L-isoleucine-producing microorganisms and methods for producing L-isoleucine using the same

Abstract

The present application relates to a microorganism capable of producing L-isoleucine, into which a gene encoding an exogenous glutamate dehydrogenase has been introduced, a method for producing L-isoleucine using the microorganism, and a composition for producing L-isoleucine containing the microorganism.

Description

Technical Field

[0001] This application relates to a microorganism having the ability to produce L-isoleucine into which a gene encoding an exogenous glutamate dehydrogenase has been introduced, a method for producing L-isoleucine using the microorganism, and a composition for producing L-isoleucine containing the microorganism.

Background Art

[0002] L-isoleucine is one of the branched-chain amino acids among a total of 20 kinds of amino acids and is classified as an essential amino acid and is used in the fields of animal feed, food additives, and pharmaceuticals. L-isoleucine functions in energy production after metabolism, hemoglobin production, blood sugar regulation, muscle production and repair, etc., so its use is increasing not only in infusion solutions, nutritional agents, sports nutritional agents, but also in animal feed.

[0003] For the production of L-isoleucine, Corynebacterium glutamicum and Escherichia coli are used as representative microorganisms. In these microorganisms, L-isoleucine shares the main biosynthetic pathway with L-valine and L-leucine, which are different branched-chain amino acids. When examining the biosynthetic pathway of L-isoleucine, 2-ketobutyrate generated from pyruvate produced in the glycolysis process and L-threonine, an amino acid derived from aspartate, is used as a precursor, and finally L-isoleucine is produced.

[0004] For the production of L-amino acids, methods using various microorganisms and their mutants are known (Patent Document 1). However, in the case of the method using mutants, a large number of by-products other than L-isoleucine are generated, which has the problem of reducing the purity of L-isoleucine in the purification step.

[0005] In this regard, methods for purifying L-isoleucine are known to increase its purity (Patent Document 2), but these purification methods have the disadvantage of requiring additional purification processes, and there is currently a need to develop methods to increase the purity of L-isoleucine.

[0006] On the other hand, L-glutamic acid is a precursor that provides an amine group in the synthesis of amino acids, including L-isoleucine. While strengthening the synthesis of L-glutamic acid, the precursor, is essential for amino acid production, when strengthening glutamate dehydrogenase, which is known to produce glutamic acid in microorganisms that produce L-isoleucine, a side reaction occurs that produces α-aminobutyric acid (AABA) as a byproduct through the dehydrogenation reaction of 2-ketobutyrate, an intermediate of isoleucine (Non-Patent Literature 1). This leads to problems in that the purity and biosynthesis efficiency of L-isoleucine decrease. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] U.S. Patent No. 10113190 [Patent Document 2] U.S. Patent No. 6072083 [Patent Document 3] U.S. Patent No. 7662943 [Patent Document 4] U.S. Patent No. 10584338 [Patent Document 5] U.S. Patent No. 10273491 [Patent Document 6] Korean Published Patent No. 10-2020-0136813 [Patent Document 7] Korean Registered Patent Publication No. 10-1996769 [License 8] Korean Registration Patent No. 10-1335789 [Non-licensed literature]

[0008] [Non-licensed Document 1] Microb Cell Fact.2017 Mar23;16(1):51 [Non-licensed Document 2] Pearson et al(1988)[Proc.Natl.Acad.Sci.USA 85]:2444 [Non-licensed Document 3] Rice et al., 2000, Trends Genet.16:276-277 [Non-licensed Document 4] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-licensed Document 5] Devereux,J.,et al,Nucleic Acids Research 12:387(1984) [Non-licensed Document 6] Atschul,[S.][F.,][ET AL,J MOLEC BIOL 215]:403(1990) [Non-licensed Document 7] Guide to Huge Computers,Martin J.Bishop,[ED.,]Academic Press,San Diego, 1994 [Non-licensed Document 8] [CARILLO ETA / . ](1988)SIAM J Applied Math 48:1073 [Non-licensed Document 9] Smith and Waterman,Adv.Appl.Math(1981)2:482 [Non-licensed Document 10] Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) [Non-Patent Document 11] Gribskov et al (1986) Nucl.Acids Res.14:6745 [Non-Patent Document 12] Sitnicka et al.Functional Analysis of Genes.Advances in Cell Biology.2010,Vol.2.1-16 [Non-Patent Document 13] Sambrook et al.Molecular Cloning2012 [Non-Patent Document 14] J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989. [Non-Patent Document 15] FMAsubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8 [Non-Patent Document 16] Journal of Biotechnology104,5-25 Jorn Kalinowski et al, 2003 [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The inventors of the present application have developed a microorganism having the ability to produce L-isoleucine into which a gene encoding an exogenous glutamate dehydrogenase has been introduced, a method for producing L-isoleucine using the microorganism, and a composition for producing L-isoleucine containing the microorganism, and completed the present application.

Means for Solving the Problems

[0010] One object of the present application is to provide a microorganism having the ability to produce L-isoleucine into which a gene encoding an exogenous glutamate dehydrogenase derived from Bacillus subtilis or Rhodospirillales has been introduced.

[0011] Another object of the present application is to provide a method for producing L-isoleucine including a step of culturing the microorganism in a medium. Another object of the present application is to provide a composition for producing L-isoleucine containing the microorganism.

Effects of the Invention

[0012] The microorganism having the ability to produce L-isoleucine into which the gene encoding the exogenous glutamate dehydrogenase of the present application has been introduced produces few by-products, can produce L-isoleucine in a high yield, and can be usefully utilized for the industrial production of L-isoleucine.

Modes for Carrying Out the Invention

[0013] Specifically explaining this, it is as follows. On the one hand, each explanation and embodiment disclosed in the present application can also be applied to each different explanation and embodiment. That is, all combinations of various elements disclosed in the present application belong to the scope of the present application. Also, it is not considered that the category of the present application is limited by the following specific description.

[0014] Furthermore, a person with ordinary skill in the art can recognize or confirm many equivalents relating to specific aspects of the present invention described in this application by ordinary experimentation alone. Such equivalents are intended to be included in this application.

[0015] One aspect of this application provides a microorganism capable of producing L-isoleucine into which a gene encoding an exogenous glutamate dehydrogenase derived from Bacillus subtilis or Rhodospirillales has been introduced.

[0016] In this application, the term "L-isoleucine" refers to an L-amino acid with the chemical formula HO2CCH(NH2)CH(CH3)CH2CH3, which is one of the essential amino acids and structurally, along with L-valine and L-leucine, is a branched-chain amino acid.

[0017] In this application, the term "strain (or microorganism)" includes all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and is a microorganism in which a specific mechanism is weakened or strengthened due to causes such as the insertion of external genes or the enhancement or inactivation of the activity of endogenous genes, and may be a microorganism that includes genetic modification for the production of the target polypeptide, protein or product.

[0018] In this application, the term "microorganism having L-isoleucine production ability" means a microorganism that naturally has the ability to produce L-isoleucine or a microorganism in which the ability to produce L-isoleucine has been conferred to a parent strain that does not have the ability to produce L-isoleucine. Specifically, the microorganism may, but is not limited to, a microorganism that produces L-isoleucine into which a gene encoding an exogenous glutamate dehydrogenase derived from Bacillus subtilis or Rhodospirillales has been introduced.

[0019] Specifically, the "microorganisms that produce L-isoleucine" include all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification. More specifically, they are microorganisms in which a specific mechanism has been weakened or strengthened due to causes such as the insertion of an external gene or the strengthening or inactivation of the activity of an endogenous gene, and may also be microorganisms in which genetic mutations have occurred or L-isoleucine production activity has been strengthened for the purpose of producing the desired L-isoleucine. For the purposes of this application, the microorganisms having L-isoleucine production ability may be, but are not limited to, genetically modified or recombinant microorganisms characterized by the introduction of a gene encoding an exogenous glutamate dehydrogenase derived from Bacillus subtilis or Rhodospirillales, thereby increasing the desired L-isoleucine production ability.

[0020] In this application, the term "introduction" of activity means that a gene not originally present in the microorganism is expressed within that microorganism, thereby exhibiting activity of a specific protein, or exhibiting increased or improved activity compared to the intrinsic activity or pre-modification activity of the protein. For example, this may involve introducing a polynucleotide encoding a specific protein into a chromosome within the microorganism, or introducing a vector containing a polynucleotide encoding a specific protein into the microorganism, thereby exhibiting its activity.

[0021] For example, the recombinant strain with increased production capacity may have an increase of approximately 1% or more compared to the L-isoleucine production capacity of the parent strain before mutation or a non-myxoid microorganism with intrinsic gdh protein activity. Specifically, this could be approximately 1% or more, approximately 2% or more, approximately 3% or more, approximately 4% or more, approximately 5% or more, approximately 6% or more, approximately 7% or more, approximately 8% or more, approximately 8.1% or more, approximately 8.2% or more, or approximately 8.3% or more (there are no special restrictions on the upper limit; for example, it may be approximately 100% or less, approximately 50% or less, approximately 25% or less, approximately 20% or less, approximately 15% or less, or approximately 10% or less). However, it is not limited to this as long as it has a positive increase compared to the production capacity of the parent strain before mutation or the non-myxoid microorganism. In other examples, the recombinant strain with increased production capacity may have an L-isoleucine production capacity increased by approximately 1.01 times or more, approximately 1.02 times or more, approximately 1.03 times or more, approximately 1.04 times or more, approximately 1.05 times or more, approximately 1.06 times or more, approximately 1.07 times or more, or approximately 1.08 times or more compared to the parent strain or non-myxoid before mutation (there is no special limit on the upper limit; for example, it may be approximately 10 times or less, approximately 5 times or less, approximately 3 times or less, approximately 2 times or less, approximately 1.5 times or less, or approximately 1.1 times or less), but is not limited to these values.

[0022] In this application, the term "non-myxoid microorganism" means a strain that is either wild-type or naturally occurring, or a strain before its characteristics are altered by genetic mutation due to natural or artificial factors, and does not exclude strains containing naturally occurring mutations in microorganisms. For example, the non-myxoid microorganism means a strain before the introduction of the foreign glutamate dehydrogenase gene described herein. The term "non-myxoid microorganism" may be used interchangeably with "pre-deformation strain," "pre-deformation microorganism," "non-mutant strain," "non-myxoid strain," "non-mutant microorganism," or "reference microorganism."

[0023] As yet another example of this application, the microorganisms of this application may be microorganisms capable of producing L-isoleucine, and are not particularly limited in type. The microorganisms of this application may be either prokaryotic or eukaryotic cells, but specifically may be prokaryotic cells. The prokaryotic cells may include, for example, microbial strains belonging to the genera Corynebacterium, Escherichia, Erwinia, Serratia, Providencia, and Brevibacterium, and specifically may be microorganisms of the genus Corynebacterium.

[0024] In this application, "microorganisms of the genus Corynebacterium" can include all microorganisms of the genus Corynebacterium. Specifically, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes It may also be Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens, and more specifically, Corynebacterium glutamicum.

[0025] In this application, the term "glutamate dehydrogenase" refers to an enzyme that synthesizes glutamate, a precursor of L-isoleucine biosynthesis, and in this application, "glutamate dehydrogenase" may be used interchangeably with "gdh" and "rocG".

[0026] In this application, the terms "protein having glutamate dehydrogenase activity" and "gene encoding glutamate dehydrogenase" may, without limitation, include any protein having glutamate dehydrogenase activity as described above and any gene encoding it. Specifically, the glutamate dehydrogenase is publicly known in the art, and the protein and gene sequences of the glutamate dehydrogenase can be obtained from publicly known databases, such as NCBI's GenBank, but are not limited thereto.

[0027] On the other hand, L-glutamic acid is a precursor that provides an amine group in the synthesis of amino acids, including L-isoleucine. While strengthening the synthesis of L-glutamic acid, the precursor, is essential for amino acid production, when strengthening glutamate dehydrogenase, which is known to produce glutamic acid from microorganisms that produce L-isoleucine, a side reaction occurs that produces α-aminobutyric acid (AABA) as a byproduct through the dehydrogenation reaction of 2-ketobutyrate, an intermediate of isoleucine (Microb Cell Fact. 2017 Mar23;16(1):51). This leads to problems in that the purity and biosynthesis efficiency of L-isoleucine decrease.

[0028] The L-isoleucine-producing microorganisms of this application exhibit reduced byproduct production when a gene encoding an exogenous glutamate dehydrogenase derived from Bacillus subtilis or Rhodospirillales is introduced. The byproduct may be α-aminobutyric acid (AABA). The reduction in byproduct production means, but is not limited to, a decrease in the production of α-aminobutyric acid (AABA) compared to the L-isoleucine production compared to wild-type microorganisms.

[0029] For the purposes of this application, the protein having glutamate dehydrogenase activity may be derived from Bacillus subtilis or Rhodospirillales. Specifically, the glutamate dehydrogenase may have and / or contain the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 3, or be essentially composed of the said amino acid sequence.

[0030] Furthermore, the glutamate dehydrogenase of this application may include amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with the amino acid sequence described in Sequence ID No. 1 or Sequence ID No. 3. It is also obvious that glutamate dehydrogenases having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted, or added are also included within the scope of this application, as long as they have such homology or identity and exhibit the efficacy corresponding to the glutamate dehydrogenase of this application.

[0031] Even if this application describes a polypeptide or protein containing an amino acid sequence described by a specific sequence number, a polypeptide or protein consisting of an amino acid sequence described by a specific sequence number, or a polypeptide or protein having an amino acid sequence described by a specific sequence number, it is obvious that proteins having amino acid sequences in which some sequences are deleted, modified, substituted, conservedly substituted, or added may also be used in this application, as long as they have the same or equivalent activity as the polypeptide consisting of the amino acid sequence of the said sequence number. For example, this includes cases where the N-terminus and / or C-terminus of the amino acid sequence have added sequences that do not change the function of the protein, naturally occurring mutations, silent mutations, or conserved substitutions.

[0032] For example, the amino acid sequence may have additions or deletions of sequences that do not alter the function of the glutamate dehydrogenase of this application, spontaneous mutations, silent mutations, or conservative substitutions at its N-terminus, C-terminus, and / or within it.

[0033] In this application, the term “conservative substitution” means replacing one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartate; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Furthermore, amino acids can be classified into those with electrically charged side chains and those with uncharged side chains. Amino acids with electrically charged side chains include aspartic acid, glutamic acid, lysine, arginine, and histidine. Amino acids with uncharged side chains can be further classified into nonpolar amino acids and polar amino acids. Nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Typically, conservative substitutions have little to no effect on the activity of the resulting polypeptide. Typically, conservative substitutions have little to no effect on the activity of a protein or polypeptide.

[0034] Furthermore, glutamate dehydrogenase may include the deletion or addition of amino acids that have minimal effect on the polypeptide's properties and secondary structure. For example, the polypeptide can be conjugated with a protein N-terminal signal (or leader) sequence involved in protein transfer co-translationally or post-translationally. The polypeptide can also be conjugated with other sequences or linkers to enable the polypeptide to be identified, purified, or synthesized.

[0035] In this application, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid sequences or base sequences, and may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0036] The homology or identity of sequences of conserved polynucleotides or polypeptides is determined by standard sequencing algorithms, and a default gap penalty established by the program used is available. Substantially homologous or identical sequences can generally be hybridized, in whole or in part, under moderate to high stringent conditions. It is obvious that hybridization also includes hybridization with polynucleotides containing codons in general or codon degeneracy in polynucleotides.

[0037] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using known computer algorithms such as the "FASTA" program, utilizing default parameters as described, for example, in Pearson et al (1988) [Proc.Natl.Acad.Sci.USA85]:2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277) (version 5.0.0 or later) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.][F.,][ET AL, J MOLEC BIOL215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San (Including Diego, 1994, and [CARILLO ETA / .] (1988) SIAM J Applied Math 48:1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information Databases.

[0038] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as Needleman et al. (1970), J Mol Biol. 48:443, as is known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In summary, the GAP program can be defined as the total number of symbols in the shorter of two sequences divided by the number of similarly sequenced symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program may include: (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and a weighted comparison matrix of Gribskov et al (1986) Nucl. Acids Res. 14:6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (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 opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0039] In this application, the term "corresponding to" refers to an amino acid residue at a position listed in the polypeptide, or an amino acid residue that is similar, identical, or homologous to a residue listed in the polypeptide. Identifying the amino acid at the corresponding position may also mean determining a specific amino acid in a sequence that references a particular sequence. As used in this application, "corresponding region" generally refers to a similar or corresponding position in a related protein or reference protein.

[0040] For example, any amino acid sequence can be aligned with sequence number 1, and based on this, each amino acid residue in the amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue in sequence number 1. For example, a sequence alignment algorithm such as the one described in this application can be used to verify the position of amino acids, or the position where deformations such as substitution, insertion, or deletion occur, by comparing them with a query sequence (also called a "reference sequence").

[0041] For such sorting, one can use, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453), the Needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277), but is not limited to these. Any sequence sorting program or pairwise sequence comparison algorithm known in this field can be used appropriately.

[0042] In this application, the term "intrinsic" activity means the activity state of a protein that a microorganism inherently possesses in its natural state or before the protein is modified. This may be used interchangeably with "pre-modification activity."

[0043] In this application, the term "enhancement" of polypeptide activity means that the activity of the polypeptide increases compared to its intrinsic activity. This enhancement may be used interchangeably with terms such as activation, upregulation, overexpression, and increase. Here, activation, enhancement, upregulation, overexpression, and increase can all include exhibiting activity that was not originally present, or exhibiting activity that is improved compared to the intrinsic activity or the activity before the mutation. "Intrinsic activity" refers to the activity of a specific polypeptide that was originally present in the parent strain or non-mutant microorganism before the mutation occurred, in cases where the trait changes due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before the mutation." "Enhancement," "upregulation," "overexpression," or "increase" of polypeptide activity compared to its intrinsic activity means that the activity and / or concentration (expression level) of the specific polypeptide that was originally present in the parent strain or non-mutant microorganism before the mutation occurred is improved.

[0044] The aforementioned enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. Whether or not the polypeptide's activity has been enhanced can be confirmed by an increase in the polypeptide's activity level, expression level, or the amount of product excreted from the polypeptide.

[0045] The enhancement of the activity of the polypeptide can be achieved by applying a variety of methods well known in the field, and is not limited as long as it can enhance the activity of the target polypeptide compared to the microorganism before deformation. Specifically, this may involve, but is not limited to, the use of gene engineering and / or protein engineering, which are routine methods in molecular biology and are well known to ordinary technicians in this field (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol.2.1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0046] Specifically, the strengthening of the polypeptide in this application is 1) Increase in the intracellular copy number of polynucleotides encoding polypeptides; 2) Modification of gene expression regulatory regions on chromosomes that encode polypeptides (e.g., mutation within the regulatory region, replacement with a more active sequence, or insertion of a more active sequence); 3) Modification of the nucleotide sequence encoding the start codon or 5'UTR region of a polypeptide-encoding gene transcript; 4) Modification of the amino acid sequence of the polypeptide so as to enhance polypeptide activity; 5) Modification of the polynucleotide sequence encoding the polypeptide so as to enhance polypeptide activity (for example, modification of the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified to enhance polypeptide activity); 6) Introduction of a foreign polypeptide exhibiting polypeptide activity or a foreign polynucleotide encoding such activity; 7) Codon optimization of polynucleotides encoding polypeptides; 8) Analyze the tertiary structure of the polypeptide, select exposed sites, and deform or chemically modify them; or 9) A combination of two or more selected from items 1) to 8) above is also acceptable, but is not particularly limited thereto.

[0047] More specifically, The increase in the intracellular copy number of the polynucleotide encoding the polypeptide described in 1) above may be achieved by introducing into the host cell a vector that is operablely linked to the polynucleotide encoding the polypeptide, replicates independently of the host, and functions. Alternatively, it may be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide into the chromosomes within the host cell. The introduction into the chromosome is performed by introducing into the host cell a vector that causes the polynucleotide to be inserted into the chromosomes within the host cell, but is not limited to this. The vector is as described above.

[0048] The replacement of a gene expression regulatory region (or regulatory sequence) on a chromosome encoding a polypeptide with a more potent sequence may, for example, involve sequence mutation by deletion, insertion, non-conservative or conservative substitution or a combination thereof, or replacement with a sequence having stronger activity, in order to further enhance the activity of the regulatory region. The regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates the termination of transcription and decoding. For example, the original promoter may be replaced with a potent promoter.

[0049] Examples of well-known strong promoters include, but are not limited to, the CJ1-CJ7 promoters (US Patent No. 7,662,943), the lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Patent No. 1,0584,338), O2 promoter (US Patent No. 1,0273,491), tkt promoter, and yccA promoter.

[0050] The sequence modification encoding the start codon or 5'UTR region of the polypeptide-encoding gene transcript described in 3) above may, but is not limited to, substitution with a sequence encoding another start codon that has a higher polypeptide expression rate than the endogenous start codon.

[0051] The modifications of the amino acid sequence or polynucleotide sequence described in 4) and 5) above may be, but are not limited to, deletion, insertion, non-conservative or conservative substitution or combination thereof of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide, thereby causing sequence mutations, or replacement with an improved amino acid sequence or polynucleotide sequence that has stronger activity or an improved amino acid sequence or polynucleotide sequence that has increased activity. Specifically, the replacement may be, but is not limited to, insertion of a polynucleotide into the chromosome by homologous recombination. The vector used in this case may further include a selection marker for confirming the presence or absence of chromosomal insertion. The selection marker is as described above.

[0052] The introduction of a foreign polynucleotide exhibiting polypeptide activity (6) above may be the introduction of a foreign polynucleotide encoding a polypeptide exhibiting the same or similar activity as the polypeptide into the host cell. The foreign polynucleotide is not restricted in its origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The method used for the introduction can be appropriately selected by a person skilled in the art from known transformation methods, and the introduction of the polynucleotide into the host cell can generate the polypeptide, thereby increasing its activity.

[0053] The codon optimization of the polynucleotide encoding the polypeptide described in 7) above may be codon optimization of the endogenous polynucleotide so that transcription or translation increases in the host cell, or optimization of the codon of the exogenous polynucleotide so that optimized transcription or translation occurs in the host cell.

[0054] 8) Analyzing the tertiary structure of a polypeptide and selecting exposed sites to deform or chemically modify may, for example, involve comparing the sequence information of the polypeptide to be analyzed with a database containing sequence information of known proteins to determine candidate template proteins according to the degree of sequence similarity, confirming the structure based on these candidates, and selecting exposed sites to deform or chemically modify.

[0055] Such enhancement of polypeptide activity may be achieved by increasing the activity or concentration of the corresponding polypeptide based on the activity or concentration of the polypeptide expressed in the wild-type or pre-deformation microbial strain, or by increasing the amount of product produced from the polypeptide, but is not limited to these methods.

[0056] Specifically, for the purposes of this application, the microorganism may be subjected to the introduction of an exogenous polynucleotide having the activity of glutamate hydrogenase derived from Bacillus subtilis or Rhodospirillales in order to enhance the protein activity of glutamate dehydrogenase.

[0057] In this application, the term "polynucleotide" means a polymer of nucleotides in which nucleotide units (monomers) are covalently linked together in a long chain, and is a DNA or RNA chain of a certain length or longer. More specifically, it means a polynucleotide fragment that encodes the aforementioned variant.

[0058] The polynucleotides described in this application are characterized by being derived from Bacillus subtilis or Rhodospirillales, and more specifically, the polynucleotides may include a base sequence encoding the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and more specifically, the genes encoding foreign glutamate dehydrogenase from Bacillus subtilis and the genes encoding foreign glutamate dehydrogenase from Rhodospirillales may have the nucleotide sequences of SEQ ID NO: 2 and 4, respectively, but are not limited thereto. The base sequences of SEQ ID NO: 2 or SEQ ID NO: 4 can be obtained from known databases, such as NCBI's GenBank, but are not limited thereto.

[0059] In this application, the gene containing the base sequence of SEQ ID NO: 2 or SEQ ID NO: 4 may be mixed with a polynucleotide containing the base sequence of SEQ ID NO: 2 or SEQ ID NO: 4, a gene or polynucleotide having the base sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or a gene or polynucleotide consisting of the base sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0060] The polynucleotides of this application undergo various modifications to the coding region, taking into consideration the degeneracy of codons or the preferred codons in organisms that intend to express the variants of this application, while not altering the amino acid sequence of the variants of this application. Specifically, the polynucleotides of this application have, or include, a base sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or consist of, or are essentially composed of, a base sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0061] Furthermore, the polynucleotides of this application may include, without limitation, any probes produced from known gene sequences, such as sequences that can hybridize under stringent conditions with complementary sequences to all or part of the polynucleotide sequences of this application. “Stringent conditions” means conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; FMAusubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, we can list conditions in which polynucleotides with high homology or identity hybridize with each other, with homology or identity levels of 70% or more, 75% or more, 6% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, but do not hybridize with polynucleotides with lower homology or identity levels. Alternatively, we can list conditions in which the polynucleotides are washed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of a normal Southern hybridization: 60°C, 1×SSC, 0.1% SDS, more specifically 60°C, 0.1×SSC, 0.1% SDS, or more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0062] Hybridization requires that two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationships between nucleotide bases that can hybridize with each other. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of this application may also include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the overall sequence.

[0063] Specifically, polynucleotides homologous or identical to the polynucleotides of this application can be detected using hybridization conditions that include a hybridization step at a Tm value of 55°C, and under the conditions described above. The Tm value may also be 60°C, 63°C, or 65°C, but is not limited thereto and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0064] The appropriate stringency for hybridizing the aforementioned polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are well known in the art (e.g., J. Sambrook et al., ibid.).

[0065] For the purposes of this application, the microorganism may, but is not limited to, a microorganism having enhanced protein activity of glutamate dehydrogenase compared to its endogenous activity by including an expression vector for expressing the foreign polynucleotide in the host.

[0066] The vector of this application may include a DNA product comprising a polynucleotide sequence encoding a target polypeptide, operably linked to a suitable regulatory region (or regulatory sequence) for expressing the target polypeptide in a suitable host. The regulatory region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences regulating the termination of transcription and decoding. After being transformed into a suitable host cell, the vector may replicate or function independently of the host genome and may be integrated into the genome itself.

[0067] The vectors used in this application are not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pSK, pSKH, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pSK, pSKH130, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0068] As an example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a chromosome insertion vector within a cell. The insertion of the polynucleotide into the chromosome may be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. A selection marker may further be included to confirm the presence or absence of the chromosome insertion. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of the target nucleic acid molecule insertion, and markers that confer selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of surface polypeptides are used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes, thus allowing for the selection of transformed cells.

[0069] In this application, the term "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include all of them, regardless of whether they are inserted into or extrachromosomal regions of the host cell, as long as they can be expressed in the host cell. The polynucleotide also includes DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced into the host cell in any form that allows for its introduction and expression. For example, the polynucleotide 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 autonomous expression. The expression cassette may typically include 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 sequences necessary for expression in the host cell, but is not limited to this.

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

[0071] Modification of some or all of the polynucleotides in the microorganisms of this application may be induced by (a) homologous recombination using a chromosome insertion vector within the microorganism or genome editing using an engineered nuclease (e.g., CRISPR-Cas9) and / or (b) light and / or chemical treatment such as ultraviolet light and radiation. The method for modifying some or all of the genes may include methods using DNA recombination technology. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene is injected into the microorganism to induce homologous recombination, thereby deleting some or all of the genes. The injected nucleotide sequence or vector may, but is not limited to, contain a dominant selection marker.

[0072] On the other hand, in specific embodiments of this application, the Corynebacterium microorganisms may include enzymes involved in the L-isoleucine biosynthesis pathway with enhanced activity. In this application, the term "enzymes involved in the L-isoleucine biosynthesis pathway" may include, but is not limited to, aspartate kinase (lysC gene), aspartate-β-semialdehyde dehydrogenase (asd gene), homoserine dehydrogenase (hom gene), homoserine kinase (thrB gene), threonine synthase (thrC gene), threonine dehydratase (ilvA gene), aminotransferase (ilvE gene), and others.

[0073] Another aspect of this application provides a method for producing L-isoleucine, comprising the step of culturing the microorganism in a culture medium. The aforementioned microorganism, L-isoleucine, is as described above.

[0074] Specifically, the gene encoding foreign glutamate dehydrogenase derived from Bacillus subtilis and the gene encoding foreign glutamate dehydrogenase derived from the Rhodospirillales order may have, but are not limited to, the nucleotide sequences of SEQ ID NOs: 2 and 4, respectively.

[0075] Specifically, the above method may result in a decrease in the amount of α-aminobutyric acid (AABA) produced compared to the amount of L-isoleucine produced, but is not limited to this.

[0076] In this application, the term "culture" means growing the microorganisms of this application under appropriately controlled environmental conditions. The culture process of this application is carried out according to suitable culture media and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culture may be batch, continuous, or fed-batch.

[0077] In this application, the term "culture medium" refers to a substance that is a mixture of nutrients necessary for culturing the microorganisms of this application, supplying nutrients and growth factors, including water, which is essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the microorganisms of this application can be any culture medium used for culturing ordinary microorganisms without any special restrictions, but the microorganisms of this application can be cultured under aerobic conditions in an ordinary culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, while adjusting the temperature, pH, etc.

[0078] In this application, the carbon sources may include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn maceration can also be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and other appropriate amounts of carbon sources can be used in a variety of ways without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited to these uses.

[0079] The nitrogen sources used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids like glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn maceration, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products. These nitrogen sources may be used individually or in combination of two or more, and are not limited to these uses.

[0080] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or corresponding sodium-containing salts. Inorganic compounds such as sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate may be used, along with amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the culture medium in batches or continuously, but are not limited thereto.

[0081] In this application, during microbial cultivation, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture. Furthermore, during cultivation, the formation of bubbles can be suppressed using an antifoaming agent such as fatty acid polyglycol ester. In addition, oxygen or oxygen-containing gas can be injected into the culture to maintain an aerobic state, or nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection to maintain an anaerobic or microaerobic state, but this is not limited to these methods.

[0082] The temperature of the culture water may be 25°C to 40°C, or more specifically, 28°C to 37°C, but is not limited to this. The culture period can be continued until the desired amount of useful substance is produced, and may be 1 hour to 100 hours, but is not limited to this.

[0083] L-isoleucine produced by the culture described in this application is either secreted into the culture medium or remains within the cells. The method for producing L-isoleucine according to this application may further include, for example, a step of preparing the microorganism of this application, a step of preparing a culture medium for culturing the microorganism, or a combination thereof (in any order), before the culturing step.

[0084] The method for producing L-isoleucine according to this application may further include a step of recovering L-isoleucine from the culture medium (the culture medium in which the culture was performed) or microorganisms. The recovery step may be included after the culture step.

[0085] The aforementioned recovery may involve collecting the target L-isoleucine using appropriate methods known in the art, such as the microbial culture methods of this application, for example, batch, continuous, or fed-batch culture methods. For example, various chromatography methods such as centrifugation, filtration, treatment with a crystallizing protein precipitant (salting-out method), extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof may be used, and the target L-isoleucine can be recovered from the culture medium or microorganism using appropriate methods known in the art.

[0086] Furthermore, the L-isoleucine production method of this application may additionally include a purification step. The purification can be carried out using appropriate methods known in the art. For example, if the L-isoleucine 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, simultaneously or integrated into a single step, regardless of the procedure.

[0087] Another aspect of this application provides a composition for L-isoleucine production containing the microorganism. The aforementioned microorganism, L-isoleucine, is as described above.

[0088] Specifically, the genes encoding foreign glutamate dehydrogenase from Bacillus subtilis and the genes encoding foreign glutamate dehydrogenase from the Rhodospirillales order may have the nucleotide sequences of SEQ ID NOs. 2 and 4, respectively, but are not limited thereto.

[0089] Specifically, the above method may result in a decrease in the amount of α-aminobutyric acid (AABA) produced compared to the amount of L-isoleucine produced, but is not limited to this.

[0090] The composition of this application may further contain any suitable excipients commonly used in compositions for L-isoleucine production, such excipients may include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents.

[0091] Another aspect of this application is to provide an application for L-isoleucine production of a microorganism capable of producing L-isoleucine into which a gene encoding an exogenous glutamate dehydrogenase derived from Bacillus subtilis or Rhodospirillales has been introduced. [Examples]

[0092] The present application will be described in more detail below through examples. However, these examples are for illustrative purposes only, and the scope of the present application is not limited to these examples.

[0093] Example 1: Fabrication of a recombinant vector for introducing exogenous glutamate dehydrogenase. To insert an exogenous gdh overexpression vector into the Corynebacterium glutamicum chromosome, NCgl2872, a gene known to encode a transposon in Corynebacterium glutamicum, was used as the insertion site (Journal of Biotechnology 104, 5-25 Jorn Kalinowski et al, 2003). To replace the NCgl2872 gene with exogenous gdh, NCgl2872 deletion and target gene insertion vectors were constructed. To construct the vectors, PCR was performed using ATCC13032 chromosomes as templates with primer pairs of SEQ ID NOs.5 and SEQ ID NOs.6, and SEQ ID NOs.7 and SEQ ID NOs.8. PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction, and 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. These denaturation, annealing, and polymerization reactions were repeated 28 times. As a result, DNA fragments of 623 bp and 620 bp were obtained, respectively. The obtained DNA products were purified using a PCR purification kit (QUIAGEN), and the NCgl2872 deletion vector and the target gene insertion vector pDCM2ΔN2872 were produced by cloning them using a heat-treated pDCM2 vector (Korean Published Patent No. 10-2020-0136813) and an infusion cloning kit (TaKaRa) according to the provided manual.

[0094] To produce strains into which an exogenous gdh possessing the gdh promoter of the parent strain Corynebacterium glutamicum was introduced, PCR was performed using chromosomes of Corynebacterium glutamicum ATC13032, Escherichia coli, Bacillus subtilis, Rhodospirillales, and Mycobacterium smegmatis as templates, respectively, with primers corresponding to SEQ ID NOs. 9 and SEQ ID NOs. 10; or SEQ ID NOs. 9 and SEQ ID NOs. 11; or SEQ ID NOs. 12 and SEQ ID NOs. 13; or SEQ ID NOs. 14 and SEQ ID NOs. 15; or SEQ ID NOs. 16 and SEQ ID NOs. 17; or SEQ ID NOs. 18 and SEQ ID NOs. 19; respectively. The primer sequences used for each of the aforementioned PCRs are shown in Table 1 below.

[0095] [Table 1]

[0096] 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. These denaturation, annealing, and polymerization reactions were repeated 28 times. As a result, a 519 bp DNA fragment of the gdh promoter site, an 1882 bp DNA fragment of the gdh site of Corynebacterium glutamicum ATC13032 containing the promoter, a 1382 bp DNA fragment of the gdh site of Escherichia coli, a 1313 bp DNA fragment of the gdh(rocG) site of Bacillus subtilis, a 1424 bp DNA fragment of the gdh site of Rhodospirillales, and a 1388 bp DNA fragment of the gdh site of Mychobacterium smegmatis were obtained.

[0097] PCR was performed using the amplified promoter and exogenous gdh DNA section as templates with primers of SEQ ID NO: 9 and SEQ ID NO: 13; or SEQ ID NO: 9 and SEQ ID NO: 15; or SEQ ID NO: 9 and SEQ ID NO: 17; or SEQ ID NO: 9 and SEQ ID NO: 19;. The PCR conditions were denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 2 minutes, repeated 28 times, followed by polymerization at 72°C for 5 minutes.

[0098] As a result, a 2Kb foreign gdh DNA fragment encoding foreign glutamate dehydrogenase was amplified using the Corynebacterium glutamicum ATC13032 gdh promoter. The amplified product was purified using a PCR purification kit (QUIAGEN) and used as an insertion DNA fragment for vector construction. After treating the purified amplification product with the restriction enzyme smaI, the molar concentration (M) ratio of the pDCM2ΔN2872 vector to the amplified insert DNA fragment was adjusted to 1:2. Using the Infusion Cloning Kit (TaKaRa), the vectors pDCM2ΔN2872::Pn_gdh(c.gl), pDCM2ΔN2872::Pn_gdh(E.coli), pDCM2ΔN2872::Pn_rocG(B.su), pDCM2ΔN2872::Pn_gdh(rhodospirillales), and pDCM2ΔN2872::Pn_gdh(m.sm) were prepared by cloning according to the manual provided, using the Infusion Cloning Kit (TaKaRa) to introduce exogenous gdh onto the chromosome.

[0099] Example 2: Production of Corynebacterium strains capable of producing L-isoleucine Wild-type Corynebacterium glutamicum has the ability to produce L-isoleucine, but does not overproduce it. In contrast, to identify the genetic trait that increases L-isoleucine production capacity in accordance with the purpose of this application, we decided to use a strain with increased L-isoleucine production capacity.

[0100] First, an L-isoleucine-producing strain was developed from the wild-type Corynebacterium glutamicum ATCC13032. Specifically, to eliminate the feedback inhibition of threonine, a precursor of isoleucine, in the L-isoleucine biosynthesis pathway, the gene hom, which encodes homoserine dehydrogenase, was mutated, and the 407th amino acid of homoserine dehydrogenase, arginine, was replaced with histidine (Korean Patent Publication No. 10-1996769). Specifically, the polynucleotide sequence encoding hom(R407H) is shown in SEQ ID NO: 20.

[0101] Specifically, in order to produce strains into which the hom(R407H) mutation was introduced, PCR was performed using the chromosome of Corynebacterium glutamicum ATCC13032 as a template, with primers sequence numbers 21 and 22, or sequence numbers 23 and 24. The primer sequences used for each of the above PCRs are shown in Table 2 below.

[0102] [Table 2]

[0103] For the PCR reaction, PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used. The PCR conditions were: denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute. These denaturation, annealing, and polymerization reactions were repeated 28 times. As a result, 1000 bp DNA fragments were obtained from the upper 5' region and the lower 3' region, respectively, focusing on mutations in the hom gene.

[0104] PCR was performed using two types of amplified DNA sections as templates with primers SEQ ID NO: 21 and SEQ ID NO: 24. The PCR conditions were as follows: denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 2 minutes, repeated 28 times, followed by polymerization at 72°C for 5 minutes.

[0105] As a result, a 2kb DNA fragment containing a mutation in the hom gene encoding a homoserine dehydrogenase mutant in which the 407th arginine is replaced with histidine was amplified. The amplified product was purified using a PCR purification kit (QUIAGEN) and used as an insertion DNA fragment for vector construction. After treating the purified amplified product with the restriction enzyme smaI, the pDCM2 vector was heat-treated at 65°C for 20 minutes. The molar concentration (M) ratio of the pDCM2 vector to the aforementioned amplified insertion DNA fragment was adjusted to 1:2, and the vector pDCM2-R407H was constructed by cloning using an infusion cloning kit (TaKaRa) according to the provided manual to introduce the hom(R407H) mutation onto the chromosome.

[0106] The fabricated vector was transformed into Corynebacterium glutamicum ATCC13032 by electroporation, and a strain containing the hom(R407H) mutation on the chromosome was obtained through a secondary crossover process, which was named Corynebacterium glutamicum ATCC13032 hom(R407H).

[0107] To enhance the feedback release and activity of L-isoleucine in the fabricated ATCC13032 hom(R407H) strain, the ilvA gene encoding L-threonine dehydratase was mutated. The 381st amino acid (threonine) of L-threonine dehydratase (SEQ ID NO: 25) was replaced with alanine, and the 383rd amino acid (phenylalanine) was replaced with alanine. Additionally, a strain with ilvA(T381A+F383A) introduced was fabricated.

[0108] Specifically, in order to produce the bacterial strain into which the ilvA(T381A+F383A) mutation was introduced, PCR was performed using the chromosome of Corynebacterium glutamicum ATCC13032 as a template, with the primers of SEQ ID NOs. 26 and 27, or SEQ ID NOs. 28 and 29, respectively. The primer sequences used for each of the aforementioned PCRs are shown in Table 3 below.

[0109] [Table 3]

[0110] For the PCR reaction, PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used. 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. These denaturation, annealing, and polymerization reactions were repeated 28 times. As a result, a 1126 bp DNA fragment at the 5' upper end and a 286 bp DNA fragment at the 3' lower end were obtained, mainly due to mutations in the ilvA gene.

[0111] PCR was performed using two types of amplified DNA sections as templates with primers of SEQ ID NO: 26 and SEQ ID NO: 29. The PCR conditions were as follows: denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 2 minutes, repeated 28 times, followed by polymerization at 72°C for 5 minutes.

[0112] As a result, a 1.4kb DNA fragment containing a mutation in the ilvA gene encoding an L-threonine dehydratase mutant in which threonine at position 381 is replaced with alanine and phenylalanine at position 383 is replaced with alanine was amplified. The amplified product was purified using a PCR purification kit (QUIAGEN) and used as an insertion DNA fragment for vector construction. After treating the purified amplified product with the restriction enzyme smaI, the pDCM2 vector was heat-treated at 65°C for 20 minutes. The molar concentration (M) ratio of the pDCM2 vector to the aforementioned insertion DNA fragment was adjusted to 1:2, and the vector pDCM2-ilvA(T381A+F383A) was constructed by cloning using an infusion cloning kit (TaKaRa) according to the provided manual to introduce the ilvA(T381A+F383A) mutation onto the chromosome.

[0113] The prepared vector was transformed into Corynebacterium glutamicum ATCC13032 hom(R407H) by electroporation, and after a secondary crossover process, a strain containing the ilvA(T381A+F383A) mutation on the chromosome was obtained and named Corynebacterium glutamicum CA10-3101.

[0114] For reference, to confirm whether introducing the aforementioned ilvA(T381A+F383A) into an L-isoleucine-producing strain increases the efficiency of L-isoleucine production by releasing feedback and increasing activity for L-isoleucine, the following experiment was conducted. Specifically, the concentrations of L-isoleucine and L-threonine in the culture medium of the KCCM11248P / pECCG117-ilvA(T381A+F383A) strain, which was created by introducing the aforementioned ilvA(T381A+F383A) mutation into the KCCM11248P (KCCM11248P, Korean Registered Patent No. 10-1335789), an NTG (N-Methyl-N'-nitro-N-nitrosoguanidine)-treated L-isoleucine-producing strain KCCM11248P / pECCG117-ilvA(T381A+F383A) using the electropulse method, were measured and the results are shown in Table 4 below.

[0115] [Table 4]

[0116] As shown in Table 3 above, the KCCM11248P / pECCG117-ilvA(T381A+F383A) strain, into which the ilvA(T381A+F383A) mutation was introduced, showed a significant increase in L-isoleucine production and a higher L-threonine degradation rate compared to the KCCM11248P or KCCM11248P / pECCG117-ilvA(F383A) strains. In other words, it was confirmed that the ilvA(T381A+F383A) mutation was introduced to release feedback and increase activity for L-isoleucine, for the purposes of this application.

[0117] Example 3: Production of L-isoleucine strains into which foreign gdh has been introduced and evaluation of their isoleucine production capacity. The vector prepared in Example 1 was used to transform Corynebacterium glutamicum CA10-3101 prepared in Example 2 by electroporation. A strain with foreign gdh introduced on the chromosome was obtained through a secondary cross-reaction, and the presence or absence of introduction was confirmed via SEQ ID NO: 30 and SEQ ID NO: 31. The primer sequences used for the PCR are shown in the table below.

[0118] [Table 5]

[0119] The strain into which CA10-3101ΔN2872 was introduced was named CA10-3135, the strain into which CA10-3101ΔN2872::Pn_gdh(C.gl) was introduced was named CA10-3136, the strain into which CA10-3101ΔN2872::Pn_gdh(eco) was introduced was named CA10-3137, the strain into which CA10-3101ΔN2872::Pn_rocG(B.su) was introduced was named CA10-3138, the strain into which CA10-3101ΔN2872::Pn_gdh(rhodospirillales) was introduced was named CA10-3139, and the strain into which CA10-3101ΔN2872::Pn_gdh(m.sm) was introduced was named CA10-3140.

[0120] To confirm the increased L-isoleucine production and reduced by-product α-aminobutyric acid (AABA) of the six strains produced, the fermentation activity of each strain was evaluated using the following method. The parent strain and the mutant strains were inoculated into 250 ml corner baffle flasks containing 25 ml of isoleucine production medium, and then cultured at 32°C for 60 hours with shaking at 200 rpm to produce L-isoleucine.

[0121] The composition of the culture medium used in this example is as follows. <Production culture medium> Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, monopotassium phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, ferrous sulfate heptahydrate 10mg / l, manganese sulfate monohydrate 10mg / l, biotin 200μg / l, pH 7.2 After the culturing was complete, the production amounts of L-isoleucine and alpha-aminobutyric acid (AABA) were measured using high-performance liquid chromatography (HPLC), and the concentrations are shown in Table 6 below.

[0122] [Table 6]

[0123] As a result, as shown in Table 6 above, it was confirmed that L-isoleucine increased by 8.3% in CA10-3136 and CA10-3138, by 4.2% in CA10-3135, CA10-3137, and CA10-3139, and by 12.5% ​​in CA10-3140 compared to the parent strain. The by-product AABA increased by 100% in CA10-3135, 75% in CA10-3136, 112.5% ​​in CA10-3137, and 162.5% in CA10-3140 compared to the parent strain, while CA10-3138 remained the same as the parent strain, and CA10-3139 decreased by 12.5%.

[0124] This confirmed that, compared to the parent strain Corynebacterium glutamicum CA10-3101, the production of α-aminobutyric acid (AABA) decreased in the rocG(b.su) and gdh(rhodospirillales)-introduced strains (CA10-3138, CA10-3139) compared to the production of L-isoleucine. Furthermore, while the production of L-isoleucine was at a similar level compared to the Corynebacterium glutamicum ATC13032 gdh-introduced strain CA10-3135, the production of α-aminobutyric acid (AABA) decreased. Therefore, it was confirmed that when genes encoding glutamate dehydrogenase derived from rocG(b.su) and gdh(rhodospirillales) are introduced among various exogenous gdh genes, L-isoleucine production is maintained while by-products are reduced, and the fermentation purity of L-isoleucine is increased.

[0125] Example 4: Production and evaluation of an exogenous gdh-enhanced strain of Corynebacterium glutamicum KCCM11248P, a L-isoleucine-producing bacterial strain. rocG (derived from b. su) and gdh (derived from rhodospirillales), which were effective in increasing L-isoleucine production capacity and reducing by-products as confirmed in Example 3, were introduced by electropulse into the KCJI-38 (KCCM11248P, Korean Registered Patent No. 10-1335789) strain, an L-isoleucine-producing strain treated with NTG (N-Methyl-N'-nitro-N-nitrosoguanidine). The strain was then spread onto a selection medium containing 25 mg / L kanamycin for transformation, and a strain with introduced foreign gdh was obtained on the chromosome through a secondary crossover process. Subsequently, the concentrations of L-isoleucine and by-products in the culture medium were measured in the same manner as in Example 3, and the results are shown in Table 7 below.

[0126] [Table 7]

[0127] As shown in Table 7 above, it was confirmed that the byproduct AABA decreased in the rocG(b.su) and gdh(rhodospirillales) introduced strains compared to the parent strain KCCM11248P. Specifically, unlike the KCCM11248PΔN2872::Pn_gdh(C.gl) strain, in which both L-isoleucine and AABA concentrations increased, it was confirmed that the KCCM11248PΔN2872::Pn_rocG(B.su) and KCCM11248PΔN2872::Pn_gdh(rhodospirillales) strains maintained L-isoleucine productivity, decreased the byproduct AABA, and improved the fermentation purity of L-isoleucine.

[0128] This confirms that microorganisms into which the gene encoding foreign glutamate dehydrogenase derived from Bacillus subtilis or Rhodospirillales has been introduced can increase the purity of L-isoleucine and produce L-isoleucine in high yield.

[0129] From the above description, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are merely illustrative and not limiting. The scope of this application should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, as described below, and their equivalent concepts, rather than from the above detailed description.

Claims

1. A microorganism having the ability to produce L-isoleucine, into which a gene encoding foreign glutamate dehydrogenase derived from the order Rhodospirillales has been introduced, wherein the amount of α-aminobutyric acid (AABA) produced relative to the amount of L-isoleucine produced is reduced compared to the microorganism before the introduction of the foreign glutamate dehydrogenase gene, and the microorganism is Corynebacterium glutamicum.

2. The microorganism according to claim 1, wherein the gene encoding an exogenous glutamate dehydrogenase derived from the order Rhodospirillales has the nucleotide sequence of Sequence ID No.

4.

3. A method for producing L-isoleucine, comprising the step of culturing a microorganism capable of producing L-isoleucine into a culture medium, into which a gene encoding foreign glutamate dehydrogenase derived from Bacillus subtilis or Rhodospirillales has been introduced, wherein the amount of α-aminobutyric acid (AABA) produced relative to the amount of L-isoleucine produced is reduced compared to the case of the microorganism before the introduction of the foreign glutamate dehydrogenase gene, wherein the microorganism is Corynebacterium glutamicum.

4. The method according to claim 3, further comprising the step of recovering L-isoleucine from the cultured microorganism or culture medium.

5. The method according to claim 3, wherein the gene encoding foreign glutamate dehydrogenase from Bacillus subtilis and the gene encoding foreign glutamate dehydrogenase from the Rhodospirillales order each have the nucleotide sequences of SEQ ID NOs: 2 and 4, respectively.

6. A composition for producing L-isoleucine, comprising a microorganism having the ability to produce L-isoleucine into which a gene encoding foreign glutamate dehydrogenase derived from Bacillus subtilis or Rhodospirillales has been introduced, wherein the amount of α-aminobutyric acid (AABA) produced relative to the amount of L-isoleucine produced is reduced compared to the case of the microorganism before the introduction of the foreign glutamate dehydrogenase gene, wherein the microorganism is Corynebacterium glutamicum.

7. The composition according to claim 6, wherein the gene encoding foreign glutamate dehydrogenase from Bacillus subtilis and the gene encoding foreign glutamate dehydrogenase from the Rhodospirillales order each have the nucleotide sequences of SEQ ID NOs: 2 and 4, respectively.

Images