Protein variants and methods of producing l-isoleucine using the same

Abstract

This disclosure relates to: a protein variant; a polynucleotide encoding the protein variant; a Corynebacterium microorganism comprising at least one selected from the group consisting of the protein variant and the polynucleotide; and a method for producing L-isoleucine using the microorganism.

Description

[0001] This invention is a divisional application of Chinese invention patent application 202580004950.8, entitled "Protein variant and method for producing L-isoleucine using the same", filed on May 15, 2025. Technical Field

[0002] This disclosure claims the priority of Korean Patent Application No. 10-2024-0066104, filed on May 21, 2024, and Korean Patent Application No. 10-2024-0133999, filed on October 2, 2024, and all the contents disclosed in the documents of the respective Korean patent applications are incorporated herein by reference.

[0003] Throughout this disclosure, numerous papers and patent documents are cited, and their citations are indicated. The contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which this application pertains and the content of this application.

[0004] This disclosure relates to a novel protein variant, a polynucleotide encoding the protein variant, a Corynebacterium microorganism comprising at least one selected from the group consisting of the protein variant and the polynucleotide encoding the protein variant, and a method for producing L-isoleucine using the microorganism. Background Technology

[0005] L-Isoleucine (Ile, I) is classified as an essential amino acid, one of the twenty essential amino acids, and is a branched-chain amino acid used in the manufacture of various products, such as animal feed, food additives, and pharmaceuticals. L-Isoleucine is increasingly used in intravenous solutions, nutritional supplements, sports nutrition supplements, and animal feed because it functions in post-metabolism energy production, hemoglobin production, blood sugar regulation, muscle building, and repair.

[0006] Based on this trend, various attempts have been made to improve the production capacity of methods for producing L-isoleucine using various microorganisms, including Corynebacterium spp. (US6072083A).

[0007] Despite these efforts, there is still a need to develop technologies to improve L-isoleucine production capacity. Summary of the Invention

[0008] Technical issues

[0009] One object of this disclosure is to provide a novel protein variant.

[0010] Another object of this disclosure is to provide a polypeptide encoding the protein variant.

[0011] Another object of this disclosure is to provide a microorganism of the genus Corynebacterium that comprises at least one selected from the group consisting of said protein variants and polynucleotides encoding them.

[0012] Another object of this disclosure is to provide a composition for the production of L-isoleucine, comprising the aforementioned Corynebacterium genus microorganism.

[0013] Another object of this disclosure is to provide a method for producing L-isoleucine, which includes culturing the microorganism in a culture medium.

[0014] Another object of this disclosure is to provide the use of the microorganism for the production of L-isoleucine.

[0015] Technical solution

[0016] Its detailed description is as follows. On the other hand, the various descriptions, aspects, and embodiments disclosed in this disclosure can also be applied to other descriptions, aspects, and embodiments. In other words, all combinations of the various elements disclosed in this disclosure fall within the scope of this disclosure. Furthermore, the scope of this disclosure should not be considered limited to the specific descriptions below. Moreover, those skilled in the art can recognize or confirm many equivalent substitutions for the specific aspects of this disclosure using only ordinary experiments. Furthermore, these equivalent substitutions are intended to be included in this disclosure.

[0017] Furthermore, numerous academic papers and patent documents are cited throughout this disclosure, and their citations are clearly indicated. The contents of the cited papers and patent documents are incorporated into this disclosure in their entirety by reference, in order to more clearly describe the level of the technical field to which this disclosure pertains and the content of this disclosure.

[0018] One aspect of this disclosure provides a protein variant comprising: wherein an amino acid sequence of histidine (His, H), glutamic acid (Glu, E), and glycine (Gly, G) is inserted between the 15th amino acid from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 52 and the 16th amino acid from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 52.

[0019] In this disclosure, the sequence of SEQ ID NO: 52 is shown in Table 1 below.

[0020] [Table 1]

[0021]

[0022] In one embodiment, the protein variant of this disclosure may substantially consist of the following: wherein an amino acid sequence of histidine (His, H), glutamic acid (Glu, E), and glycine (Gly, G) is inserted between the 15th amino acid position from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 52 and the 16th amino acid position from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 52. In another embodiment, the protein variant of this disclosure may consist of the following: wherein an amino acid sequence of histidine (His, H), glutamic acid (Glu, E), and glycine (Gly, G) is inserted between the 15th amino acid position from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 52 and the 16th amino acid position from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 52.

[0023] Histidine (His, H), glutamic acid (Glu, E), and glycine (Gly, G) can be inserted in the order of histidine, glutamic acid, and glycine.

[0024] In this disclosure, the amino acid sequence of SEQ ID NO: 52 may be the amino acid sequence of a transport protein. In one embodiment, the protein may be encoded by the NCgl2828 gene.

[0025] In one embodiment, the amino acid at position 15 from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 52 may be alanine (Ala, A), but is not limited thereto.

[0026] In one embodiment, the amino acid at position 16 from the N-terminus in the amino acid sequence corresponding to SEQ ID NO: 52 may be histidine (His, H), but is not limited thereto.

[0027] The protein variant disclosed herein may have activities that increase L-isoleucine production activity compared to wild-type proteins (peptides).

[0028] As used in this disclosure, the term "variant" refers to a polypeptide in which one or more amino acids or nucleotides have been conservedly substituted and / or modified, such that the variant differs from the original amino acid or polynucleotide sequence while retaining its function or properties. Such variants are typically identified by modifying one or more amino acids in the amino acid sequence of the polypeptide or one or more nucleotides in the nucleotide sequence of the polynucleotide, and by assessing the properties of the modified polypeptide or polynucleotide. That is, the capabilities of the variant may be increased, unchanged, or decreased relative to the original polypeptide or polynucleotide. Furthermore, some variants may include variants in which one or more portions (such as an N-terminal leader sequence or transmembrane domain, or a nucleotide sequence encoding a leader sequence or transmembrane domain) have been removed. Other variants may include variants in which a portion of the N- and / or C-terminus of the mature protein or a nucleotide sequence encoding that portion has been removed. The term "variant" may be used interchangeably with, and is not limited to, terms such as modification, modified polypeptide, modified protein, modified polynucleotide, mutant, mutant protein, and divergent, as long as the term is used to denote a form of modification or mutation.

[0029] Furthermore, variants can contain the deletion or addition of amino acids or nucleotides (in the case of polynucleotides encoding polypeptides) that have minimal impact on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence or the nucleotide sequence encoding it can be bound to the N-terminus, said signal (or leader) sequence being associated with co-translation or post-translational translocation of the protein. Additionally, variants can be bound to another sequence or adapter for identification, purification, or synthesis.

[0030] In one embodiment, the protein variants described above in this disclosure may have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.25%, 99.275%, or 99.28% or more and less than 100% homology or identity with the amino acid sequence of SEQ ID NO: 52.

[0031] More specifically, the protein variants of this disclosure may comprise an amino acid sequence of histidine (His, H), glutamic acid (Glu, E), and glycine (Gly, G) inserted between the 15th amino acid from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 52 and the 16th amino acid from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 52, and may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, at least 99.1%, 99.2%, 99.25%, 99.275% or 99.28% or more and less than 100% homology or identity with the amino acid sequence of SEQ ID NO: 52.

[0032] Furthermore, it is evident that variants of amino acid sequences with some of these sequence deletions, modifications, substitutions, conserved substitutions, or additions are included within the scope of this disclosure, provided that they are amino acid sequences that have such homology or identity and exhibit the efficacy (efficacy of increasing L-isoleucine production) corresponding to the variants of this disclosure.

[0033] For example, the variants may include sequence additions or deletions at the N-terminus, C-terminus, and / or amino acid sequence that do not alter the function of the variants disclosed herein, as well as naturally occurring mutations, silent mutations, or conserved substitutions.

[0034] "Conservative substitution" refers to the replacement of an amino acid with an amino acid that has a similar structure and / or chemical characteristics, different from the original amino acid. Such amino acid substitutions typically occur based on the similarity of residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties. Generally, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0035] Another aspect of this disclosure provides a protein variant comprising at least one amino acid sequence selected from the group consisting of amino acid sequences selected from SEQ ID NO: 1 to 5. In one embodiment, the protein variant may be a protein variant comprising the amino acid sequence of SEQ ID NO: 4. In other embodiments, the protein variant may consist substantially of the amino acid sequence of SEQ ID NO: 4.

[0036] In this disclosure, the amino acid sequence of SEQ ID NO: 1 may be a sequence of a protein variant in which the 118th amino acid of the protein (membrane protein) encoded by the NCgl0214 gene is replaced by alanine (Ala, A) with valine (Val, V).

[0037] In this disclosure, the amino acid sequence of SEQ ID NO: 2 may be a sequence of a protein variant in which the 38th amino acid of the protein (hypothetical protein) encoded by the NCgl0216 gene is replaced by alanine (Ala, A) with threonine (Thr, T).

[0038] In this disclosure, the amino acid sequence of SEQ ID NO: 3 may be a sequence of a protein variant in which the 303rd amino acid of the protein (ABC type transporter, repeating ATPase subunit) encoded by the NCgl2176 gene is replaced by methionine (Met, M) instead of valine (Val, V).

[0039] In this disclosure, the amino acid sequence of SEQ ID NO: 4 may be a sequence of a protein variant wherein histidine (His, H), glutamic acid (Glu, E), and glycine (Gly, G) are inserted between the 15th amino acid alanine (Ala, A) and the 16th amino acid histidine (His, H) of the protein (transporter) encoded by the NCgl2828 gene.

[0040] In this disclosure, the amino acid sequence of SEQ ID NO: 5 may be the sequence of a protein variant of a branched acid mutase encoded by the NCgl0819 gene (csm), which is generated by replacing the first nucleotide of the NCgl0819 gene (csm) with G.

[0041] Protein variants of this disclosure may comprise any amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NO: 1 to 5 (e.g., the amino acid sequence of SEQ ID NO: 4), or may comprise an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with said amino acid sequence. Furthermore, it is apparent that variants having amino acid sequences with deletions, modifications, substitutions, conserved substitutions, or additions thereof are included within the scope of this disclosure, provided that they possess such homology or identity and exhibit an amino acid sequence corresponding to the efficacy of the variants of this disclosure (the efficacy of increased L-isoleucine production).

[0042] For example, the variants may include sequence additions or deletions at the N-terminus, C-terminus, and / or amino acid sequence that do not alter the function of the variants disclosed herein, as well as naturally occurring mutations, silent mutations, or conserved substitutions.

[0043] Conservative substitutions and variants of protein variants are described above.

[0044] Another aspect of this disclosure provides a polynucleotide variant comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 11 to 14. The polynucleotide variant may encode rRNA. The rRNA encoded by the polynucleotide variant may exhibit enhanced L-isoleucine production capacity (e.g., increased production, increased productivity, increased yield, etc.) compared to the pre-mutant RNA (e.g., the corresponding wild-type RNA).

[0045] In one embodiment, the polynucleotide variant of this disclosure may consist substantially of a nucleotide sequence selected from the group consisting of SEQ ID NO: 11 to 14. In another embodiment, the polynucleotide variant of this disclosure may consist of a nucleotide sequence selected from the group consisting of SEQ ID NO: 11 to 14.

[0046] The polynucleotide variants of this disclosure may comprise nucleotide sequences selected from the group consisting of SEQ ID NO: 11 to 14, or may comprise nucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity with said nucleotide sequences. Furthermore, it is apparent that any nucleotide sequence having such homology or identity and exhibiting activity corresponding to the variants of this disclosure (increased L-isoleucine production activity) is included within the scope of this disclosure, even if a portion of said sequence is deleted, modified, substituted, conservedly substituted, or added.

[0047] For example, the variants may include sequence additions or deletions at the 5′-end, 3′-end, and / or nucleotide sequences that do not alter the function of the variants disclosed herein, as well as naturally occurring mutations, silent mutations, or conserved substitutions.

[0048] Conservative substitutions and variants of polynucleotide variants are described above.

[0049] In this disclosure, the terms “homology” or “identity” refer to the degree of similarity between two given amino acid sequences or nucleotide sequences, and may be expressed as a percentage. The terms homology and identity are generally used interchangeably.

[0050] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined using standard alignment algorithms and can be achieved using default gap penalties established by the procedure used. Essentially, homologous or identical sequences can typically hybridize with all or part of the sequence under moderate to highly stringent conditions. Clearly, hybridization also includes hybridization of polynucleotides with polynucleotides containing a common codon or codons that take into account codon degeneracy.

[0051] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using known computer algorithms, such as the “FASTA” program with default parameters, as described in, for example, Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol.Biol.48: 443-453) can be used to determine the Needleman program (version 5.0.0 or later) in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277) (GCG package (including 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 HugeComputers, Martin J. Bishop, [ED.,] Academic Press, San Francisco). This was performed as described in Diego, 1994 and [CARILLOETA / .] (1988) SIAM J Applied Math 48: 1073. For example, BLAST or ClustalW from the National Biotechnology Information Database Center can be used to determine homology, similarity, or identity.

[0052] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using, for example, the GAP computer program, as known in Needleman et al. (1970), J Mol Biol. 48:443, and, for example, Smith and Waterman, Adv. Appl. Math (1981) 2:482. In summary, the GAP program can be defined as the value of the total number of symbols in the shorter sequence of two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). The default parameters of the GAP procedure may include: (1) a binary comparison matrix (containing a value of 1 for identity and a value of 0 for non-identity), and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14: 6745, disclosed in Schwartz and Dayhoff, eds., Atlas Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) permutation matrix); (2) a penalty of 3.0 for each vacancy and an additional penalty of 0.10 for each symbol in each vacancy (or a penalty of 10 for vacancy opening and 0.5 for vacancy extension); and (3) no penalty for terminal vacancy.

[0053] As an example of this disclosure, variants of this disclosure may have activity that increases L-isoleucine production capacity compared to wild-type proteins (peptides).

[0054] In this disclosure, the term "corresponding to" refers to an amino acid residue or nucleotide at a position listed in a polypeptide or polynucleotide, or an amino acid residue or nucleotide that is similar to, identical to, or homologous to a residue or nucleotide listed in a polypeptide or polynucleotide. Identifying an amino acid or nucleotide at a corresponding position can be done by referring to a specific sequence to determine the specific amino acid or nucleotide in the sequence. As used in this disclosure, "corresponding region" generally refers to a similar or corresponding position in a related protein, nucleic acid molecule, or a reference protein or nucleic acid molecule.

[0055] For example, any amino acid sequence; or nucleotide sequence can be compared with the following: an amino acid sequence in which histidine (His, H), glutamic acid (Glu, E), and glycine (Gly, G) are inserted between the 15th amino acid from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 52 and the 16th amino acid from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 52, or any amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 5 (e.g., the amino acid sequence of SEQ ID NO: 4); or any nucleotide sequence selected from the group consisting of SEQ ID NO: 6 to 14, and based thereon, each amino acid residue or nucleotide of the amino acid sequence can be numbered with reference to the following amino acid residue or nucleotide numbering positions: in which histidine (His, H), glutamic acid (Glu, E), and glycine (Gly, G) are inserted between the 15th amino acid from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 52 and the 16th amino acid from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 52. The amino acid sequence of G), or any amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 5 (e.g., the amino acid sequence of SEQ ID NO: 4); or any nucleotide sequence selected from the group consisting of SEQ ID NO: 6 to 14. For example, the sequence alignment algorithm described in this disclosure can identify the position of an amino acid or nucleotide compared to a query sequence (also called a “reference sequence”), or the position of a modification such as substitution, insertion, or deletion.

[0056] For such alignments, one can use, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), but is not limited to these, and one can also use sequence alignment programs known in the art, pairwise sequence comparison algorithms, etc.

[0057] Other aspects of this disclosure provide polynucleotides encoding protein variants of this disclosure.

[0058] In this disclosure, the term "polynucleotide" refers to a DNA or RNA chain of a certain length or longer, which is a polymer of nucleotides in which nucleotide monomers are linked together by covalent bonds to form a long chain, and more specifically, to a polynucleotide fragment encoding a variant.

[0059] In one embodiment, the polynucleotide may be a polynucleotide encoding a protein variant comprising an amino acid sequence of histidine (His, H), glutamic acid (Glu, E), and glycine (Gly, G) inserted between the 15th amino acid from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 52 and the 16th amino acid from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 52.

[0060] In one embodiment, the polynucleotide may include any polynucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NO: 6 to 10, for example, the nucleotide sequence of SEQ ID NO: 9.

[0061] The polynucleotide encoding a protein variant of this disclosure may have or comprise any nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NO: 6 to 10, for example, the nucleotide sequence of SEQ ID NO: 9. In another embodiment, the polynucleotide may comprise a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with the nucleotide sequence. Furthermore, it is apparent that polynucleotides having nucleotide sequences with some of these sequences deleted, modified, substituted, conserved substitutions, or added are included within the scope of this disclosure, provided that it is a sequence having such homology or identity and exhibiting the efficacy (efficacy of increased L-isoleucine production) corresponding to the variant of this disclosure.

[0062] In one embodiment, the nucleotide or amino acid sequence provided in this specification may include a nucleotide or amino acid sequence modified by common mutagenesis, such as direct evolution and / or site-directed mutagenesis, to the extent that its original or desired function is maintained. In one embodiment, a polynucleotide or polypeptide “comprising a specific nucleotide or amino acid sequence” may mean that the polynucleotide or polypeptide (i) consists of, or substantially comprises, the specific nucleotide or amino acid sequence, or (ii) is composed of, 60% or more, 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, or 95% or more. It consists of an amino acid sequence that is 9% or more, 99.5% or more, or 99.9% or more homologous to the particular nucleotide sequence or amino acid sequence, or substantially contains an amino acid sequence that is 60% or more, 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 homologous to the particular nucleotide sequence or amino acid sequence, and retains the original function and / or the desired function.

[0063] Considering the degeneracy of codons or the preferred codons in the organism to express variants of this disclosure, the polynucleotides of this disclosure may have various modifications in the coding region within a range that does not alter the amino acid sequence of the variants of this disclosure. Specifically, the polynucleotides of this disclosure may have or comprise any nucleotide sequence of the group consisting of nucleotide sequences selected from SEQ ID NO: 6 to 10 (e.g., the nucleotide sequence of SEQ ID NO: 9) having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, or 98% or more and less than 100% homology or identity with said nucleotide sequence, or may consist of or substantially consist of nucleotide sequences having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, or 98% or more and less than 100% homology or identity with said nucleotide sequence, but are not limited thereto.

[0064] Furthermore, the polynucleotides disclosed herein may comprise probes that can be prepared from known gene sequences, for example, any sequence capable of hybridizing under stringent conditions with all or part of the polynucleotide sequence of this disclosure without limitation. “Stringent conditions” refers to conditions that allow specific hybridization between polynucleotides. For example, polynucleotides with high homology or identity, i.e., polynucleotides with 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity hybridization, and polynucleotides with lower homology or identity not hybridizing, or typical Southern hybridization washing conditions, i.e., washing once, specifically two to three times, at a salt concentration and temperature corresponding to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0065] Hybridization requires two nucleotides to have complementary sequences, but due to the strictness of hybridization, mismatches between bases are permitted. The term "complementary" can be used to describe the relationship between nucleotide bases that are capable of hybridizing with each other. For example, in the case of DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of this disclosure can not only contain substantially similar nucleotide sequences, but also contain separate nucleic acid fragments complementary to the whole sequence.

[0066] Specifically, hybridization conditions including a hybridization step with a Tm value of 55°C can be used, and the aforementioned conditions can be used to detect polynucleotides that are homologous or identical to the polynucleotides disclosed herein. Additionally, the Tm value can be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by those skilled in the art for the purpose.

[0067] The appropriate stringency of polynucleotide hybridization depends on the length and complementarity of the polynucleotides, and the variables are well known in the field.

[0068] Other aspects of this disclosure provide vectors that contain the polynucleotides or polynucleotide variants of this disclosure. The vectors may be expression vectors that express the polynucleotides or polynucleotide variants in host cells, but are not limited thereto.

[0069] The vector disclosed herein may comprise a DNA product containing a nucleotide sequence of a polynucleotide encoding a target polypeptide, the polynucleotide being operatively linked to an expression regulatory region (or expression regulatory sequence) suitable for expression of the target polypeptide in a suitable host. The expression regulatory region may contain a promoter capable of initiating transcription, any operon sequence regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and / or a nucleic acid sequence regulating transcription and translation termination. The vector may be transformed into a suitable host cell and then replicate or function independently of the host genome, and integrate into the genome itself.

[0070] There are no particular limitations on the vectors used in this disclosure, and any vector known in the art may be used. Examples of commonly used vectors may include plasmids, granules, viruses, and bacteriophages in their native or recombinant states. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or granule vectors, while pDZ-based vectors, pBR-based vectors, pUC-based vectors, pBluescriptII-based vectors, pGEM-based vectors, pTZ-based vectors, pCL-based vectors, and pET-based vectors can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, or pDC24 vectors may be used.

[0071] As an example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using a vector for chromosome insertion in cells. The polynucleotide can be inserted into the chromosome by any method known in the art, such as homologous recombination, but is not limited thereto. Selection markers may also be included to confirm whether chromosome insertion has occurred. These selection markers are used to select cells transformed by the vector, i.e., to confirm whether the target nucleic acid molecule has been inserted, and may use markers that confer selectable phenotypes (such as drug resistance, auxotrophic phenotype, cytotoxic agent resistance, or surface protein expression). In an environment treated with a selection agent, only cells expressing the selection marker survive or exhibit other phenotypes, thus allowing for the selection of transformed cells.

[0072] In this disclosure, the term "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism, such that the polypeptide encoded by the polynucleotide is expressed in the host cell. All such polynucleotides may be included, regardless of whether they are inserted into the host cell's chromosome or located extrachromosomally, as long as the transformed polynucleotide can be expressed in the host cell. Furthermore, the polynucleotide contains DNA and / or RNA encoding the target polypeptide. The polynucleotide can be introduced in any form, as long as it can be introduced into the host cell and expressed. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a genetic structure containing all the elements required for autonomous expression. An expression cassette may typically contain a promoter, transcription termination signal, ribosome binding site, and translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. Furthermore, the polynucleotide may be introduced into the host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

[0073] Furthermore, in the above text, the term "operably linked" refers to the functional linking of a polynucleotide sequence to a promoter sequence that initiates and mediates the transcription of a polynucleotide encoding the target variant of this disclosure.

[0074] Other aspects of this disclosure provide Corynebacterium microorganisms comprising at least one selected from the group consisting of protein variants, polynucleotides, and polynucleotide variants of this disclosure.

[0075] The microorganisms disclosed herein may include a vector containing a protein variant of the present disclosure, a polynucleotide encoding the protein variant, a polynucleotide of the present disclosure, or a polynucleotide variant of the present disclosure.

[0076] In this disclosure, the term "microorganism (or strain)" includes all wild-type microorganisms or naturally or artificially genetically modified microorganisms, and can be microorganisms whose specific mechanisms are weakened or enhanced due to reasons such as the insertion of exogenous genes or the enhancement or inactivation of endogenous gene activity, and which contain genetic modifications for the production of target peptides, proteins or products.

[0077] The microorganisms disclosed herein may be any one or more of the following: protein variants, polynucleotides, polynucleotide variants, and vectors containing polynucleotides or polynucleotide variants disclosed herein; microorganisms modified to express protein variants, polynucleotides, or polynucleotide variants disclosed herein; microorganisms expressing variants, polynucleotides, or polynucleotide variants disclosed herein (e.g., recombinant microorganisms); or microorganisms having the activity of protein variants or polynucleotide variants disclosed herein (e.g., recombinant microorganisms), but are not limited thereto.

[0078] The microorganisms disclosed herein may be microorganisms capable of producing L-isoleucine.

[0079] The microorganisms disclosed herein may have increased L-isoleucine production capacity compared to Corynebacterium microorganisms that do not contain at least one of the protein variants selected from the group consisting of the protein variants of this disclosure, the polynucleotides encoding said protein variants, and the polynucleotide variants of this disclosure (i.e., microorganisms that do not express protein variants, said protein variants comprising: an amino acid sequence in which histidine (His, H), glutamic acid (Glu, E), and glycine (Gly, G) are inserted between the 15th and 16th amino acids from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 52; or an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 5, such as the amino acid sequence of SEQ ID NO: 4; microorganisms that do not contain the nucleotide sequences of SEQ ID NO: 6 to 14; microorganisms that express wild-type proteins corresponding to the protein variants; or microorganisms that express wild-type polynucleotides corresponding to the polynucleotide variants).

[0080] The microorganisms disclosed herein may be microorganisms naturally capable of producing L-isoleucine, or microorganisms in which a protein variant of the present disclosure, a polynucleotide encoded therein, or a polynucleotide variant of the present disclosure (or a vector containing said polynucleotide or polynucleotide variant) is introduced into a parent strain that does not have the ability to produce L-isoleucine and / or microorganisms that are endowed with the ability to produce L-isoleucine, but are not limited thereto.

[0081] As an example, the microorganism of this disclosure is a cell or microorganism expressing a variant of the protein of this disclosure, transformed by a vector containing a polynucleotide of this disclosure, a polynucleotide encoding a variant of this disclosure, or a polynucleotide variant of this disclosure, and for the purposes of this disclosure, the microorganism of this disclosure may include all microorganisms capable of producing L-isoleucine by containing a variant of this disclosure. For example, the microorganism of this disclosure may be a recombinant microorganism with increased L-isoleucine production capacity, obtained by introducing a polynucleotide encoding a protein variant of this disclosure or a polynucleotide variant of this disclosure into a naturally occurring wild-type microorganism or an L-isoleucine-producing microorganism.

[0082] In one specific embodiment, the microorganism of this disclosure may have increased L-isoleucine production capacity compared to Corynebacterium microorganisms that do not contain at least one of the group consisting of protein variants selected from the group consisting of polynucleotides encoding said protein variants and polynucleotide variants of this disclosure. The Corynebacterium microorganism that does not contain at least one of the group consisting of said protein variants may be naturally occurring wild-type microorganisms or unmodified microorganisms, and may also be referred to as parental strains.

[0083] In this application, the microorganism serving as the host cell (the unmodified microorganism) can be (1) a microorganism naturally capable of producing L-isoleucine, (2) a microorganism naturally lacking or having significantly low L-isoleucine production capacity, and / or (3) a microorganism in which a mutation is introduced (transformed) into a microorganism naturally capable of producing L-isoleucine or lacking or having significantly low L-isoleucine production capacity, thereby acquiring or exhibiting enhanced L-isoleucine production capacity. In one embodiment, the microorganism serving as the host cell can be *Corynebacterium glutamicum* KCCM12739P or *Corynebacterium glutamicum* KCCM11248P (KR 10-1335789 B), but is not limited thereto.

[0084] In one specific embodiment, the microorganism (a variant microorganism or a microorganism serving as a host cell) may be a microorganism in which the biosynthetic pathway of L-isoleucine is additionally enhanced to increase L-isoleucine production, but is not limited thereto.

[0085] In one specific embodiment, the microorganism may be a microorganism in which feedback inhibition of L-threonine dehydratase is relieved and / or feedback inhibition of homoserine dehydrogenase is relieved.

[0086] In one specific implementation, the microorganism may be a microorganism in which feedback inhibition of L-threonine dehydratase is relieved.

[0087] In this disclosure, the term "threonine dehydratase (EC 4.3.1.19)" refers to an enzyme that produces 2-ketobutyrate from threonine, encoded by the ilvA gene, and known to be subject to feedback inhibition by L-isoleucine. Corynebacteria use pyruvate and 2-ketobutyrate as precursors to synthesize L-isoleucine via three intermediate metabolites. The amino acid sequence of threonine dehydratase can be obtained from the known database NCBI GenBank or from US 2023-0098971 A1 and US 10982244 B2.

[0088] In another specific embodiment, the microorganism may be a microorganism in which feedback inhibition of homoserine dehydrogenase is relieved.

[0089] In this disclosure, the term "homoserine dehydrogenase (EC:1.1.1.3)" refers to the enzyme encoded by the hom gene that catalyzes the synthesis of homoserine. Homoserine dehydrogenase is known to be subject to feedback inhibition by isoleucine. The amino acid sequence of homoserine dehydrogenase can be obtained from the known database NCBI GenBank or from US 10982244 B2.

[0090] In one embodiment, the microorganism may be one in which feedback inhibition of aspartate kinase is relieved. In this disclosure, the term "aspartate kinase (EC: 2.7.2.4)" is encoded by the lysC gene, and the amino acid sequence of aspartate kinase can be obtained from the known database NCBI's GenBank or from US 10662450 B2.

[0091] In this disclosure, the term "relief of feedback inhibition" can refer to an increase or enhancement in the activity of a polypeptide, protein, or enzyme compared to its inherent activity, or the removal of inhibition relative to its inherent activity. In one embodiment, the microorganism may be a microorganism further comprising one or more of the following mutations: additionally introducing a genetic mutation (R407H) into the hom gene (US 10982244 B2); additionally introducing a genetic mutation (T381A, F383A, and / or V323A) into the ilvA gene (US 2023-0098971 A1, US 10982244 B2); and additionally introducing a genetic mutation (L377K) into the lysC gene encoding aspartate kinase (US 10662450 B2), but is not limited thereto.

[0092] In one embodiment, the microorganism of this disclosure having increased L-isoleucine production may exhibit an increase of about 1% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 42.5% or more, about 42.75% or more, or about 42.8% or more (no particular upper limit is imposed, and may be, for example, about 200% or less, about 150% or less, about 100% or less, or about 50% or less) of L-isoleucine production capacity, but is not limited thereto. In another embodiment, the microorganism of this disclosure having increased L-isoleucine production may exhibit an increase in L-isoleucine production capacity of about 1.01 times or more, about 1.1 times or more, about 1.2 times or more, about 1.3 times or more, about 1.4 times or more, about 1.425 times or more, about 1.4275 times or more, or about 1.428 times or more (no particular upper limit is imposed, and may be, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less), but is not limited thereto.

[0093] The term “about” includes all ranges such as ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes, but is not limited to, all values ​​that are the same as or similar to the values ​​that appear after the term “about”.

[0094] In one embodiment, the microorganisms disclosed herein may be *Corynebacterium glutamicum*, *Corynebacterium crudilactis*, *Corynebacterium deserti*, *Corynebacterium efficiens*, *Corynebacterium callunae*, *Corynebacterium stationis*, *Corynebacterium singulare*, *Corynebacterium halotolerans*, *Corynebacterium striatum*, *Corynebacterium ammoniagenes*, *Corynebacterium pollutisoli*, *Corynebacterium imitans*, *Corynebacterium testudinoris*, or *Corynebacterium*. More specifically, it can be *Bacillus flavescens*, or more specifically, *Corynebacterium glutamicum*.

[0095] In this disclosure, the term "attenuation" of a polypeptide is a concept that includes reduced or absent activity compared to its inherent activity. Attenuation may be used interchangeably with terms such as inactivation, lack, downregulation, reduction, decrease, and weakening.

[0096] Attenuation can also include situations where the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide inherent in the original microorganism due to mutations in the polynucleotide encoding the polypeptide; situations where the overall polypeptide activity level and / or concentration (expression level) is lower than that of the native strain in the cell due to suppression of gene expression encoding the polynucleotide or suppression of its translation into a polypeptide; situations where polynucleotides are not expressed at all; and / or situations where the polypeptide has no activity even if polynucleotides are expressed. "Inherent activity" refers to the activity of a specific polypeptide originally possessed by the parental strain or unmodified microorganism before the trait change, when the trait is altered due to genetic mutations caused by natural or artificial factors. This can be used interchangeably with "activity before modification." "Inactivation, lack, reduction, downregulation, decrease, or weakening" of polypeptide activity compared to inherent activity means that the activity is lower than the activity of the specific polypeptide originally possessed by the parental strain or unmodified microorganism before the trait change.

[0097] This reduction in peptide activity can be achieved by any method known in the art, but is not limited thereto, and can be achieved by applying a variety of methods well known in the art (e.g., Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793; Sambrook et al., Molecular Cloning 2012, etc.).

[0098] Specifically, the attenuation of peptides can be

[0099] 1) The gene encoding all or part of the polypeptide is missing;

[0100] 2) Modify expression regulatory regions (or expression regulatory sequences) to reduce the expression of genes encoding polypeptides;

[0101] 3) Modify the amino acid sequence that makes up the polypeptide (e.g., delete / replace / add at least one amino acid in the amino acid sequence) to remove or weaken the activity of the polypeptide;

[0102] 4) Modify the gene sequence encoding the polypeptide (e.g., add / replace / add at least one nucleic acid base to the nucleic acid base sequence of the polypeptide gene to encode the modified polypeptide, thereby removing or weakening the activity of the polypeptide), thereby removing or weakening the activity of the polypeptide.

[0103] 5) Modify the start codon or 5'-UTR sequence of the encoded gene transcriptome (which encodes a polypeptide);

[0104] 6) Introduce antisense oligonucleotides (e.g., antisense RNA) that bind complementary to the transcriptome of the gene encoding the polypeptide.

[0105] 7) Add a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence in the gene encoding the polypeptide to form a secondary structure that cannot attach to the ribosome.

[0106] 8) Add a promoter that transcribes in the opposite direction to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE); or

[0107] 9) Regulating the cellular localization of proteins (peptides); or

[0108] 10) A combination of at least two of 1) to 9), but not limited thereto.

[0109] For example,

[0110] 1) A gene that is missing all or part of the coding polypeptide can be a polynucleotide that has been removed from the chromosome and that is replaced with a polynucleotide that has been missing some nucleotides, or a marker gene that has been replaced.

[0111] Furthermore, 2) Modification of the expression regulatory region (or expression regulatory sequence) can be achieved through mutations in the expression regulatory region (or expression regulatory sequence) via deletion, insertion, non-conservative or conserved substitution, or a combination thereof, or by substitution with a sequence having weaker activity. The expression regulatory region includes, but is not limited to, promoters, operon sequences, sequences encoding ribosome binding sites, and sequences regulating transcription and translation termination.

[0112] In addition, 3) modifying the start codon or 5'-UTR region of the encoded gene transcriptome (which encodes a polypeptide) can be, for example, by replacing it with a base sequence that encodes another start codon that has a lower polypeptide expression rate compared to the intrinsic start codon, but is not limited thereto.

[0113] Furthermore, modifications to amino acid sequences or polynucleotide sequences in 4) and 5) can be achieved by mutations in the sequence through deletion, insertion, non-conservative or conserved substitutions, or combinations thereof, or by substitution with amino acid sequences or polynucleotide sequences that are modified to have weaker activity or to be modified to have no activity, but are not limited thereto. For example, gene expression can be suppressed or weakened by introducing mutations into polynucleotide sequences to form stop codons, but are not limited thereto.

[0114] 6) Antisense oligonucleotides (e.g., antisense RNA) that are complementary to the transcriptome of the gene encoding the polypeptide can be referenced, for example, in the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

[0115] 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence in the gene encoding the polypeptide creates a secondary structure that cannot attach to the ribosome, which can prevent mRNA from being translated or inhibit its translation rate.

[0116] 8) Adding a promoter that transcribes in the opposite direction to the 3' end of the ORF (open reading frame) of a gene sequence encoding a polypeptide (reverse transcription engineering, RTE) can produce antisense nucleotides that are complementary to the transcriptome of the gene encoding the polypeptide, thereby reducing its activity.

[0117] 9) Regulating the cellular localization of proteins (peptides) can target proteins (peptides) to specific organelles or intracellular spaces within the cell. For example, by adding or removing a leader sequence that functions in the target protein (peptide), it can be targeted to the periplasm or cytoplasm, but is not limited to this.

[0118] This reduction in peptide activity may be based on, but is not limited to, a decrease in the activity or concentration of the peptide expressed in the wild-type or pre-transformation microbial strain, a decrease in the activity or concentration of the corresponding peptide, or a reduction in the amount of product produced by the corresponding peptide.

[0119] In this disclosure, the term "enhanced" peptide activity means an increase in peptide activity compared to its intrinsic activity. Enhancement may be used interchangeably with terms such as activation, upregulation, overexpression, and increase. In this document, activation, upregulation, overexpression, and increase may include exhibiting activity not originally present, or exhibiting increased activity compared to intrinsic activity or activity before modification. "Intrinsic activity" refers to the activity of a specific peptide that was originally present in the parental strain or unmodified microorganism before the trait change, when the trait is altered due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before modification." "Enhanced," "upregulated," "overexpressed," or "increased" peptide activity compared to intrinsic activity means an increase in activity and / or concentration (expression level) of a specific peptide that was originally present in the parental strain or unmodified microorganism before the trait change.

[0120] Enhancement can be achieved by introducing exogenous peptides or by increasing the activity and / or concentration (expression level) of intrinsic peptides. Whether the activity of a peptide is enhanced can be confirmed by an increase in the activity level, expression level, or amount of product released by the peptide.

[0121] To enhance the activity of peptides, various methods well known in the art can be applied, and there are no limitations, as long as the activity of the target peptide can be enhanced compared to the unmodified microorganism. Specifically, genetic engineering and / or protein engineering methods well known to those skilled in the art can be used, which are routine methods in molecular biology, but are 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.).

[0122] Specifically, the enhancement of the peptides disclosed herein can be

[0123] 1) Increase the copy number of polynucleotides encoding polypeptides in cells;

[0124] 2) Replace the gene expression regulatory region on the chromosome encoding the polypeptide with a highly active sequence;

[0125] 3) Modify the start codon or 5'-UTR sequence of the encoded gene transcriptome (which encodes a polypeptide);

[0126] 4) Modifying the amino acid sequence of peptides to enhance their activity;

[0127] 5) Modify the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide (e.g., modify the polynucleotide of the polypeptide gene to encode a modified polypeptide to enhance polypeptide activity).

[0128] 6) Import exogenous peptides that exhibit peptide activity or exogenous polynucleotides encoding them;

[0129] 7) Codon optimization for polynucleotides encoding polypeptides;

[0130] 8) Modify or chemically modify the peptide by analyzing its tertiary structure and selecting exposure sites;

[0131] 9) Regulating the cellular localization of proteins (peptides); or

[0132] 10) A combination of at least two of 1) to 9), but not limited thereto.

[0133] 1) Increasing the copy number of polynucleotides encoding polypeptides in cells can be achieved by introducing a vector that can replicate and function independently of the host, wherein the polynucleotide encoding the corresponding polypeptide is operatively linked to the vector. Alternatively, this can be achieved by introducing one, two, or more copies of the polynucleotide encoding the corresponding polypeptide into the chromosome of a host cell. Introduction into the chromosome can be performed by introducing a vector capable of inserting polynucleotides into the host cell's chromosome, but is not limited to this. Vectors are as described above.

[0134] 2) Replacing the gene expression regulatory region (or expression regulatory sequence) on the chromosome encoding the polypeptide with a sequence of high activity can be achieved, for example, by mutation in the sequence through deletion, insertion, non-conserved or conserved substitution, or a combination thereof, to further enhance the activity of the expression regulatory region, or by replacing it with a sequence of even greater activity. The expression regulatory region is not particularly limited to this, but can include promoters, operon sequences, sequences encoding ribosome binding sites, and sequences regulating transcription and translation termination, etc. As an example, a strong promoter can be used for replacement, but it is not limited to this.

[0135] Examples of known strong promoters include, but are not limited to, the CJ1 to CJ7 promoters (US Patent 7662943 B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the λ phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13(sm3) promoter (US Patent 10584338 B2), the O2 promoter (US Patent 10273491 B2), the tkt promoter, and the yccA promoter.

[0136] 3) Modifying the start codon or 5'-UTR region of the encoded gene transcriptome (which encodes a polypeptide) can be, for example, by replacing it with a base sequence that encodes another start codon that has a higher polypeptide expression rate compared to the intrinsic start codon, but is not limited thereto.

[0137] 4) and 5) Modification of the amino acid sequence or polynucleotide sequence may be achieved by mutation in the sequence through deletion, insertion, non-conservative or conserved substitution, or a combination thereof, or by substitution with an amino acid sequence or polynucleotide sequence that has been modified to have stronger activity for the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, to enhance the activity of the polypeptide, but is not limited thereto. Substitution may be specifically carried out by inserting polynucleotides into the chromosome through homologous recombination, but is not limited thereto. The vector then used may also contain selection markers for confirming chromosomal insertion. The selection markers are as described above.

[0138] 6) The introduction of a foreign polypeptide exhibiting peptide activity can be achieved by introducing a foreign polynucleotide encoding a polypeptide exhibiting the same / similar activity as the stated polypeptide into a host cell. The source or sequence of the foreign polynucleotide is not limited, as long as it exhibits the same / similar activity as the stated polypeptide. The method used for introduction can be performed by those skilled in the art by appropriately selecting known transformation methods, and by expressing the introduced polynucleotide in the host cell, the polypeptide can be produced and its activity can be increased.

[0139] 7) Codon optimization of polynucleotides encoding polypeptides can be codon optimization that increases the transcription or translation of intrinsic polynucleotides in the host cell, or codon optimization that optimizes exogenous polynucleotides in the host cell to achieve transcription and translation.

[0140] 8) By analyzing the tertiary structure of the peptide and selecting exposure sites for modification or chemical modification, the sequence information of the peptide to be analyzed can be compared, and template protein candidates can be determined according to the degree of sequence similarity. Based on this, the structure can be confirmed, the exposure sites to be modified or chemically modified can be selected, and then transformed or modified.

[0141] 9) Regulating the cellular localization of proteins (peptides) can target proteins (peptides) to specific organelles or intracellular spaces within the cell. For example, by adding or removing a leader sequence that functions in the target protein (peptide), it can be targeted to the periplasm or cytoplasm, but is not limited to this.

[0142] This enhancement of peptide activity means, but is not limited to, increasing the activity or concentration level of the corresponding peptide, or increasing the amount of product produced by the corresponding peptide, based on the activity or concentration of the peptide expressed in the wild-type or pre-transformation microbial strain.

[0143] In the microorganisms disclosed herein, modifications to some or all polynucleotides (e.g., modifications encoding the protein variants described above) can be induced by: (a) homologous recombination using a vector for chromosomal insertion in the microorganism or genome editing using genetic scissors (engineered nucleases, such as CRISPR-Cas9) and / or (b) treatment with light such as ultraviolet light and radiation, and / or chemicals, but are not limited thereto. Methods for modifying some or all genes can include methods using DNA recombination techniques. For example, deletion of some or all genes can be achieved by injecting a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism to induce homologous recombination. The injected nucleotide sequence or vector may contain, but is not limited to, a dominant selection marker.

[0144] In the microorganisms disclosed herein, the protein variants, polynucleotides, and L-isoleucine, etc., are as described in other aspects above.

[0145] Other aspects of this disclosure provide a method for producing L-isoleucine, comprising culturing in a culture medium a Corynebacterium microorganism comprising: protein variants of this disclosure, polynucleotides of this disclosure, or polynucleotide variants thereof.

[0146] The method for producing L-isoleucine disclosed herein may include culturing Corynebacterium microorganisms comprising the following in a culture medium: protein variants of the present disclosure, polynucleotides of the present disclosure, polynucleotide variants of the present disclosure, or vectors of the present disclosure.

[0147] In this disclosure, "culture" refers to the growth of the Corynebacterium genus microorganisms disclosed herein under appropriately regulated environmental conditions. The culture process of this disclosure can be carried out according to suitable culture media and culture conditions known in the art. Such a culture process can be easily adapted and used by those skilled in the art based on the selected strain. Specifically, the culture can be batch, continuous, and / or fed-batch, but is not limited thereto.

[0148] In this disclosure, the term "culture medium" refers to a material in which nutrients required for culturing Corynebacterium spp. are mixed as the main components, and nutrients and growth factors, including water necessary for survival and growth, are provided. Specifically, the culture medium and other culture conditions used for culturing the Corynebacterium spp. of this disclosure can be used, as long as it is a culture medium for culturing ordinary microorganisms without particular limitation. However, the Corynebacterium spp. of this disclosure can be cultured under aerobic conditions in an ordinary culture medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, by adjusting temperature, pH, etc.

[0149] Specifically, culture media for Corynebacterium species can be found in the literature [(American Society for Bacteriology, "Manual of Methods for General Bacteriology" (Washington DC, USA, 1981)].

[0150] In this disclosure, carbon sources 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.; and amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrate, molasses, brown molasses, rice bran, cassava, sugarcane residue, and corn steep liquor may be used. Specifically, carbohydrates such as glucose and aseptically pretreated molasses (i.e., molasses converted with reducing sugars) may be used, and various other carbon sources may be used in unlimited quantities. These carbon sources may be used alone or in combination of at least two, but are not limited thereto.

[0151] Inorganic nitrogen sources, such as ammonia, ammonium sulfate, ammonium chloride, ammonium citrate, ammonium phosphate, ammonium carbonate, and ammonium nitrate, can be used; organic nitrogen sources, such as amino acids, including glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn steep liquor, casein hydrate, fish or its degradation products, defatted soybean meal or its degradation products, etc., can be used alone or in combination of at least two, but are not limited to these.

[0152] As a phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or their corresponding sodium-containing salts may be included. As inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc., may be used, and in addition, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added in batches or continuously. However, the method is not limited to these.

[0153] Furthermore, during the cultivation of the Corynebacterium spp. disclosed herein using appropriate methods, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid. Additionally, antifoaming agents such as polyethylene glycol fatty acids can be used to suppress foam formation during cultivation. Furthermore, to maintain an aerobic state in the culture medium, oxygen or oxygen-containing gas can be injected; or to maintain an anaerobic or microaerobic state, no gas may be injected, or nitrogen, hydrogen, or carbon dioxide gas may be injected, but these are not limited to these methods.

[0154] The culture temperature in this disclosure can be from 20 to 45°C, specifically 25°C to 40°C, 25°C to 40°C, 25°C to 37°C, 25°C to 35°C, 27°C to 40°C, 27°C to 37°C, 27°C to 35°C, 30°C to 40°C, 30°C to 37°C, or 30°C to 35°C, but is not limited thereto. The culture time in this disclosure can be from approximately 10 to 160 hours, but is not limited thereto.

[0155] The L-amino acids produced by the culture method disclosed herein can be released into the culture medium or retained in the cells.

[0156] The method for producing L-amino acids disclosed herein may further include preparing the Corynebacterium genus of the present disclosure, preparing a culture medium for culturing the strain, or a combination thereof (in any order), for example prior to culturing.

[0157] The method for producing L-amino acids disclosed herein may further include recovering L-amino acids from a culture medium (the culture medium in which the culture is carried out) based on the culture of Corynebacterium spp. microorganisms. Recovery may also be included after the culture.

[0158] According to the microbial culture methods disclosed herein, such as batch, continuous, or fed-batch culture methods, the recovery can be performed using suitable methods known in the art to collect the target L-amino acid. For example, centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.), HPLC, or combinations of these methods, and using suitable methods known in the art, can recover the target L-amino acid from the culture medium or microorganism.

[0159] Furthermore, the method for producing L-amino acids disclosed herein may additionally include a purification step. Purification can be performed using suitable methods known in the art. In one embodiment, when the method for producing L-amino acids disclosed herein includes both a recovery step and a purification step, the recovery step and the purification step may be performed sequentially or discontinuously without regard to the order, or may be integrated into a single step, but are not limited thereto.

[0160] In the methods disclosed herein, the variants, polynucleotides, polynucleotide variants, vectors, and strains, etc., are described in other aspects above.

[0161] Other aspects of this disclosure provide compositions for the production of L-isoleucine comprising Corynebacterium microorganisms, said microorganisms comprising at least one selected from the group consisting of protein variants of this disclosure, polynucleotides encoding said protein variants, polynucleotide variants of this disclosure, vectors comprising said polynucleotides or polynucleotide variants; a culture medium for culturing the microorganisms; or a combination of two or more thereof.

[0162] The compositions disclosed herein may also contain any suitable excipients commonly used in compositions for the production of amino acids, and such excipients may be, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers or isotonic agents, etc., but are not limited thereto.

[0163] In the compositions disclosed herein, the variants, polynucleotides, vectors, strains, culture media, and L-isoleucine, etc., are as described in other aspects above.

[0164] In another aspect of this disclosure, methods are provided for increasing the L-isoleucine production capacity of microorganisms, methods for conferring L-isoleucine production capacity on microorganisms, or methods for preparing microorganisms with increased L-isoleucine production capacity, the methods comprising introducing a novel protein variant of this disclosure, a polynucleotide encoding the variant, a polynucleotide variant of this disclosure, and / or a recombinant vector containing the polynucleotide or polynucleotide variant into microorganisms (e.g., transforming microorganisms therein).

[0165] In the method for preparing the microorganisms disclosed herein, details regarding the protein variants, the polynucleotides encoding them, the polynucleotide variants, the recombinant vectors, and the microorganisms are as described above.

[0166] According to other aspects of this disclosure, this disclosure provides uses for at least one selected from the group consisting of: for producing L-isoleucine, for preparing microorganisms for producing L-isoleucine, and / or for conferring and / or increasing the L-isoleucine production capacity of microorganisms; protein variants of this disclosure; polynucleotides encoding said protein variants; recombinant vectors containing said polynucleotides; and microorganisms containing said protein variants, polynucleotides encoding said protein variants, and / or recombinant vectors.

[0167] Regarding the above-described uses of L-isoleucine production, preparation of L-isoleucine-producing microorganisms, and / or conferring and / or increasing the L-isoleucine-producing capacity of microorganisms, details regarding the protein variants, polynucleotides, recombinant vectors, and microorganisms are as described above.

[0168] According to other aspects of this disclosure, this disclosure provides a composition, method, product, process, or use characterized by at least one element disclosed in this disclosure.

[0169] Beneficial effects

[0170] When culturing Corynebacterium microorganisms containing variants of this disclosure, it is possible to produce L-isoleucine in high yields compared to microorganisms with conventional unmodified peptides. Detailed Implementation

[0171] The present disclosure will now be described in more detail with reference to the following embodiments. However, the following embodiments are provided merely to illustrate the present disclosure and are not intended to limit the scope of the disclosure. Furthermore, those skilled in the art or similar fields will fully understand and readily implement technical matters not specifically described herein.

[0172] Throughout this instruction manual, unless otherwise stated, the "%" used to indicate the concentration of a particular substance refers to (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid.

[0173] Example

[0174] Example 1: Selecting mutant strains with increased isoleucine productivity through artificial mutation

[0175] Example 1-1: Random mutagenesis induced by UV irradiation

[0176] To select mutant strains with increased isoleucine production capacity, the L-isoleucine-producing strain *Corynebacterium glutamicum* KCCM12739P was plated on nutrient medium containing agar and incubated at 30°C for 16 hours. Colonies obtained in this manner were then irradiated with UV light at room temperature (UV mutagenesis), inducing random mutations in the strain's genome.

[0177] <Nutritional medium (pH 7.2)>

[0178] 10g glucose, 5g meat extract, 10g peptone, 2.5g sodium chloride, 5g yeast extract, 20g agar, 2g urea (based on 1 liter of distilled water)

[0179] Examples 1-2: Selection of strains with improved L-isoleucine productivity

[0180] To select mutant strains with increased L-isoleucine production compared to the parent strain KCCM12739P, KCCM12739P strain and randomly induced mutant strains were cultured as follows.

[0181] Each strain was inoculated into 96-well (Bioneer) plates containing 400 µL of seed medium and cultured at 1200 rpm for approximately 48 hours in a 32°C plate shaker incubator (TAITEC). L-isoleucine concentrations were measured using NIR (near-infrared spectroscopy) for approximately 3,000 cultured strains, and the top five mutant strains showing increased L-isoleucine production compared to the parental strain KCCM12739P were selected.

[0182] Seed culture medium (pH 7.0)

[0183] Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH2PO4 4 g, K2HPO4 8 g, MgSO4•7H2O 0.5 g, biotin 100 μg, HCl thiamine 1000 μg, calcium pantothenate 2000 μg, nicotinamide 2000 μg (based on 1 liter of distilled water)

[0184] To ultimately select strains with reproducibly increased L-isoleucine production capacity from five selected mutant strains, they were cultured and evaluated using the following methods.

[0185] The parental strain and the aforementioned mutant strain were inoculated into 250 mL baffled Erlenmeyer flasks containing 25 mL of isoleucine production medium, and then cultured at 32 °C with shaking at 200 rpm for 60 hours. After the culture was completed, the L-isoleucine concentration in the culture medium was analyzed by HPLC. The L-isoleucine production concentration of each mutant strain is shown in Table 2 below.

[0186] <Production medium (pH 7.2)>

[0187] Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, ferric sulfate heptahydrate 10 mg / L, manganese sulfate monohydrate 10 mg / L, biotin 200 μg / L (based on 1 L of distilled water)

[0188] [Table 2]

[0189]

[0190] Among the five mutant strains selected, KCCM12739P_mt4 was ultimately chosen as the strain with significantly improved L-isoleucine production capacity.

[0191] Example 2. Mutation identification using whole-genome sequencing (WGS)

[0192] Whole-genome sequencing (WGS) was performed on the KCCM12739P_mt4 strain selected in Examples 1-2 to analyze its sequence, and nucleotide variations appearing in the CDS region or rRNA region were identified by comparison with the parental strain KCCM12739P. The respective substitution types are shown in Tables 3 to 5 below.

[0193] [Table 3]

[0194] Variations in the CDS (coding sequence) region

[0195]

[0196] [Table 4]

[0197] Variations in the rRNA region

[0198]

[0199] [Table 5]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205] In the examples below, the effects of the variants shown in Tables 3 to 5 on the L-isoleucine production capacity of Corynebacterium spp. were evaluated to identify the effective factors affecting L-isoleucine production capacity.

[0206] Example 3: Preparation of L-isoleucine-producing strains with introduced variant genes

[0207] Example 3-1: Construction of a recombinant vector for introducing variant genes

[0208] To confirm the role of the variants identified in Example 2, vectors capable of introducing them into chromosomes were constructed.

[0209] Specifically, vectors containing the target mutations were constructed, and the respective gene variants of NCgl0214, NCgl0216, NCgl2176, NCgl2828, NCgl0819, NCg1r05, NCg1r09, NCg1r16 and NCg1r19 were inserted into KCCM12739P.

[0210] Specifically, genomic DNA was extracted from strain KCCM12739P_mt4 using the G-spin Total DNA Extraction Mini Kit (Intron, catalog number 17045) according to the kit's protocol, and the genomic DNA was used as a template for PCR. Solg™ Pfu-X DNA polymerase was used as the DNA polymerase. PCR conditions were as follows: initial denaturation at 95°C for 4 minutes; 27 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 2 minutes; followed by a final extension at 72°C for 5 minutes. PCR products were obtained using primer pairs SEQ ID NO: 16 and 17; 18 and 19; 20 and 21; 22 and 23; 24 and 25; 26 and 27; 28 and 29; 30 and 31; or 32 and 33. The primer sequences used in the experiment are shown in Table 7 below.

[0211] Recombinant plasmids were obtained by cloning mutant fragments using the Gibson assembly method (DG Gibson et al., Nature Methods, Vol. 6, No. 5, May 2009; NEBuilder HiFi DNA Assembly Master Mix) and by using the pDC24 vector (SEQ ID NO: 15, WO2024-242409 A1, Table 6) treated with the restriction enzyme SmaI. The resulting vectors containing each mutant fragment were named pDC24-NCgl0214*, pDC24-NCgl0216*, pDC24-NCgl2176*, pDC24-NCgl2828*, pDC24-NCgl0819*, pDC24-NCg1r05*, pDC24-NCg1r09*, pDC24-NCg1r16*, and pDC24-NCg1r19*.

[0212] [Table 6]

[0213]

[0214]

[0215] [Table 7]

[0216]

[0217] Example 3-2: Preparation of L-isoleucine-producing strains with introduced variant genes

[0218] Nine vectors constructed in Example 3-1 were transformed into L-isoleucine-producing strain KCCM12739P via electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541–545), and strains in which the vectors were inserted into the chromosome via homologous recombination were selected on kanamycin-containing medium. Subsequently, the transformed individuals that had undergone secondary recombination were analyzed by PCR using primer pairs SEQ ID NO: 34 and 35; 36 and 37; 38 and 39; 40 and 41; 42 and 43; 44 and 45; 46 and 47; 48 and 49; or 50 and 51 to confirm the introduction of the variant gene. PCR was performed in the same manner as in Example 3-1, and the primer pairs used for confirmation are shown in Table 8 below.

[0219] [Table 8]

[0220]

[0221] The recombinant strains were named KCCM12739P△NCgl0214::NCgl0214*, KCCM12739P△NCgl0216::NCgl0216*, KCCM12739P△NCgl2176::NCgl2176*, KCCM12739P△NCgl2828::NCgl2828*, KCCM12739P△NCgl0819::NCgl0819*, KCCM12739P△NCg1r05::NCgl1r05*, KCCM12739P△NCg1r09::NCgl1r09*, KCCM12739P△NCg1r16::NCgl1r16*, and KCCM12739P△NCg1r19::NCgl1r19*.

[0222] Example 4. Evaluation of L-isoleucine production capacity of strains with introduced variant genes.

[0223] To evaluate the L-isoleucine production capacity of the strains prepared in Example 3-2 at the flask level, the strains were cultured and analyzed as follows.

[0224] The parental strain and the mutant strain were inoculated into 250 mL baffled Erlenmeyer flasks containing 25 mL of L-isoleucine production medium and cultured at 32 °C with shaking at 200 rpm for 60 hours. After culture, the amount of L-isoleucine produced was measured by high performance liquid chromatography (HPLC), and the results are shown in Table 9 below.

[0225] <Production medium (pH 7.2)>

[0226] Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, ferric sulfate heptahydrate 10 mg / L, manganese sulfate monohydrate 10 mg / L, biotin 200 μg / L (based on 1 L of distilled water)

[0227] [Table 9]

[0228]

[0229] As shown in Table 9, some mutant strains (KCCM12739P△NCgl0819::NCgl0819* and KCCM12739P△NCg1r05::NCgl1r05*) exhibited L-isoleucine production capacity comparable to the parental strain, while all the remaining mutant strains showed superior L-isoleucine production capacity compared to the parental strain. In particular, KCCM12739P△NCgl2828::NCgl2828* showed a significantly increased L-isoleucine production capacity compared to the parental strain KCCM12739P.

[0230] These results confirm that protein variants in which histidine (His, H), glutamic acid (Glu, E), and glycine (Gly, G) are inserted between the 15th and 16th amino acids of the transporter encoded by the NCgl2828 gene can produce L-isoleucine more efficiently.

[0231] Based on the above description, those skilled in the art will understand that this application can be implemented in other specific forms without altering its technical concept or essential features. In this regard, it should be understood that the above embodiments are illustrative in all respects and not restrictive. The scope of this application should be interpreted to include all variations or modifications derived from the meaning and scope of the appended claims and their equivalents, and not the detailed description above.

Claims

1. A protein variant comprising an amino acid sequence having at least 90% or higher sequence identity with the amino acid sequence of SEQ ID NO:

3. The amino acid corresponding to position 303 is methionine.

2. The protein variant of claim 1, wherein the protein variant comprises the amino acid sequence of SEQ ID NO:

3.

3. The protein variant of claim 1, wherein the protein variant has an activity that increases L-isoleucine production activity compared to the wild-type protein encoded by the NCgl2176 gene.

4. The protein variant according to claim 1, comprising an amino acid sequence having at least 95% or higher sequence identity with the amino acid sequence of SEQ ID NO:

3.

5. The protein variant of claim 1, comprising an amino acid sequence having at least 99% or higher sequence identity with the amino acid sequence of SEQ ID NO:

3.

6. A polynucleotide encoding a protein variant according to claim 1.

7. A Corynebacterium microorganism comprising at least one selected from the group consisting of a protein variant according to claim 1 and a polynucleotide encoding said protein variant.

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

9. The microorganism of claim 7, wherein the microorganism has increased L-isoleucine productivity compared to Corynebacterium microorganisms that do not contain any of the group consisting of the protein variant and the polynucleotide encoding the protein variant.

10. A method for producing L-isoleucine, comprising culturing the microorganism according to claim 7 in a culture medium.

11. The method for producing L-isoleucine according to claim 10, further comprising recovering L-isoleucine from a culture medium or cultured microorganisms.

12. Use of the microorganism according to any one of claims 7 to 9 for the production of L-isoleucine.

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