Microorganisms with improved activity of pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase and method for producing L-amino acids using the same

Abstract

This application relates to a microorganism in which the activity of pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase is improved, and to a method for producing L-amino acids using the same.

Description

Technical Field

[0001] This application relates to a microorganism with improved activity of pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase and an L - amino acid production method using the same.

Background Art

[0002] Microorganisms belonging to the genus Corynebacterium are Gram - positive microorganisms widely used in L - amino acid production.

[0003] To produce L - amino acids and other useful substances, various studies have been conducted for the development of highly efficient production microorganisms. For example, for the production of L - amino acids, in Corynebacterium strains, approaches specific to the target substances, such as increasing the expression of genes encoding enzymes mainly involved in L - amino acid biosynthesis or removing genes unnecessary for L - amino acid biosynthesis, are mainly used (Patent Document 1).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

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

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

Non-licensed Document 17

Non-Patent Document 18

Non-Patent Document 19

Non-Patent Document 20

Non-Patent Document 21

Summary of the Invention

Problems to be Solved by the Invention

[0006] Research is required on a method for efficiently producing L-amino acids in high yields. [[ID=2�]]

Means for Solving the Problems

[0007] One aspect of the present application provides a microorganism with improved activity of pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase (AceF).

[0008] In one specific example, the microorganism is a microorganism that produces L-amino acids.

[0009] In other specific examples, the microorganisms possess pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase (AceF) activity that is enhanced compared to endogenous activity.

[0010] In microorganisms according to any of the specific examples described above, the L-amino acids include glutamate products, O-acetylhomoserine, homoserine, and methionine.

[0011] In a microorganism according to any of the above-mentioned specific examples, the L-amino acid is at least one selected from glutamine, glutamic acid, ornithine, citrulline, arginine, proline, O-acetylhomoserine, homoserine, and methionine.

[0012] In any of the above-mentioned specific examples of microorganisms, the microorganism may be a microorganism of the genus Corynebacterium.

[0013] In any of the above-mentioned specific examples of microorganisms, the Corynebacterium genus microorganism may be Corynebacterium glutamicum.

[0014] In microorganisms according to any of the above-mentioned specific examples, the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase may be derived from Corynebacterium glutamicum.

[0015] In a microorganism according to any of the above-mentioned specific examples, the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase may contain the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 32.

[0016] In microorganisms according to any of the specific examples described above, the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase may be encoded by the polynucleotide of SEQ ID NO: 2 or SEQ ID NO: 33.

[0017] In any of the above-mentioned specific examples of microorganisms, the microorganism may have improved L-amino acid production capacity compared to the unmodified microorganism.

[0018] In any of the above-mentioned specific examples of microorganisms, the microorganism may be one in which the activity of pyruvate dehydrogenase complex subunit E1 is enhanced compared to its endogenous activity.

[0019] In any of the microorganisms described above, the pyruvate dehydrogenase complex subunit E1 may include the amino acid sequence of SEQ ID NO: 10.

[0020] In any of the microorganisms described above, the pyruvate dehydrogenase complex subunit E1 may be encoded by the polynucleotide of SEQ ID NO: 11 or SEQ ID NO: 39.

[0021] Another aspect of this application provides a method for producing L-amino acids, comprising the step of culturing a microorganism in which the activity of pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase is enhanced in a culture medium.

[0022] In one specific example, the method may further include the step of recovering L-amino acids from the cultured microorganism, the culture of the microorganism, the fermented product of the microorganism, or the culture medium.

[0023] A further aspect of this application provides a composition for L-amino acid production comprising a microorganism in which pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase activity is improved from endogenous activity, a culture of the microorganism, a fermented product of the microorganism, or a combination of at least two thereof.

[0024] Further aspects of this application provide applications for L-amino acid production in microorganisms in which the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase activity is improved compared to endogenous activity.

[0025] Further aspects of this application provide a method for producing L-amino acid-producing microorganisms, comprising a step of modifying the microorganism so that the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase activity of this application is improved compared to endogenous activity. [Effects of the Invention]

[0026] The microorganism described in this application can be used to produce L-amino acids in high yield. [Modes for carrying out the invention]

[0027] These will be explained in detail below. Note that each description and embodiment disclosed in this application applies to other descriptions and embodiments. That is, any combination of the various elements disclosed in this application is included. Furthermore, this application is not limited to the following specific descriptions. In addition, numerous papers and patent documents are referenced throughout this specification, and their citations are indicated. The disclosures of the cited papers and patent documents are incorporated in their entirety as references in this specification, thereby more clearly explaining the level of the art to which this application belongs and the content of this application.

[0028] definition As used in the specification and claims of this application, singular articles ("a," "an," and "the") include plural subjects unless otherwise specified. Also, unless otherwise specified, plural terms include singular terms. Furthermore, in the specification and claims of this application, unless otherwise specified, "or" is used to include "and / or."

[0029] In this application, "about" is used before a specific number. In this application, "about" includes not only the exact number that follows the term "about," but also a range that is approximately that number or close to it. Considering the context in which the number is used, it is possible to determine whether it is close to or approximately that specific number. For example, "about" indicates a range of -10% to +10% of a given number. Another example is that "about" indicates a range of -5% to +5% of a given number. However, it is not limited to these examples.

[0030] In this application, terms such as "first, second, third," "i), ii), iii)," and "(a), (b), (c), (d)," are used to distinguish similar configurations and do not imply that they are continuous or performed in order. For example, when the above terms are used in relation to steps of a method, use, or analysis, there may be no time interval between those steps, they may be performed simultaneously, or they may be performed with intervals of a few seconds, a few minutes, a few hours, a few days, or a few months.

[0031] In this application, "consisting essentially of" means that the presence of the unspecified component is not substantially affected by the presence of the unspecified component in the manner in which the features of the subject matter claimed by this application are claimed.

[0032] In this application, "consisting of" means that the proportion of the specific components referred to by the above term totals 100%. The components or features referred to as "consisting of" are either essential or mandatory. In one specific example, any other optional or non-essential components other than those referred to as "consisting of" are excluded.

[0033] In this application, “comprising” means that the features, steps, or components referred to by the above terms are present, and does not exclude the presence or addition of one or more features, steps, or components. While the components or features referred to as “comprising” in this application are essential or mandatory, in a specific example, other optional or non-essential components or features may be further included.

[0034] Proteins, polypeptides In this application, "protein" or "polypeptide" means a polymer or oligomer of continuous amino acid residues. In this application, "polypeptide," "protein," and "peptide" are used interchangeably with "amino acid sequence."

[0035] In some cases, an active protein, polypeptide, or peptide is referred to as an "enzyme." In this application, unless otherwise specified, amino acid sequences are described in the direction from the N-terminus to the C-terminus.

[0036] In this application, "mature polypeptide" refers to a polypeptide in a form that lacks a signal sequence or propeptide sequence. A mature protein / polypeptide / peptide may also be a functional form of a protein / polypeptide / peptide. A mature polypeptide may also be the final form after translation or post-translational modification. Examples of post-translational modification include, but are not limited to, alteration of the N or C terminus, glycosylation, phosphorylation, and removal of a leader sequence.

[0037] In this application, with respect to amino acid sequences, polypeptides or proteins that "contain" an amino acid sequence represented by a specific sequence number, polypeptides or proteins that "consist" of an amino acid sequence represented by a specific sequence number, or polypeptides or proteins that "have" an amino acid sequence represented by a specific sequence number, include polypeptides or proteins in which some amino acids are deleted, modified, substituted, conservatively substituted, or added, as long as they have the same or equivalent activity as the polypeptide or protein consisting of the amino acid sequence of the said sequence number. For example, the polypeptides or proteins also include polypeptides or proteins that have additions or deletions of sequences that do not change the function of the protein, spontaneously occurring mutations, silent mutations, or conservative substitutions within or before / after (N-terminus or C-terminus) the polypeptide or protein sequence, as long as they have the same or equivalent activity.

[0038] As a specific example, polypeptides (proteins) bound to an N-terminal signal (or leader) sequence involved in the translocation of polypeptides (proteins) co-translationally or post-translationally, as well as polypeptides (proteins) bound to other sequences or linkers so that polypeptides (proteins) can be identified, purified, or synthesized, are also included in the polypeptides of the amino acid sequence represented by the specific sequence number.

[0039] In this application, "conservative substitution" means that one amino acid is replaced by another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; amino acids with nonpolar side chains (nonpolar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Other examples include amino acids with electrically charged side chains (arginine, lysine, histidine, glutamic acid, and aspartic acid) and amino acids with uncharged side chains (also called neutral amino acids) (glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine). Further examples include phenylalanine, tryptophan, and tyrosine, which are classified as aromatic amino acids. Still further examples include valine, leucine, and isoleucine, which are classified as branched amino acids.As another example, the 20 amino acids are classified by size and, in order of increasing relative volume, into five groups: glycine, alanine, and serine; cysteine, proline, threonine, aspartic acid, and asparagine; valine, histidine, glutamic acid, and glutamine; isoleucine, leucine, methionine, lysine, and arginine; and phenylalanine, tryptophan, and tyrosine. Conservative substitutions typically have little to no effect on polypeptide activity.

[0040] Polynucleotides In this application, "gene" means a polynucleotide that codes for a polypeptide and a polynucleotide that includes the regions before and after the coding region. In one specific example, the gene may have sequences (introns) inserted between each coding region (exon).

[0041] In this application, "polynucleotide, nucleic acid, or nucleic acid molecule" means a polymer of nucleotides in which nucleotide monomers are covalently linked together in a long chain, and which is a DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA) chain longer than a predetermined length.

[0042] homology, identity In this application, "homology" or "identity" refers to the degree of similarity between corresponding sequences in two given amino acid or nucleotide sequences, expressed as a percentage. Homology and identity are often used interchangeably.

[0043] The sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard sequencing algorithms, which may also be used in conjunction with a default gap penalty established by the program used. Substantively, homologous or identical sequences generally hybridize with at least 50%, 60%, 70%, 80%, or 90% of the entire sequence or its total length under moderate to high stringent conditions. Hybridization also includes hybridization with polynucleotides that have common codons or codons considering codon degeneracy.

[0044] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined, for example, using default parameters as described in Non-Patent Document 1 and known computer algorithms such as the "FASTA" program. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 3), as performed in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later) (including the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, and FASTA (Non-Patent Documents 5, 6, and 7)). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information.

[0045] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as Non-Patent Document 3, as disclosed in Non-Patent Document 8, for example. In summary, the GAP program is defined as the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of two sequences. Default parameters for the GAP program include (1) unitary matrices (where identity is 1 and non-identity is 0), a PAM Matrix (see disclosure in Non-Patent Document 9), and a weighted comparison matrix from Non-Patent Document 10 (or an EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix), (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extended penalty of 0.5), and (3) no penalty for terminal gaps.

[0046] Furthermore, whether any two polynucleotide sequences are homologous, similar, or identical can be confirmed by comparing the sequences in a Southern hybridization experiment under defined stringent conditions. The defined appropriate hybridization conditions are within the scope of the art and are determined by methods well known to those skilled in the art (e.g., Non-Patent Documents 11 and 12), but are not limited to these.

[0047] In this application, "stringent condition" refers to conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Document 13). For example, this could involve hybridizing polynucleotides with high homology or identity, such as 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, while not hybridizing polynucleotides with lower homology or identity. Alternatively, it could involve washing once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of a typical Southern hybridization: 60°C, 1×SSC, 0.1%SDS, more specifically 60°C, 0.1×SSC, 0.1%SDS, or more specifically 68°C, 0.1×SSC, 0.1%SDS.

[0048] The aforementioned hybridization requires that the two nucleotides have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of this application may include not only substantially similar base sequences, but also isolated nucleic acid fragments whose entire sequences are complementary.

[0049] For example, polynucleotides homologous or identical to the polynucleotides of this application can be detected using hybridization conditions in which the hybridization step is performed at a Tm value of 55°C and the conditions described above. The Tm value may be 60°C, 63°C, or 65°C, but is not limited to these, and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0050] The appropriate stringency for hybridizing the polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are known in the art (e.g., Non-Patent Document 11).

[0051] nucleic acid constructs, vectors, transformations In this application, "nucleic acid construct" means a single-stranded or double-stranded nucleic acid molecule that includes at least one regulatory sequence and is either artificially synthesized, manipulated to include a specific sequence in a manner not found in nature, or isolated from nature.

[0052] In this application, “vector” means a DNA product for delivering a target polynucleotide into a suitable host or host cell. For example, it may include a polynucleotide sequence encoding the target polypeptide operably linked to a suitable regulatory region (or regulatory sequence) so that the target polypeptide can be expressed in a suitable host. The regulatory region includes a promoter to initiate transcription, an optional operator sequence to regulate the transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences to regulate the termination of transcription and translation. Once transformed into a suitable host cell, the vector can replicate and function independently of the host genome and is integrated into the genome itself.

[0053] The vectors used in this application are not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state. For example, as phage vectors or cosmid vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used, and as plasmid vectors, pDZ series, pDC series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, pET series, etc. can be used. Specifically, vectors such as pDZ, pDC, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, and pDCM2 (Patent Document 2) can be used.

[0054] For example, a target polynucleotide can be inserted into a chromosome using an intracellular chromosome insertion vector. The insertion of the polynucleotide into the chromosome can be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. The vector may further include a selection marker to confirm whether or not the polynucleotide has been inserted into the chromosome. The selection marker is used to select cells transformed by the vector, that is, to confirm whether or not the target nucleic acid molecule has been inserted, and markers that confer selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, and expression of surface polypeptides are used. In an environment treated with a selective agent, only cells expressing the selection marker will survive or exhibit different phenotypes, thus allowing for the selection of transformed cells.

[0055] In this application, "transformation" means altering the genetic traits of a host cell by introducing a vector containing a target polynucleotide into the host cell. The transformed polynucleotide may be located within the host cell's chromosomes or outside of them. The polynucleotide may also contain DNA or RNA. The polynucleotide may be introduced in a form appropriate to the purpose of introduction. For example, a polynucleotide for expressing a target polypeptide may be introduced into the host cell in the form of an expression cassette, which is a gene structure containing all the elements necessary for its expression. Typically, the expression cassette includes a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may also be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form and operably linked to the sequence necessary for expression in the host cell, but is not limited to this.

[0056] In this application, "operably linked" means a configuration in which a regulatory sequence is positioned appropriately so that the regulatory sequence indicates the expression of a coding sequence. Therefore, "operably linked" includes a regulatory region of a well-known or desired functional domain, such as a promoter, terminator, signal sequence, or enhancer region, that is attached to or linked to a target (gene or polypeptide) so that its expression, secretion, or function can be regulated according to its well-known or desired activity. For example, it means a promoter sequence that initiates and mediates the transcription of a polynucleotide encoding a polypeptide, and the polynucleotide sequence is functionally linked to the polynucleotide sequence.

[0057] In this application, "expression" includes, but is not limited to, any steps involved in the production of polypeptides, such as transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0058] In this application, "expression vector" means a linear or cyclic nucleic acid molecule comprising a target polynucleotide sequence and a regulatory sequence operably linked for its expression.

[0059] In this application, "regulatory sequence" means a polynucleotide sequence necessary for regulating the expression of a target polynucleotide sequence. Each regulatory sequence may be a sequence native to the coding sequence (of the same origin), a foreign sequence (derived from another gene), a variant thereof, or another artificial sequence. Examples of regulatory sequences include leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, operator sequences, sequences encoding ribosome binding sites, and sequences that regulate transcription and translation termination. The smallest unit of a regulatory sequence includes a promoter and transcription and translation termination sequences.

[0060] With respect to cells, polynucleotides, polypeptides, or vectors, “recombinant” in this application means that cells, polynucleotides, polypeptides, or vectors are modified by the introduction of heterologous nucleic acids or polypeptides, or by alteration of native polynucleotides or polypeptides, or that cells are derived from such modified cells. For example, recombinant cells express genes that are not found in the cell’s native (non-recombinant) form, or they express genes that are not expressed at all, or they express native genes that are abnormally expressed.

[0061] microorganisms In this application, "microorganism (or strain)" includes all wild-type microorganisms and prokaryotic or eukaryotic microorganisms that have been genetically modified naturally or artificially. These microorganisms have had specific mechanisms weakened or strengthened due to causes such as the insertion of external genes or the enhancement or inactivation of endogenous gene activity, and have been genetically modified for the production of a target polypeptide, protein, or product. In this application, "microorganism" and "strain" are used interchangeably and are used together.

[0062] For example, the microorganisms of this application are microorganisms (e.g., recombinant strains) in which the acetyltransferase activity of the pyruvate dehydrogenase complex dihydrolipoyllysine residue is enhanced compared to endogenous activity.

[0063] In this application, "microorganisms having L-amino acid production ability" refers to microorganisms that produce L-amino acids within their bodies, and includes all microorganisms that have been given L-amino acid production ability from those that originally lacked it, as well as microorganisms that originally possessed L-amino acid production ability. L-amino acid production ability can be conferred or improved through selective breeding.

[0064] In this application, "unmodified microorganism (strain)" does not exclude microorganisms (strains) that include naturally occurring mutations, but rather refers to wild-type microorganisms (strains) or natural microorganisms (strains) themselves, or microorganisms (strains) before their traits change due to genetic mutation caused by natural or artificial factors. The aforementioned "unmodified microorganism (strain)" is used interchangeably with "pre-modification microorganism (strain)," "non-mutant microorganism (strain)," "parent microorganism," "parent strain," "wild-type microorganism (strain)," "reference microorganism (strain)," or "standard microorganism (strain)." In this application, "unmodified microorganism" refers to, but is not limited to, microorganisms in which the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase activity in this application has not been improved from endogenous activity, or before it has been improved.

[0065] Furthermore, in this application, an unmodified microorganism is a microorganism containing an amino acid sequence consisting of SEQ ID NO: 1 or SEQ ID NO: 32, or a polynucleotide consisting of SEQ ID NO: 2 or SEQ ID NO: 33, but is not limited to these.

[0066] Improvement of polypeptide activity In this application, "increase" of polypeptide activity means that the activity of the polypeptide in the host cell is improved compared to its endogenous activity. This increase is used interchangeably with activation, upregulation, overexpression, enhancement, etc. The host may be a prokaryotic or eukaryotic microorganism.

[0067] The aforementioned improvement in polypeptide activity includes both the host microorganism exhibiting polypeptide activity it did not inherently possess, and the polypeptide activity being improved compared to the endogenous activity or the activity before modification.

[0068] For example, the aforementioned "exhibiting activity that was not originally present" is due to "introduction of polypeptides (proteins)," but is not limited to this. The introduction of polypeptides (proteins) means causing the activity of a specific polypeptide (protein) to appear as a result of the expression of a gene that was not originally present in the microorganism, or causing an increase or improvement in the activity of the polypeptide (protein) compared to its endogenous activity or the activity before modification. For example, this may involve introducing a polynucleotide encoding a specific polypeptide (protein) into the chromosome of the microorganism, or introducing a vector containing a polynucleotide encoding a specific polypeptide (protein) into the microorganism, resulting in the appearance of its activity.

[0069] The aforementioned "endogenous activity" refers to the activity of a specific polypeptide that was originally present in the microorganism before the trait change or in the unmodified microorganism when a trait changes due to genetic mutation caused by natural or artificial factors. This is often used interchangeably with "activity before modification."

[0070] An increase in polypeptide activity compared to endogenous activity means that the activity and / or concentration (expression level) of a specific polypeptide is higher than that originally present in the microorganism before the trait change or in the unmodified microorganism.

[0071] For example, the aforementioned improvement means that activity that was not present in the corresponding protein / polypeptide becomes apparent, or that the activity or concentration increases by approximately 1%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, or 500%, and up to approximately 1000% or 2000% or more, compared to the activity or concentration in the initial microbial strain, but is not limited to these.

[0072] The activity of the polypeptide can be improved by introducing an exogenous polypeptide or by improving the activity of an endogenous polypeptide. Whether or not the activity of the polypeptide has improved can be confirmed by the degree of the polypeptide's activity, its expression level, or the increase in the amount of the product produced by the polypeptide's activity.

[0073] Various methods known in the field can be applied to improve the activity of the polypeptide, and any method that improves the activity of the target polypeptide compared to the microorganism before modification is acceptable. Specifically, this includes, but is not limited to, conventional methods in molecular biology, including genetic engineering and / or protein engineering known to those with ordinary skill in the field (see, for example, Non-Patent Documents 14, 15, etc.).

[0074] Specifically, the improvement of the polypeptide activity of this application is achieved by 1) increasing the intracellular copy number of the polynucleotide encoding the polypeptide, 2) modifying the expression regulatory region of the gene on the chromosome encoding the polypeptide (for example, by inducing mutations in the expression regulatory region, substituting with a sequence having higher activity, or inserting a sequence having higher activity), 3) modifying the base sequence encoding the start codon or 5'UTR region of the gene transcript encoding the polypeptide, 4) modifying the amino acid sequence of the polypeptide to improve polypeptide activity, 5) modifying the polynucleotide sequence encoding the polypeptide to improve polypeptide activity (for example, by modifying the polynucleotide sequence of the polypeptide gene to encode a polypeptide modified to improve polypeptide activity), 6) introducing an exogenous polypeptide exhibiting polypeptide activity or an exogenous polynucleotide encoding it, 7) optimizing the codon of the polynucleotide encoding the polypeptide, 8) analyzing the tertiary structure of the polypeptide and selectively modifying or chemically modifying exposed regions, 9) regulating the cellular localization of the protein (polypeptide), or 10) a combination of two or more selected from 1) to 9) above, but is not limited to these.

[0075] For example, increasing the intracellular copy number of the polynucleotide encoding the polypeptide (as described in 1) above may be carried out by introducing a vector containing the polynucleotide encoding the polypeptide operably linked to a suitable regulatory sequence into a host cell. The vector may be a vector that replicates and functions independently of the host. Alternatively, it may be carried out by introducing one or more copies of the polynucleotide encoding the polypeptide operably linked to a suitable regulatory sequence into a chromosome in a host cell. The introduction into the chromosome is carried out by introducing a vector into the host cell that is capable of inserting the polynucleotide into a chromosome in the host cell, but is not limited to this. The vector is as described above. The regulatory sequence may be a sequence native (of the same origin) to the encoding polynucleotide sequence, a foreign (derived from another gene) sequence, a variant thereof, or another artificial sequence, and may induce the expression of the polynucleotide in the host cell.

[0076] 2) The substitution of a gene expression regulatory region (or expression regulatory sequence) on a chromosome encoding a polypeptide with a more potent sequence may be carried out, for example, by causing a sequence mutation through deletion, insertion, substitution, or a combination thereof, or by substituting it with a sequence having higher activity, so as to further improve the activity of the expression regulatory region. The expression regulatory region includes, but is not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, sequences regulating transcription and translation termination, etc. For example, this may be carried out by substituting the original promoter with a potent promoter, but is not limited to this.

[0077] Examples of known strong promoters include, but are not limited to, the cj1-cj7 promoter (Patent Document 3), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (Patent Document 4), the O2 promoter (Patent Document 5), the tkt promoter, and the yccA promoter.

[0078] 3) Modifying the base sequence of the start codon or 5'UTR region of the polypeptide-encoding gene is performed, for example, by substituting it with another start codon that has a higher polypeptide expression rate compared to the endogenous start codon, but is not limited to this.

[0079] Modifying the amino acid sequence or polynucleotide sequence of the polypeptides described in 4) and 5) above is performed by causing sequence mutations through deletion, insertion, non-conservative or conservative substitution, or combination thereof, of the amino acid sequence or polynucleotide sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, in order to improve the activity of the polypeptide, or by substituting it with an improved amino acid sequence or polynucleotide sequence that has higher activity, or an improved amino acid sequence or polynucleotide sequence that has improved activity. Specifically, the substitution is performed by inserting the polynucleotide into the chromosome by homologous recombination, but is not limited to this. The vector used here may further include a selection marker to confirm whether or not it has been inserted into the chromosome. The selection marker is as described above.

[0080] The introduction of a foreign polynucleotide exhibiting polypeptide activity (6) above may be carried out by introducing a foreign polynucleotide encoding a polypeptide exhibiting identical or similar activity to the polypeptide into the host cell. The foreign polynucleotide may have any origin or sequence, as long as it exhibits identical or similar activity to the polypeptide. The introduction can be carried out by a person skilled in the art using a known transformation method as appropriate, and as described above, the introduction of the polynucleotide into the host cell results in the production of the polypeptide and improvement of its activity.

[0081] The optimization of codons of polynucleotides encoding polypeptides described in 7) above may be performed by optimizing endogenous polynucleotide codons so as to increase transcription or translation within the host cell, or by optimizing exogenous polynucleotide codons so as to perform optimized transcription or translation within the host cell.

[0082] The 8) analysis of the tertiary structure of the polypeptide, and the selection and modification or chemical modification of exposed portions may be carried out, for example, by comparing the sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored, determining candidate template proteins according to the degree of sequence similarity, confirming the structure based on these, and selecting and modifying or chemically modifying exposed portions.

[0083] 9) The regulation of the intracellular location of a protein (polypeptide) may be carried out by targeting the protein (polypeptide) to a specific intracellular organelle or specific intracellular space. For example, this can be done by adding or removing a leader sequence that functions to target the protein (polypeptide), thereby targeting the periplasm or cytoplasm, but is not limited to these.

[0084] Such improvement in polypeptide activity is achieved by increasing the activity or concentration of the corresponding polypeptide compared to the activity or concentration of the polypeptide expressed in the wild-type or pre-modification microorganism, or by increasing the amount of product produced from the polypeptide, but is not limited to these methods.

[0085] Weakening of polypeptide activity In this application, "weakening" of the activity of polypeptides (including proteins identified by the names of each enzyme) is a concept that encompasses all cases where the activity is reduced compared to endogenous activity or where the activity is eliminated. The term "weakening" is used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation.

[0086] The aforementioned weakening includes at least one of the following: the activity of the polypeptide itself is reduced or eliminated compared to the original polypeptide activity of the microorganism due to mutations in the polynucleotide encoding the polypeptide; the overall degree and / or concentration (expression level) of polypeptide activity within the cell is reduced compared to the natural strain due to inhibition of the expression of the gene encoding the polynucleotide or inhibition of translation into the polypeptide; there is no expression of the polynucleotide at all; and even if the polynucleotide is expressed, there is no polypeptide activity. "Inactivation," "deficiency," "reduction," "downregulation," "decrease," or "attenuation" of polypeptide activity compared to endogenous activity means that it is reduced compared to the activity of the specific polypeptide that was originally present in the parent strain or unmodified microorganism before the trait change.

[0087] Such weakening of polypeptide activity is not limited to these methods and can be achieved by applying various methods well known in the field (e.g., Non-Patent Documents 15, 16, etc.).

[0088] Specifically, weakening the activity of the polypeptide in this application involves: 1) deleting all or part of the gene encoding the polypeptide; 2) modifying the expression regulatory region (or expression regulatory sequence) so that the expression of the gene encoding the polypeptide is reduced; 3) modifying the amino acid sequence constituting the polypeptide so that the activity of the polypeptide is deleted or weakened (for example, deleting / substituting / adding one or more amino acids in the amino acid sequence); and 4) modifying the gene sequence encoding the polypeptide so that the activity of the polypeptide is deleted or weakened (for example, modifying the gene sequence to encode a polypeptide that has been modified so that the activity of the polypeptide is deleted or weakened). 1) Deleting / substituting / adding one or more nucleic acid bases in the nucleic acid base sequence of a peptide gene; 5) Modifying the base sequence encoding the start codon or 5'UTR region of the polypeptide-encoding gene transcript; 6) Introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementaryly to the polypeptide-encoding gene transcript; 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the polypeptide-encoding gene so that a secondary structure is formed that prevents ribosome attachment; 8) Adding a promoter to the 3' end of the open reading frame (ORF) of the polypeptide-encoding gene sequence to reverse transcription (Reverse transcription engineering, RTE); 9) Regulating the cellular localization of the protein (polypeptide); or 10) Combining two or more of the above 1) to 9), but not limited to these.

[0089] For example, the deletion of part or all of the gene encoding the polypeptide described in 1) above may be carried out by deleting the entire polynucleotide encoding the endogenous target polypeptide within the chromosome, or by substituting it with a polynucleotide or marker gene in which some nucleotides are deleted.

[0090] Furthermore, modifying the regulatory expression region (or regulatory expression sequence) described in 2) above may be carried out by causing a mutation in the regulatory expression region (or regulatory expression sequence) through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by substituting it with a sequence having lower activity. The regulatory expression region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates the termination of transcription and translation.

[0091] Modifying the amino acid sequence or polynucleotide sequence described in 3) and 4) above is carried out by introducing a sequence mutation through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, so as to weaken the activity of the polypeptide, or by substituting it with an amino acid sequence or polynucleotide sequence modified to have lower activity, or an amino acid sequence or polynucleotide sequence modified to eliminate activity, but is not limited to these methods. For example, gene expression can be inhibited or weakened by introducing a mutation into a polynucleotide sequence to form a stop codon, but is not limited to these methods.

[0092] Modification of the start codon or 5'UTR region of the gene transcript encoding the polypeptide (as described in 5) above is performed, for example, by substituting it with a nucleotide sequence encoding another start codon that has a lower polypeptide expression rate compared to the endogenous start codon, but is not limited to this.

[0093] The introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcript encoding the polypeptide described in 6) above may be carried out, for example, as described in Non-Patent Document 17.

[0094] 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence in the polypeptide-coding gene so that a secondary structure is formed that makes ribosome attachment impossible may be done by making mRNA translation impossible or slowing down the rate of mRNA translation.

[0095] Furthermore, the reverse transcription engineering (RTE) of the open reading frame (ORF) of the gene sequence encoding the polypeptide (8) above may be performed by creating an antisense nucleotide complementary to the gene transcript encoding the polypeptide and weakening its activity.

[0096] 9) This may be carried out by targeting specific organelles or spaces within specific cells with the protein (polypeptide). For example, this may be done by adding or removing a leader sequence that functions to target the protein (polypeptide), thereby targeting the periplasm or cytoplasm, but is not limited to these methods.

[0097] Such weakening of polypeptide activity may be, but is not limited to, a reduction in the activity, concentration, or expression level of the corresponding polypeptide compared to the activity or concentration of the polypeptide expressed in the wild-type or pre-modification microbial strain, or a decrease in the amount of product produced from the polypeptide.

[0098] In the microorganisms of this application, modification of part or all of the polynucleotides can be induced by (a) homologous recombination using a chromosome introduction vector in the microorganism, or genome editing using an engineered nuclease (e.g., CRISPR-Cas9), and / or (b) light and / or chemical treatment such as ultraviolet light or radiation.

[0099] culture In this application, "cultivation" means growing microorganisms under appropriately controlled environmental conditions. The cultivation process can be carried out using suitable culture media and cultivation conditions known in the art. Such a cultivation process can be easily adjusted and used by those skilled in the art depending on the selected microorganism. Specifically, the cultivation is batch, continuous, and / or fed-batch culture, but is not limited to these.

[0100] In this application, "culture medium" refers to a substance that is a mixture mainly composed of nutrients necessary for culturing microorganisms, and supplies nutrients and growth factors, including water, which are essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the microorganisms in this application may be any that are normally used for culturing microorganisms, and the microorganisms in this application can be cultured in a normal culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, under aerobic conditions, with the temperature, pH, etc. adjusted. For example, a culture medium for Corynebacterium microorganisms is disclosed in Non-Patent Document 18.

[0101] In this application, the carbon source can be carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn maceration liquid can be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and any other carbon source in an appropriate amount may be used. These carbon sources can be used individually or in combination of two or more, but are not limited to these uses.

[0102] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate can be used, as well as organic nitrogen sources such as amino acids like glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn maceration liquid, casein hydrolysates, fish or their decomposition products, defatted soybean cake or its decomposition products. These nitrogen sources can be used individually or in combination of two or more, but are not limited to these uses.

[0103] As the phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or equivalent sodium-containing salts can be used. As inorganic compounds, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc., can be used, and in addition, amino acids, vitamins, and / or suitable precursors can be used. These components or precursors can be added to the culture medium in batches or continuously, but are not limited to these.

[0104] Furthermore, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture medium in a suitable manner during the cultivation of the microorganisms of this application. In addition, during cultivation, the formation of bubbles can be suppressed using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, oxygen or oxygen-containing gas may be injected into the culture medium to maintain an aerobic state, and to maintain an anaerobic and microaerobic state, it is not necessary to inject gas, but nitrogen, hydrogen, or carbon dioxide gas may be injected, but the invention is not limited to these.

[0105] In the culture described in this application, the culture temperature is maintained at 20-45°C, specifically 25-40°C, and the culture is performed for approximately 10-160 hours, but is not limited to these values.

[0106] In this application, "culture" means a culture solution, concentrated culture solution, dried culture solution, culture filtrate, concentrated culture filtrate, or dried culture filtrate obtained by culturing a specific microorganism in a culture medium, wherein the culture solution means one containing the specific microorganism, and the culture filtrate means one that substantially does not contain the specific microorganism (here, this means substantially removing the specific microorganism separated by filtration, etc., and does not mean completely removing microorganisms from the filtrate). The culture may be in any dosage form, for example, a liquid, emulsion, or solid.

[0107] In this application, "fermentation" refers to the process by which microorganisms use their enzymes to decompose organic matter, excluding putrefaction reactions. Fermentation and putrefaction reactions proceed through similar processes, but if useful substances are produced as a result of the decomposition, it is called fermentation, while if foul odors are produced or harmful substances are created, it is called putrefaction.

[0108] In this application, the method for obtaining the fermented product from the microorganism is not particularly limited and can be obtained by methods commonly used in the art or similar fields.

[0109] In this application, "fermented product" includes not only the fermented substance itself, but also all kinds of substances containing fermented products generated from the microorganisms, such as a microbial culture medium in which microorganisms and cultures coexist, a fermented product obtained by filtering microorganisms from the culture medium, a fermented product obtained by filtering sterilized microorganisms from the culture medium, an extract obtained by extracting the fermented product or a culture medium containing the same, a diluted solution or concentrate obtained by diluting the fermented product or its extract, a dried product obtained by drying the fermented product or its extract, and a lysate obtained by collecting and crushing the microbial cells of the microorganisms.

[0110] Specific description of this application The following provides a more detailed explanation of a specific example of this application.

[0111] One aspect of this application provides a microorganism in which the acetyltransferase activity of the pyruvate dehydrogenase complex dihydrolipoyllysine residue is improved compared to its endogenous activity.

[0112] As an example of this application, the microorganism described herein may have the ability to produce L-amino acids.

[0113] The improved pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase activity is defined as the improved L-amino acid production capacity of the microorganisms of this application compared to the production capacity of natural wild-type microorganisms or unmodified microorganisms (for example, microorganisms expressing polypeptides having wild-type pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase activity (e.g., the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 32), or microorganisms in which the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase activity of this application is not improved from or before improvement occurs). However, it is not limited to this definition.

[0114] For example, the acetyltransferase activity of the pyruvate dehydrogenase complex dihydrolipoyllysine residue can be determined by measuring the L-amino acid production capacity or yield, but is not limited to this.

[0115] In this application, "pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase" refers to one component of the pyruvate dehydrogenase complex that has dihydrolipoyllysine residue acetyltransferase activity, that is, activity to transfer an acetyl group to CoA.

[0116] The pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase of this application is used interchangeably with "AceF". The pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase is known in the art and is specifically encoded by aceF, but is not limited thereto. The amino acid and polynucleotide sequences of the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase are obtained from known databases, such as NCBI's GenBank, but are not limited thereto.

[0117] For example, the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase may contain the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having 60% or more homology or identity thereto. However, any protein that has pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase activity is acceptable. Specifically, even if a protein contains a sequence in which some of the amino acid sequences of SEQ ID NO: 1 are deleted, modified, substituted, or added, any protein that exhibits efficacy equivalent to the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase is included in the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase. Furthermore, any protein that has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 32, or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with the aforementioned sequence, or that contains the aforementioned amino acid sequence, or consists of the aforementioned amino acid sequence, or is substantially composed of the aforementioned amino acid sequence, and exhibits efficacy equivalent to that of the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase, is included in the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase. For example, the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase is either an exogenous protein or a protein endogenously present in microorganisms of the genus Corynebacterium or Corynebacterium glutamicum, but is not limited to these. Specifically, it is a pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 32, which is endogenously present in microorganisms of the genus Corynebacterium or Corynebacterium glutamicum, but is not limited to these.Examples include NCBIReference No.WP_011014958.1, WP_060564822.1, and WP_074492848.1.

[0118] For example, the AceF, pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase of this application may also exhibit the activity of 2-oxoglutarate dehydrogenase, the E2 component, or dihydrolipoamide succinyltransferase. For example, the gene encoding AceF of this application is the gene sucB. However, it is not limited to this.

[0119] Furthermore, polynucleotides encoding pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase 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 based on codon information known in the art. For example, the protein is encoded by a polynucleotide having, or containing, or consisting of, or substantially composed of, the sequence of SEQ ID NO: 2 or 33, or a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the sequence of SEQ ID NO: 2 or 33.

[0120] In this application, a gene containing the base sequence represented by a specific sequence number is used in combination with a polynucleotide containing the base sequence represented by a specific sequence number.

[0121] The polynucleotides of this application can be modified in various ways in the coding region, either through codon degeneracy or by considering codons preferred in organisms that intend to express the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase of this application, as long as the amino acid sequence of the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase of this application does not change. Therefore, it goes without saying that polynucleotides translated by codon degeneracy into polypeptides consisting of the amino acid sequence of the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase of this application, or polypeptides homologous or identical thereto, are also included in the polynucleotides of this application. As an example, the polynucleotides of this application may be SEQ ID NO: 2, SEQ ID NO: 33, or their degenerated sequences.

[0122] As another example, the polynucleotides of this application have a base sequence that is homologous or identical to Sequence ID No. 2 by 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and less than 100%, or include such a base sequence, consist of such a base sequence, or are substantially composed of such a base sequence.

[0123] Furthermore, the polynucleotide of this application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a complementary sequence to all or part of the polynucleotide sequence of this application, and encodes the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase of this application.

[0124] In this application, "L-amino acids" include both proteinaceous and non-proteinaceous amino acids. In this application, the L-amino acids include glutamate products, O-acetylhomoserine, homoserine, and methionine.

[0125] In this application, "glutamate-based products" include glutamic acid and all L-amino acids that are biosynthesized using glutamic acid as a precursor. For example, L-amino acids produced by the ornithine cycle using glutamic acid as a precursor include ornithine, citrulline, arginine, and proline. Furthermore, glutamine is an L-amino acid that is converted using glutamic acid as a precursor. In addition, any other L-amino acid that is biosynthesized using glutamic acid as a precursor is included in the glutamate-based products of this application.

[0126] As a specific example, the L-amino acid of this application may be at least one selected from glutamine, glutamic acid, ornithine, citrulline, arginine, proline, O-acetylhomoserine, homoserine, and L-methionine.

[0127] For the purposes of this application, the microorganisms described herein include all microorganisms that produce the target L-amino acid by having improved pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase activity compared to endogenous activity. For example, the microorganisms described herein are characterized by improved L-amino acid production capacity due to improved pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase activity compared to endogenous activity, and are genetically modified or recombinant microorganisms, but are not limited to these. Specifically, the recombinant microorganisms with improved L-amino acid production capacity are microorganisms that have improved L-amino acid production capacity compared to natural wild-type microorganisms or unmodified microorganisms with endogenous activity of pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase, but are not limited to these.

[0128] For example, microorganisms capable of producing L-amino acids are prokaryotic or eukaryotic microorganisms that produce L-amino acids within their bodies, and include both microorganisms that inherently possess L-amino acid production ability and microorganisms that lacked L-amino acid production ability intrinsically but have been given L-amino acid production ability. L-amino acid production ability can be conferred or improved through the improved pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase activity and selective breeding described in this application.

[0129] For example, the recombinant microorganisms having L-amino acid production ability of this application include any microorganism that, when transformed by a vector, have improved pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase activity and produce L-amino acids.

[0130] For example, the microorganism producing the L-amino acid may be a microorganism that intrinsically contains a protein consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 32, or a protein consisting of an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with SEQ ID NO: 1 or SEQ ID NO: 32.

[0131] For example, the microorganism producing the L-amino acid may be a microorganism that intrinsically contains a polynucleotide sequence encoding a protein having at least 60% homology to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 32, the base sequence of SEQ ID NO: 2 or SEQ ID NO: 33, or a base sequence having 60% or more homology or identity with the base sequence of SEQ ID NO: 2 or SEQ ID NO: 33, 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.

[0132] The microorganisms described in this application include all microorganisms in which the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase activity has been improved from its endogenous activity by various known methods.

[0133] As a specific example, a microorganism in which the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase activity is improved compared to endogenous activity is a microorganism in which the protein expression is increased and the activity is improved by replacing the sequence that regulates the expression of the sequence encoding the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase with another sequence, or by modifying the base sequence that encodes the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase, or by substituting the start codon, but is not limited to these. As a specific example, a microorganism in which the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase activity is improved compared to endogenous activity may be a microorganism in which the protein expression is increased and the activity is improved by replacing the sequence that regulates the expression of the sequence encoding the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase with a sequence that has higher activity than the endogenous expression regulatory sequence, but is not limited to these.

[0134] For example, the microorganisms with improved L-amino acid production capacity in this application are, but are not limited to, microorganisms that have improved L-amino acid production capacity compared to unmodified microorganisms. For example, the unmodified microorganisms used for comparison to determine whether or not the L-amino acid production capacity has improved are the ATCC13032 or ATCC13869 strains, but are not limited to these.

[0135] As an example, the microorganism with improved L-amino acid production capacity is one in which the L-amino acid production capacity has improved by approximately 1% or more, specifically by approximately 1% or more, approximately 2.5% or more, or approximately 5% or more, compared to the L-amino acid production capacity of the parent microorganism (parent strain) before mutation or the unmodified microorganism. However, any microorganism that has a positive increase in production capacity compared to the parent microorganism (parent strain) before mutation or the unmodified microorganism is acceptable. As another example, the recombinant microorganism with improved L-amino acid production capacity is one in which the L-amino acid production capacity has improved by approximately 1.01 times or more, 1.02 times or more, 1.03 times or more, 1.04 times or more, 1.05 times or more, 1.06 times or more, 1.07 times or more, 1.08 times or more, 1.09 times or more, or approximately 1.1 times or more, compared to the parent microorganism (parent strain) before mutation or the unmodified microorganism.

[0136] For example, the microorganism having the ability to produce L-amino acids may be either a prokaryotic cell or a eukaryotic cell, and more specifically, it may be a prokaryotic cell. The prokaryotic cells include, for example, microbial strains belonging to the genera Escherichia, Erwinia, Serratia, Providencia, Corynebacterium, Pseudomonas, Leptospira, Salmonella, Brevibacterium, Hypomononas, Chromobacterium, and Norcardia, or fungi or yeast. Specifically, these are microbial strains and yeasts belonging to the genera Escherichia, Corynebacterium, and Leptospira. More specifically, they are microbial strains of the genus Corynebacterium.

[0137] In any of the above-mentioned specific examples of microorganisms, the microorganism of this application may be a microorganism of the genus Corynebacterium.

[0138] As an example of this application, the microorganisms of this application are Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium The microorganisms may be Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens. Specifically, the microorganisms of this application are microorganisms of the genus Corynebacterium, and more specifically, Corynebacterium glutamicum, but are not limited to these.

[0139] The Corynebacterium microorganisms possessing L-amino acid production ability described in this application include the naturally occurring wild-type microorganisms themselves, Corynebacterium microorganisms whose L-amino acid production ability has been improved by enhancing or reducing the activity of genes related to the L-amino acid production mechanism, and Corynebacterium microorganisms whose L-amino acid production ability has been improved by introducing or enhancing the activity of external genes.

[0140] The microorganisms possessing L-amino acid production ability according to this application may further have pyruvate dehydrogenase complex subunit E1 whose activity is enhanced compared to its endogenous activity.

[0141] In this application, "Pyruvate Dehydrogenase Complex subunit E1" is a component of the pyruvate dehydrogenase complex, and may be a polypeptide containing an E1 active site that converts pyruvate to acetyl-CoA and CO2.

[0142] The pyruvate dehydrogenase complex subunit E1 of this application is used interchangeably with "aceE". The pyruvate dehydrogenase complex subunit E1 is known in the art and is specifically encoded by aceE, but is not limited thereto. The amino acid and polynucleotide sequences of the pyruvate dehydrogenase complex subunit E1 are obtained from known databases, such as NCBI's GenBank, but are not limited thereto.

[0143] For example, the pyruvate dehydrogenase complex subunit E1 may contain the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having 60% or more homology or identity thereto. However, any protein that has the same activity as pyruvate dehydrogenase complex subunit E1 is acceptable. Specifically, even if a protein contains a sequence in which some of the amino acid sequences of SEQ ID NO: 10 are deleted, modified, substituted, or added, any protein that exhibits efficacy equivalent to that of pyruvate dehydrogenase complex subunit E1 is included in the pyruvate dehydrogenase complex subunit E1. Furthermore, any protein that exhibits efficacy equivalent to that of pyruvate dehydrogenase complex subunit E1, and has an amino acid sequence of at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with the amino acid sequence of sequence 10, or contains the amino acid sequence, consists of the amino acid sequence, or is essentially composed of the amino acid sequence, is included in pyruvate dehydrogenase complex subunit E1. For example, pyruvate dehydrogenase complex subunit E1 may be an exogenous protein or a protein endogenously present in Corynebacterium microorganisms or Corynebacterium glutamicum, but is not limited to these. Specifically, it may be pyruvate dehydrogenase complex subunit E1 consisting of the amino acid sequence of sequence 10 endogenously present in Corynebacterium microorganisms or Corynebacterium glutamicum, but is not limited to these. For example, NCBIReference No.WP_011014985.1 is one such example.

[0144] Furthermore, polynucleotides encoding pyruvate dehydrogenase complex subunit E1 having the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having 60% or more homology or identity thereto, can be prepared based on codon information known in the art. For example, the protein is encoded by a polynucleotide having, or containing, or consisting of, or substantially composed of, the sequence of SEQ ID NO: 11 or SEQ ID NO: 39, or a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the sequence of SEQ ID NO: 11 or SEQ ID NO: 39.

[0145] The microorganism having L-amino acid production ability of this application may further include modifications to improve the target L-amino acid production ability.

[0146] For example, the microorganism of this application may be a microorganism in which the activity of arginine repressor (ArgR) is weakened. For example, the microorganism of this application may be a microorganism in which all or part of the argR gene is further deleted. For example, the microorganism of this application may be a microorganism in which the 54th methionine of acetylglutamate kinase (ArgB) is replaced with valine.

[0147] For example, the microorganism of this application may be a microorganism in which the activity of N-acetyl-γ-glutamyl-phosphate reductase (hereinafter referred to as ArgC) is enhanced compared to its endogenous activity.

[0148] For example, the microorganism of this application may be a microorganism in which the activity of argininosuccinate synthetase (ArgG) is attenuated. For example, the microorganism of this application may be a microorganism in which all or part of the argG gene is further deleted.

[0149] For example, the microorganism of this application may be a microorganism in which the activity of the L-lysine efflux gene LysE is weakened. For example, it may be a microorganism in which all or part of the lysE gene encoding the L-lysine efflux gene is missing.

[0150] For example, the microorganism of this application may be a microorganism in which the activity of carbamoyl phosphate synthase is weakened. For example, it may be a microorganism in which the activity of the small subunit (CarA) and / or large subunit (CarB) of carbamoyl phosphate synthase is weakened.

[0151] For example, the microorganism of this application may be a microorganism in which the activity of ornithine carbamoyltransferase F (ArgF) is weakened. For example, the microorganism of this application may be a microorganism in which all or part of the argF gene is missing.

[0152] For example, the microorganism of this application may be a microorganism into which an exogenous ornithine efflux protein has been introduced. For example, it may be a microorganism into which a membrane protein derived from a microorganism of the genus Shewanella has been introduced. For example, it may be a microorganism into which a membrane protein derived from Shewanella oneidensis has been introduced.

[0153] For example, the microorganism of this application may be one in which the inner membrane protein YjeH has been modified. Patent Document 6 is referenced for this.

[0154] For example, the microorganism of this application may be a microorganism in which the activity of O-acetyl-homoserinetransferase (MetX) is enhanced compared to its endogenous activity.

[0155] For example, the microorganism of this application may be modified to enhance the expression of aspart kinase (LysC) and / or to remove feedback inhibition. Patent Document 7 is referenced for this.

[0156] For example, the microorganism of this application may be a microorganism in which the activity of cystathionine gamma-synthase is attenuated. For example, the microorganism of this application may be a microorganism in which the activity of the metB gene encoding cystathionine gamma-synthase is attenuated. For example, the microorganism may be a microorganism in which all or part of the metB gene is missing.

[0157] For example, the microorganism of this application may be a microorganism in which the activity of O-acetylhomoserine(thiol)-lyase is attenuated. For example, the microorganism of this application may be a microorganism in which the activity of the metY gene encoding O-acetylhomoserine(thiol)-lyase is attenuated. For example, the microorganism may be a microorganism in which all or part of the metY gene is missing.

[0158] For example, the microorganism of this application may be a microorganism in which the activity of the endogenous protein Ncgl0616 is weakened. For example, it may be a microorganism in which part or all of the gene encoding Ncgl0616 is missing.

[0159] Another aspect of this application provides a method for producing L-amino acids, comprising the step of culturing a microorganism in which the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase activity of this application is improved compared to its endogenous activity, in a culture medium.

[0160] In the method of this application, any culture conditions and methods known in the art are used for culturing microorganisms. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected strain.

[0161] The L-amino acids produced by the culture described in this application are either secreted into the culture medium or remain within the cells.

[0162] In one specific example, the L-amino acid production method of the present application may further include, for example, the steps of preparing the microorganism of the present application, preparing a culture medium for culturing the strain, or a combination thereof (in any order) before the culturing step.

[0163] The method for producing L-amino acids according to this application may further include a step of recovering a target substance, specifically an L-amino acid, from the cultured microorganism, the culture of the microorganism, the fermented product of the microorganism, or the culture medium. The recovery step may further include a step after the culture step.

[0164] The aforementioned recovery may involve collecting the target L-amino acids using a suitable method known in the art, depending on the microorganism culture method of this application, such as batch, continuous, or fed-batch culture. For example, various chromatography methods such as centrifugation, filtration, crystallization, treatment with protein precipitants (salting-out method), extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof can be used, and the target substance, specifically L-amino acids, can be recovered from the culture medium or microorganism using a suitable method known in the art.

[0165] Furthermore, the L-amino acid production method of this application may further include a purification step. The purification can be carried out by a preferred method known in the art. For example, if the L-amino acid production method of this application includes both a recovery step and a purification step, the recovery step and the purification step may be carried out sequentially or discontinuously, regardless of order, simultaneously, or integrated as a single step, but are not limited thereto.

[0166] In the method of this application, the improvement of acetyltransferase activity of the pyruvate dehydrogenase complex dihydrolipoyllysine residue, L-amino acids, etc., are as described above.

[0167] A further aspect of this application provides a composition for L-amino acid production comprising a microorganism in which the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase activity of this application is improved from endogenous activity, a culture of the microorganism, a ferment of the microorganism, or at least two combinations thereof.

[0168] The composition of this application may further contain any suitable excipients commonly used in compositions for L-amino acid production. Examples of such excipients include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0169] As a specific example, each component present in the composition of this application is included in a microbiologically effective amount or in an appropriate amount for production.

[0170] In the composition of this application, the improvement of acetyltransferase activity of the pyruvate dehydrogenase complex dihydrolipoyllysine residue, L-amino acids, etc., is as described above.

[0171] Further aspects of this application provide applications for L-amino acid production in microorganisms in which the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase activity of this application is improved compared to endogenous activity.

[0172] In the application described in this application, the improvement of acetyltransferase activity of the pyruvate dehydrogenase complex dihydrolipoyllysine residue, L-amino acids, etc., are as described above.

[0173] Further aspects of this application provide a method for producing L-amino acid-producing microorganisms, comprising a step of modifying the microorganism so that the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase activity of this application is improved compared to endogenous activity.

[0174] In the microbial production method of this application, the improvement of acetyltransferase activity of the pyruvate dehydrogenase complex dihydrolipoyllysine residue, L-amino acids, etc., are as described above. [Examples]

[0175] The present application will be described in more detail below with reference to examples and experimental cases. However, these examples and experimental cases are merely illustrative of the present application and the present application is not limited to these examples and experimental cases. [Example 1]

[0176] Plasmid construction for enhancing the activity of pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase. Example 1-1: Plasmid preparation for promoter substitution To determine the effectiveness of the gene encoding the Corynebacterium glutamicum pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase (NCBI registration number WP_060564822.1), a vector was created to enhance BBD29_RS10495 (SEQ ID NO: 2) derived from Corynebacterium glutamicum ATCC13869. Specifically, in order to create a pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase-enhancing vector, a plasmid was created that enhances aceF activity by substituting the wild-type promoter of the aceF gene with the Pcj7 promoter (Patent Document 3) (SEQ ID NO: 3), which is known as a strong promoter.

[0177] The upstream and downstream regions of the aceF gene were obtained. Specifically, to create a strain into which aceF possessing the Pcj7 promoter was introduced, chromosomal DNA from Corynebacterium glutamicum ATCC13869 was used as a template. PCR of the gene fragment of the upstream region of the aceF gene was performed using primers SEQ ID NO: 4 and SEQ ID NO: 5, and PCR of the gene fragment of the downstream region of the aceF gene was performed using primers SEQ ID NO: 6 and SEQ ID NO: 7. In addition, the Pcj7 promoter fragment was obtained using pDCM2-Pcj7 as a template and SEQ ID NO: 8 and SEQ ID NO: 9. The primer sequences used for each of the above PCRs are shown in Table 1.

[0178] [Table 1]

[0179] PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction. The PCR conditions were denaturation at 95°C for 30 seconds, denaturation at 55°C for 30 seconds, and polymerization at 72°C for 1 minute. This denaturation, annealing, and polymerization reaction was repeated 28 times. As a result, a 318 bp DNA fragment of the Pcj7 promoter region, a 526 bp DNA fragment upstream of Corynebacterium glutamicum ATCC13869aceF, and a 529 bp DNA fragment downstream were obtained. Using the amplified promoter and DNA fragments as templates, PCR was performed with primers SEQ ID NO: 4 and SEQ ID NO: 7. The PCR conditions were denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 2 minutes, repeated 28 times, and then polymerization at 72°C for 5 minutes. The two fragments obtained in this way underwent DNA purification, and then a plasmid was obtained by fusion cloning using the In-Fusion® HD cloning kit (Clontech) with a pDCM2 vector treated with SmaI restriction enzyme (Patent Document 2) according to the attached manual. The resulting vector was named pDCM2-Pcj7_aceF.

[0180] Examples 1-2: Plasmid preparation for enhancing the activity of pyruvate dehydrogenase complex subunit E1 A vector was created to enhance the activity of pyruvate dehydrogenase complex subunit E1 (NCBI registration number BBD29_RS10685, hereafter referred to as aceE). Specifically, in order to create a vector to enhance pyruvate dehydrogenase complex subunit E1 (BBD29_RS10685, SEQ ID NO: 11), a plasmid was created to enhance aceE activity by replacing the wild-type promoter of the aceE gene with Pcj7, a promoter known as a strong promoter (Patent Document 3) (SEQ ID NO: 3), using the Pcj7 promoter.

[0181] The upstream and downstream regions of the aceE gene were obtained. Specifically, to create a strain into which aceE possessing the Pcj7 promoter was introduced, chromosomal DNA from Corynebacterium glutamicum ATCC13869 was used as a template. PCR was performed to amplify the gene fragment of the upstream region of the aceE gene using primers SEQ ID NO: 12 and SEQ ID NO: 13, and PCR was performed to amplify the gene fragment of the downstream region of the aceE gene using primers SEQ ID NO: 14 and SEQ ID NO: 15. In addition, the Pcj7 promoter fragment was obtained using pDCM2-Pcj7 as a template and SEQ ID NO: 8 and SEQ ID NO: 9. The primer sequences used for each of the above PCRs are shown in Table 2.

[0182] [Table 2]

[0183] PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction. The PCR conditions were denaturation at 95°C for 30 seconds, denaturation at 55°C for 30 seconds, and polymerization at 72°C for 1 minute. This denaturation, annealing, and polymerization reaction was repeated 28 times. As a result, a 318 bp DNA fragment of the Pcj7 promoter region, a 526 bp DNA fragment upstream of Corynebacterium glutamicum ATCC13869aceE, and a 527 bp DNA fragment downstream were obtained. Using the amplified promoter and DNA fragments as templates, PCR was performed with primers of SEQ ID NO: 12 and SEQ ID NO: 15. The PCR conditions were denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 2 minutes, repeated 28 times, and then polymerization at 72°C for 5 minutes. The two fragments obtained in this way underwent DNA purification, and then a plasmid was obtained by fusion cloning using the In-Fusion® HD cloning kit (Clontech) with a pDCM2 vector treated with SmaI restriction enzyme (Patent Document 2) according to the attached manual. The resulting vector was named pDCM2-Pcj7_aceE. [Example 2]

[0184] Creation of microorganisms with enhanced pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase activity, and evaluation of their L-amino acid production capacity. Example 2-1. Preparation of Corynebacterium glutamicum CJR2 strain To evaluate the L-arginine, citrulline, and ornithine production capacity, the Corynebacterium glutamicum strain CR2 was prepared.

[0185] This is done by introducing the mutation ((ΔargR,argB(M54V)) into the wild-type Corynebacterium glutamicum ATCC13869 (Non-Patent Literature 19).

[0186] First, vectors introducing argR-deficient and argB(M54V) mutations were prepared. Using the genome DA of Corynebacterium glutamicum ATCC13869 as a template, homologous recombination fragments containing the argR-deficient mutant sequence were obtained by PCR using the primer pairs in Table 3 (SEQ ID NOs. 16 and 17, SEQ ID NOs. 18 and 19), and by overlapping PCR using the primer pairs of SEQ ID NOs. 16 and 19. Similarly, to prepare homologous recombination fragments containing the argB(M54V) mutation, PCR was performed using the primer pairs in Table 3 (SEQ ID NOs. 20 and 21, SEQ ID NOs. 22 and 23), and by overlapping PCR using SEQ ID NOs. 20 and 23. The PCR reaction consisted of 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 2 minutes. Fusion cloning was performed on the linearized pDCM2 vector and each homologous recombination fragment, similar to Example 2. The prepared vectors (recombinant plasmids) were named pDCM2-ΔargR and pDCM2-argB(M54V), respectively.

[0187] Next, an argR-deficient mutation was introduced into wild-type Corynebacterium glutamicum ATCC13869. Transformation was performed using the pDCM2-ΔargR plasmid prepared as described above, via electrotherapy (Non-Patent Literature 20). Secondary recombination was then performed on solid agar plates containing 4% sucrose. PCR using primer pairs (SEQ ID NO: 16 and SEQ ID NO: 19) was then performed on the transformed strains after secondary recombination to confirm the introduction of a deletion mutation into the argR gene on the chromosome. The PCR reaction consisted of 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 2 minutes. The transformed strain was named CJR1. <Composite agar plate medium (pH 7.0)> Glucose 10g, Peptone 10g, Beef extract 5g, Yeast extract 5g, Brain Heart Infusion 18.5g, NaCl 2.5g, Urea 2g, Sorbitol 91g, Agar 20g (per liter of distilled water)

[0188] Next, the argB(M54V) mutation was introduced into Corynebacterium glutamicum CJR1 as described above. Using the pDCM2-argB(M54V) plasmid prepared as described above, the introduction of the M54V mutation into the argB gene on the chromosome was confirmed by PCR using primer pairs (SEQ ID NO: 20 and SEQ ID NO: 23) in the transformed strain after secondary recombination. The transformed strain was named CJR2.

[0189] [Table 3]

[0190] Example 2-2. Preparation of Corynebacterium glutamicum CJR100 strain Based on the CJR2 strain prepared in Example 2-1, we attempted to create a CJR100 strain in which the N-acetyl-γ-glutamyl-phosphate reductase (argC) gene was enhanced.

[0191] To enhance the activity of argC (SEQ ID NO: 25, NCBI registration number BBD29, RS07530), an N-acetyl-Γ-glutamyl phosphate reductase, plasmids were created to enhance argC activity by replacing the wild-type promoter of the argC gene with the Po2 promoter (Patent Document 5), which is known as a strong promoter. The upstream and downstream regions of the argC gene were obtained. Specifically, to create a bacterial strain into which argC with the Po2 promoter was introduced, chromosomal DNA of Corynebacterium glutamicum ATCC13869 was used as a template, and PCR was performed to amplify the gene fragment of the upstream region of the argC gene using primers SEQ ID NO: 26 and SEQ ID NO: 27, and PCR was performed to amplify the gene fragment of the downstream region of the argC gene using primers SEQ ID NO: 28 and SEQ ID NO: 29. In addition, using pDCM2-Po2 as a template, Po2 promoter fragments were obtained using SEQ ID NO: 30 and SEQ ID NO: 31. PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction. The PCR conditions were denaturation at 95°C for 30 seconds, denaturation at 55°C for 30 seconds, and polymerization at 72°C for 1 minute. This denaturation, annealing, and polymerization reaction was repeated 28 times. As a result, an 86 bp DNA fragment of the Po2 promoter region, a 610 bp DNA fragment upstream of Corynebacterium glutamicum ATCC13869argC, and a 1086 bp DNA fragment downstream were obtained. Using the amplified promoter and DNA fragments as templates, PCR was performed with primers of SEQ ID NO: 26 and SEQ ID NO: 29. The PCR conditions were denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 2 minutes, repeated 28 times, and then polymerization at 72°C for 5 minutes. The two fragments obtained in this way underwent DNA purification, and then fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech) to ligate them to a pDCM2 plasmid treated with SmaI restriction enzyme.The resulting vector was named pDCM2-Po2-argC.

[0192] Next, the CJR2 strain prepared in Example 2-1 was transformed using the pDCM2-Po2-argC plasmid prepared as described above by electrolysis (Non-Patent Literature 20). Then, secondary recombination was performed on solid agar plates containing 4% sucrose, and the transformed strains after secondary recombination were examined by PCR using primer pairs (SEQ ID NO: 26 and SEQ ID NO: 31) to confirm that the argC gene was enhanced by the Po2 promoter on the chromosome. The PCR reaction consisted of 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 2 minutes. The transformed strain was named CJR100. <Composite agar plate medium (pH 7.0)> Glucose 10g, Peptone 10g, Beef extract 5g, Yeast extract 5g, Brain Heart Infusion 18.5g, NaCl 2.5g, Urea 2g, Sorbitol 91g, Agar 20g (per liter of distilled water)

[0193] Table 4 shows the sequence and detailed information of the primers used in this example.

[0194] [Table 4]

[0195] Examples 2-3. Preparation of bacterial strains with enhanced pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase and pyruvate dehydrogenase complex subunit E1 activity, and evaluation of arginine, citrulline, and ornithine production capacity. Based on the CJR100 strain prepared in Example 2-2, transformation was performed using the pDCM2-Pcj7_aceF plasmid prepared in Example 1 by electrophoretic pulse method (Non-Patent Literature 20). Next, secondary recombination was performed on solid plate medium containing 4% sucrose. After secondary recombination, PCR using primer pairs (SEQ ID NO: 4 and SEQ ID NO: 9) was performed on the transformed strain to confirm that the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase was enhanced by the Pcj7 promoter. The PCR reaction consisted of 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 2 minutes. The transformed strain was named CJR101 (CJR100-Pcj7_aceF).

[0196] Furthermore, based on the aceF-enhanced CJR101 strain, transformation was performed using the pDCM2-Pcj7_aceE plasmid prepared in Example 1-2 by electrophoretic pulse method (Non-Patent Literature 20). Next, secondary recombination was performed on solid plate medium containing 4% sucrose, and PCR using primer pairs (SEQ ID NO: 12 and SEQ ID NO: 19) was performed on the transformed strain after secondary recombination to confirm that the pyruvate dehydrogenase complex subunit E1 was enhanced by the Pcj7 promoter on the chromosome. The PCR reaction consisted of 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 2 minutes. The transformed strain was named CJR103 (CJR101-Pcj7_aceE).

[0197] The following production mediums were placed in 250 ml corner baffled flasks, to which the parent strain Corynebacterium glutamicum CJR2 and the strains CJR101, CJR102, and CJR103 prepared in Example 2-3 were inoculated. The cultures were then incubated at 30°C and 200 rpm for 44 hours with shaking. The composition of each production medium was as follows: <Composite agar plate medium (pH 7.0)> Glucose 10g, Peptone 10g, Beef extract 5g, Yeast extract 5g, Brain Heart Infusion 18.5g, NaCl 2.5g, Urea 2g, Sorbitol 91g, Agar 20g (per liter of distilled water) <Production medium (pH 7.2)> Sucrose 50g, Ammonium sulfate 57g, Magnesium sulfate heptahydrate 2g, Beet molasses 5g, Calcium chloride 1mg, Cobalt chloride 1mg, Potassium dihydrogen phosphate 2g, Biotin 0.01mg, Thiamine HCl 0.1mg, Calcium pantothenate 2mg, Nicotinamide 3mg, Ferrous sulfate 10mg, Manganese sulfate 10mg, Zinc sulfate 0.02mg, Copper sulfate 0.5mg, Calcium carbonate 30g (per 1 liter of distilled water)

[0198] After the culturing was complete, the L-arginine production capacity (concentration) was analyzed using HPLC (Waters 2478). The analyzed L-arginine and L-citrulline concentrations and the degree of improvement in L-arginine yield are shown in Table 5.

[0199] [Table 5]

[0200] As a result, it was confirmed that in strain CJR101, in which aceF was enhanced by the Pcj7 promoter, L-arginine production capacity was improved by 8-10% compared to the unenhanced strain CJR100. Furthermore, in strain CJR103, in which aceE within the aceF-enhanced CJR101 strain was enhanced by the Pcj7 promoter, L-arginine production capacity was improved by 110% compared to the parent strain CJR101 and by 118% compared to CJR100.

[0201] Furthermore, it was confirmed that the CJR101 and CJR103 strains exhibited improved L-citrulline and L-ornithine production compared to the control group (CJR2, CJR100) strains. These results indicate that enhancement of the pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase is effective for L-arginine production in Corynebacterium L-arginine-producing strains, and further, enhancement of the pyruvate dehydrogenase complex subunit E1 is effective for arginine production. It also appears to be effective not only for arginine production but also for citrulline and ornithine production. [Example 3]

[0202] Preparation of citrulline-producing bacterial strains with enhanced pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase and pyruvate dehydrogenase complex subunit E1 activity, and evaluation of citrulline production capacity. Example 3-1. Preparation of citrulline-producing bacterial strains A bacterial strain was prepared to be used as a control group in experiments to evaluate citrulline production capacity.

[0203] A vector was constructed in which the glutamic acid at position 47 of the argR (ANU33619.1) protein sequence was replaced with a stop codon. Using the genome of wild-type C. glutamicum ATCC13869 as a template, the homologous recombinant A arm was amplified using the primer pair of SEQ ID NOs. 40 and 41, and the homologous recombinant B arm was amplified using the primer pair of SEQ ID NOs. 42 and 43. A plasmid was then obtained in the same manner as in Example 1. This plasmid was named pDCM2-argR(E47*).

[0204] To create a microorganism with further improved L-citrulline production capacity, a vector was constructed in which the phenylalanine at position 68 of the argG (ANU33620.1) protein sequence was replaced with a stop codon. Using the genome of C. glutamicum ATCC13869 as a template, the homologous recombinant A arm was amplified using primers SEQ ID NO: 44 and SEQ ID NO: 45, and the homologous recombinant B arm was amplified using primers SEQ ID NO: 46 and SEQ ID NO: 47. Subsequently, a plasmid was obtained as described above. This plasmid was named pDCM2-argG(F68*).

[0205] The primer sequences used here are shown in Table 6.

[0206] [Table 6]

[0207] Using the constructed pDCM2-argR(E47*) vector, wild-type C. glutamicum ATCC13869 was transformed by electroporation (Non-Patent Literature 20). Subsequently, after a secondary cross-reaction, a strain was obtained in which the 139th base sequence of argR was replaced from guanine (g) to thymine (t), and the 47th protein sequence was replaced with a stop codon. Using primer pairs of SEQ ID NOs. 40 and 43, which amplify the adjacent region including the insertion site of the gene, PCR and sequence analysis were performed to confirm the genetic manipulation. The microorganism thus obtained was named C. gl::argR*.

[0208] To create a microorganism with further enhanced citrulline production in C.gl::argR*, we obtained the microorganism using the pDCM2-argG(F68*) vector as described above. Using primer pairs of SEQ ID NOs. 44 and 47, which amplify the adjacent region including the insertion site of the gene, PCR and sequencing analysis were performed to confirm the genetic manipulation. The microorganism thus obtained was named C.gl::argR*_argG*.

[0209] Example 3-2. Preparation of a citrulline-producing bacterial strain with enhanced activity of pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase and pyruvate dehydrogenase complex subunit E1. To confirm whether there is an effect of enhancing citrulline production capacity in the pyruvate dehydrogenase complex activity-enhanced strain of Corynebacterium glutamicum, which has the ability to produce L-citrulline, a strain was prepared in which aceF activity was enhanced in the L-citrulline-producing strain C.gl::argR*_argG* prepared in Example 3-1.

[0210] Specifically, the L-citrulline-producing bacterial strain C.gl::argR*_argG* was transformed using the pDCM2-Pcj7_aceF plasmid vector prepared in Example 1 by electropulse (Non-Patent Literature 20). The transformed strain was named C.gl::argR*_argG*-Pcj7_aceF.

[0211] Based on the aforementioned C.gl::argR*_argG*-Pcj7_aceF strain, a strain with further enhanced aceE activity was created. Specifically, the L-citrulline-producing strain C.gl::argR*_argG*_Pcj7_aceF was transformed using the pDCM2-Pcj7_aceE plasmid vector prepared in Example 1-2 by electropulse (Non-Patent Literature 20). The transformed strain was named C.gl::argR*_argG*-Pcj7_aceF-Pcj7_aceE.

[0212] Example 3-3. Evaluation of citrulline production capacity of strains with enhanced pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase and pyruvate dehydrogenase complex subunit E1 activity. To compare the citrulline production capacity of the citrulline-producing strains Corynebacterium glutamicum C.gl::argR*_argG*, C.gl::argR*_argG*-Pcj7_-aceF, and C.gl::argR*_argG*-Pcj7_aceF-Pcj7_aceE, flask evaluations were performed. First, each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of seed medium and cultured with shaking at 30°C and 200 rpm for 20 hours. Next, 1 ml of seed culture solution was inoculated into a 250 ml corner baffle flask containing 24 ml of production medium and cultured with shaking at 33°C and 200 rpm for 42 hours. After the culture period, the amount of L-citrulline produced was measured by HPLC. The results are shown in Table 7. <Seed culture medium (pH 7.0)> Glucose 20g, Peptone 10g, Yeast extract 5g, Urea 1.5g, KH2PO4 4g, K2HPO4 8g, MgSO4·7H2O 0.5g, Biotin 0.1mg, Thiamine HCl 1mg, Calcium pantothenate 22mg, Nicotinamide 2mg (per liter of distilled water) <Production medium (pH 7.2)> Raw sugar 50g, (NH4)2SO4 30g, yeast extract 1g, KH2PO4 1.1g, MgSO4·7H2O 1.2g, L-arginine 0.2g, biotin 1mg, thiamine hydrochloride 5mg, calcium pantothenate 5mg, nicotinamide 15mg, MnSO4 10mg, FeSO4 10mg, ZnSO4 0.5mg, CuSO4 0.5mg, CaCO3 30g (in 1 liter of distilled water)

[0213] [Table 7]

[0214] As shown in Table 7, compared to the parent strain C.gl::argR*_argG*, the C.gl::argR*_argG*-Pcj7_aceF strain, in which the pyruvate dehydrogenase complex dihydrolipoyllysine-residueacetyltransferase is enhanced by the Pcj7 promoter, showed a 108% improvement in citrulline production capacity. Furthermore, in the C.gl::argR*_argG*-Pcj7_aceF-Pcj7_aceE strain, in which the pyruvate dehydrogenase complex subunit E1 in the C.gl::argR*_argG*-Pcj7_aceF strain is enhanced by the Pcj7 promoter, it was confirmed that the citrulline production capacity was 110% higher than that of the parent strain C.gl::argR*_argG*-Pcj7_aceF strain. In the C.gl::argR*_argG*-Pcj7_aceF-Pcj7_aceE strain, it was confirmed that citrulline production capacity was improved by 117% compared to C.gl::argR*_argG*.

[0215] These results indicate that enhancement of the pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase is effective in citrulline production in Corynebacterium citrulline-producing strains, and further, that enhancement of the pyruvate dehydrogenase complex subunit E1 is effective in citrulline production. [Example 4]

[0216] Preparation of ornithine-producing bacterial strains with enhanced pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase and pyruvate dehydrogenase complex subunit E1 activity, and evaluation of ornithine production capacity. Example 4-1. Preparation of ornithine-producing bacterial strains The following bacterial strains were prepared for evaluation of ornithine production capacity.

[0217] Example 4-1-1. Introduction of Shewanella oneidensis membrane protein and creation of a lysE-deficient strain. Example 4-1-1-1. Preparation of a Shewanella oneidensis membrane protein-transformed plasmid. First, we selected the LysE / ArgO family amino acid transporter (WP_011072781.1) (SEQ ID NO: 48), a membrane protein derived from Shewanella oneidensis MR-1 that exhibits high L-ornithine efflux activity, and then constructed a plasmid for introducing Shewanella oneidensis membrane proteins.

[0218] To amplify the base sequence of the gene encoding the aforementioned protein, information on the gene encoding the membrane protein and its neighboring nucleic acid sequences (NC_004347.2) was obtained from the National Institutes of Health (NIH) GenBank. Based on this sequence information, DNA synthesis was performed (Cosmo Genetech, Korea).

[0219] As mentioned above, we created a vector for introducing the selected membrane protein derived from Shewanella oneidensis MR-1 into an L-ornithine-producing strain.

[0220] ANU34435.1, one of the transposases present in the genome of wild-type Corynebacterium glutamicum ATCC13869 (NZ_CP016335.1), was used as the insertion site. Using the genome of wild-type Corynebacterium glutamicum ATCC13869 as a template, the homologous recombinant A arm was amplified using the primer pair of SEQ ID NO: 49 and SEQ ID NO: 50, and the homologous recombinant B arm was amplified using the primer pair of SEQ ID NO: 51 and SEQ ID NO: 52.

[0221] To secure the gene fragment encoding the membrane protein derived from Shewanella oneidensis MR-1 (SEQ ID NO: 48), synthesized DNA was used as a template, and PCR was performed using primer pairs of SEQ ID NO: 53 and SEQ ID NO: 54.

[0222] To secure the gapA promoter, PCR was performed using the genomic DNA (NC_006958.1) of wild-type Corynebacterium glutamicum ATCC13032 as a template, with primer pairs of SEQ ID NO: 55 and SEQ ID NO: 56. The denaturation process involved 25 cycles of denaturation at 95°C for 2 minutes, denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, polymerization at 72°C for 1 minute, followed by polymerization at 72°C for 5 minutes. SolgTMPfu-X DNA polymerase was used for the PCR.

[0223] The amplified gapA promoter region, a gene fragment encoding a membrane protein derived from Shewanella oneidensis MR-1, a homologous recombinant arm gene fragment, and the vector pDCM2 (WO2021-187781 A1) cleaved with SalI and BamHI restriction enzymes were ligated using the Gibson assembly method (Non-Patent Literature 21), and then transformed into Escherichia coli DH5α, which was then streaked onto LB solid medium containing kanamycin (25 mg / l).

[0224] PCR was performed using primer pairs SEQ ID NO: 57 and SEQ ID NO: 58 to select colonies transformed with a vector containing a gene encoding a membrane protein derived from Shewanella oneidensis MR-1. Plasmids were extracted from the selected colonies using the plasmidprep kit (QIAGEN). The plasmid was named pDCM2-PgapA-Son.

[0225] The primer information used here is shown in Table 8.

[0226] [Table 8]

[0227] Example 4-1-1-2. Preparation of lysE-deficient plasmid Next, we created a plasmid to eliminate lysE.

[0228] To construct a vector deleting the open reading frame (ORF) of wild-type Corynebacterium glutamicum LysE (ANU33473.1) (SEQ ID NO: 59), the genome of wild-type Corynebacterium glutamicum ATCC13869 was used as a template. The homologous recombinant A arm was amplified using primer pairs of SEQ ID NO: 68 and 69, and the homologous recombinant B arm was amplified using primer pairs of SEQ ID NO: 70 and 71. The homologous recombinant arm gene fragments were cloned into vector pDCM2, which was cut with SalI and BamHI restriction enzymes, as in 4-1-1-1. The resulting cells were transformed into E. coli DH5α, and PCR was performed using primer pairs of SEQ ID NO: 57 and 58 to select transformed colonies. Plasmids were extracted from the selected colonies using the plasmidprep kit (QIAGEN). The plasmid was named pDCM2-Δlys.

[0229] The primer information used here is as follows:

[0230] [Table 9]

[0231] Example 4-1-1-3. Preparation of argF* plasmid To create an L-ornithine-producing strain, a vector was prepared by substituting the serine at position 55 from the N-terminus of the amino acid sequence of wild-type Corynebacterium glutamicum-derived ArgF (ANU33618.1) (SEQ ID NO: 64) with a stop codon. Using the genome of wild-type Corynebacterium glutamicum ATCC13869 as a template, the homologous recombinant A arm was amplified using the primer pairs of SEQ ID NO: 60 and 61, and the homologous recombinant B arm was amplified using the primer pairs of SEQ ID NO: 62 and 63. Subsequently, a plasmid was obtained in the same manner as in Example 4-1-1-1. This plasmid was named pDCM2-argF(S55*).

[0232] Example 4-1-1-4. Preparation of C.gl::argF*_argR*_PgapA-Son and C.gl::argF*_argR*_ΔlysE_PgapA-Son strains Wild-type Corynebacterium glutamicum ATCC13869 was transformed using the pDCM2-argF(S55*) vector by electroporation (Non-Patent Literature 20). Subsequently, a strain was obtained in which the serine at position 55 from the N-terminus of the ArgF amino acid sequence was replaced with a stop codon through a secondary crossover process. The genetic manipulation was confirmed by PCR and nucleotide sequence analysis using primer pairs of SEQ ID NOs. 60 and 63, which amplify the adjacent region including the insertion site of the gene. This strain was named C.gl::argF*.

[0233] C.gl::argF* was transformed with the pDCM2-argR(E47*) vector prepared in Example 3-1 to obtain a strain in which the glutamic acid at position 47 from the N-terminus of the ArgR amino acid sequence was replaced with a stop codon, as described above. PCR and nucleotide sequence analysis were performed using primer pairs of SEQ ID NOs. 40 and 43, which amplify the adjacent region including the insertion site of the gene, to confirm the genetic manipulation. The strain thus obtained was named C.gl::argF*_argR*.

[0234] C.gl::argF*_argR* was transformed by electroporation with the pDCM2-ΔlysE plasmid prepared in Example 4-1-1-2, and then, after a secondary cross-reaction, a strain lacking the gene encoding LysE (lysE) was obtained. The genetic manipulation was confirmed by PCR and nucleotide sequence analysis using primer pairs of SEQ ID NOs. 68 and 71, which amplify the adjacent region including the insertion site of the missing gene. The strain thus obtained was named C.gl::argF*_argR*_Δlys.

[0235] The primer information used here is as follows:

[0236] [Table 10]

[0237] The pDCM2-PgapA-Son vector prepared in Example 4-1-1-1 was used to transform C.gl::argF*_argR* and lysE-deficient strains, C.gl::argF*_argR*_ΔlysE, by electroporation. Subsequently, a secondary cross-reaction was performed to obtain strains in which PgapA-Son was inserted. Using primer pairs of Sequence ID No. 68 and Sequence ID No. 71, which amplify the adjacent region including the insertion site of the gene, PCR and nucleotide sequence analysis were performed to confirm the genetic manipulation.

[0238] The strains obtained in this way were named C.gl::argF*_argR*_PgapA-Son and C.gl::argF*_argR*_ΔlysE_PgapA-Son, respectively.

[0239] Example 4-1-2. Preparation of a bacterial strain with weakened carbamoyl phosphate synthase activity. Example 4-1-2-1. Preparation of a vector for carAB promoter substitution. A vector was constructed to replace the promoter of the carA gene, which encodes the carbamoyl phosphate synthase small subunit (CarA). Information on the carAB gene (sequence number 67), which encodes CarA (ANU33813.1, sequence number 65), and nearby nucleic acid sequences (NZ_CP016335.1) was obtained from the National Institutes of Health Gene Bank (NIH GenBank). Using this information, a vector was constructed to replace the promoter site of carA with the promoter site of betP (ANU33153.1) (sequence number 72).

[0240] Using the genome of wild-type C. glutamicum ATCC13869 as a template, the betP promoter was amplified by PCR (SolTM Pfu-X DNA polymerase) using the primer pair of SEQ ID NO: 77 and SEQ ID NO: 78, the homologousrecombinant A arm was amplified by PCR (SolTM Pfu-X DNA polymerase) using the primer pair of SEQ ID NO: 79 and SEQ ID NO: 80, and the homologousrecombinant B arm was amplified by PCR (SolTM Pfu-X DNA polymerase) using the primer pair of SEQ ID NO: 81 and SEQ ID NO: 82, thereby obtaining DNA fragments from each.

[0241] As described above, the obtained promoter and recombinant arm gene fragments were cleaved with SalI and BamHI restriction enzymes to produce the vector pDCM2 (Patent Document 8), and the vector was prepared by cloning using the Gibson assembly method (Non-Patent Document 21). The prepared vector was used to transform E. coli DH5α, and the cells were streaked onto LB solid medium containing kanamycin (25 mg / l). PCR was performed using the primer pairs of SEQ ID NO: 83 and SEQ ID NO: 84 to select colonies transformed with the vector. Plasmids were obtained from the selected colonies using a well-known plasmid extraction method. As described above, the obtained plasmid was named pDCM2-PbetP-carA.

[0242] The primer information used here is as follows.

[0243]

Table 11

[0244] Preparation of vector for carA start codon mutation in Example 4-1-2-2 A vector was prepared to replace the start codon gtg of the gene carA with ttg. Using the genome of wild-type C. glutamicum ATCC13869 as a template, the homologous recombinant A arm was amplified using the primer pair of SEQ ID NO: 79 and SEQ ID NO: 85, and the homologous recombinant B arm was amplified using the primer pair of SEQ ID NO: 82 and SEQ ID NO: 86. Then, a plasmid was obtained as described above and named pDCM2-carA(g1t).

[0245] The primer information used here is as follows.

[0246]

Table 12

[0247] Preparation of vector for carB start codon mutation in Example 4-1-2-3 A vector was constructed to replace the start codon atg in the carB gene with ttg. Information on the carAB gene (sequence number 67), which encodes the carbamoyl phosphate synthase large subunit (sequence number 66), and nearby nucleic acid sequences (NZ_CP016335.1) was obtained from the National Institutes of Health gene bank. Using the genome of wild-type C. glutamicum ATCC13869 as a template, the homologous recombinant A arm was amplified using primer pairs of sequence numbers 73 and 74, and the homologous recombinant B arm was amplified using primer pairs of sequence numbers 75 and 76. Subsequently, a plasmid was obtained as described above and named pDCM2-carB(a1t).

[0248] The primer information used here is as follows:

[0249] [Table 13]

[0250] Example 4-1-2-4. Preparation of a bacterial strain with weakened carbamoyl phosphate synthase activity. The pDCM2-PbetP-carA and pDCM2-carA(g1t) vectors prepared in Examples 4-1-2-1 to 4-1-2-2 were used to transform wild-type C. glutamicum ATCC13869 and C. gl::argF* and C. gl::argF*_argR* prepared in 4-1-1-3, respectively, by electroporation. Subsequently, secondary cross-reactivity was performed to obtain the bacterial strains. PCR and nucleotide sequence analysis were performed using primer pairs of SEQ ID NO: 78 and SEQ ID NO: 81 to confirm the genetic manipulation. The strains obtained in this way were named C.gl::PbetP-carA, C.gl::carA(g1t), C.gl::argF*_PbetP-carA, C.gl::argF*_carA(g1t), C.gl::argF*_argR*_PbetP-carA, and C.gl::argF*_argR*_carA(g1t), respectively.

[0251] To produce promoter and start codon substitution strains for carB, the plasmid pDCM2-carB(a1t) vector obtained in Example 4-1-2-3 was used to transform wild-type C. glutamicum ATCC13869, C. gl::argF*, and C. gl::argF*_argR* by electroporation, and then a secondary cross-reactivity was performed to obtain the strains. Using primer pairs of SEQ ID NOs. 87 and 90, which amplify the adjacent region including the insertion site of the gene, PCR and nucleotide sequence analysis were performed to confirm the genetic manipulation. The strains thus obtained were named C. gl::carB(a1t), C. gl::argF*_carB(a1t), and C. gl::argF*_argR*_carB(a1t), respectively.

[0252] Example 4-2. Preparation of ornithine-producing bacterial strains with enhanced activity of pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase and pyruvate dehydrogenase complex subunit E1. To confirm whether the activity-enhancing strains have an effect on improving the production capacity of Corynebacterium glutamicum strains capable of producing L-ornithine, strains with enhanced aceF activity were prepared from the Corynebacterium glutamicum L-ornithine-producing strains C.gl::argF*_argR*_PgapA-Son, C.gl::argF*_argR*_ΔlysE_PgapA-Son, C.gl::argF*_argR*_carA(g1t), and C.gl::argF*_argR*_carB(a1t) prepared in Example 4-1.

[0253] Specifically, the vector pDCM2-Pcj7_aceF prepared in Example 1 was transformed into Corynebacterium glutamicum C.gl::argF*_argR*_PgapA-Son, C.gl::argF*_argR*_ΔlysE_PgapA-Son, C.gl::argF*_argR*_carA(g1t), and C.gl::argF*_argR*_carB(a1t) by homologous recombination on the chromosome, respectively (Non-Patent Literature 20).

[0254] The recombinant strains were named C.gl::argF*_argR*_PgapA-Son-Pcj7_aceF, C.gl::argF*_argR*_ΔlysE_PgapA-Son-Pcj7_aceF, C.gl::argF*_argR*_carA(g1t)-Pcj7_aceF, and C.gl::argF*_argR*_carB(a1t)-Pcj7_aceF.

[0255] Furthermore, based on the strains enhanced with aceF, the vector pDCM2-Pcj7_aceE prepared in Examples 1-2 was transformed into Corynebacterium glutamicum C.gl::argF*_argR*_PgapA-Son-Pcj7_aceF, C.gl::argF*_argR*_ΔlysE_PgapA-Son-Pcj7_aceF, C.gl::argF*_argR*_carA(g1t)-Pcj7_aceF, and C.gl::argF*_argR*_carB(a1t)-Pcj7_aceF, respectively, by homologous recombination on the chromosome (Non-Patent Literature 20). The recombinant strains were named C.gl::argF*_argR*_PgapA-Son-Pcj7_aceF-Pcj7_aceE, C.gl::argF*_argR*_ΔlysE_PgapA-Son-Pcj7_aceF-Pcj7_aceE, C.gl::argF*_argR*_carA(g1t)-Pcj7_aceF-Pcj7_aceE, and C.gl::argF*_argR*_carB(a1t)-Pcj7_aceF-Pcj7_aceE.

[0256] Example 4-3. Evaluation of the production capacity of ornithine strains with enhanced pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase and pyruvate dehydrogenase complex subunit E1 activity. To compare the ornithine production capacity of the ornithine-producing bacterial strains prepared in Example 4-2, flask evaluations were performed. First, each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of seed medium and cultured with shaking at 30°C and 200 rpm for 20 hours. 1 ml of seed culture solution was inoculated into a 250 ml corner baffle flask containing 24 ml of production medium and cultured with shaking at 33°C and 200 rpm for 42 hours. After the culture was completed, the amount of L-ornithine produced was measured by HPLC. The results are shown in Table 14. <Seed culture medium (pH 7.0)> 20 g of glucose, 10 g of peptone, 5 g of yeast extract, 1.5 g of urea, 4 g of KH2PO4, 8 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.1 mg of biotin, 1 mg of thiamine HCl, 22 mg of calcium pantothenate, 2 mg of nicotinamide (in 1 liter of distilled water) <Production medium (pH 7.0)> 50 g of raw sugar, 25 g of (NH4)2SO4, 1 g of yeast extract, 0.55 g of KH2PO4, 0.6 g of MgSO4·7H2O, 0.2 g of L-arginine, 0.9 mg of biotin, 4.5 mg of thiamine hydrochloride, 4.5 mg of calcium pantothenate, 30 mg of nicotinamide, 9 mg of MnSO4, 9 mg of FeSO4, 0.45 mg of ZnSO4, 0.45 mg of CuSO4, 30 g of CaCO3 (in 1 liter of distilled water)

[0257]

Table 表14

[0258] As shown in Table 14, in the L-ornithine-producing strain in which the pyruvate dehydrogenase complex dihydrolipoamide lysine residue acetyltransferase (aceF) was enhanced compared with the L-ornithine-producing strain, it was confirmed that the increase was about 112 - 115% compared with the parent strain. Also, in the L-ornithine-producing strain in which the pyruvate dehydrogenase complex subunit E1 (Pyruvate Dehydrogenase Complex subunit E1, aceE) was further enhanced based on the strain in which the pyruvate dehydrogenase complex dihydrolipoamide lysine residue acetyltransferase (aceF) was enhanced, the increase was 111% - 114% compared with the strain in which the pyruvate dehydrogenase complex dihydrolipoamide lysine residue acetyltransferase (aceF) was enhanced, which was the parent strain, and it was confirmed that the increase was 125 - 134% compared with the parent strain.

[0259] These results indicate that enhancement of the pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase is effective in L-ornithine production in Corynebacterium L-arginine-producing strains, and further, that enhancement of pyruvate dehydrogenase complex subunit E1 activity is also effective in L-ornithine production. [Example 5]

[0260] Preparation of O-acetylhomoserine and homoserine-producing strains with enhanced activity of pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase and pyruvate dehydrogenase complex subunit E1, and evaluation of their production capacity. Example 5-1. Preparation of O-acetylhomoserine and homoserine-producing bacterial strains Example 5-1-1. Defect of metB The metB gene, which encodes cystathionine gamma-synthase in the O-acetylhomoserine degradation pathway, was obtained by PCR using chromosomal DNA from Corynebacterium glutamicum ATCC13032 as a template. The nucleotide sequence information for the metB gene (NCBI registration number Ncgl2360, SEQ ID NO: 91) was obtained from the National Institutes of Health (NIH) GenBank, and primers containing the N-terminus and linker portion of the metB gene (SEQ ID NO: 87 and SEQ ID NO: 88) and primers containing the C-terminus and linker portion (SEQ ID NO: 89 and SEQ ID NO: 90) were synthesized based on this information. The primer sequences are shown in Table 15.

[0261] [Table 15]

[0262] PCR was performed using chromosomal DNA from ATCC13032 as a template, with primers number 87, 88, 89, and 90. PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as the polymerase. The PCR conditions consisted of denaturation at 96°C for 30 seconds, annealing at 53°C for 30 seconds, and polymerization at 72°C for 1 minute, repeated for 30 cycles. As a result, two amplified genes were obtained: a 558 bp gene containing the N-terminal and linker portions of the metB gene, and a 527 bp gene containing the C-terminal and linker portions of the metB gene.

[0263] As described above, PCR was performed using the two amplified genes obtained as templates. The PCR conditions were denaturation at 96°C for 60 seconds, annealing at 50°C for 60 seconds, polymerization at 72°C for 1 minute, followed by 10 cycles of polymerization. Then, SEQ ID NOs. 92 and 95 were added, and the polymerization reaction was carried out for another 20 cycles. As a result, an inactivated cassette of 1064 bp containing the N-terminus, linker, and C-terminus of the metB gene was obtained.

[0264] The pDCM2 vector was treated with SmaI, and the PCR product (1064 bp) obtained as described above was fusion cloned with the pDCM2 vector treated with SmaI restriction enzyme using the In-Fusion® HD Cloning Kit (Clontech). The cloned vector was transformed into E. coli DH5α, and the transformed E. coli were streaked onto LB solid medium containing 25 mg / l kanamycin. Colonies transformed with plasmids were selected from the LB medium, and plasmids were then obtained by plasmid extraction (Patent Document 9), and finally, a pDCM2-ΔmetB recombinant vector with a cloned metB gene-deficient cassette was constructed.

[0265] The prepared pDCM2-ΔmetB vector was transformed into the ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L) (KCCM12634P; Patent Document 6) strain using electropulse technology. After a secondary crossover process, ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetB was obtained in which the metB gene was inactivated on the chromosome. Whether or not the metB gene was inactivated was confirmed by PCR using primers of sequence numbers 87 and 90, and then by comparison with ATCC13032 in which the metB gene was not inactivated.

[0266] Example 5-1-2. Defect of metY The metY gene, which encodes O-acetylhomoserine (thiol)-lyase in the O-acetylhomoserine degradation pathway, was obtained by PCR using chromosomal DNA from Corynebacterium glutamicum ATCC13032 as a template. The nucleotide sequence information for the metY gene (NCBI registration number Ncgl0625, SEQ ID NO: 92) was obtained from the National Institutes of Health (NIH) GenBank, and primers containing the N-terminus and linker portion of the metY gene (SEQ ID NO: 93 and 94), and primers containing the C-terminus and linker portion (SEQ ID NO: 95 and 96) were synthesized based on this information. The primer sequences are shown in Table 16.

[0267] [Table 16]

[0268] PCR was performed using chromosomal DNA from ATCC13032 as a template, with primers numbered SEQ ID NOs. 93, 94, 95, and 96. PfuUltra polymerase was used. TMHigh-fidelity DNA polymerase (Stratagene) was used. The PCR conditions were denaturation at 96°C for 30 seconds, annealing at 53°C for 30 seconds, and polymerization at 72°C for 1 minute, repeated for 30 cycles. As a result, a 548 bp amplified gene containing the N-terminus and linker of the metY gene and a 550 bp amplified gene containing the C-terminus and linker of the metY gene were obtained. PCR was performed using the two amplified genes obtained as described above as templates. The PCR conditions were denaturation at 96°C for 60 seconds, annealing at 50°C for 60 seconds, and polymerization at 72°C for 1 minute, repeated for 10 cycles, after which sequence numbers 97 and 100 were added and the polymerization reaction was repeated for another 20 cycles. As a result, an inactivated cassette of 1077 bp containing the N-terminus, linker, and C-terminus of the metY gene was obtained.

[0269] The pDCM2 vector was treated with SmaI, and the PCR product (1077 bp) obtained as described above was fusion cloned with the pDCM2 vector treated with SmaI restriction enzyme using the In-Fusion® HD Cloning Kit (Clontech). The cloned vector was transformed into E. coli DH5α, and the transformed E. coli were streaked onto LB solid medium containing 25 mg / l kanamycin. Colonies transformed with plasmids were selected from the LB medium, and plasmids were then obtained by plasmid extraction (Patent Document 9), and finally, a pDCM2-ΔmetY recombinant vector with a meT gene-deficient cassette cloned was constructed.

[0270] The prepared pDCM2-ΔmetY vector was transformed into ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetB using electrolysis. After a secondary crossover, ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetY was obtained in which the metY gene was further inactivated on the chromosome. Whether or not the metY gene was inactivated was confirmed by PCR using primers of sequence numbers 93 and 96, and then by comparing it with ATCC13032 in which the metY gene was not inactivated.

[0271] Example 5-1-3. Introduction of lysC (L377K) A mutation (L377K) (Patent Document 7) was introduced into the lysC gene (SEQ ID NO: 97), which encodes aspart kinase derived from Corynebacterium glutamicum ATCC13032, to enhance lysC gene expression and remove feedback inhibition to L-lysine and L-threonine. To construct a vector containing the mutant lysC gene, a pair of primers (SEQ ID NO: 98 and 99) for amplification upstream of the 5' end and a pair of primers (SEQ ID NO: 100 and 101) for amplification downstream of the 3' end were designed, centered around the mutation site. The primer sequences are shown in Table 17.

[0272] [Table 17]

[0273] PCR was performed using the chromosome of ATCC13032 as a template, with primers SEQ ID NOs. 98 and 99, and SEQ ID NOs. 100 and 101. The PCR conditions were denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 30 seconds, for 30 cycles, followed by polymerization at 72°C for 7 minutes. As a result, a 512 bp DNA fragment upstream of the 5' end and a 522 bp DNA fragment downstream of the 3' end were obtained, mainly due to mutations in the lysC gene.

[0274] PCR was performed using the two amplified DNA fragments as templates with primers SEQ ID NO: 98 and SEQ ID NO: 101. The PCR conditions were denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 60 seconds, for 30 cycles, followed by polymerization at 72°C for 7 minutes. As a result, a 1011 bp DNA fragment containing the mutant lysC(L377K) gene encoding an aspart kinase variant in which leucine at position 377 is replaced with lysine was amplified.

[0275] The pDCM2 vector was treated with SmaI, and the PCR product (1011 bp) obtained as described above was fusion cloned with the pDCM2 vector treated with SmaI restriction enzyme using the In-Fusion® HD Cloning Kit (Clontech). The cloned vector was used to transform E. coli DH5α, and the transformed E. coli were streaked onto LB solid medium containing 25 mg / l kanamycin. Colonies transformed with the plasmid were selected from the LB medium, and the plasmid was then obtained by plasmid extraction (Patent Document 9). Finally, a pDCM2-lysC(L377K) recombinant vector was constructed, in which a cassette with the lysC(L377K) gene substituted was cloned.

[0276] The prepared pDCM2-lysC(L377K) vector was transformed into the bacterial strain ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetY using electrotherapy. After a secondary crossover, Corynebacterium glutamicum ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetYlysC(L377K) was obtained, in which a nucleotide mutation was introduced into the lysC gene on the chromosome. Whether or not a nucleotide mutation had been introduced was confirmed by PCR using primers of SEQ ID NO: 98 and SEQ ID NO: 101, followed by sequencing and final confirmation by comparing with the sequence of the wild-type lysC gene.

[0277] Example 5-1-4. Defect of NCgl0616 A vector was created to delete the endogenous gene NCgl0616 (SEQ ID NO: 38) in Corynebacterium glutamicum ATCC13032.

[0278] Specifically, focusing on the NCgl0616 locus of SEQ ID NO: 38, we designed one pair of primers for amplifying the upstream 5' end (SEQ ID NO: 102 and 103) and one pair of primers for amplifying the downstream 3' end (SEQ ID NO: 104 and 105).

[0279] [Table 18]

[0280] PCR was performed using ATCC13032 wild-type (WT) chromosomes as templates and primers SEQ ID NOs. 102, 103, 104, and 105. The PCR conditions were denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 30 seconds, for 30 cycles, followed by polymerization at 72°C for 7 minutes. As a result, a 701 bp DNA fragment upstream of the 5' end and a 699 bp DNA fragment downstream of the 3' end were obtained, centered around the deletion site of the NCgl0616 gene.

[0281] PCR was performed using the two amplified DNA fragments as templates with primers SEQ ID NO: 102 and SEQ ID NO: 105. The PCR conditions were denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 90 seconds, for 30 cycles, followed by polymerization at 72°C for 7 minutes. As a result, a 1410 bp DNA fragment containing the deletion site of the NCgl0616 gene was amplified.

[0282] The pDCM2 vector was treated with SmaI, and the PCR product (2912 bp DNA fragment) obtained as described above was fusion cloned with the pDCM2 vector treated with SmaI restriction enzyme using the In-Fusion® HD Cloning Kit (Clontech). The cloned vector was transformed into E. coli DH5α, and the transformed E. coli were streaked onto LB solid medium containing 25 mg / l kanamycin. Colonies transformed with the plasmid were selected from the LB solid medium, and the plasmid was then obtained by plasmid extraction (Patent Document 9), and finally, a pDCM2-ΔNCgl0616 recombinant vector with a cloned NCgl0616-deficient cassette was constructed.

[0283] [Table 19]

[0284] The prepared pDCM2-ΔNCgl0616 vector was used to transform a bacterial strain with improved O-acetylhomoserine production capacity using the electro-pulse method. After a secondary crossover process, ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetYlysC(L377K)ΔNCgl0616 was obtained, which lacks the NCgl0616 gene on the chromosome.

[0285] Whether or not the NCgl0616 gene was inactivated was determined by PCR using primers for SEQ ID NO: 106 and SEQ ID NO: 107, and then final confirmation was made by comparing it with ATCC13032, in which the NCgl0616 gene was not inactivated.

[0286] Example 5-2. Preparation of O-acetylhomoserine and homoserine-producing strains with enhanced pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase and pyruvate dehydrogenase complex subunit E1 activity. To confirm whether the enhanced activity strain has an effect on improving the production capacity of Corynebacterium glutamicum strains capable of producing O-acetylhomoserine and homoserine, the Corynebacterium glutamicum O-acetylhomoserine and homoserine-producing strain ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetYlysC(L377K)ΔNCgl0616 prepared in Example 5-1 was used to investigate the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase (pyruvate dehydrogenase complex We created a strain of NCgl2126 (sequence number 33) derived from Corynebacterium glutamicum ATCC13032 with enhanced activity (NCBI registration number NCgl2126, hereafter referred to as NCgl2126, sequence number 32) and evaluated its effectiveness.

[0287] Specifically, in order to create a pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase-enhanced vector, plasmids enhancing NCgl2126 activity were constructed by substituting the wild-type promoter of the aceF gene with the Pcj7 promoter (Patent Document 3) (SEQ ID NO: 3), which is known as a strong promoter. PCR was performed to obtain the upstream and downstream regions of the NCgl2126 gene. Specifically, in order to create a strain into which the Pcj7 promoter was introduced, chromosomal DNA of Corynebacterium glutamicum ATCC13032 was used as a template, and PCR was performed to amplify the gene fragment of the upstream region of the NCgl2126 gene using primers SEQ ID NO: 4 and SEQ ID NO: 5, and PCR was performed to amplify the gene fragment of the downstream region of the NCgl2126 gene using primers SEQ ID NO: 6 and SEQ ID NO: 34. Furthermore, using pDCM2-Pcj7 as a template, we obtained the Pcj7 promoter fragment using SEQ ID NOs: 8 and 9.

[0288] PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction. The PCR conditions were denaturation at 95°C for 30 seconds, denaturation at 55°C for 30 seconds, and polymerization at 72°C for 1 minute. This denaturation, annealing, and polymerization reaction was repeated for 28 cycles. As a result, a 318 bp DNA fragment of the Pcj7 promoter region, a 526 bp DNA fragment of the upstream (upstream) and a 529 bp DNA fragment of the downstream (downsteam) of Corynebacterium glutamicum ATC13032NCgl2126 were obtained. Using the amplified promoter and DNA fragments as templates, PCR was performed with primers of SEQ ID NO: 4 and SEQ ID NO: 34. The PCR conditions involved denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 2 minutes, repeated 28 times, and then polymerization at 72°C for 5 minutes. The two resulting fragments were subjected to DNA purification, and then a plasmid was obtained by fusion cloning using the In-Fusion® HD cloning kit (Clontech) with a pDCM2 vector treated with SmaI restriction enzyme (Patent Document 2) according to the attached manual. The resulting vector was named pDCM2-Pcj7_NCgl2126. The sequences and detailed information of the primers used in this example are shown in Table 20.

[0289] [Table 20]

[0290] As described above, the prepared pDCM2-Pcj7_NCgl2126 vector was transformed into the Corynebacterium glutamicum strain ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetYlysC(L377K)ΔNCgl0616 by homologous recombination on the chromosome (Non-Patent Literature 20). The recombinant strain ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetYlysC(L377K)ΔNCgl0616-Pcj7_NCgl2126 was named CM04-8000.

[0291] Furthermore, the vector pDCM2-Pcj7_aceE prepared in Examples 1-2 was used to transform the CM04-8000 strain (Non-Patent Literature 20). The recombinant strain (ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetYlysC(L377K)ΔNCgl0616-Pcj7_NCgl2126-Pcj7_aceE) was named CM04-8002.

[0292] Example 5-3. Evaluation of the production capacity of O-acetylhomoserine and homoserine strains with enhanced pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase and pyruvate dehydrogenase complex subunit E1 activity. Flask evaluations were performed to compare the production capacity of O-acetylhomoserine and homoserine-producing strains Corynebacterium glutamicum ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetYlysC(L377K)ΔNCgl0616, CM04-8000, and CM04-8002.

[0293] The bacterial strain was inoculated onto a 250 ml corner baffle flask containing 25 ml of the following culture medium using one platinum loop, and incubated at 33°C and 200 rpm for 20 hours with shaking. The O-acetylhomoserine concentration was analyzed by HPLC. The analyzed concentrations are shown in Table 21. O-acetylhomoserine production medium (pH 7.2) Glucose 30g, KH2PO4 2g, Urea 3g, (NH4)2SO4 40g, Peptone 2.5g, CSL (Sigma) 5g (10ml), MgSO4·7H2O 0.5g, CaCO3 20g (in 1 liter of distilled water)

[0294] [Table 21]

[0295] As shown in Table 21, compared to O-acetylhomoserine-producing strains, the concentration of O-acetylhomoserine was confirmed to increase by approximately 110-124% in O-acetylhomoserine-producing strains with enhanced pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase and pyruvate dehydrogenase complex subunit E1 activity.

[0296] Example 5-4. Preparation of O-acetylhomoserinetransferase (MetX) introduced plasmid To amplify the gene encoding O-acetylhomoserinetransferase (MetX), the nucleotide sequence information of the metX gene (NCBI registration number NCgl0624, SEQ ID NO: 35) was obtained from the National Institutes of Health (NIH) GenBank. Based on this, primers (SEQ ID NO: 36 and SEQ ID NO: 37) were constructed to amplify the region from the promoter site (300 bp upstream of the start codon) to the terminator site (100 bp downstream of the stop codon). The primer sequences were designed by inserting BamHI restriction enzyme sites at both ends. The PCR conditions were denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 90 seconds, and then polymerization at 72°C for 7 minutes, repeated for 30 cycles. As a result, a 1546 bp DNA fragment of the metX gene encoding region was obtained. The pECCG117 vector (Patent Document 10) and a metX DNA fragment were treated with the restriction enzyme BamHI, ligated using DNA ligase, and then cloned to obtain a plasmid, which was named pECCG117-metX WT. The primer sequences are shown in Table 22.

[0297] [Table 22]

[0298] The prepared pECCG117-metX WT vector was introduced into the bacterial strains ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetYlysC(L377K)ΔNCgl0616 and CM04-8000, prepared in Example 5-2, by electropulse. These strains were then spread onto a selective medium containing 25 mg / L of kanamycin to obtain the respective transformed strains.

[0299] As mentioned above, in order to compare the O-acetylhomoserine production capacity of the prepared bacterial strains, they were cultured using the following method, and the O-acetylhomoserine in the culture medium was analyzed.

[0300] The bacterial strain was inoculated using one platinum loop (inoculation loop) into a 250 ml corner baffled flask containing 25 ml of the following culture medium, and incubated with shaking at 33°C and 200 rpm for 20 hours. The O-acetylhomoserine concentration was analyzed by HPLC. The analyzed concentrations are shown in Table 23. O-acetylhomoserine production medium (pH 7.2) Glucose 30g, KH2PO4 2g, Urea 3g, (NH4)2SO4 40g, Peptone 2.5g, CSL (Sigma) 5g (10ml), MgSO4·7H2O 0.5g, Methionine 400mg, CaCO3 20g (per 1 liter of distilled water)

[0301] [Table 23]

[0302] As a result, as shown in Table 23, when the ATCC13032ΔNCgl2335::PCJ7-yjeH(eco,F351L)ΔmetBΔmetYlysC(L377K)ΔNCgl0616 / pECCG117-metX WT strain was cultured, 2.83 g / L of O-acetyl-L-homoserine accumulated, and when CM04-8002 was cultured, O-acetyl-L-homoserine reached 3.68 g / L, confirming a 130% improvement in production. In O-acetyl-L-homoserine-producing strains, enhancing the activity of the pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase improved O-acetyl-L-homoserine production capacity. Furthermore, enhancing the activity of the pyruvate dehydrogenase complex subunit E1 similarly improved O-acetyl-L-homoserine production capacity.

[0303] These results indicate that in Corynebacterium strains producing O-acetyl-L-homoserine and homoserine, enhancement of pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase activity is effective in O-acetyl-L-homoserine and homoserine production, and further, enhancement of pyruvate dehydrogenase complex subunit E1 is also effective in O-acetyl-L-homoserine and homoserine production.

[0304] From the above explanation, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. It should be understood that the above embodiments are merely illustrative and not limiting. This application should be interpreted as including all modified or altered forms derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. The activity of pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase (AceF) was improved compared to its endogenous activity. Microorganisms that produce L-amino acids.

2. The L-amino acid is at least one selected from glutamine, glutamic acid, ornithine, citrulline, arginine, proline, O-acetylhomoserine, homoserine, and methionine. A microorganism that produces the L-amino acid described in claim 1.

3. The aforementioned microorganism belongs to the genus Corynebacterium. The microorganism described in claim 1.

4. The aforementioned microorganism of the genus Corynebacterium is Corynebacterium glutamicum. The microorganism described in claim 3.

5. The pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase is derived from Corynebacterium glutamicum. A microorganism that produces the L-amino acid described in claim 1.

6. The pyruvate dehydrogenase complex dihydrolipoyl lysine residue acetyltransferase contains the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

32. A microorganism that produces the L-amino acid described in claim 1.

7. The pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase is encoded by the polynucleotide of SEQ ID NO: 2 or SEQ ID NO:

33. A microorganism that produces the L-amino acid described in claim 6.

8. The aforementioned microorganisms exhibit enhanced activity of the pyruvate dehydrogenase complex subunit E1 compared to its endogenous activity. The microorganism according to any one of claims 1 to 7.

9. The pyruvate dehydrogenase complex subunit E1 contains the amino acid sequence of Sequence ID No.

10. The microorganism according to claim 8.

10. The aforementioned pyruvate dehydrogenase complex subunit E1 is encoded by the polynucleotide of Sequence ID No. 11 or Sequence ID No.

39. The microorganism according to claim 9.

11. The process includes the step of culturing microorganisms in which the activity of pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase (AceF) is enhanced compared to its endogenous activity, in a culture medium. Method for producing L-amino acids.

12. The L-amino acid is at least one selected from glutamine, glutamic acid, ornithine, citrulline, arginine, proline, O-acetylhomoserine, homoserine, and methionine. The method for producing L-amino acids according to claim 11.

13. The aforementioned microorganisms exhibit enhanced activity of the pyruvate dehydrogenase complex subunit E1 compared to its endogenous activity. The method for producing L-amino acids according to claim 9.

14. The further step includes recovering L-amino acids from the cultured microorganism, the culture of the microorganism, the fermented product of the microorganism, or the culture medium. The method according to claim 9.

15. A microorganism in which the pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase activity is improved compared to its endogenous activity, a culture of the said microorganism, a fermented product of the said microorganism, or a combination of at least two of the above. Composition for L-amino acid production.

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