L-isoleucine producing microorganism and method 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] 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. [Background technology]

[0002] L-isoleucine is one of the branched-chain amino acids out of a total of 20 amino acids, and is classified as an essential amino acid and is used in animal feed, food additives, and pharmaceutical fields. L-isoleucine is increasingly used not only in infusions, nutritional supplements, and sports nutritional supplements, but also in animal feed, as it has functions such as energy production after metabolism, hemoglobin production, blood sugar regulation, and muscle production and repair.

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

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

[0005] In this regard, a method for purifying L-isoleucine is known to increase the purity of L-isoleucine (Patent Document 2), but this purification method has the disadvantage of requiring an additional purification process, and there is currently a need to develop a method for increasing the purity of L-isoleucine.

[0006] Meanwhile, L-glutamic acid is a precursor that provides an amine group in the synthesis of amino acids including L-isoleucine. In the production of amino acids, it is essential to enhance the synthesis of L-glutamic acid, which is a precursor. However, when glutamate dehydrogenase, which is known to produce glutamic acid in microorganisms that produce L-isoleucine, is enhanced, a side reaction occurs in which α-aminobutyric acid (AABA) is produced as a by-product through the dehydrogenation of 2-ketobutyrate, an intermediate of isoleucine (Non-Patent Document 1), resulting in a problem of reduced purity and biosynthesis efficiency of L-isoleucine. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 10113190 [Patent Document 2] U.S. Patent No. 6,072,083 [Patent Document 3] U.S. Patent No. 7,662,943 [Patent Document 4] U.S. Pat. No. 1,058,438 [Patent Document 5] U.S. Pat. No. 10,273,491 [Patent Document 6] Korean Patent Publication No. 10-2020-0136813 [Patent Document 7] Korean Patent No. 10-1996769

Patent Document 8

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

[0009] The present inventors have developed 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, and have completed the present application. [Means for solving the problem]

[0010] One object of the present application is to provide 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.

[0011] Another object of the present application is to provide a method for producing L-isoleucine, comprising culturing said microorganism in a medium. Another object of the present application is to provide a composition for producing L-isoleucine, comprising the microorganism. Effect of the Invention

[0012] The microorganism having L-isoleucine producing ability into which a gene encoding an exogenous glutamate dehydrogenase of the present application has been introduced can produce L-isoleucine in high yield with little by-product production, and can be usefully utilized for industrial production of L-isoleucine. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in this application may be applied to each different description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. Also, the following specific description is not considered to limit the category of this application.

[0014] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein, and such equivalents are intended to be encompassed by this application.

[0015] One aspect of the present 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 corresponds to a branched-chain amino acid along with L-valine and L-leucine.

[0017] In the present application, the term "strain (or microorganism)" includes all wild-type microorganisms and microorganisms that have been genetically modified, either naturally or artificially, in which a specific mechanism has been weakened or enhanced by, for example, inserting an exogenous gene or enhancing or inactivating the activity of an endogenous gene, and may also be a microorganism that contains a genetic modification for the production of a desired polypeptide, protein, or product.

[0018] In the present application, the term "microorganism capable of producing L-isoleucine" refers to a microorganism naturally having the ability to produce L-isoleucine or a microorganism in which the ability to produce L-isoleucine has been imparted to a parent strain that does not have the ability to produce L-isoleucine. Specifically, the microorganism may be, but is not limited to, 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.

[0019] Specifically, the "microorganism capable of producing L-isoleucine" includes all wild-type microorganisms and microorganisms genetically modified naturally or artificially. More specifically, it may be a microorganism in which a specific mechanism is weakened or enhanced by inserting an exogenous gene or enhancing or inactivating the activity of an endogenous gene, and may be a microorganism in which a genetic mutation has occurred or L-isoleucine production activity has been enhanced for the production of the desired L-isoleucine. For the purposes of this application, the microorganism capable of producing L-isoleucine may be a genetically modified microorganism or a recombinant microorganism, characterized in that a gene encoding an exogenous glutamate dehydrogenase derived from Bacillus subtilis or Rhodospirillales has been introduced to increase the ability to produce the desired L-isoleucine, but is not limited thereto.

[0020] In this application, the term "introduction" of an activity means that a gene not originally possessed by a microorganism is expressed in the microorganism to exhibit a specific protein activity, or that the protein exhibits an increased or improved activity compared to its endogenous activity or the activity before modification. For example, it may mean that a polynucleotide encoding a specific protein is introduced into a chromosome in a microorganism, or a vector containing a polynucleotide encoding a specific protein is introduced into a microorganism, and the activity is exhibited.

[0021] As an example, the recombinant strain having increased production ability may have an L-isoleucine production ability increased by about 1% or more, specifically, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 8.1% or more, about 8.2% or more, or about 8.3% or more (there is no particular limit on the upper limit, and it may be, for example, about 100% or less, about 50% or less, about 25% or less, about 20% or less, about 15% or less, or about 10% or less), compared to the L-isoleucine production ability of the parent strain before mutation or a non-modified microorganism having the endogenous activity of the gdh protein, but is not limited thereto as long as there is an increase in the + value compared to the production ability of the parent strain or non-modified microorganism before mutation. In other examples, the recombinant strain with increased production ability may have an L-isoleucine production ability increased by about 1.01-fold or more, about 1.02-fold or more, about 1.03-fold or more, about 1.04-fold or more, about 1.05-fold or more, about 1.06-fold or more, about 1.07-fold or more, or about 1.08-fold or more (there is no particular upper limit, and the increase may be, for example, about 10-fold or less, about 5-fold or less, about 3-fold or less, about 2-fold or less, about 1.5-fold or less, or about 1.1-fold or less) compared to the parent strain or non-modified microorganism before mutation, but is not limited thereto.

[0022] In the present application, the term "non-modified microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, but refers to a wild-type or naturally occurring strain itself, or a strain before its characteristics are changed due to genetic mutations caused by natural or artificial factors. For example, the non-modified microorganism refers to a strain before an exogenous glutamate dehydrogenase gene described herein is introduced. The term "non-modified microorganism" may be used interchangeably with "pre-modified strain," "pre-modified microorganism," "non-mutated strain," "non-modified strain," "non-mutated microorganism," or "reference microorganism."

[0023] In yet another embodiment of the present application, the microorganism of the present application may be a microorganism capable of producing L-isoleucine, and is not particularly limited by its type. The microorganism of the present application may be either a prokaryotic cell or a eukaryotic cell, and specifically may be a prokaryotic cell. The prokaryotic cell may include, for example, a microbial strain belonging to the genera Corynebacterium, Escherichia, Erwinia, Serratia, Providencia, and Brevibacterium, and specifically may be a microorganism of the genus Corynebacterium.

[0024] In the present application, the term "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, 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 glutamic acid, which is a precursor of L-isoleucine biosynthesis, and in this application, the term "glutamate dehydrogenase" may be used interchangeably with "gdh" and "rocG".

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

[0027] Meanwhile, L-glutamic acid is a precursor that provides an amine group in the synthesis of amino acids including L-isoleucine. In the production of amino acids, it is essential to enhance the synthesis of L-glutamic acid, which is a precursor. However, when glutamate dehydrogenase, which is known to produce glutamic acid from microorganisms that produce L-isoleucine, is enhanced, a side reaction occurs in which α-aminobutyric acid (AABA) is produced as a by-product through the dehydrogenation of 2-ketobutyrate, an intermediate of isoleucine (Microb Cell Fact. 2017 Mar23; 16(1): 51), which results in a problem of reduced purity and biosynthesis efficiency of L-isoleucine.

[0028] The microorganism capable of producing L-isoleucine of the present application is introduced with a gene encoding an exogenous glutamate dehydrogenase derived from Bacillus subtilis or Rhodospirillales, thereby reducing the amount of by-product produced. The by-product may be α-aminobutyric acid (AABA). The reduction in the amount of by-product produced means, but is not limited to, a reduction in the amount of α-aminobutyric acid (AABA) produced relative to the amount of L-isoleucine produced, compared to a wild-type microorganism.

[0029] For purposes of this application, the protein having glutamate dehydrogenase activity may be from Bacillus subtilis or the order Rhodospirillales. Specifically, the glutamate dehydrogenase may have and / or comprise, or essentially consist of, the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:3.

[0030] Furthermore, the glutamate dehydrogenase of the present application may include an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3. It is also obvious that glutamate dehydrogenase having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted or added is also included within the scope of the present application, so long as the amino acid sequence has such homology or identity and exhibits an efficacy corresponding to the glutamate dehydrogenase of the present application.

[0031] Even if the present application describes "a polypeptide or protein comprising an amino acid sequence set forth in a specific SEQ ID NO," "a polypeptide or protein consisting of an amino acid sequence set forth in a specific SEQ ID NO," or "a polypeptide or protein having an amino acid sequence set forth in a specific SEQ ID NO," it is self-evident that the present application also uses a protein having an amino acid sequence in which a part of the sequence has been deleted, modified, substituted, conservatively substituted, or added, so long as it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of the SEQ ID NO. For example, the N-terminus and / or C-terminus of the amino acid sequence may have an addition of a sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation, or a conservative substitution.

[0032] For example, the amino acid sequence may have an addition or deletion of a sequence at the N-terminus, C-terminus and / or within the amino acid sequence that does not change the function of the glutamate dehydrogenase of the present application, a naturally occurring mutation, a silent mutation or a conservative substitution.

[0033] In this application, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions generally occur on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartate; aromatic amino acids include phenylalanine, tryptophan, and tyrosine, and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Amino acids can also be classified as those with electrically charged side chains and those with uncharged side chains, with the charged side chains including aspartic acid, glutamic acid, lysine, arginine, and histidine, and the uncharged side chains again can be classified as nonpolar or polar, with the nonpolar amino acids including glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, and the polar amino acids including serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Conservative substitutions usually have little or no effect on the activity of the resulting polypeptide. Conservative substitutions usually have little or no effect on the activity of the protein or polypeptide.

[0034] The glutamate dehydrogenase can also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide can be conjugated to a signal (or leader) sequence at the N-terminus of a protein that is involved in co-translationally or post-translationally protein transfer. The polypeptide can also be conjugated to other sequences or linkers that allow the polypeptide to be identified, purified, or synthesized.

[0035] In this application, the term "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, which can be expressed as a percentage. The terms homology and identity can often be used interchangeably.

[0036] Homology or identity of conserved polynucleotide or polypeptide sequences can be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences can generally hybridize to all or a portion of the sequence under moderate or high stringent conditions. Hybridization obviously includes hybridization to polynucleotides containing common codons in polynucleotides or codons that take into account codon degeneracy.

[0037] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms, such as the "FASTA" program using default parameters, for example, as in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as implemented 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 versions) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48:1073. For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0038] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, e.g., Needleman et al. (1970), J Mol Biol. 48: 443, as known in, e.g., 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 the two sequences divided by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include: (1) a binary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (the 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 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.

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

[0040] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, based on which each amino acid residue in the amino acid sequence is numbered with reference to the numeric position of the corresponding amino acid residue in SEQ ID NO: 1. For example, sequence alignment algorithms such as those described in this application can identify the position of an amino acid relative to a query sequence (also referred to as a "reference sequence"), or the position at which a modification, such as a substitution, insertion or deletion, occurs.

[0041] For such alignment, 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), etc. can be used, but are not limited thereto, and sequence alignment programs, pairwise sequence comparison algorithms, etc. known in the art can be appropriately used.

[0042] In the present application, the term "intrinsic" activity refers to the activity state of a protein that a microorganism originally has in its natural state or before the protein is modified. This term may be used interchangeably with "activity before modification."

[0043] In the present application, the term "enhancement" of a polypeptide activity means that the activity of the polypeptide is increased compared to the endogenous activity. The term "enhancement" may be used interchangeably with terms such as "activation," "up-regulation," "overexpression," and "increase." Here, the terms "activation," "enhancement," "up-regulation," "overexpression," and "increase" may all include the display of an activity that was not originally possessed, or the display of an activity that is improved compared to the endogenous activity or the activity before the transformation. The term "endogenous activity" refers to the activity of a specific polypeptide that was originally possessed by a parent strain or a non-transformed microorganism before the transformation, in the case where the trait is changed due to a genetic mutation caused by natural or artificial factors. This term may be used interchangeably with "activity before the transformation." The term "enhancement," "up-regulation," "overexpression," or "increase" of a polypeptide activity compared to the endogenous activity means that the activity and / or concentration (expression level) of a specific polypeptide is improved compared to the activity and / or concentration (expression level) of a specific polypeptide that was originally possessed by a parent strain or a non-transformed microorganism before the transformation.

[0044] The enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. The enhancement of the activity of the polypeptide can be confirmed by an increase in the activity, expression level, or amount of a product secreted from the polypeptide.

[0045] The activity of the polypeptide can be enhanced by various methods well known in the art, and is not limited as long as the activity of the target polypeptide can be enhanced compared to that of the microorganism before transformation. Specifically, the activity of the polypeptide can be enhanced by using genetic engineering and / or protein engineering, which are routine methods in molecular biology and are well known to those skilled in the art, but is not limited thereto (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 enhancement of the polypeptide of the present application is 1) increasing the intracellular copy number of a polynucleotide encoding a polypeptide; 2) modification of the expression regulatory region of the gene on the chromosome encoding the polypeptide (e.g., mutation in the expression regulatory region, replacement with a sequence having stronger activity, or insertion of a sequence having stronger activity); 3) a modification of the nucleotide sequence encoding the initiation codon or 5'UTR region of a gene transcript encoding a polypeptide; 4) modifying the amino acid sequence of the polypeptide so that the activity of the polypeptide is enhanced; 5) modifying a polynucleotide sequence encoding a polypeptide so as to enhance the activity of the polypeptide (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the activity of the polypeptide); 6) introduction of a foreign polypeptide exhibiting a polypeptide activity or a foreign polynucleotide encoding the same; 7) codon optimization of a polynucleotide encoding a polypeptide; 8) analyzing the tertiary structure of a polypeptide and selectively altering or chemically modifying exposed sites; or 9) It may be a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0047] More specifically, The 1) increase in the intracellular copy number of a polynucleotide encoding a polypeptide may be achieved by introducing into a host cell a vector to which a polynucleotide encoding the polypeptide is operably linked, the vector being capable of replicating and functioning independently of a host. Alternatively, the increase may be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide into a chromosome in the host cell. The introduction into a chromosome is achieved by introducing into the host cell a vector that inserts the polynucleotide into a chromosome in the host cell, but is not limited thereto. The vector is as described above.

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

[0049] Examples of known strong promoters include, but are not limited to, the CJ1 to CJ7 promoters (U.S. Pat. No. 7,662,943), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (U.S. Pat. No. 10,584,338), O2 promoter (U.S. Pat. No. 10,273,491), tkt promoter, and yccA promoter.

[0050] The modification of the base sequence encoding the initiation codon or 5'UTR region of the gene transcript encoding the polypeptide may be, for example, a replacement with a base sequence encoding another initiation codon that has a higher polypeptide expression rate than the endogenous initiation codon, but is not limited thereto.

[0051] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) may be, but is not limited to, a sequence mutation caused by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide, or an amino acid sequence or polynucleotide sequence improved to have stronger activity or an amino acid sequence or polynucleotide sequence improved to increase activity. Specifically, the replacement may be performed by inserting the polynucleotide into a chromosome by homologous recombination, but is not limited to this. The vector used in this case may further include a selection marker for checking the presence or absence of insertion into the chromosome. The selection marker is as described above.

[0052] The introduction of a foreign polynucleotide exhibiting the activity of the polypeptide 6) may be the introduction into a host cell of a foreign polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide. The foreign polynucleotide is not limited in origin or sequence as long as it exhibits the same / similar activity as the polypeptide. The method used for the introduction is a publicly known transformation method appropriately selected by a person skilled in the art, and the introduced polynucleotide is expressed in the host cell to produce a polypeptide and increase its activity.

[0053] The codon optimization of the polynucleotide encoding the polypeptide (7) may be codon optimization of an endogenous polynucleotide so as to increase transcription or translation in a host cell, or may be codon optimization of an exogenous polynucleotide so as to optimize transcription or translation in a host cell.

[0054] The above 8) analyzing the tertiary structure of a polypeptide and selecting and modifying or chemically modifying exposed sites may involve, for example, comparing sequence information of a polypeptide to be analyzed with a database storing sequence information of known proteins, determining candidates for template proteins according to the degree of sequence similarity, confirming the structure based on the candidates, and selecting and modifying exposed sites to be modified or chemically modified.

[0055] Such an enhancement of polypeptide activity may be, but is not limited to, an increase in the activity or expression level of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in a wild-type or untransformed microbial strain, or an increase in the amount of a product produced from the polypeptide.

[0056] Specifically, for purposes of this application, the microorganism can be introduced with an exogenous polynucleotide having activity of glutamate dehydrogenase from Bacillus subtilis or Rhodospirillales to enhance the protein activity of glutamate dehydrogenase.

[0057] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and a DNA or RNA chain of a certain length or more, and more specifically, refers to a polynucleotide fragment encoding the mutant.

[0058] The polynucleotide according to the present application is characterized in that it is derived from Bacillus subtilis or Rhodospirillales, and specifically, the polynucleotide may include a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and more specifically, the gene encoding the foreign glutamate dehydrogenase derived from Bacillus subtilis and the gene encoding the foreign glutamate dehydrogenase derived from Rhodospirillales may have the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 4, respectively, but are not limited thereto. The nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 can be obtained from a known database, such as, but not limited to, GenBank of NCBI.

[0059] In the present 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 polynucleotide of the present application may be modified in various ways in the coding region without changing the amino acid sequence of the variant of the present application, taking into consideration the degeneracy of codons or the codons preferred in an organism in which the variant of the present application is to be expressed. Specifically, the polynucleotide of the present application may have or include a base sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity to the sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or may consist of or essentially consist of a base sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity to the sequence of SEQ ID NO: 2 or SEQ ID NO: 4, but is not limited thereto.

[0061] Furthermore, the polynucleotide of the present application may include, without limitation, a sequence that can hybridize under stringent conditions with a probe prepared from a known gene sequence, for example, a sequence complementary to the entire or partial polynucleotide sequence of the present application. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, conditions include conditions under which polynucleotides having high homology or identity, such as polynucleotides having a homology or identity 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, hybridize with each other, but polynucleotides having lower homology or identity do not hybridize with each other, or conditions under which washing is performed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions for normal Southern hybridization, such as 60°C, 1 x SSC, 0.1% SDS, specifically 60°C, 0.1 x SSC, 0.1% SDS, more specifically 68°C, 0.1 x SSC, 0.1% SDS.

[0062] Hybridization requires that two nucleic acids have complementary sequences, even though mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to one another. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Thus, the polynucleotides of the present application can also include isolated nucleic acid fragments that are complementary to an entire sequence, as well as substantially similar nucleic acid sequences.

[0063] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the above-mentioned hybridization conditions including a hybridization step at a Tm value of 55° C. The Tm value may be 60° C., 63° C., or 65° C., but is not limited thereto, and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0064] The appropriate stringency for hybridizing such polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables well known in the art (eg, J. Sambrook et al., supra).

[0065] For the purposes of this application, the microorganism may include an expression vector for expressing the exogenous polynucleotide in a host, thereby enhancing the protein activity of the glutamate dehydrogenase compared to the endogenous activity, but is not limited thereto.

[0066] The vector of the present application can comprise a DNA construct comprising a base sequence of a polynucleotide encoding a polypeptide of interest operably linked to an expression control region (or expression control sequence) suitable for expressing said polypeptide of interest in a suitable host. The expression control region can comprise a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After being transformed into a suitable host cell, the vector can replicate or function independently of the host genome, or can be integrated into the genome itself.

[0067] The vector used in the present application is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a 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 series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, pSK series, pSKH series, and pET series 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] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome through a vector for chromosomal insertion in a cell. The polynucleotide can be inserted into a chromosome by any method known in the art, for example, but not limited to, homologous recombination. A selection marker for checking the presence or absence of insertion into the chromosome may be further included. The selection marker is used to select cells transformed with the vector, i.e., to check the presence or absence of insertion of a target nucleic acid molecule, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to a cytotoxic agent, or expression of a surface polypeptide is used. In an environment treated with a selective agent, only cells expressing the selection marker survive or show other phenotypes, so that transformed cells can be selected.

[0069] In the present application, the term "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or a microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include all of the above, regardless of whether it is inserted into the chromosome of the host cell or located extrachromosomally, as long as it can be expressed in the host cell. The polynucleotide may also include DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic structure containing all elements necessary for autonomous expression. The expression cassette may usually 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 be in the form of an expression vector capable of self-replicating. The polynucleotide may also be introduced into the host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0070] In addition, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target mutant of the present application.

[0071] In the microorganism of the present application, modification of a part or the whole of a polynucleotide may be induced by, but is not limited to, (a) homologous recombination using a vector for chromosomal insertion in the microorganism or genome editing using engineered nuclease (e.g., CRISPR-Cas9) and / or (b) light and / or chemical treatment such as ultraviolet light and radiation. A method for modifying a part or the whole of the gene may include a method using DNA recombination technology. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene is injected into the microorganism to cause homologous recombination, thereby deleting a part or the whole of the gene. The injected nucleotide sequence or vector may contain a dominant selection marker, but is not limited to this.

[0072] Meanwhile, in a specific embodiment of the present application, the Corynebacterium microorganism may further comprise an enzyme involved in the L-isoleucine biosynthetic pathway with enhanced activity. In the present application, the term "enzyme involved in the L-isoleucine biosynthetic 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 the like.

[0073] Another aspect of the present application provides a method for producing L-isoleucine, comprising culturing the microorganism in a medium. The microorganism and L-isoleucine are as described above.

[0074] Specifically, the gene encoding the foreign glutamate dehydrogenase derived from Bacillus subtilis and the gene encoding the foreign glutamate dehydrogenase derived from Rhodospirillales may have the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 4, respectively, but are not limited thereto.

[0075] Specifically, the method may involve, but is not limited to, decreasing the amount of α-aminobutyric acid (AABA) produced relative to the amount of L-isoleucine produced.

[0076] In the present application, the term "culture" means growing the microorganism of the present application under appropriately controlled environmental conditions. The culture process of the present application is carried out according to appropriate 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, but is not limited to, a batch, continuous, or fed-batch type.

[0077] In the present application, the term "culture medium" refers to a substance that is a mixture of nutrients required for culturing the microorganism of the present application as the main component, and supplies nutrients and growth factors, including water essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the microorganism of the present application may be any medium used for culturing ordinary microorganisms without any particular limitation, and the microorganism of the present application may be cultured under aerobic conditions by adjusting the temperature, pH, etc. in an ordinary culture medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins, etc.

[0078] In the present application, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid may be used, specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and other appropriate amounts of carbon sources may be used in a variety of ways without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited thereto.

[0079] Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more kinds, and are not limited thereto.

[0080] The phosphorus source may include potassium monophosphate, potassium diphosphate, or a corresponding sodium-containing salt. The inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and may further include amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the medium in a batch or continuous manner. However, the present invention is not limited thereto.

[0081] In the present application, during the cultivation of microorganisms, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. may be added to the culture in an appropriate manner to adjust the pH of the culture. Also, during the cultivation, foam generation may be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. Also, to maintain the aerobic state of the culture, oxygen or an oxygen-containing gas may be injected into the culture, or to maintain anaerobic and microaerobic states, no gas may be injected, or nitrogen, hydrogen, or carbon dioxide gas may be injected, but is not limited thereto.

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

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

[0084] The method for producing L-isoleucine of the present application may further include a step of recovering L-isoleucine from the culture medium (the medium in which the culture was carried out) or the microorganism. The recovery step may be further included after the culture step.

[0085] The recovery may be a collection of the target L-isoleucine using a suitable method known in the art, such as the method for culturing the microorganism of the present application, for example, a batch, continuous or fed-batch culture method, etc. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographies such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination of these methods may be used, and the target L-isoleucine can be recovered from the medium or the microorganism using a suitable method known in the art.

[0086] In addition, the L-isoleucine production method of the present application may additionally include a purification step. The purification may be performed using a suitable method known in the art. In one example, when the L-isoleucine production method of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the procedure, or may be performed simultaneously or integrated into one step, but is not limited thereto.

[0087] Another aspect of the present application provides a composition for producing L-isoleucine, comprising the microorganism. The microorganism and L-isoleucine are as described above.

[0088] Specifically, the gene encoding the foreign glutamate dehydrogenase derived from Bacillus subtilis and the gene encoding the foreign glutamate dehydrogenase derived from Rhodospirillales may have the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 4, respectively, but are not limited thereto.

[0089] Specifically, the method may involve, but is not limited to, decreasing the amount of α-aminobutyric acid (AABA) produced relative to the amount of L-isoleucine produced.

[0090] The composition of the present application may further contain any suitable excipient that is commonly used in compositions for producing L-isoleucine, such excipients may be, for example, but are not limited to, a preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer, or isotonicity agent.

[0091] Another aspect of the present application is to provide a use of a microorganism having an ability to produce L-isoleucine, into which a gene encoding an exogenous glutamate dehydrogenase derived from Bacillus subtilis or Rhodospirillales of the present application has been introduced, for producing L-isoleucine. EXAMPLES

[0092] The present application will be described in more detail with reference to the following examples. However, these examples are merely for illustrative purposes and are not intended to limit the scope of the present application.

[0093] Example 1: Construction of a recombinant vector for introducing foreign glutamate dehydrogenase To insert a foreign gdh overexpression vector into the Corynebacterium glutamicum chromosome, NCgl2872, known as a gene encoding a transposon in Corynebacterium glutamicum, was used as an insertion site (Journal of Biotechnology 104, 5-25 Jorn Kalinowski et al., 2003). To replace the NCgl2872 gene with foreign gdh, NCgl2872 deletion and target gene insertion vectors were constructed. To construct the vectors, PCR was performed using the chromosome of ATCC13032 as a template and the primer pairs of SEQ ID NO:5 and SEQ ID NO:6, and SEQ ID NO:7 and SEQ ID NO:8, respectively. The polymerase used for the PCR reaction was PfuUltraTM high fidelity DNA polymerase (Stratagene). The PCR conditions were denaturation at 95°C for 30 seconds; denaturation at 55°C for 30 seconds; and polymerization at 72°C for 1 minute. The denaturation, annealing, and polymerization reactions under these conditions 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 (QIAGEN), and heat-treated pDCM2 vector (Korean Patent Publication No. 10-2020-0136813) and an infusion cloning kit (TaKaRa) were used to clone the vector pDCM2ΔN2872 for NCgl2872 deletion and target gene insertion according to the manual provided.

[0094] In order to prepare a strain into which an exogenous gdh having a gdh promoter of the parent strain Corynebacterium glutamicum was introduced, PCR was performed using the chromosomes of Corynebacterium glutamicum ATC13032, E. coli, Bacillus subtilis, Rhodospirillales, and Mycobacterium smegmatis as templates, respectively, and the primers SEQ ID NO: 9 and SEQ ID NO: 10; or SEQ ID NO: 9 and SEQ ID NO: 11; or SEQ ID NO: 12 and SEQ ID NO: 13; or SEQ ID NO: 14 and SEQ ID NO: 15; or SEQ ID NO: 16 and SEQ ID NO: 17; or SEQ ID NO: 18 and SEQ ID NO: 19; respectively. The primer sequences used for each PCR are shown in Table 1 below.

[0095] [Table 1]

[0096] The polymerase used for PCR was PfuUltraTM high fidelity DNA polymerase (Stratagene), 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 conditions were repeated 28 times. As a result, a 519 bp DNA fragment of the gdh promoter site, a 1882 bp DNA fragment of the promoter-containing Corynebacterium glutamicum ATC13032 gdh site, a 1382 bp DNA fragment of the E. coli gdh site, a 1313 bp DNA fragment of the Bacillus subtilis gdh(rocG) site, a 1424 bp DNA fragment of the Rhodospirillales gdh site, and a 1388 bp DNA fragment of the Mychobacterium smegmatis gdh site were obtained, respectively.

[0097] Using the amplified promoter and exogenous gdh DNA fragment as templates, PCR was performed 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 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 2 minutes, followed by polymerization reaction at 72°C for 5 minutes.

[0098] As a result, a 2 Kb foreign gdh DNA fragment encoding foreign glutamate dehydrogenase was amplified with the Corynebacterium glutamicum ATC13032 gdh promoter. The amplified product was purified using a PCR Purification kit (QIAGEN) and used as an insert DNA fragment for vector construction. The purified amplified product was treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes so that the molar concentration (M) ratio of the pDCM2ΔN2872 vector to the amplified product, the inserted DNA fragment, was 1:2. 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 produced for introducing the exogenous gdh into the chromosome by cloning according to the manual provided with the Infusion Cloning Kit (TaKaRa).

[0099] Example 2: Construction of Corynebacterium sp. strain capable of producing L-isoleucine Wild-type Corynebacterium glutamicum has the ability to produce L-isoleucine but does not overproduce it. In response to this, we decided to use a strain with increased L-isoleucine production ability to confirm genetic traits that increase L-isoleucine production ability according to the purpose of this application.

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

[0101] Specifically, to prepare a strain having the hom(R407H) mutation, PCR was performed using the chromosome of Corynebacterium glutamicum ATCC13032 as a template and primers of SEQ ID NO: 21 and SEQ ID NO: 22, or SEQ ID NO: 23 and SEQ ID NO: 24. The primer sequences used for each PCR are shown in Table 2 below.

[0102] [Table 2]

[0103] The polymerase used for the PCR reaction was PfuUltraTM high fidelity DNA polymerase (Stratagene), and 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, and these denaturation, annealing, and polymerization reactions were repeated 28 times. As a result, a 1000 bp DNA fragment at the 5' upper site and a 1000 bp DNA fragment at the 3' lower site were obtained, centered on the mutation of the hom gene.

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

[0105] As a result, a 2 kb DNA fragment containing a mutation in the hom gene encoding a homoserine dehydrogenase mutant in which arginine at position 407 was replaced with histidine was amplified. The amplified product was purified using a PCR Purification kit (QIAGEN) and used as an insert DNA fragment for vector construction. The purified amplified product was treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes so that the molar concentration (M) ratio of the amplified product, the insert DNA fragment, was 1:2. The vector pDCM2-R407H for introducing the hom (R407H) mutation into the chromosome was constructed by cloning using an Infusion Cloning Kit (TaKaRa) according to the manual provided.

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

[0107] In order to increase the feedback release and activity of L-isoleucine in the ATCC13032 hom (R407H) strain, the ilvA gene encoding L-threonine dehydratase was mutated to replace the 381st amino acid threonine with alanine and the 383rd amino acid phenylalanine with alanine in L-threonine dehydratase (SEQ ID NO: 25). In addition, a strain into which ilvA (T381A + F383A) was introduced was prepared.

[0108] Specifically, to prepare a strain having the ilvA(T381A+F383A) mutation, PCR was performed using the chromosome of Corynebacterium glutamicum ATCC13032 as a template and primers of SEQ ID NO: 26 and SEQ ID NO: 27, or SEQ ID NO: 28 and SEQ ID NO: 29. The primer sequences used for each PCR are shown in Table 3 below.

[0109] [Table 3]

[0110] The polymerase used for the PCR reaction was PfuUltraTM high fidelity DNA polymerase (Stratagene), 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. The denaturation, annealing, and polymerization reactions under these conditions 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, each of which was centered on the mutation in the ilvA gene.

[0111] Using the two types of amplified DNA fragments as templates, PCR was performed 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, followed by 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 2 minutes, followed by polymerization reaction at 72°C for 5 minutes.

[0112] As a result, a 1.4 kb DNA fragment containing a mutation in the ilvA gene encoding an L-threonine dehydratase mutant in which the 381st threonine was replaced by alanine and the 383rd phenylalanine was replaced by alanine was amplified. The amplified product was purified using a PCR Purification kit (QIAGEN) and used as an insert DNA fragment for vector construction. The purified amplified product was treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes so that the molar concentration (M) ratio of the amplified product, the insert DNA fragment, was 1:2. The vector pDCM2-ilvA(T381A+F383A) for introducing the ilvA(T381A+F383A) mutation into the chromosome was constructed by cloning using an Infusion Cloning Kit (TaKaRa) according to the manual provided.

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

[0114] For reference, the following experiment was carried out to confirm whether the introduction of ilvA(T381A+F383A) into an L-isoleucine producing strain increases the productivity of L-isoleucine by releasing the feedback loop and increasing the activity of L-isoleucine. Specifically, the ilvA(T381A+F383A) mutation was introduced into KCJI-38 (KCCM11248P, Korean Patent No. 10-1335789), an L-isoleucine producing strain treated with NTG (N-Methyl-N'-nitro-N-nitrosoguanidine), by electric pulse method, and the L-isoleucine and L-threonine concentrations in the culture medium of KCCM11248P / pECCG117-ilvA(T381A+F383A) strain were measured. The results are shown in Table 4 below.

[0115] [Table 4]

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

[0117] Example 3: Construction of L-isoleucine strains with exogenous gdh and evaluation of isoleucine production ability The vector prepared in Example 1 was transformed into the Corynebacterium glutamicum CA10-3101 prepared in Example 2 by electroporation, and a strain in which an exogenous gdh was introduced on the chromosome was obtained through a secondary crossover process, and the presence or absence of the exogenous gdh was confirmed by SEQ ID NO: 30 and SEQ ID NO: 31. The primer sequences used for the PCR are shown in the following table.

[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] In order to confirm the effect of increasing L-isoleucine productivity and reducing the by-product α-aminobutyric acid (AABA) of the six strains prepared, the fermentation titer of each strain was evaluated as follows: The parent strain and the mutant strain were inoculated into a 250 ml corner baffle flask 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 production medium used in this example is as follows. <Production medium> Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, potassium phosphate monobasic 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 cultivation was completed, the amounts of L-isoleucine and alpha-aminobutyric acid (AABA) produced 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, it was confirmed that L-isoleucine was increased by 8.3% in CA10-3136 and CA10-3138, 4.2% in CA10-3135, CA10-3137, and CA10-3139, and 12.5% ​​in CA10-3140 compared to the parent strain. It was confirmed that the by-product AABA was 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 was the same as the parent strain and CA10-3139 was decreased by 12.5%.

[0124] This confirmed that the amount of α-aminobutyric acid (AABA) produced was reduced compared to the amount of L-isoleucine produced in the rocG(b.su) and gdh(rhodospirillales)-introduced strains (CA10-3138, CA10-3139) compared to the parent strain Corynebacterium glutamicum CA10-3101. In addition, it was confirmed that the amount of L-isoleucine produced was at the same level as that of the Corynebacterium glutamicum ATC13032 gdh-introduced strain CA10-3135, but the amount of α-aminobutyric acid (AABA) produced was reduced. Therefore, it was confirmed that when the genes encoding glutamate dehydrogenase derived from rocG(b.su) and gdh(rhodospirillales) were introduced among various foreign gdh genes, the production of L-isoleucine was maintained while by-products were reduced and the fermentation purity of L-isoleucine was increased.

[0125] Example 4: Construction and evaluation of an exogenous gdh-enhanced strain in the L-isoleucine producing strain Corynebacterium glutamicum KCCM11248P rocG (from b.su) and gdh (from rhodospirillales), which are effective in increasing L-isoleucine productivity and reducing by-products as confirmed in Example 3, were introduced into KCJI-38 (KCCM11248P, Korean Patent No. 10-1335789), an L-isoleucine producing strain treated with NTG (N-Methyl-N'-nitro-N-nitrosoguanidine), by electric pulse method, and then the strain was transformed by plating on a selection medium containing 25 mg / L kanamycin, and a strain with exogenous gdh introduced on the chromosome was obtained through a second crossover process. Thereafter, the L-isoleucine and by-product concentrations in the culture solution 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, it was confirmed that the by-product AABA was reduced 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 the L-isoleucine and AABA concentrations increased, the KCCM11248PΔN2872::Pn_rocG(B.su) and KCCM11248PΔN2872::Pn_gdh(rhodospirillales) strains maintained L-isoleucine productivity, reduced the by-product AABA, and increased the fermentation purity of L-isoleucine.

[0128] As a result, it was confirmed that the microorganism into which the gene encoding the foreign glutamate dehydrogenase derived from Bacillus subtilis or Rhodospirillales of the present application has been introduced can increase the purity of L-isoleucine and produce L-isoleucine in a high yield.

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

Claims

1. A microorganism capable of producing L-isoleucine, into which a gene encoding a foreign glutamate dehydrogenase derived from Bacillus subtilis or Rhodospirillales has been introduced.

2. The microorganism according to claim 1, wherein the gene encoding the foreign glutamate dehydrogenase derived from Bacillus subtilis; and the gene encoding the foreign glutamate dehydrogenase derived from Rhodospirillales have the nucleotide sequences of SEQ ID NOs: 2 and 4, respectively.

3. The microorganism according to claim 1, wherein the amount of α-aminobutyric acid (AABA) produced by the microorganism is reduced compared to the amount of L-isoleucine produced by the microorganism.

4. The microorganism according to claim 1 , wherein the microorganism is a Corynebacterium genus microorganism.

5. The microorganism according to claim 4, wherein the Corynebacterium microorganism is Corynebacterium glutamicum.

6. A method for producing L-isoleucine, comprising the step of culturing in a medium a microorganism having L-isoleucine producing ability, into which a gene encoding a foreign glutamate dehydrogenase derived from Bacillus subtilis or Rhodospirillales has been introduced.

7. 7. The method of claim 6, further comprising recovering L-isoleucine from the cultured microorganism or the culture medium.

8. The method according to claim 6, wherein the gene encoding the foreign glutamate dehydrogenase derived from Bacillus subtilis; and the gene encoding the foreign glutamate dehydrogenase derived from Rhodospirillales have the nucleotide sequences of SEQ ID NOs: 2 and 4, respectively.

9. The method according to claim 6, wherein the amount of α-aminobutyric acid (AABA) produced is reduced relative to the amount of L-isoleucine produced.

10. A composition for producing L-isoleucine, comprising a microorganism having an ability to produce L-isoleucine, into which a gene encoding a foreign glutamate dehydrogenase derived from Bacillus subtilis or Rhodospirillales has been introduced.

11. The composition according to claim 10, wherein the gene encoding the foreign glutamate dehydrogenase derived from Bacillus subtilis; and the gene encoding the foreign glutamate dehydrogenase derived from Rhodospirillales have the nucleotide sequences of SEQ ID NOs: 2 and 4, respectively.

12. The composition according to claim 11, wherein the composition reduces the amount of α-aminobutyric acid (AABA) produced compared to the amount of L-isoleucine produced.

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