cellular localization of a protein (polypeptide) can be achieved by targeting the protein (polypeptide) to a specific organelle or a specific space within the cell. For example, the protein (polypeptide) can be targeted to the periplasm or cytoplasm by adding or removing a leader sequence that works in targeting the protein (polypeptide), but is not limited thereto.

[0125] Such weakening of the activity of the polypeptide can mean that the activity or concentration of the corresponding polypeptide is weakened relative to the activity or concentration of the polypeptide expressed in the wild-type strain or the microbial strain before modification, or the amount of product produced by the corresponding polypeptide is reduced, but is not limited thereto.

[0126] The modification of part or all of the polynucleotides in the microorganism of the present disclosure can be achieved by (a) homologous recombination using a vector for chromosomal insertion in the microorganism or genome editing using an engineered nuclease (e.g., CRISPR-Cas9), and / or (b) can be induced by light such as ultraviolet rays and irradiation, etc., and / or chemical treatment, but is not limited thereto.

[0127] Culturing

[0128] As used herein, the term "culturing" refers to growing a microorganism under suitably controlled environmental conditions. The culturing process of the present disclosure can be performed under suitable culture media and culture conditions known in the art. Such a culturing process can be easily adjusted for use by those skilled in the art depending on the microorganism to be selected. Specifically, the culturing can be batch culture, continuous culture, and / or fed-batch culture, but is not limited thereto.

[0129] As used herein, the term "culture medium" refers to a mixture of substances containing nutrients required for culturing a microorganism as a main component, which provides nutrients and growth factors, as well as water necessary for survival and growth. Specifically, the culture medium and other culture conditions for culturing the microorganism of the present disclosure can be any culture medium used for conventional culturing of microorganisms, without any particular limitation. However, the microorganism of the present disclosure can be cultured in a conventional culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin, etc., under aerobic conditions, while adjusting the temperature, pH, etc. For example, the culture medium for a microorganism of the genus Corynebacterium can be found in the literature [“Manual of Methods for General Bacteriology” (American Society for Bacteriology, Washington D.C., USA, 1981)].

[0130] In the present disclosure, the carbon source can include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. In addition, the carbon source can include natural organic nutrients such as starch hydrolysate, molasses, jaggery paste, rice bran, cassava, cane molasses, and corn steep liquor, etc. Specifically, carbohydrates such as glucose and sterile pretreated molasses (i.e., molasses converted into reducing sugar) can be used, and in addition, an appropriate amount of various other carbon sources can be used without limitation. These carbon sources can be used alone or in combination of two or more, but are not limited thereto.

[0131] The nitrogen source can include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; amino acids such as glutamic acid, methionine, glutamine, etc.; and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0132] The phosphorus source can include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or a corresponding sodium-containing salt, etc. Examples of the inorganic compound can include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and / or suitable precursors, etc. can be included. These constituent ingredients or precursors can be added to the culture medium in a batch or continuous manner, but the phosphorus source is not limited thereto.

[0133] In addition, in the process of culturing the microorganism of the present disclosure in an appropriate manner, the pH of the culture medium can be adjusted by adding a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid, etc. In addition, an antifoaming agent such as a fatty acid polyethylene glycol ester can be used to prevent the formation of bubbles during the culturing process. In addition, oxygen or an oxygen-containing gas can be injected into the culture medium to maintain aerobic conditions of the culture medium; or nitrogen, hydrogen, or carbon dioxide or no gas can be injected to maintain anaerobic or microaerophilic conditions, but the gas is not limited thereto.

[0134] The temperature during the culturing process of the present disclosure can be in the range of 20°C to 45°C, specifically 25°C to 40°C, and the culturing can last for about 10 hours to 160 hours, but is not limited thereto.

[0135] As used herein, the term "culture" refers to a culture broth, a concentrated culture broth, a dried product of the culture broth, a culture filtrate, a concentrated culture filtrate, or a dried product of the culture filtrate, obtained by culturing a specific microorganism in a culture medium, and refers to the culture broth can contain the specific microorganism, while the culture filtrate substantially does not contain the specific microorganism (specifically, it substantially refers to excluding the specific microorganism isolated by filtration, etc., but does not mean that the microorganism is completely excluded from the filtrate). The dosage form of the culture is not limited, and can be, for example, a liquid, an emulsion, or a solid.

[0136] As used herein, the term "fermentation" refers to the process in which a strain does not spoil in decomposing organic matter using its own enzymes. Fermentation and spoilage reactions are carried out by similar processes, but when decomposition produces useful substances, it is called fermentation, and when it produces odor or harmful substances, it is called spoilage.

[0137] In the present disclosure, the method of obtaining a fermentation product from a microorganism is not particularly limited, and can be obtained according to the method commonly used in the art or a similar field.

[0138] As used herein, the term "fermentation product" can include not only the material itself after fermentation, but also all kinds of materials including the fermentation product produced by the strain, such as a strain culture medium in which the strain and the culture coexist, a fermentation product produced from the culture medium, a fermentation product obtained by filtering the microorganism from the culture medium, a fermentation product obtained by sterilizing the microorganism from the culture medium and filtering the microorganism, an extract obtained by extracting the fermentation product or the culture medium containing the fermentation product, a dilution solution obtained by diluting the fermentation product or the extract thereof, a concentrated solution, a dried product obtained by drying the fermentation product or the extract thereof, and a lysate obtained by collecting and lysing the microorganism cells, and the like.

[0139] Detailed description of the disclosure

[0140] Hereinafter, embodiments of the present disclosure will be described in detail as follows:

[0141] One aspect of the present disclosure provides a microorganism in which the activity of pyruvate dehydrogenase complex subunit E1 is increased compared to its endogenous activity.

[0142] In one example of the present disclosure, the microorganism of the present disclosure can have the ability to produce O-acetylhomoserine or a derivative thereof.

[0143] The increase in the activity of pyruvate dehydrogenase complex subunit E1 can be defined as that the ability of the microorganism of the present disclosure to produce O-acetylhomoserine or a derivative thereof is increased compared to the production ability of a natural wild-type microorganism or an unmodified microorganism (e.g., a microorganism expressing a polypeptide having the activity of pyruvate dehydrogenase complex subunit E1 (e.g., a polypeptide of SEQ ID NO: 1), or a microorganism in which the activity of pyruvate dehydrogenase complex subunit E1 is not increased compared to its endogenous activity, or a microorganism before the increase in activity), but is not limited thereto.

[0144] For example, the activity of pyruvate dehydrogenase complex subunit E1 is determined by measuring the production ability or yield of O-acetylhomoserine or a derivative thereof, but is not limited thereto.

[0145] As used herein, the term "pyruvate dehydrogenase complex subunit E1" as a component of the pyruvate dehydrogenase complex can be a polypeptide including an E1 active site that converts pyruvate to acetyl CoA and CO2 in the action of pyruvate dehydrogenase.

[0146] The pyruvate dehydrogenase complex subunit E1 of the present disclosure can be used interchangeably with “AceE”. The pyruvate dehydrogenase complex subunit E1 is known in the art and can be specifically encoded by aceE, but is not limited thereto. The amino acid and polynucleotide sequences of the pyruvate dehydrogenase complex subunit E1 can be obtained from known databases such as GenBank of NCBI, but are not limited thereto.

[0147] In one example, the pyruvate dehydrogenase complex subunit E1 can include the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having 60% or more homology or identity thereto, but is not limited thereto, as long as it has the same activity as the pyruvate dehydrogenase complex subunit E1. Specifically, any protein having the amino acid sequence of SEQ ID NO: 1, in which part of the sequence is deleted, modified, substituted, or added, as long as it exhibits the corresponding efficacy of the pyruvate dehydrogenase complex subunit E1, can fall within the scope of the pyruvate dehydrogenase complex subunit E1. In addition, any protein having or including the amino acid sequence of SEQ 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 to SEQ ID NO: 1, consisting of the above-described amino acid sequence, or consisting essentially of the above-described amino acid sequence, and exhibiting the corresponding efficacy of the pyruvate dehydrogenase complex subunit E1, can fall within the scope of the pyruvate dehydrogenase complex subunit E1. In one example, the pyruvate dehydrogenase complex subunit E1 can be an exogenous protein, or a protein endogenously present in a coryneform microorganism or C. glutamicum, but is not limited thereto. Specifically, it can be the pyruvate dehydrogenase complex subunit E1 consisting of the amino acid sequence of SEQ ID NO: 1, which is endogenously present in a coryneform microorganism or C. glutamicum, but is not limited thereto. Examples thereof can include NCBI Reference No. WP_011014985.1, etc.

[0148] In addition, a 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 prepared, for example, based on codon information known in the art. In one example, the protein can be encoded by a polynucleotide which can have or include the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 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, 99% or more, and less than 100% homology or identity to the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3, can consist of the aforementioned nucleotide sequence, or can consist essentially of the aforementioned nucleotide sequence, but is not limited thereto. In addition, the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3 can be obtained from known databases such as GenBank of NCBI, but is not limited thereto.

[0149] In the present disclosure, a gene including a nucleotide sequence shown by a specific sequence number can be used interchangeably with a polynucleotide including a nucleotide sequence shown by a specific sequence number.

[0150] Due to codon degeneracy or in consideration of preferred codons in an organism in which the pyruvate dehydrogenase complex subunit E1 of the present disclosure is to be expressed, the polynucleotide of the present disclosure can be variously modified in the coding region without changing the amino acid sequence of the pyruvate dehydrogenase complex subunit E1 of the present disclosure. Therefore, based on codon degeneracy, it is obvious that a polynucleotide which can be translated into a polypeptide consisting of the amino acid sequence of the pyruvate dehydrogenase complex subunit E1 of the present disclosure or a polypeptide having homology or identity thereto can also be included in the scope of the polynucleotide of the present disclosure. For example, the polynucleotide of the present disclosure can be SEQ ID NO: 2, SEQ ID NO: 3, or a degenerate sequence thereof.

[0151] In another example, the polynucleotide of the present disclosure can have or include 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 to SEQ ID NO: 2 or SEQ ID NO: 3, or can consist of or consist essentially of 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 to SEQ ID NO: 2 or SEQ ID NO: 3, but is not limited thereto.

[0152] Further, the polynucleotide of the present disclosure can include a probe that can be prepared from a known gene sequence, for example, any sequence that can hybridize to the complement of all or a portion of the polynucleotide sequence of the present disclosure under stringent conditions to encode the pyruvate dehydrogenase complex subunit E1 of the present disclosure, without limitation.

[0153] As used herein, the term "O-acetylhomoserine or a derivative thereof" includes O- acetylhomoserine and products that can be produced using O-acetylhomoserine as a precursor. In one specific example, the O-acetylhomoserine or a derivative thereof can be any one or more selected from the group consisting of O-acetylhomoserine, L-methionine, and L-homoserine.

[0154] For the purpose of the present disclosure, the microorganism of the present disclosure can include all microorganisms capable of producing the desired O-acetylhomoserine or a derivative thereof by increasing the activity of the pyruvate dehydrogenase complex subunit E1 compared to its endogenous activity. For example, the microorganism of the present disclosure is characterized in that the activity of the pyruvate dehydrogenase complex subunit E1 is increased compared to its endogenous activity, thereby increasing the ability to produce O-acetylhomoserine or a derivative thereof, and it can be a genetically modified microorganism or a recombinant microorganism, but is not limited thereto. Specifically, the recombinant microorganism having an increased ability to produce O-acetylhomoserine or a derivative thereof can be a microorganism having an increased ability to produce O-acetylhomoserine compared to a natural wild-type microorganism or an unmodified microorganism having the endogenous activity of the pyruvate dehydrogenase complex subunit E1, but is not limited thereto.

[0155] In one example, the microorganism having the ability to produce O-acetylhomoserine or a derivative thereof is a prokaryotic or eukaryotic microorganism capable of producing O-acetylhomoserine or a derivative thereof in the organism, which can include all of the following: a microorganism endogenously having the ability to produce O-acetylhomoserine or a derivative thereof, or a microorganism in which the ability to produce O-acetylhomoserine or a derivative thereof has been conferred to a microorganism that does not endogenously have the ability to produce O-acetylhomoserine or a derivative thereof. The ability to produce O-acetylhomoserine or a derivative thereof can be conferred or enhanced by the activity of pyruvate dehydrogenase complex subunit El or species modification.

[0156] In one example, the recombinant microorganism having the ability to produce O-acetylhomoserine or a derivative thereof can include all of the following: a microorganism that can be transformed by a vector and thus is capable of producing O-acetylhomoserine or a derivative thereof by increasing the activity of pyruvate dehydrogenase complex subunit El.

[0157] For example, the microorganism producing O-acetylhomoserine or a derivative thereof can be a microorganism endogenously including 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 to SEQ ID NO: 1.

[0158] For example, the microorganism producing O-acetylhomoserine or a derivative thereof can be a microorganism endogenously including a polynucleotide sequence capable of encoding a protein comprising an amino acid sequence having at least 60% homology to SEQ ID NO: 1; or a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or a polynucleotide 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, or 98% or more homology or identity to the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

[0159] The microorganism of the present disclosure can include all microorganisms in which the activity of pyruvate dehydrogenase complex subunit El is increased compared to its endogenous activity by various known methods.

[0160] In one embodiment, the microorganism in which the activity of pyruvate dehydrogenase complex subunit El is increased compared to its endogenous activity can be a microorganism having increased activity by replacing a sequence regulating expression of a sequence encoding pyruvate dehydrogenase complex subunit El with another sequence, modifying a nucleotide sequence encoding pyruvate dehydrogenase complex subunit El, or replacing a start codon, thereby increasing expression of the protein, but is not limited thereto. In one embodiment, the microorganism in which the activity of pyruvate dehydrogenase complex subunit El is increased compared to its endogenous activity can be a microorganism having increased activity by replacing a sequence regulating expression of a sequence encoding pyruvate dehydrogenase complex subunit El with a sequence having stronger activity than an endogenous expression regulating sequence, thereby increasing expression of the protein, but is not limited thereto.

[0161] In one example, the microorganism having increased ability to produce O-acetylhomoserine or a derivative thereof of the present disclosure can be a microorganism having increased ability to produce O-acetylhomoserine or a derivative thereof compared to an unmodified microorganism, but is not limited thereto. In one example, the unmodified microorganism, which is a target strain for comparison of the increased ability to produce O-acetylhomoserine or a derivative thereof, can be an ATCC 13032 strain, but is not limited thereto.

[0162] In one example, the microorganism having increased ability to produce O-acetylhomoserine or a derivative thereof can have an increased ability to produce O-acetylhomoserine or a derivative thereof by about 1% or more, specifically, about 1% or more, about 2.5% or more, or about 5% or more compared to the ability of the parent microorganism before modification (parent strain) or the unmodified microorganism to produce O-acetylhomoserine or a derivative thereof, but is not limited thereto, as long as it has a positive value of increase compared to the production ability of the parent microorganism before modification (parent strain) or the unmodified microorganism. In another example, the recombinant microorganism having increased ability to produce O-acetylhomoserine or a derivative thereof can have an increased ability to produce O-acetylhomoserine or a derivative thereof by about 1.01-fold or more, about 1.02-fold or more, about 1.03-fold or more, about 1.04-fold or more, about 1.05-fold or more, about 1.06-fold or more, about 1.07-fold or more, about 1.08-fold or more, about 1.09-fold or more, or about 1.1-fold or more compared to the ability of the parent microorganism before modification (parent strain) or the unmodified microorganism to produce O-acetylhomoserine or a derivative thereof, but is not limited thereto.

[0163] In one example, the microorganism having the ability to produce O-acetylhomoserine or a derivative thereof can be a prokaryotic cell or a eukaryotic cell, but can specifically be a prokaryotic cell. The prokaryotic cell can include, for example, a microbial strain of the genus Escherichia, Erwinia, Serratia, Providencia, Corynebacterium, Pseudomonas, Leptospira, Salmonella, Brevibacteria, Hypomononas, Chromobacterium, and Norcardia, or a fungus or a yeast, but is not limited thereto. Specifically, it can be a microbial strain of the genus Escherichia, Corynebacterium, Leptospira, and a yeast. More specifically, it can be a microbial strain of the genus Corynebacterium.

[0164] As the microorganism according to any one of the above-described embodiments, the microorganism of the present disclosure can be a microbial strain of the genus Corynebacterium.

[0165] In one example, the microorganism of the present disclosure can be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens. Specifically, the microorganism of the present disclosure can be a microbial strain of the genus Corynebacterium, more specifically, Corynebacterium glutamicum, but is not limited thereto.

[0166] Further, the microorganism of the present disclosure having the ability to produce O-acetylhomoserine or a derivative thereof can include all of the microorganisms: a natural wild-type microorganism as it is; a microorganism of the genus Corynebacterium in which the activity of a gene related to the production mechanism of O-acetylhomoserine or a derivative thereof is increased or decreased, thereby having an enhanced ability to produce O-acetylhomoserine or a derivative thereof; or a microorganism of the genus Corynebacterium in which the activity of an exogenous gene is introduced or increased, thereby having an enhanced ability to produce O-acetylhomoserine or a derivative thereof.

[0167] The microorganism of the present disclosure having the ability to produce O-acetylhomoserine or a derivative thereof can additionally include a modification that increases the ability to produce the desired O-acetylhomoserine or a derivative thereof.

[0168] In one example, the microorganism of the present disclosure can be a microorganism in which the activity of O-acetylhomoserine transferase (MetX) is increased compared to its endogenous activity.

[0169] In another example, the microorganism of the present disclosure can include a modification of the inner membrane protein YjeH. The contents of WO 2021-125896 Al can be incorporated herein by reference.

[0170] In another example, the microorganism of the present disclosure can include a modification in which the expression of aspartate kinase (LysC) is enhanced and / or feedback inhibition is eliminated. In this regard, the contents of US 10662450 B2 can be incorporated herein by reference.

[0171] In another example, the microorganism of the present disclosure can be a microorganism in which the activity of cystathionine gamma-synthase is attenuated. For example, the microorganism of the present disclosure can be a microorganism in which the activity of the metB gene encoding cystathionine gamma-synthase is attenuated. For example, the microorganism can be a microorganism in which all or part of the metB gene is deleted.

[0172] In another example, the microorganism of the present disclosure can be a microorganism in which the activity of O-acetylhomoserine(thiol)-lyase is attenuated. For example, the microorganism of the present disclosure can be a microorganism in which the activity of the metY gene encoding O-acetylhomoserine(thiol)-lyase is attenuated. For example, the microorganism can be a microorganism in which all or part of the metY gene is deleted.

[0173] In another example, the microorganism of the present disclosure can be a microorganism in which the activity of the endogenous protein Ncgl0616 is attenuated. For example, the microorganism can be a microorganism in which all or part of the gene encoding Ncgl0616 is deleted.

[0174] Another aspect of the present disclosure provides a method for producing O-acetylhomoserine or a derivative thereof, which comprises: culturing a microorganism in which the activity of pyruvate dehydrogenase complex subunit E1 is increased as compared to its endogenous activity in a culture medium.

[0175] In the method of the present disclosure, the culturing of the microorganism can be performed using any culture conditions and culture methods known in the art. Such a culturing process can be easily adjusted for use by those skilled in the art depending on the strain to be selected.

[0176] O-acetylhomoserine or a derivative thereof produced by the culturing of the present disclosure can be released into the culture medium or retained in the cells.

[0177] In one embodiment, the method for producing O-acetylhomoserine or a derivative thereof of the present disclosure can further include a step of preparing the microorganism of the present disclosure, a step of preparing a culture medium for culturing the strain, or a combination thereof (regardless of the order, in any order), for example, prior to the culturing step.

[0178] The method for producing O-acetylhomoserine or a derivative thereof of the present disclosure can further include a step of recovering the desired substance (specifically, O-acetylhomoserine or a derivative thereof) from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism, or the culture medium. The recovery step can also be included after the culturing step.

[0179] In the recovery step, the desired O-acetylhomoserine or a derivative thereof can be collected using a method for culturing the microorganism of the present disclosure, for example, according to a batch culture, a continuous culture, or a fed-batch culture method, using a suitable method known in the art. For example, methods such as centrifugation, filtration, treatment with a protein crystallization precipitant (salting-out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.), HPLC, etc., or a combination thereof can be used, and the desired substance, specifically, O-acetylhomoserine or a derivative thereof, can be recovered from the culture medium or the microorganism using a suitable method known in the art.

[0180] In addition, the method for producing O-acetylhomoserine or a derivative thereof of the present disclosure can further include a purification step, which can be performed using a suitable method known in the art. In one example, when the method for producing O-acetylhomoserine or a derivative thereof of the present disclosure includes both a recovery step and a purification step, the recovery step and the purification step can be performed continuously or intermittently regardless of the order, or can be performed simultaneously, or can be integrated into one step, but the method is not limited thereto.

[0181] In the method of the present disclosure, the increase in the activity of pyruvate dehydrogenase complex subunit E1, and O-acetylhomoserine or derivatives thereof, etc. are as described in other aspects above.

[0182] Another aspect of the present disclosure provides a composition for producing O-acetylhomoserine or derivatives thereof, 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 fermentation product of the microorganism; or a combination of two or more thereof.

[0183] The composition of the present disclosure can further include any suitable excipient generally used in a composition for producing O-acetylhomoserine or derivatives thereof, and such an excipient can include, for example, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffering agent, a stabilizer, or an isotonic agent, etc., but is not limited thereto.

[0184] In one embodiment, each component present in the composition of the present disclosure can be included in a microbiologically effective amount, or in an amount that can be appropriately present in a composition for production.

[0185] In the composition of the present disclosure, the increase in the activity of pyruvate dehydrogenase complex subunit E1, and O-acetylhomoserine or derivatives thereof, etc. are as described in other aspects above.

[0186] Another aspect of the present disclosure provides the use of a microorganism in which the activity of pyruvate dehydrogenase complex subunit E1 is increased compared to its endogenous activity for producing O-acetylhomoserine or derivatives thereof.

[0187] In the use of the present disclosure, the increase in the activity of pyruvate dehydrogenase complex subunit E1, and O-acetylhomoserine or derivatives thereof, etc. are as described in other aspects above.

[0188] Another aspect of the present disclosure provides a method of preparing a microorganism for producing O-acetylhomoserine or derivatives thereof, comprising modifying a microorganism to have increased pyruvate dehydrogenase complex subunit E1 activity compared to its endogenous activity.

[0189] In the method of preparing a microorganism of the present disclosure, the increase in the activity of pyruvate dehydrogenase complex subunit E1, and O-acetylhomoserine or derivatives thereof, etc. are as described in other aspects above.

[0190] Mode for carrying out the invention

[0191] Example 1. Preparation of a strain for producing O-acetylhomoserine or homoserine

[0192] A strain for evaluating the ability to produce O-acetylhomoserine and homoserine was prepared.

[0193] Example 1-1. Deletion of metB

[0194] Using the chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template, the metB gene encoding cystathionine γ-synthase involved in the O-acetylhomoserine degradation pathway was obtained by PCR. Information on the nucleotide sequence of the metB gene (NCBI No. Ncgl2360, SEQ ID NO: 4) was obtained from GenBank of the National Institutes of Health (USA), based on which primers including the N-terminal end of the metB gene and a linker (SEQ ID NO: 6 and 7) and primers including the C-terminal end and a linker (SEQ ID NO: 8 and 9) were synthesized. The primer sequences are shown in Table 1 below.

[0195] [Table 1]

[0196]

[0197] PCR was performed using the primers of SEQ ID NO: 6 and 7 and SEQ ID NO: 8 and 9 based on the chromosomal DNA of ATCC 13032 as a template. PfuUltra TM High-Fidelity DNA Polymerase (Stratagene) was used as a polymerase, and PCR was performed by repeating 30 cycles of denaturation at 96°C for 30 seconds, annealing at 53°C for 30 seconds, and polymerization at 72°C for 1 minute. As a result, an amplified gene containing the N-terminal end of the metB gene and a linker (558 bp) and an amplified gene containing the C-terminal end of the metB gene and a linker (527 bp) were obtained, respectively. Based on the two amplified genes obtained as described above as templates, PCR was performed by repeating 10 cycles of denaturation at 96°C for 60 seconds, annealing at 50°C for 60 seconds, and polymerization at 72°C for 1 minute, followed by the addition of the primers of SEQ ID NO: 6 and 9 thereto, followed by 20 cycles of polymerization. As a result, an inactivation cassette including the N-terminal end-linker-C-terminal end of the metB gene (1064 bp) was obtained.

[0198] The pDCM2 vector was treated with the restriction enzyme Smal, and the obtained PCR product (1064 bp) was fused and cloned with the pDCM2 vector treated with the restriction enzyme Smal using the In-Fusion® HD Cloning Kit (Clontech). The cloned vector was transformed with E. coli DH5α, and the transformed E. coli was spread on LB solid medium containing kanamycin (25 mg / L). The colonies transformed with the plasmid on the LB medium were selected, and the plasmid was obtained using a plasmid extraction method (US5981235 A). Finally, the pDCM2-ΔmetB recombinant vector into which the metB gene deletion cassette was cloned was prepared.

[0199] The pDCM2-ΔmetB vector thus prepared was transformed with the ATCC13032 ΔNCgl2335::PCJ7-yjeH (eco, F351L) (KCCM12634P; WO2021-125896 A1) strain using an electroporation method, and a secondary crossover process was performed to obtain ATCC13032 ΔNCgl2335::PCJ7-yjeH (eco, F351L) ΔmetB in which the metB gene is inactivated in the chromosome. After performing PCR using primers of SEQ ID NO: 6 and 9, the inactivation of the metB gene was finally confirmed by comparison with ATCC13032 in which the metB gene is not inactivated.

[0200] Example 1-2. Deletion of metY

[0201] The metY gene encoding O-acetylhomoserine (thiol)-lyase involved in the O- acetylhomoserine degradation pathway was obtained by PCR using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template. Information on the nucleotide sequence of the metY gene (NCBI No. Ncgl0625, SEQ ID NO: 19) was obtained from GenBank of the National Institutes of Health (USA), based on which primers including the N-terminal of the metY gene and a linker (SEQ ID NO: 20 and 21) and primers including the C-terminal and a linker (SEQ ID NO: 22 and 23) were synthesized. The primer sequences are shown in Table 2 below.

[0202] [Table 2]

[0203]

[0204] Using ATCC13032 chromosomal DNA as a template, PCR was performed using primers from SEQ ID NO: 20 and 21, and SEQ ID NO: 22 and 23. PfuUltra was used. TM Using a high-reliability DNA polymerase (Stratagene), PCR was performed by repeating 30 cycles of denaturation at 96°C for 30 seconds, annealing at 53°C for 30 seconds, and polymerization at 72°C for 1 minute. This yielded amplified genes containing the N-terminus and adapter of the metY gene (548 bp) and amplified genes containing the C-terminus and adapter of the metY gene (550 bp), respectively. Based on these two amplified genes as templates, PCR was performed by repeating 10 cycles of denaturation at 96°C for 60 seconds, annealing at 50°C for 60 seconds, and polymerization at 72°C for 1 minute, followed by the addition of primers SEQ ID NO: 20 and 23, and then repeating polymerization for 20 cycles. This yielded an inactivation cassette containing the N-terminus-adaptor-C-terminus of the metY gene (1077 bp).

[0205] The pDCM2 vector was treated with the restriction enzyme SmaI, and the obtained PCR product (1077 bp) was fused with the SmaI-treated pDCM2 vector using the In-Fusion® HD Cloning Kit (Clontech). The cloned vector was transformed with *E. coli* DH5α, and the transformed *E. coli* were plated on LB agar containing kanamycin (25 mg / L). Colonies transformed with the plasmid on LB agar were selected, and the plasmid was obtained using the plasmid extraction method (US5981235 A). Finally, the pDCM2-ΔmetY recombinant vector containing the metY gene deletion cassette was prepared.

[0206] The pDCM2-ΔmetY vector prepared therefrom was transformed using the electroporation method with ATCC13032 ΔNCgl2335::PCJ7-yjeH(eco,F351L) ΔmetB, followed by a second exchange process to obtain ATCC13032 ΔNCgl2335::PCJ7-yjeH(eco,F351L) ΔmetB ΔmetY, in which the metY gene is additionally inactivated in the chromosome.

[0207] After PCR was performed using primers of SEQ ID NO: 20 and 23, the inactivation of the metY gene was finally confirmed by comparison with ATCC13032, in which the metY gene was not inactivated.

[0208] Examples 1-3. Importing lysC (L377K)

[0209] To introduce a mutation (L377K) (US 10662450 B2) into the lysC gene (SEQ ID NO: 24) encoding aspartokinase derived from C. glutamicum ATCC 13032 to enhance the expression of the lysC gene and to relieve the feedback inhibition of L-lysine and L-threonine (US 10662450 B2), and to prepare a vector containing the variant lysC gene, a primer pair (SEQ ID NOs: 25 and 26) for amplifying the 5' upstream region and a primer pair (SEQ ID NOs: 27 and 28) for amplifying the 3' downstream region (relative to the mutation position) were designed. The primer sequences are shown in Table 3 below.

[0210] [Table 3]

[0211]

[0212] PCR was performed using the primers of SEQ ID NOs: 25 and 26 and SEQ ID NOs: 27 and 28 based on the chromosome of C. glutamicum ATCC 13032 as a template. The PCR was performed under the following conditions: after denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds, followed by polymerization at 72°C for 7 minutes. As a result, a 512 bp DNA fragment of the 5' upstream region and a 522 bp DNA fragment of the 3' downstream region relative to the lysC gene mutation were obtained. Thereafter, PCR was performed using the primers of SEQ ID NOs: 25 and 28 based on the two amplified DNA fragments as templates. The PCR was performed under the following conditions: after denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds, followed by polymerization at 72°C for 7 minutes. As a result, a 1011 bp DNA fragment was amplified, which includes the variant lysC (L377K) gene encoding an aspartokinase variant in which the leucine at position 377 is replaced with lysine.

[0213] The pDCM2 vector was treated with the restriction enzyme SmaI, and the obtained PCR product (1011 bp) was fused and cloned with the pDCM2 vector treated with the restriction enzyme SmaI using the HD Cloning Kit (Clontech). The cloned vector was transformed with Escherichia coli DH5a, and the transformed E. coli was spread on LB solid medium containing kanamycin (25 mg / L). The colonies transformed with the plasmid on the LB medium were selected, and the plasmid was obtained using a plasmid extraction method (US 5981235 A). Finally, the pDpDCM2-lysC(L377K) recombinant vector in which the expression cassette containing the lysC (L377K) gene replacement was cloned was prepared.

[0214] The pDCM2-lysC(L377K) vector thus prepared was transformed with ATCC 13032 ΔNCgl2335::PCJ7-yjeH(eco, F351L) ΔmetB ΔmetY using the electroporation method, and a second exchange process was performed to obtain C. glutamicum ATCC 13032 ΔNCgl2335::PCJ7-yjeH(eco, F351L) ΔmetB ΔmetY lysC(L377K) in which a nucleotide mutation was introduced in the lysC gene on the chromosome. Introduction of the nucleotide mutation was finally determined by performing PCR using primers of SEQ ID NO: 25 and 28, and then comparing the sequence with that of the wild-type lysC gene by sequencing.

[0215] Examples 1-4. Deletion of NCgl0616

[0216] A vector was prepared to delete the endogenous gene NCgl0616 (SEQ ID NO: 29) in C. glutamicum ATCC 13032.

[0217] Specifically, a primer pair for amplifying the 5' upstream region (SEQ ID NO: 30 and 31) and a primer pair for amplifying the 3' downstream region (with respect to the NCgl0616 gene position of SEQ ID NO: 29) were designed (SEQ ID NO: 32 and 33). The primer sequences are shown in Table 4 below.

[0218] [Table 4]

[0219]

[0220] PCR was performed using primers of SEQ ID NO: 30 and 31 and SEQ ID NO: 32 and 33 based on the wild-type ATCC 13032 chromosome as a template. The PCR was performed under the following conditions: after denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds, and then polymerization at 72°C for 7 minutes. As a result, a 701 bp DNA fragment of the 5' upstream region and a 699 bp DNA fragment of the 3' downstream region with respect to the deletion position of the NCgl0616 gene were obtained.

[0221] Thereafter, PCR was performed using primers of SEQ ID NO: 30 and 33 based on the two amplified DNA fragments as templates. The PCR was performed under the following conditions: after denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 90 seconds, followed by polymerization at 72°C for 7 minutes. As a result, a 1410 bp DNA fragment, which includes a region in which the NCgl0616 gene can be deleted, was amplified.

[0222] The obtained PCR product (a 2912 bp DNA fragment) was fused and cloned with the pDCM2 vector treated with the restriction enzyme Smal using the HD Cloning Kit (Clontech). The cloned vector was transformed with Escherichia coli DH5a, and the transformed Escherichia coli was spread on LB solid medium containing kanamycin (25 mg / L). The colonies transformed with the plasmid on the LB medium were selected, and the plasmid was obtained using a plasmid extraction method (US5981235 A). Finally, the pDCM2-ΔNCgl0616 recombinant vector into which the NCgl0616 deletion cassette was cloned was prepared.

[0223] [Table 5]

[0224]

[0225] The pDCM2-ΔNCgl0616 vector thus prepared was transformed using an electroporation method, and a second exchange process was performed to obtain ATCC13032 ΔNCgl2335::PCJ7-yjeH(eco, F351L) ΔmetB ΔmetY lysC(L377K) ΔNCgl0616 in which the NCgl0616 gene is deleted in the chromosome. The inactivation of the NCgl0616 gene was finally confirmed by comparison with ATCC13032 in which the NCgl0616 gene is not inactivated after PCR was performed using primers of SEQ ID NO: 34 and 35.

[0226] Example 2. Preparation of a plasmid for enhancing the activity of pyruvate dehydrogenase complex subunit E1

[0227] A vector was prepared to enhance the activity of pyruvate dehydrogenase complex subunit El (NCgl2167, hereinafter referred to as aceE). Specifically, to prepare a vector for enhancing pyruvate dehydrogenase complex subunit El (NCgl2167, SEQ ID NO: 1), a strong promoter called Pcj7 promoter (US 7662943 B2) (SEQ ID NO: 3) was used to replace the wild-type promoter of the aceE gene (SEQ ID NO: 2), thereby preparing a plasmid for enhancing the activity of aceE. The upstream and downstream regions of the aceE gene were obtained. Specifically, to prepare a strain into which aceE having the Pcj7 promoter was introduced, PCR was performed using the chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template, and the upstream region of the aceE gene was amplified using primers of SEQ ID NO: 12 and SEQ ID NO: 13, and the downstream region of the aceE gene was amplified using primers of SEQ ID NO: 14 and SEQ ID NO: 15. In addition, the Pcj7 promoter fragment was obtained using SEQ ID NO: 10 and SEQ ID NO: 11 based on pDCM2-Pcj7 as a template. The primer sequences used to perform each PCR are shown in Table 6 below.

[0228] [Table 6]

[0229]

[0230] PfuUltra TM PfuUltra II High-Fidelity DNA Polymerase (Agilent) was used as a polymerase for the PCR reaction, and PCR was performed by repeating 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute.

[0231] As a result, a 318 bp DNA fragment of the Pcj7 promoter region, a 526 bp DNA fragment upstream of aceE of C. glutamicum ATCC13032, and a 527 bp DNA fragment downstream thereof were obtained, respectively. PCR was performed using the amplified promoter and DNA fragments as templates using primers of SEQ ID NO: 12 and SEQ ID NO: 15. PCR was performed under the following conditions: after denaturation at 95℃ for 5 minutes, 28 cycles of denaturation at 95℃ for 30 seconds, annealing at 55℃ for 30 seconds, and polymerization at 72℃ for 2 minutes, followed by polymerization at 72℃ for 5 minutes. The two fragments obtained above were subjected to DNA purification, and then fusion cloning was performed using an HD Cloning Kit (Clontech) based on a pDCM2 vector (Korean Patent No. WO2021-187781 A1) treated with a SmaI restriction enzyme to obtain a plasmid according to the provided manual. The resulting vector was named pDCM2-Pcj7_aceE.

[0232] Example 3. Preparation of O-acetylhomoserine strain with enhanced pyruvate dehydrogenase complex subunit E1 activity

[0233] To determine whether the strain with enhanced activity has an effect of increasing the production capacity in a C. glutamicum strain having the ability to produce O-acetylhomoserine and homoserine, the pDCM2-Pcj7_aceE vector prepared in Example 2 (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999) was transformed into ATCC13032 ΔNCgl2335::PCJ7-yjeH(eco,F351L) ΔmetB ΔmetY lysC(L377K) ΔNCgl0616, which is an O-acetylhomoserine and homoserine-producing C. glutamicum strain prepared by homologous recombination in the chromosome in Example 1.

[0234] The resulting recombinant strain was named CM04-8001 (ATCC13032 ΔNCgl2335::PCJ7-yjeH(eco,F351L) ΔmetB ΔmetY lysC(L377K) ΔNCgl0616-Pcj7_aceE).

[0235] Example 4. Evaluation of production capacity of O-acetylhomoserine and homoserine strain with enhanced pyruvate dehydrogenase complex subunit E1 activity

[0236] Flask evaluation was performed to compare the production ability of C. glutamicum ATCC13032 ΔNCgl2335::PCJ7-yjeH (eco, F351L) ΔmetB ΔmetY lysC(L377K) ΔNCgl0616 and CM04-8001, which are O-acetylhomoserine and homoserine production strains.

[0237] One inoculation loop of each strain was inoculated into a 250 mL corner-baffled flask containing 25 mL of the following medium, and then incubated at 33°C for 20 hours with shaking at 200 rpm. The concentration of O-acetylhomoserine was analyzed using HPLC, and the analyzed concentrations are shown in Table 7.

[0238] O-acetylhomoserine production medium (pH 7.2)

[0239] 30 g glucose, 2 g KH2PO4, 3 g urea, 40 g (NH4)2SO4, 2.5 g peptone, 5 g (10 ml) corn steep liquor (CSL, Sigma), 0.5 g MgSO4·7H2O, and 20 g CaCO3 (based on 1 L distilled water)

[0240] [Table 7]

[0241]

[0242] As shown in Table 7 above, it was confirmed that the concentration of O-acetylhomoserine increased in the O-acetylhomoserine production strain with enhanced pyruvate dehydrogenase complex subunit El activity compared to ATCC13032 and ATCC13032 ΔNCgl2335::PCJ7-yjeH (eco, F351L) ΔmetB ΔmetY lysC(L377K) ΔNCgl0616.

[0243] Example 5. Preparation of plasmid introducing O-acetylhomoserine transferase (MetX)

[0244] To amplify the gene encoding O-acetylhomoserine transferase (metX), information on the nucleotide sequence of the metX gene (NCBI No. NCgl0624, SEQ ID NO: 6) was obtained from GenBank of the National Institutes of Health (USA), and a BamHI restriction enzyme site was inserted at both ends of each primer (SEQ ID NOs: 17 and 18) for amplification from the promoter region (located about 300 bp upstream of the start codon) to the terminator region (located about 100 bp downstream of the stop codon) on this basis. PCR was performed under the following conditions: after denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 90 seconds, followed by polymerization at 72°C for 7 minutes. As a result, a 1546 bp DNA fragment of the metX gene coding region was obtained. The pECCG117 vector (KR 10-0057684 B1) and the metX DNA fragment were treated with the restriction enzyme BamHI, ligated using a DNA ligase, and cloned to obtain a plasmid, which was named pECCG117-metX WT. The primer sequences are shown in Table 8 below.

[0245] [Table 8]

[0246]

[0247] After introducing the pECCG117-metX WT vector prepared as described above into ATCC 13032 ΔNCgl2335::PCJ7-yjeH(eco, F351L) ΔmetB ΔmetY lysC(L377K) ΔNCgl0616 and CM04-8001 using the electroporation method, the strains were spread on a selective medium containing kanamycin (25 mg / L) and cultured to obtain respective transformants.

[0248] To compare the O-acetylhomoserine production ability of the strains prepared as described above, the strains were cultured by the following method, and the O-acetylhomoserine contained in the culture solution was analyzed.

[0249] One inoculation loop of each strain was inoculated into a 250 mL baffled flask containing 25 mL of the following medium, and then cultured at 33°C with shaking at 200 rpm for 20 hours. The concentration of O-acetylhomoserine was analyzed using HPLC, and the analyzed concentrations are shown in Table 9.

[0250] O-acetylhomoserine production medium (pH 7.2)

[0251] 30 g glucose, 2 g KH2PO4, 3 g urea, 40 g (NH4)2SO4, 2.5 g peptone, 5 g (10 ml) corn steep liquor (CSL, Sigma), 0.5 g MgSO4·7H2O, 400 mg methionine, and 20 g CaCO3(based on 1 L distilled water)

[0252] [Table 9]

[0253]

[0254] As a result, it was confirmed that O-acetyl-L-homoserine accumulated at a concentration of 2.83 g / L when ATCC13032 ΔNCgl2335::PCJ7-yjeH(eco, F351L) ΔmetB ΔmetY lysC(L377K) ΔNCgl0616 / pECCG117-metX WT was cultured, and that the concentration of O-acetylhomoserine was 3.68 g / L when CM04-8002 was cultured, and the yield was increased by 130%.

[0255] Accordingly, when the activity of pyruvate dehydrogenase complex subunit E1 was enhanced in a strain in which the ability to produce O-acetylhomoserine was increased, an increase in the yield of O-acetyl-L-homoserine was confirmed.

[0256] Accordingly, when the activity of pyruvate dehydrogenase complex subunit E1 was enhanced in a strain in which the ability to produce O-acetylhomoserine was increased, an increase in the yield of O-acetyl-L-homoserine was confirmed.

[0257] The above results indicate that, in a coryneform O-acetyl-L-homoserine and homoserine producing strain, enhancement of the activity of pyruvate dehydrogenase complex subunit E1 is effective in the production of O-acetyl-L-homoserine and derivatives thereof.

[0258] Based on the above description, those skilled in the art will understand that the present disclosure can be implemented in different specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are not restrictive, but are illustrative in all aspects. The scope of the present disclosure is defined by the appended claims, not by the specification, and thus all changes and modifications falling within the boundaries and scope of the claims, or equivalent replacements of these boundaries and scope, are encompassed by the claims.

Claims

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

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

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

4. The method of claim 1, wherein the pyruvate dehydrogenase complex subunit El is derived from Corynebacterium glutamicum.

5. The method of claim 1, wherein the pyruvate dehydrogenase complex subunit El comprises the amino acid sequence of SEQ ID NO:

1.

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

3.

7. The method of claim 1, wherein the O-acetylhomoserine or derivative thereof is any one or more selected from the group consisting of O-acetylhomoserine, L-methionine, and L-homoserine.

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

9. A microorganism for producing O-acetylhomoserine or a derivative thereof, in which the activity of pyruvate dehydrogenase complex subunit El is increased compared to its endogenous activity.

10. A composition for producing O-acetylhomoserine or a derivative thereof, comprising: a microorganism in which the activity of pyruvate dehydrogenase complex subunit El is increased compared to its endogenous activity; a culture of the microorganism; a fermentation product of the microorganism; or a combination of two or more thereof.

11. Use of a microorganism in which the activity of pyruvate dehydrogenase complex subunit El is increased compared to its endogenous activity, for producing O-acetylhomoserine or a derivative thereof.

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