Microorganisms expressing threonine excretion protein RhtC and their use in L-isoleucine production

Abstract

The present disclosure provides microorganisms expressing the threonine excretion protein RhtC and their use for producing L-isoleucine, methods for producing L-isoleucine, and methods for increasing L-isoleucine production.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This disclosure claims the benefit of priority based on Korean Patent Application No. 10-2023-0083501, dated June 28, 2023, and all contents disclosed in the documents of this Korean patent application are incorporated herein by reference.

[0002] Numerous papers and patent documents are referenced and citations are provided throughout this disclosure, the disclosures of which are incorporated by reference in their entirety into this disclosure to more clearly explain the state of the art to which this invention pertains and the content of the present invention.

[0003] The present disclosure relates to a microorganism expressing the threonine export protein RhtC and its use for producing L-isoleucine, and more particularly to a microorganism expressing the threonine export protein RhtC, a method for producing L-isoleucine using the microorganism, a method for increasing the L-isoleucine-producing ability of the microorganism, and / or an L-isoleucine-producing composition. [Background technology]

[0004] L-isoleucine (Ile, I) is an essential amino acid used in the production of a variety of products, including animal feed, food additives, and pharmaceuticals. L-isoleucine plays a role in metabolic energy production, hemoglobin production, blood sugar regulation, muscle building and repair, and is therefore increasingly used in fluid infusions, nutritional supplements, sports nutrition, and animal feed.

[0005] Based on this trend, various attempts have been made to improve the productivity of L-isoleucine in methods using various microorganisms, including Corynebacterium spp.

[0006] Despite these efforts, there is still a need to develop technologies to improve L-isoleucine productivity. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 10,113,190 (October 30, 2018) [Patent Document 2] U.S. Patent Application Publication No. 2023-0012923 (January 19, 2023) Summary of the Invention [Problem to be solved by the invention]

[0008] The present disclosure provides an L-isoleucine-producing microorganism that expresses the threonine excretion protein RhtC.

[0009] The present disclosure provides a composition for producing L-isoleucine, comprising at least one selected from the group consisting of the threonine excretion protein RhtC, a polynucleotide encoding the same, a vector comprising the polynucleotide, and a microorganism expressing the threonine excretion protein RhtC.

[0010] The present disclosure provides a method for producing L-isoleucine, comprising culturing a microorganism that expresses the threonine excretion protein RhtC.

[0011] The present disclosure provides a method for increasing L-isoleucine productivity of a microorganism, comprising the step of introducing a polynucleotide encoding the threonine excretion protein RhtC into the microorganism.

[0012] The present disclosure provides uses of one or more selected from the group consisting of the threonine excretion protein RhtC, a polynucleotide encoding the same, a vector comprising the polynucleotide, and a microorganism expressing the threonine excretion protein RhtC for producing L-isoleucine, producing an L-isoleucine-producing microorganism, and / or increasing the L-isoleucine-producing ability of a microorganism. [Means for solving the problem]

[0013] The present disclosure seeks to identify proteins capable of imparting and / or increasing L-isoleucine production ability to microorganisms, and by introducing these proteins into suitable host cells, develops microorganisms that have L-isoleucine production ability or have increased L-isoleucine production ability, and provides microorganisms containing the proteins, uses thereof for L-isoleucine production, and related technologies.

[0014] Definition of Terms In the present disclosure, the term "corresponding to" refers to the amino acid sequence or nucleic acid sequence of a corresponding region of a polypeptide or polynucleotide, as determined by aligning the amino acid sequence or nucleic acid sequence of the polypeptide or polynucleotide with a specific amino acid sequence or nucleic acid sequence provided herein. Such sequence alignment can be performed using conventional sequence alignment methods, such as the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), or Trends Genet. 16:276-277, but is not limited thereto. Sequence alignment programs and pairwise sequence comparison algorithms known in the art can be used as appropriate.

[0015] In the present disclosure, a polynucleotide (also referred to as a "nucleic acid molecule" or "gene") or a polypeptide (also referred to as a "protein") "having, comprising, or consisting of a specific nucleic acid sequence (base sequence) or amino acid sequence" can mean that the polynucleotide or polypeptide essentially contains the specific nucleic acid sequence (base sequence) or amino acid sequence, and can be interpreted as including "substantially equivalent sequences" in which mutations (deletions, substitutions, variations, and / or additions) have been added to the specific nucleic acid sequence (base sequence) or amino acid sequence to the extent that the original function and / or intended function of the polynucleotide or polypeptide is maintained (or the mutations are not excluded).

[0016] In one example, the nucleic acid sequences or amino acid sequences provided in the present disclosure may include those modified by conventional mutagenesis methods, such as directed evolution and / or site-directed mutagenesis, to the extent that their original or intended functions are maintained.

[0017] For example, a polynucleotide or polypeptide "comprising or consisting of a specific nucleic acid sequence (base sequence) or amino acid sequence" can mean that the polynucleotide or polypeptide (i) essentially contains the specific nucleic acid sequence (base sequence) or amino acid sequence, or (ii) consists of or essentially contains a nucleic acid sequence or amino acid sequence that has 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology or identity to the specific nucleic acid sequence (base sequence) or amino acid sequence (the upper limit may be 100% or less), and maintains its original and / or intended function. In one example, the original function may be a threonine excretion function, and the desired function may mean an L-isoleucine production ability (e.g., a function that, when introduced into a host cell (microorganism), confers and / or increases the L-isoleucine production ability compared to the parent strain (microorganism not introduced)).

[0018] As used herein, the terms "homology" or "identity" refer to the degree of relatedness between two given amino acid or nucleotide sequences, expressed as a percentage. The terms homology and identity are sometimes used interchangeably.

[0019] Furthermore, proteins having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, or added are also included within the scope of proteins to be mutated in the present disclosure, as long as the amino acid sequence has such homology or identity and exhibits a function corresponding to that of the threonine excretion protein (the original function and / or the intended function).

[0020] As used herein, the term "variant" refers to a protein or a gene encoding such a protein that differs from the sequence defined by a given SEQ ID NO due to conservative substitutions and / or modifications of one or more amino acids, but maintains the functions or properties of the protein. Variants can have a variety of sequences due to one or more (e.g., one, two, three, etc.) amino acid substitutions, deletions, or additions. Such variants are generally identified by modifying one or more amino acids in the amino acid sequence of the protein and evaluating the properties of the modified protein. The function of the variant may be maintained at the same level as, increased, or insignificantly decreased compared to the native protein. Some variants may also include variants in which one or more portions, such as the N-terminal leader sequence or transmembrane domain, have been deleted. Other variants may include variants in which portions have been deleted from the N-terminus and / or C-terminus of the mature protein. The term "mutant" can also be expressed as a mutant, a modified form, a mutated protein / polynucleotide, a mutant polypeptide / polynucleotide, etc., and is not limited to these as long as it is used to mean mutated.

[0021] For purposes of the present disclosure, the mutant may have, but is not limited to, maintained or increased activity (e.g., original activity and / or desired activity) compared to the wild-type or non-mutated protein.

[0022] Mutants of proteins disclosed herein may be generated by conservative substitutions. The term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. The variant can have, for example, one or more conservative substitutions while still retaining one or more biological activities. Such amino acid substitutions can generally be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids with electrically charged side chains include arginine, lysine, and histidine, negatively charged (acidic) amino acids include glutamic acid and aspartic acid, nonpolar amino acids with uncharged side chains include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, polar or hydrophilic amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine, and aromatic nonpolar amino acids include phenylalanine, tryptophan, and tyrosine. The variants 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 polypeptides can be conjugated to N-terminal signal (or leader) sequences of proteins involved in co- or post-translationally protein transfer, and can be conjugated to other sequences or linkers that allow the polypeptides to be identified, purified, or synthesized.

[0023] In this disclosure, "homology" refers to the percentage of identity between two polynucleotide or polypeptide moieties. Sequence homology from one moiety to another can be determined by known techniques. For example, homology can be determined by directly aligning sequence information between two polynucleotide molecules or two polypeptide molecules, such as parameters like score, identity, and similarity, using a readily available computer program. Such computer programs may include BLAST (NCBI), CLC Main Workbench (CLC bio), and MegAlign™ (DNASTAR Inc). Alternatively, polynucleotide homology can be determined by hybridizing polynucleotides under conditions that allow stable duplex formation between homologous regions, followed by digestion with a single-strand-specific nuclease and determining the size of the resulting fragments.

[0024] As used herein, "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, and can be expressed as a percentage. The terms homology and identity are sometimes used interchangeably.

[0025] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences can generally hybridize to the entire sequence or a portion thereof under moderate or high stringent conditions. Hybridization obviously includes hybridization to polynucleotides containing common codons or codons that take codon degeneracy into account.

[0026] 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, e.g., as described in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it may 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), as implemented in the 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 [CARILLOETA / .](1988) SIAM J Applied Math 48:1073. For example, BLAST from the National Database Center for Biotechnology Information, or ClustalW can be used to determine homology, similarity, or identity.

[0027] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, e.g., as known in Smith and Waterman, Adv. Appl. Math (1981) 2:482, or as described in Needleman et al. (1970), J. Mol. Biol. 48:443. Briefly, the GAP program can be defined as the number of similar aligned symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. 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 (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.

[0028] In this disclosure, the term "about" used before a numerical value is used to inclusively mean a numerical value in a range equivalent to or similar to the numerical value described thereafter. For example, the equivalent or similar range may mean a range of ±20%, ±15%, ±10%, ±5%, ±3%, ±2%, ±1% of the described numerical value, but is not limited thereto.

[0029] In the present disclosure, even when only a numerical value is described, it can be interpreted as being used to inclusively mean numerical values ​​in a range equivalent to or similar to the described numerical value. For example, the equivalent or similar range can mean a range such as ±20%, ±15%, ±10%, ±5%, ±3%, ±2%, ±1% of the described numerical value, but is not limited thereto.

[0030] The present disclosure is described in more detail below.

[0031] The present disclosure provides microorganisms that express the threonine excretion protein RhtC and techniques related to the production of L-isoleucine using the same.

[0032] The present disclosure provides a microorganism that expresses the threonine excretion protein RhtC. More specifically, the microorganism expressing the threonine excretion protein RhtC is (1) threonine efflux protein RhtC, (2) a polynucleotide encoding the threonine export protein RhtC; and (3) A vector containing the polynucleotide The composition may include one or more selected from the group consisting of: The polynucleotides (2) and / or (3) are (a) containing the gene for the threonine export protein RhtC, or (b) a gene for the threonine efflux protein RhtC and a promoter (e.g., an enhanced promoter) operably linked to the gene; It may also include.

[0033] Threonine efflux protein The threonine efflux protein may be the L-threonine exporter (RhtC). The L-threonine efflux protein RhtC mediates the extracellular export of L-threonine and is known to be an inner membrane protein with five transmembrane domains. Experimental topology analysis has shown that the C-terminus of the protein may be present in the cytoplasm. In the present disclosure, the threonine efflux protein may be used in combination with the RhtC protein or RhtC. The threonine efflux protein RhtC may be a wild-type protein or a mutant that maintains its original function and / or the desired function. The amino acid sequence of the threonine efflux protein may be obtained from publicly known databases such as NCBI's GenBank.

[0034] The threonine excretion protein RhtC may be a threonine excretion protein derived from Escherichia coli (E. coli).

[0035] In one specific example, the threonine excretion protein RhtC may comprise an amino acid sequence having 70% or more identity to either the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence in which the amino acid corresponding to position 62 of SEQ ID NO: 11 is substituted with an amino acid different from the original amino acid. More specifically, the threonine excretion protein RhtC may comprise an amino acid sequence having 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more (the upper limit may be 100% or less) identity to either the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence in which the amino acid corresponding to position 62 of SEQ ID NO: 11 is substituted with an amino acid different from the original amino acid. The activity of the threonine export protein RhtC is enhanced by substituting the amino acid corresponding to position 62 of SEQ ID NO: 11 with a different amino acid. For example, the amino acid corresponding to position 62 of SEQ ID NO: 11 may be leucine (Leu, L). For example, the different amino acid may be serine (Ser, S). For example, the amino acid sequence in which the amino acid corresponding to position 62 of SEQ ID NO: 11 is substituted with serine is represented by SEQ ID NO: 13. The activity of the threonine export protein is enhanced by substituting the amino acid corresponding to position 62 of SEQ ID NO: 11 (leucine; Leu, L) with serine (Ser, S).

[0036] In one example, the threonine export protein RhtC is understood to include proteins having 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more (the upper limit may be 100% or less) homology or identity to either the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence in which the amino acid corresponding to position 62 of SEQ ID NO: 11 is substituted with an amino acid different from the original, so long as the original function (threonine export function) and / or desired function (L-isoleucine producing ability (function of imparting and / or increasing L-isoleucine producing ability)) of the protein containing the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence in which the amino acid corresponding to position 62 of SEQ ID NO: 11 is substituted with an amino acid different from the original) is maintained.

[0037] Polynucleotide encoding the threonine efflux protein RhtC In the present disclosure, the polynucleotide encoding the threonine excretion protein RhtC can include the gene encoding the aforementioned threonine excretion protein RhtC.

[0038] The gene may be modified in various ways in the coding region without changing the amino acid sequence of the polypeptide, taking into account codon degeneracy or the codons preferred by the organism in which the protein is to be expressed (codon optimization). In one specific example, a gene encoding the threonine export protein RhtC of SEQ ID NO: 11 may have the nucleic acid sequence of SEQ ID NO: 12, and a gene encoding a protein having an amino acid sequence in which the amino acid at position 62 of SEQ ID NO: 11 is substituted with serine (e.g., SEQ ID NO: 13) may have the nucleic acid sequence of SEQ ID NO: 14. The nucleic acid sequence may also include a mutated nucleic acid sequence without changing the encoded amino acid sequence, taking into account codon degeneracy and / or codon optimization.

[0039] For example, the polynucleotide encoding the threonine excretion protein RhtC may further comprise a promoter in addition to the gene encoding the threonine excretion protein RhtC.

[0040] The promoter may be operably linked to the gene. More specifically, the polynucleotide encoding the threonine excretion protein RhtC may comprise, in a 5' to 3' direction, a promoter and the gene encoding the threonine excretion protein RhtC.

[0041] In the present disclosure, the term "operably linked" may mean that the promoter is functionally linked to the gene so as to regulate the transcription of the gene. The operably linking may be achieved using recombinant DNA techniques known in the art, for example, but not limited to, conventional site-specific DNA cleavage and ligation.

[0042] The promoter may be a strong promoter that enhances the expression (transcription) of an operably linked gene. In one embodiment, the promoter may comprise the nucleic acid sequence of SEQ ID NO: 15. In another embodiment, the promoter may be selected from known strong promoters, such as, but not limited to, one or more selected from the group consisting of cj1 to cj7 promoters (Korean Patent Registered No. 10-0620092), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL1 promoter, SPL7 promoter, SPL13 (sm3) promoter (Korean Patent Registered No. 10-1783170), O2 promoter (Korean Patent Registered No. 10-1632642), tkt promoter, yccA promoter, and PlysC promoter (e.g., PlysCP1 promoter (US8426577B2)).

[0043] A vector containing the polynucleotide In the present disclosure, a vector containing the polynucleotide may refer to a DNA construct that contains the gene or gene and promoter and is capable of replicating / expressing (transcriptional) in a host cell. The vector may be referred to as a recombinant vector or an expression vector.

[0044] The vector is not particularly limited as long as it can be replicated / transcribed in a host cell, and can be selected from any commonly used vector. For example, the vector may be present in the form of a plasmid in the host cell, or may be in the form of a vector that can be inserted into the genome (chromosome) of the host cell, but is not limited thereto.

[0045] The vector can refer to a DNA construct containing the polynucleotide operably linked to a suitable regulatory sequence to enable expression of the target protein (threonine excretion protein RhtC) in a suitable host cell. The regulatory sequence can include a promoter capable of initiating transcription, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and / or a sequence regulating the termination of transcription and / or decoding. After transformation into a suitable host cell, the vector can be expressed independently of the host cell's genome (chromosome) or integrated into the host cell's genome.

[0046] Commonly used vectors include, but are not limited to, plasmids, cosmids, viruses, bacteriophages, and the like, whether naturally occurring or recombinant. Examples of vectors include phage or cosmid vectors such as pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, as well as plasmid vectors such as pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET. Other examples of vectors include, but are not limited to, pDC24, pDCM2, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC.

[0047] The vectors usable herein may be known expression vectors and / or vectors for inserting polynucleotides into host cell chromosomes, which may be achieved by any method known in the art, such as, but not limited to, homologous recombination or the CRISPR system.

[0048] The vector may additionally contain a selection marker to confirm whether the host cell has been transformed (e.g., whether the polynucleotide has been introduced into the host cell (e.g., inserted into a chromosome)). The selection marker is used to select cells transformed with the vector, i.e., to confirm whether the polynucleotide has been introduced into the cell or inserted into a chromosome, and is selected from genes that confer a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface protein. In an environment treated with a selective agent, only cells that express the selection marker survive or exhibit other phenotypes, allowing the selection of transformed cells.

[0049] Polypeptide Enhancement In one example, the threonine efflux protein RhtC of the present disclosure can be enhanced.

[0050] As used herein, the term "enhancement" of polypeptide activity refers to an increase in polypeptide activity compared to its endogenous activity. The term "enhancement" can be used interchangeably with terms such as "activation," "up-regulation," "overexpression," and "increase." Here, activation, enhancement, up-regulation, overexpression, and increase can all refer to the development of an activity not inherently possessed, or an improvement in activity compared to the endogenous activity or the activity prior to transformation. The term "endogenous activity" refers to the activity of a specific polypeptide inherently possessed by a parent strain or an untransformed microorganism when a trait is altered by genetic mutation due to natural or artificial factors. This term can be used interchangeably with "activity prior to transformation." "Enhancement," "up-regulation," "overexpression," or "increase" of a polypeptide activity compared to its endogenous activity refers to an improvement in the activity and / or concentration (expression level) of a specific polypeptide inherently possessed by a parent strain or an untransformed microorganism prior to transformation.

[0051] The enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or increasing the concentration (expression level) of an endogenous polypeptide. Whether or not the activity of the polypeptide is enhanced can be confirmed by measuring the level of activity, expression level, or the amount of a product resulting from the activity of the polypeptide.

[0052] The activity of the polypeptide can be enhanced by various methods well known in the art, and is not limited thereto, as long as the activity of the target polypeptide can be enhanced compared to that of the microorganism before transformation. Specifically, the enhancement can be achieved by using routine methods in molecular biology, such as genetic engineering and / or protein engineering, which are well known to those skilled in the art (e.g., Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2, pp. 1-16; Sambrook et al., Molecular Cloning 2012, etc.).

[0053] Specifically, the enhancement of the polypeptides of the present disclosure 1) an increase in the intracellular copy number of a polynucleotide encoding a polypeptide; 2) Replacement of the expression regulatory region of the chromosomal gene encoding the polypeptide with a sequence with strong activity; 3) a variation in the nucleotide sequence coding for 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 polypeptide activity is enhanced; 5) modifying a polynucleotide sequence encoding the polypeptide so as to enhance the polypeptide's activity (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the polypeptide's activity); 6) introduction of a foreign polypeptide exhibiting the activity of the polypeptide 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 deforming or chemically modifying exposed sites; 9) Regulation of the intracellular localization of proteins (polypeptides); or 10) It may be a combination of two or more selected from the above 1) to 9), but is not particularly limited thereto.

[0054] More specifically, The 1) increase in intracellular copy number of a polynucleotide encoding a polypeptide can be achieved by introducing into a host cell a vector operably linked to the polynucleotide encoding the polypeptide, which can replicate and function independently of the host. Alternatively, it can 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 can be achieved by, but is not limited to, introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome of the host cell.

[0055] The replacement of the expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide with a sequence with stronger activity can be, for example, mutation of the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further enhance the activity of the expression regulatory region, or replacement with a sequence with stronger activity. The expression regulatory region can 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. One example is, but is not limited to, replacing the original promoter with a strong promoter.

[0056] 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 substitution with a base sequence encoding another initiation codon that results in a higher polypeptide expression rate than the endogenous initiation codon, but is not limited thereto.

[0057] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) above can be, but is not limited to, a mutation in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, such as deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to enhance the activity of the polypeptide, or a substitution 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. The substitution can be specifically, but is not limited to, by inserting the polynucleotide into a chromosome via homologous recombination. The vector used in this case can additionally contain a selection marker to check for chromosomal insertion.

[0058] The introduction of an exogenous polynucleotide that exhibits the activity of a polypeptide as described above in 6) above can be the introduction into a host cell of an exogenous polynucleotide that encodes a polypeptide that exhibits the same or similar activity as the polypeptide. The exogenous polynucleotide is not limited 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 selected appropriately by those skilled in the art from known transformation methods, and the introduced polynucleotide can be expressed in the host cell to produce a polypeptide and increase its activity.

[0059] 7) Codon optimization of a polynucleotide encoding a polypeptide may be codon optimization of an endogenous polynucleotide to increase transcription or translation in a host cell, or codon optimization of an exogenous polynucleotide to optimize transcription or translation in a host cell.

[0060] 8) Analyzing the tertiary structure of a polypeptide and selecting and modifying or chemically modifying exposed sites can be, for example, by comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins to determine candidate template proteins based on the degree of sequence similarity, confirming the structure based on the sequence information, and selecting and modifying or chemically modifying exposed sites.

[0061] 9) Regulation of intracellular location of a protein (polypeptide) can be targeting the protein (polypeptide) to a specific intracellular organelle or space, for example, to the periplasm or cytoplasm through the addition or removal of a leader sequence that functions in targeting the protein (polypeptide), but is not limited thereto.

[0062] Such enhanced 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 product produced from the polypeptide.

[0063] In one embodiment, the threonine excretion protein RhtC of the present disclosure may be enhanced by replacing the promoter of the gene encoding the protein with an enhanced promoter, which may be, but is not limited to, CJ1 to CJ7 promoters (U.S. Patent No. US7662943B2), 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. Patent No. US10584338B2), O2 promoter (U.S. Patent No. US10273491B2), tkt promoter, yccA promoter, or PlysC promoter (e.g., PlysCP1 promoter (U.S. Patent No. US8426577B2)).

[0064] Threonine excretion protein-expressing microorganisms As mentioned above, the present disclosure provides a microorganism that expresses the threonine excretion protein RhtC.

[0065] In the present disclosure, "a microorganism expressing the threonine efflux protein RhtC" may refer to a microorganism that did not express the threonine efflux protein RhtC but now expresses the threonine efflux protein RhtC, and / or a microorganism that has expressed the threonine efflux protein RhtC but now overexpresses the threonine efflux protein RhtC.

[0066] The microorganism is characterized in that it has an ability to produce L-isoleucine, or has an increased ability to produce L-isoleucine compared to the same microorganism or a wild-type microorganism that does not express the threonine excretion protein RhtC.

[0067] In one example, the microorganism expressing the threonine excretion protein RhtC is (1) threonine efflux protein RhtC, (2) a polynucleotide encoding the threonine export protein RhtC; and (3) A vector containing the polynucleotide The composition may include one or more selected from the group consisting of:

[0068] In other words, the microorganism expressing the threonine export protein RhtC may be a recombinant microorganism obtained by introducing (transforming) a polynucleotide encoding the threonine export protein RhtC and / or a vector containing the polynucleotide into a host cell (also referred to as a microorganism or a parent strain).

[0069] When the microorganism used as the host cell (a wild-type microorganism or a microorganism into which a polynucleotide and / or vector encoding the threonine export protein RhtC has not been introduced) is capable of producing L-isoleucine, the recombinant microorganism may have increased L-isoleucine production ability compared to a wild-type microorganism and / or a host cell into which the polynucleotide and / or vector has not been introduced.When the microorganism used as the host cell (a wild-type microorganism or a microorganism into which a polynucleotide and / or vector encoding the threonine export protein RhtC has not been introduced) does not have the ability to produce L-isoleucine, the recombinant microorganism may have been imparted with L-isoleucine production ability.

[0070] For example, an L-isoleucine-producing microorganism comprising one or more selected from the group consisting of the threonine excretion protein RhtC of the present disclosure, a polynucleotide encoding the threonine excretion protein RhtC, and a vector comprising the polynucleotide may have increased L-isoleucine productivity compared to a microorganism not comprising one or more selected from the group consisting of the threonine excretion protein RhtC, a polynucleotide encoding the threonine excretion protein, and a vector comprising the polynucleotide. The microorganism not comprising one or more selected from the group consisting of the threonine excretion protein RhtC, a polynucleotide encoding the threonine excretion protein, and a vector comprising the polynucleotide may be the same species of microorganism or a wild-type microorganism that does not express the threonine excretion protein RhtC.

[0071] In this disclosure, the term "microorganism" encompasses unicellular bacteria and is used interchangeably with "cell." In this disclosure, a microorganism before being mutated (transformed) to express the threonine excretion protein RhtC can be referred to as a "parent microorganism or parent strain" or a "host cell" to distinguish it from the mutated microorganism (recombinant microorganism).

[0072] In one example, the microorganism, e.g., a microorganism used as a host cell, may be (1) a microorganism naturally capable of producing L-isoleucine, (2) a microorganism naturally lacking or significantly reduced L-isoleucine production ability, and / or (3) a microorganism in which a mutation has been introduced (transformed) into the microorganism naturally capable of producing L-isoleucine or the microorganism lacking or significantly reduced L-isoleucine production ability, thereby imparting L-isoleucine production ability or improving L-isoleucine production ability. In one specific example, the microorganism used as a host cell may be, but is not limited to, Corynebacterium glutamicum KCCM12739P (CA10-3101, US2023-0098971A1) or Corynebacterium glutamicum KCCM11248P (Korean Patent Registered No. 10-1335789).

[0073] In one embodiment, the microorganism may be a microorganism of the genus Corynebacterium. The Corynebacterium microorganism may include, but is not limited to, Corynebacterium glutamicum, Corynebacterium ammoniagenes, Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium thermoaminogenes, Corynebacterium efficiens, etc. Specifically, the Corynebacterium microorganism may be Corynebacterium glutamicum.

[0074] In one embodiment, the microorganism may be a microorganism in which the L-isoleucine biosynthesis pathway has been further enhanced to increase the amount of L-isoleucine produced, but is not limited thereto.

[0075] In one embodiment, the microorganism may be a microorganism in which feedback inhibition of L-threonine dehydratase is desensitized.

[0076] As used herein, the term "threonine dehydratase (EC 4.3.1.19)" refers to an enzyme that synthesizes 2-ketobutyrate from threonine. The enzyme is encoded by the ilvA gene and is known to be subject to feedback inhibition by L-isoleucine. Corynebacterium microorganisms synthesize L-isoleucine using pyruvate and 2-ketobutyrate as precursors via three intermediate metabolites. The amino acid sequence of the threonine dehydratase can be obtained from publicly known databases such as NCBI GenBank or US2023-0098971A1 and US10982244B2.

[0077] In one embodiment, the microorganism may be one in which feedback inhibition of homoserine dehydrogenase is desensitized.

[0078] As used herein, the term "homoserine dehydrogenase (EC: 1.1.1.3)" refers to an enzyme encoded by the hom gene that catalyzes the synthesis of homoserine. Homoserine dehydrogenase is known to be feedback inhibited by isoleucine. The amino acid sequence of homoserine dehydrogenase can be obtained from publicly known databases such as NCBI GenBank or US10982244B2.

[0079] In one embodiment, the microorganism may be one in which feedback inhibition of aspartate kinase is desensitized. In the present disclosure, the term "aspartate kinase (EC: 2.7.2.4)" is encoded by the lysC gene, and the amino acid sequence of the aspartate kinase can be obtained from publicly known databases such as NCBI GenBank or US10662450B2.

[0080] As used herein, the term "release of feedback inhibition" may refer to an increase or enhancement of the activity of a polypeptide, protein, or enzyme relative to its endogenous activity, or to an increase in activity that is no longer inhibited relative to its endogenous activity. In one embodiment, the microorganism may be a microorganism into which a genetic mutation (R407H) has been introduced into the hom gene (US 10982244B2), a genetic mutation (T381A, F383A, and / or V323A) has been introduced into the ilvA gene (US 2023-0098971A1, US 10982244B2), and / or a genetic mutation (L377K) has been introduced into the lysC gene encoding aspartate kinase (US Patent Publication No. US 10662450B2).

[0081] Introduction of the polynucleotide or vector can be carried out by those skilled in the art using known transformation methods. In the present disclosure, the term "transformation" refers to introducing a polynucleotide encoding a target protein (the threonine export protein RhtC) or a vector containing the same into a host cell to express the protein encoded by the polynucleotide in the host cell. A transformed polynucleotide can be expressed in the host cell, regardless of whether it is located intrachromosomally or extrachromosomally. The polynucleotide can also include DNA and / or RNA encoding the target protein. The polynucleotide may be introduced into the host cell in any form, as long as it can be introduced and expressed in the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, a genetic construct containing all elements necessary for autonomous expression. The expression cassette typically includes expression control elements, such as a promoter, a transcription termination signal, a ribosome binding site, and / or a translation termination signal, operably linked to the polynucleotide. The promoter is as described above. The expression cassette may be in the form of a self-replicating expression vector. Alternatively, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell. The term "operably linked" as used herein may refer to the functional linkage of an expression regulatory element (e.g., a promoter) with a polynucleotide such that the expression regulatory element can regulate transcription (e.g., initiation of transcription) of a polynucleotide encoding a target protein (the threonine export protein RhtC). Operable linkage can be achieved using recombinant DNA techniques known in the art, such as, but not limited to, conventional site-specific DNA cleavage and ligation.

[0082] The method for transforming the polynucleotide into the host cell can be any method for introducing nucleic acid into a cell (microorganism), and can be selected from transformation techniques known in the art depending on the host cell. Examples of known transformation methods include, but are not limited to, electroporation, electropulse, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) precipitation (polyethylene glycol-mediated uptake), DEAE-dextran, cationic liposome method, lipofection, and lithium acetate-DMSO method.

[0083] The polynucleotide can be introduced (inserted) into the genome (chromosome) of a host cell using a known method appropriately selected by those skilled in the art. For example, the polynucleotide can be introduced (inserted) into the genome (chromosome) of a host cell by conventional homologous recombination or by using an RNA-guided endonuclease system (RNA-guided endonuclease system or CRISPR system; for example, a mixture containing (a) an RNA-guided endonuclease (e.g., Cas9 protein), a gene encoding the endonuclease, or a vector containing the gene; and (b) a guide RNA (e.g., single guide RNA (sgRNA)), a DNA encoding the endonuclease, or a vector containing the DNA (e.g., a mixture of an RNA-guided endonuclease protein and a guide RNA), a complex (e.g., a ribonucleic acid fusion protein (RNP), a recombinant vector (e.g., a vector containing both an RNA-guided endonuclease-encoding gene and a DNA encoding a guide RNA), etc.), but is not limited thereto.

[0084] In this disclosure, the term "vector" refers to a DNA construct containing a polynucleotide sequence encoding a protein of interest operably linked to a suitable regulatory sequence to enable expression of the protein in a suitable host. The regulatory sequence may include a promoter capable of initiating transcription, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosomal binding site, and / or a sequence regulating the termination of transcription and / or translation. After transformation into an appropriate host cell, the vector may be expressed independently of the host cell's genome (chromosome) or integrated into the host cell's genome.

[0085] L-Isoleucine Production As described above, the microorganisms expressing the threonine excretion protein RhtC provided by the present disclosure have the ability to produce L-isoleucine, or are characterized by increased L-isoleucine production ability compared to the same species of microorganism that does not express the threonine excretion protein RhtC or a wild-type microorganism.

[0086] The L-isoleucine-producing ability may include not only the L-isoleucine-producing function but also the L-isoleucine excretion / secretion function, etc. The L-isoleucine-producing ability may be confirmed by the isoleucine concentration in the medium, sugar consumption ability, etc., but is not limited thereto.

[0087] In one example, the L-isoleucine productivity of the microorganism expressing the threonine export protein RhtC is about 30% or more (i.e., about 1.3 times or more), about 40% or more (i.e., about 1.4 times or more), about 50% or more (i.e., about 1.5 times or more), about 60% or more (i.e., about 1.6 times or more), compared to the L-isoleucine productivity (e.g., L-isoleucine concentration in the medium (g / L), sugar consumption ability, L-isoleucine export ability, etc.) (100%) of the parent strain (host cell, the same microorganism that does not express the threonine export protein RhtC, or a wild-type microorganism). fold or more), about 70% or more (i.e., about 1.7 times or more), about 80% or more (i.e., about 1.8 times or more), about 90% or more (i.e., about 1.9 times or more), about 100% or more (i.e., about 2 times or more), about 110% or more (i.e., about 2.1 times or more), about 120% or more (i.e., about 2.2 times or more), or about 130% or more (i.e., about 2.3 times or more) (the upper limit is not particularly limited, and may be, for example, about 300%, about 200%, or about 150%, but is not limited to these).

[0088] Therefore, one example of the present disclosure provides a composition for producing L-isoleucine, comprising one or more selected from the group consisting of the threonine excretion protein RhtC, a polynucleotide encoding the same, a vector containing the polynucleotide, and a microorganism expressing the threonine excretion protein RhtC.

[0089] The composition of the present disclosure may additionally contain any suitable excipient commonly used in compositions for producing amino acids, and such excipients may be, for example, but are not limited to, a preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer, or isotonic agent.

[0090] In the compositions of the present disclosure, the variants, polynucleotides, vectors, strains, media, and L-amino acids are as described above in other aspects.

[0091] Another example provides a method for producing L-isoleucine, comprising culturing a microorganism that expresses the threonine excretion protein RhtC.

[0092] Another example provides a method for producing an L-isoleucine-producing microorganism, and / or a method for imparting and / or increasing L-isoleucine production ability to a microorganism, comprising the step of transforming a host cell (microorganism) by introducing a polynucleotide encoding the threonine excretion protein RhtC or a vector containing the polynucleotide.

[0093] Another example provides a use of one or more selected from the group consisting of the threonine excretion protein RhtC, a polynucleotide encoding the same, a vector containing the polynucleotide, and a microorganism expressing the threonine excretion protein RhtC for producing L-isoleucine, and / or for producing an L-isoleucine-producing microorganism, and / or for imparting and / or increasing L-isoleucine production ability to a microorganism.

[0094] The threonine export protein RhtC, the polynucleotide encoding it, the vector containing the polynucleotide, the microorganism expressing the threonine export protein RhtC, the host cell, the transformation, and the like are as described above.

[0095] In the method for producing L-isoleucine, which includes culturing a microorganism expressing the threonine excretion protein RhtC, the culturing can be carried out in a medium suitable for culturing a microorganism (e.g., microbial growth and / or L-isoleucine production). Such media are well known to those skilled in the art. The method can further include, after the culturing step, recovering L-isoleucine from the cultured microorganism, the medium, or both.

[0096] In the above method, the step of culturing the microorganism can be carried out by known methods such as batch culture, continuous culture, and fed-batch culture, but is not limited thereto. The culture conditions are not limited thereto, but include adjusting the pH to an appropriate level (e.g., pH 5-9, specifically pH 6-8, most specifically pH 7.2) using a basic compound (e.g., sodium hydroxide, potassium hydroxide, or ammonia) or an acidic compound (e.g., phosphoric acid, sulfuric acid, phosphate salts, sulfate salts, etc.), and maintaining aerobic conditions by introducing oxygen or an oxygen-containing gas mixture into the culture. The culture temperature can be maintained at 20-45°C or 25-40°C, and the culture can be performed for approximately 10-160 hours, but is not limited thereto. L-isoleucine produced by the culture can be secreted into the medium or remain intracellularly.

[0097] The media usable for the culture may include, but are not limited to, one or more carbon sources selected from the group consisting of sugars and carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose), milk fats and fats (e.g., soybean oil, sunflower seed oil, peanut oil, and coconut oil), fatty acids (e.g., palmitic acid, stearic acid, and linoleic acid), alcohols (e.g., glycerol and ethanol), and organic acids (e.g., acetic acid), either individually or in combination. The nitrogen sources may include, but are not limited to, one or more nitrogen-containing organic compounds (e.g., peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean meal, and urea), and inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate), either individually or in combination. The phosphorus source may be one or more selected from the group consisting of potassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and corresponding sodium-containing salts, and may be used individually or in combination. The medium may also contain essential growth-promoting substances such as other metal salts (e.g., magnesium sulfate, iron sulfate, manganese sulfate, etc.), amino acids, and / or vitamins.

[0098] The step of recovering L-isoleucine may involve collecting the target amino acid from the culture medium, culture solution, or microorganism using a suitable method known in the art depending on the culture method. For example, the recovery step may be performed by one or more methods selected from centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, etc. The method of recovering L-isoleucine may additionally include a purification step before, simultaneously with, or after the recovery step.

[0099] Other examples provide compositions, methods, products, processes, or uses featuring one or more elements disclosed in this disclosure. [Effects of the Invention]

[0100] The microorganisms expressing the threonine excretion protein RhtC provided by the present disclosure have improved L-isoleucine-producing ability compared to microorganisms not expressing RhtC, and can be used to effectively produce L-isoleucine. Therefore, the L-isoleucine produced by these microorganisms can be expected to be widely used in industrial applications such as food, feed, and pharmaceuticals. DETAILED DESCRIPTION OF THE INVENTION

[0101] The present invention will be described in more detail below with reference to examples, but these are merely illustrative and are not intended to limit the scope of the present invention. It will be obvious to those skilled in the art that the examples described below can be modified within the scope of the essential gist of the invention. [Example]

[0102] Example 1. Construction of a recombinant vector for introducing the threonine excretion protein RhtC Example 1-1: Construction of a plasmid for gene insertion To insert the rhtC gene encoding the threonine export protein RhtC, NCgl2533 in the host cell (Corynebacterium glutamicum ATCC13032) genome was used as the insertion site. NCgl2533 deletion and target gene insertion vectors were constructed to replace the NCgl2533 gene with the foreign rhtC. To construct the vectors, PCR was performed using the chromosome of Corynebacterium glutamicum ATCC13032 as a template and primer pairs set forth in SEQ ID NOs: 1 and 2, and SEQ ID NOs: 3 and 4, respectively. The primer sequences used for each PCR are listed in Table 1 below.

[0103] [Table 1]

[0104] PfuUltra™ High-Fidelity DNA Polymerase (Stratagene) was used as the polymerase for PCR. 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 cycles were repeated 28 times. DNA fragments of 1020 bp and 1043 bp were obtained. The resulting DNA products were purified using a PCR Purification kit (QIAGEN). The purified amplified products were digested with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes. The pDC24 vector (SEQ ID NO: 16) was cloned into the pDC24 vector (SEQ ID NO: 16) using an Infusion Cloning Kit (TaKaRa) according to the supplied instructions to create the NCgl1756 deletion and target gene insertion vector pDC24△N2533.

[0105] Example 1-2: Plasmid construction for promoter replacement and mutagenesis To enhance rhtC activity, the coding gene (SEQ ID NO: 12) of rhtC (wild-type; SEQ ID NO: 11) from E. coli W3110 (ATCC27325) and the coding gene (SEQ ID NO: 14) of a mutant based on this, rhtC(L62S; SEQ ID NO: 13), were introduced (US20230012923A1). Using the PlysCP1 promoter (US8426577B2), which has a partially modified sequence based on PlysC, the promoters of the rhtC(wild-type) and rhtC(L62S) genes were replaced with PlysCP1 (SEQ ID NO: 15), thereby constructing a plasmid that enhances rhtC activity. The proteins and DNA sequences used are as follows in Table 2:

[0106] [Table 2] TIFF2026505900000003.tif165149 TIFF2026505900000004.tif125149

[0107] Specifically, to construct strains carrying rhtC (wild-type) and rhtC (L62S) carrying the PlysCP1 promoter, a PlysCP1 promoter fragment (WO 2009 / 096689) was obtained using pDZ_lysCP1 as a template and SEQ ID NO: 5 and SEQ ID NO: 6. PCR was performed using the chromosome of E. coli W3110 as a template and primers SEQ ID NO: 7 and SEQ ID NO: 10 to obtain an rhtC (wild-type) fragment. PCR was also performed using the chromosome of E. coli W3110 as a template and primers SEQ ID NO: 7 and SEQ ID NO: 8, and SEQ ID NO: 9 and SEQ ID NO: 10 to obtain an rhtC (L62S) fragment in which the 62nd amino acid was substituted from leucine to serine. The primer sequences used for each PCR are listed in Table 3 below.

[0108] [Table 3]

[0109] PfuUltra™ High-Fidelity DNA Polymerase (Stratagene) was used as the polymerase for PCR. 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 cycles were repeated 28 times. As a result, a 243-bp DNA fragment from the PlysCP1 promoter region, a 661-bp DNA fragment from the rhtC region, and 220-bp and 471-bp DNA fragments from the rhtC(L62S) region were obtained. The amplified products were purified using a PCR Purification kit (QIAGEN) and used as insert DNA fragments for vector construction. The purified amplified product was treated with the restriction enzyme ScaI and then heat-treated at 65°C for 20 minutes. The molar ratio of the amplified insert DNA fragment to the pDC24△N2533 vector (previously heat-treated) was adjusted to 1:2. The vectors for introducing rhtC (wild type) and rhtC(L62S) mutant into the chromosome, pDC24△N2533::PlysCP1_rhtC(Ec) and pDC24△N2533::PlysCP1_rhtC(Ec, L62S), were constructed using an infusion cloning kit (TaKaRa) according to the provided instructions.

[0110] Example 2: Construction and evaluation of an rhtC-introduced strain of Corynebacterium glutamicum L-isoleucine-producing strain (1) The vector constructed in Example 1.2 was transformed into the isoleucine-producing strain Corynebacterium glutamicum KCCM12739P (CA10-3101, Korean Patent Registration No. 10-2363913) by electroporation to construct strains in which exogenous rhtC (wild-type) and rhtC (L62S) genes were introduced into the genome via homologous recombination. The resulting strains were designated CA10-3136 (rhtC (wild-type) introduced) and CA10-3135 (rhtC (L62S) introduced), respectively.

[0111] To confirm the effect of the strain on increasing or decreasing isoleucine productivity, the strain was cultured 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. The composition of the medium used in this example is as follows. <Production medium> (pH 7.2) glucose 10% (w / v), yeast extract 0.2% (w / v), ammonium sulfate 1.6% (w / v), potassium phosphate monobasic 0.1% (w / v), magnesium sulfate heptahydrate 0.1% (w / v), ferrous sulfate heptahydrate 10 mg / L, manganese sulfate monohydrate 10 mg / L, biotin 200 μg / L After the cultivation was completed, the amount of L-isoleucine produced (isoleucine content in the medium) was measured using high performance liquid chromatography (HPLC), and the concentrations are shown in Table 4 below.

[0112] [Table 4]

[0113] As a result, as shown in Table 4, the parent strain, Corynebacterium glutamicum CA10-3101, was confirmed to produce approximately 2.1 g / L of isoleucine. Strain CA10-3136, into which rhtC was introduced, produced approximately 3.7 g / L of isoleucine, an approximately 80% increase in productivity compared to the parent strain. Strain CA10-3135, into which rhtC(L62S) was introduced, increased its sugar consumption rate and produced approximately 4.5 g / L of isoleucine, an approximately 114% increase in productivity compared to the parent strain.

[0114] Example 3: Construction and evaluation of rhtC-introduced strains of Corynebacterium glutamicum L-isoleucine-producing strains (2) The vectors constructed in Example 1.2 were introduced into the NTG (N-Methyl-N'-nitro-N-nitrosoguanidine)-treated L-isoleucine-producing strain KCJI-38 (KCCM11248P, Korean Patent No. 10-1335789) by electroporation, followed by transformation by plating on a selective medium containing 25 mg / L kanamycin. Mutants carrying rhtC or rhtC(L62S) were isolated from the chromosome. The resulting strains were designated CA10-3137 and CA10-3138. The L-isoleucine concentration in the culture medium was then measured in the same manner as in Example 2, and the results are shown in Table 5 below.

[0115] [Table 5]

[0116] As a result, as shown in Table 5, the parent strain, Corynebacterium glutamicum KCCM11248P, was confirmed to produce approximately 1.0 g / L of isoleucine. The rhtC-introduced strain, CA10-3137, increased its sugar consumption rate to produce approximately 1.8 g / L of isoleucine, demonstrating an approximately 80% increase in productivity compared to the parent strain. The rhtC(L62S)-introduced strain, CA10-3138, increased its sugar consumption rate to produce approximately 2.4 g / L of isoleucine, demonstrating an approximately 140% increase in productivity compared to the parent strain.

[0117] From the above description, it should be understood by those skilled in the art to which the present disclosure pertains that the present disclosure can be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present disclosure should be interpreted as including all modifications and variations derived from the meaning and scope of the claims below, rather than the above detailed description, and equivalent concepts.

Claims

1. An L-isoleucine-producing microorganism that expresses the threonine excretion protein RhtC.

2. 2. The L-isoleucine producing microorganism according to claim 1, wherein the microorganism comprises at least one selected from the group consisting of a threonine excretion protein RhtC, a polynucleotide encoding the threonine excretion protein RhtC, and a vector comprising the polynucleotide.

3. The microorganism described in claim 1, wherein the threonine excretion protein RhtC comprises an amino acid sequence having 70% or more identity with either the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence in which the amino acid corresponding to position 62 of SEQ ID NO: 11 is replaced with an amino acid different from the original.

4. The microorganism of claim 3 , wherein the different amino acid is serine.

5. The microorganism of claim 1 , wherein the threonine excretion protein RhtC is enhanced.

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

7. The microorganism according to claim 6, wherein the Corynebacterium microorganism is Corynebacterium glutamicum.

8. The microorganism according to claim 1, wherein the microorganism has increased L-isoleucine productivity compared to a microorganism that does not contain one or more selected from the group consisting of the threonine excretion protein RhtC, a polynucleotide encoding the threonine excretion protein RhtC, and a vector comprising the polynucleotide.

9. The microorganism according to claim 1, wherein feedback inhibition of L-threonine dehydratase is desensitized.

10. The microorganism according to claim 1 , wherein the feedback inhibition of homoserine dehydrogenase is relieved.

11. A method for producing L-isoleucine, comprising culturing the microorganism of any one of claims 1 to 10 in a medium.

12. 12. The method for producing L-isoleucine according to claim 11, further comprising the step of recovering L-isoleucine from the cultured microorganism, the medium, or both.

13. A method for increasing L-isoleucine productivity in a microorganism, comprising the step of transforming a host cell by introducing a polynucleotide encoding a threonine export protein RhtC or a vector containing said polynucleotide.

14. Use of the microorganism according to any one of claims 1 to 10 for producing L-isoleucine.

15. A composition, method, product, process, or use featuring one or more elements disclosed in this disclosure.

Images