Microorganisms of Corynebacterium genus with enhanced productivity of recombinant protein

Abstract

The present application provides a microorganism of the genus Corynebacterium with weakened gene activity having 75% or more sequence homology with the nucleic acid sequence of SEQ ID NO. 1, and said microorganism has excellent recombinant protein expression ability.

Description

Technology Field

[0001] The present application relates to a microorganism of the genus Corynebacterium with increased recombinant protein production capacity and a method for producing a recombinant protein comprising the step of culturing said microorganism. Background Technology

[0003] Corynebacterium glutamicum has industrial advantages as an expression host for the production of recombinant proteins because it is free of endotoxins and has low protease activity. The recombinant proteins are widely used in the food, pharmaceutical, and chemical industries, such as in enzymes and biological agents. Since Corynebacterium glutamicum can secrete proteins produced within the cell into the supernatant of the culture medium through a unique release mechanism, recombinant proteins can be recovered from the culture medium to effectively produce recombinant proteins. Prior art literature

[0005] Korean Registered Patent Publication (KR 10-2501259 B1) The problem to be solved

[0006] The objective of the present application is to provide a microorganism with increased recombinant protein production capacity, in which the activity of a gene having 75% or more sequence homology with the nucleic acid sequence of SEQ ID NO. 1 is weakened.

[0007] Another object of the present application is to a method for producing a recombinant protein comprising the step of culturing the microorganism in a culture medium.

[0008] Another objective of the present application is to provide a composition for producing recombinant proteins comprising one or more selected from the group consisting of the microorganism and the culture medium in which the microorganism is cultured. means of solving the problem

[0010] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not to be considered limited by the specific descriptions provided below. Additionally, a person skilled in the art can recognize or identify numerous equivalents to the specific aspects of this application described in this application using only ordinary experiments. Moreover, such equivalents are intended to be included in this application.

[0012] One aspect provides a microorganism with increased production capacity of a recombinant protein, in which the activity of a gene having 75% or more sequence homology or identity with the nucleic acid sequence of SEQ ID NO. 1 is weakened.

[0013] The gene having the nucleic acid sequence of SEQ ID NO. 1 above may be the NCgl1048 gene of Corynebacterium glutamicum ATCC13032. The NCgl1048 gene may have Ref No. CYL77_05540 in the genome of Corynebacterium glutamicum ATCC13032 (GenBank: CP025533.1 or GenBank: BA000036.3), and the protein encoded by the NCgl1048 gene may have protease activity. The amino acid sequence of the protein encoded by the NCgl1048 gene can be obtained from a known database (e.g., NCBI) (NCBI Reference Sequence: WP_011014118.1 or GenBank: AUI00634.1).

[0014] The inventors of the present application have discovered that microorganisms with weakened activity of a gene having 75% or more sequence homology with the nucleic acid sequence of SEQ ID NO. 1 have increased recombinant protein production capacity.

[0015] In one example, the gene comprises 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 90.5% or more, 91% or more, 91.5% or more, 92% or more, 92.5% or more, 93% or more, 93.5% or more, 94% or more, 94.5% or more, 95% or more, 95.5% or more, 96% or more, 96.5% or more, 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, 99.5% or more, or It may include a nucleic acid sequence having 99.9% or more sequence homology or identity, or may consist of said nucleic acid sequence.

[0016] In one example, the gene comprises (i) the nucleic acid sequence of SEQ ID NO. 1 and 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 90.5% or more, 91% or more, 91.5% or more, 92% or more, 92.5% or more, 93% or more, 93.5% or more, 94% or more, 94.5% or more, 95% or more, 95.5% or more, 96% or more, 96.5% or more, 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, 99.5% (ii) having sequence homology or identity of 99.9% or more; and (ii) encoding a protein having peptidase activity.

[0017] The protein having proteolytic activity encoded by the above gene may be a proteolytic enzyme derived from Corynebacterium glutamicum, such as S1 family peptidase (NCBI Reference Sequence: WP_011014118.1) or peptidase S1 (GenBank: ANU33319.1), but is not limited thereto.

[0018] In one example, the gene may be derived from a microorganism of the genus Corynebacterium. The microorganism of the genus Corynebacterium is Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium crudilactis ( Corynebacterium crudilactis ), Corynebacterium deserti ( Corynebacterium deserti ), Corynebacterium epiphysiens ( Corynebacterium efficiens ), Corynebacterium calunae ( Corynebacterium callunae ), Corynebacterium stationaryis ( Corynebacterium stationis ), Corynebacterium singulare ( Corynebacterium singular ), Corynebacterium halotolerans ( Corynebacterium halotolerans ), Corynebacterium striatum ( Corynebacterium striatum ), Corynebacterium pollutisoli ( Corynebacterium pollutisoli ), Corynebacterium imitans ( Corynebacterium imitans ), Corynebacterium testudinoris ( Corynebacterium testudinoris ), and Corynebacterium flavescens ( Corynebacterium flavescens It may be one or more microorganisms selected from a group consisting of ), but is not limited thereto.

[0019] In one example, the gene may be of Corynebacterium glutamicum, and in one example, the gene may be of Corynebacterium glutamicum ATCC13032 or Corynebacterium glutamicum ATCC13869.

[0020] In one example, the gene may be the NCgl1048 gene (Sequence No. 1) of Corynebacterium glutamicum ATCC13032 (genome sequence, GenBank: CP025533.1 or GenBank: BA000036.3) or the BBD29_05870 gene (Sequence No. 2) derived from Corynebacterium glutamicum ATCC13869 (genome sequence, GenBank: CP016335.1).

[0021] In one embodiment, the gene may include the nucleic acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2, or be composed of the nucleic acid sequence.

[0022] The above microorganism is the nucleic acid sequence of SEQ ID NO. 1, the nucleic acid sequence of SEQ ID NO. 2, or the nucleic acid sequence of SEQ ID NO. 1 and 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 90.5% or more, 91% or more, 91.5% or more, 92% or more, 92.5% or more, 93% or more, 93.5% or more, 94% or more, 94.5% or more, 95% or more, 95.5% or more, 96% or more, 96.5% or more, 97% or more, 97.5% or more, 98% or more, 98.5% or more, It may be a microorganism in which the activity of a polypeptide encoded by a gene containing a nucleic acid sequence having 99% or more, 99.5% or more, or 99.9% or more sequence homology or identity is weakened.

[0023] In one example, the microorganism comprises the amino acid sequence of SEQ ID NO. 75, the amino acid sequence of SEQ ID NO. 76, or the amino acid sequence of SEQ ID NO. 75 and 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 90.5% or more, 91% or more, 91.5% or more, 92% or more, 92.5% or more, 93% or more, 93.5% or more, 94% or more, 94.5% or more, 95% or more, 95.5% or more, 96% or more, 96.5% or more, 97% or more, 97.5% or more, 98% or more, It may be a microorganism with weakened activity of a polypeptide containing an amino acid sequence having 98.5% or more, 99% or more, 99.5% or more, or 99.9% or more sequence homology or identity.

[0024] The above polypeptide may be a polypeptide having peptidase activity, such as S1 family peptidase (NCBI Reference Sequence: WP_011014118.1) or peptidase S1 (GenBank: ANU33319.1), but is not limited thereto.

[0025] The above polypeptide may include the amino acid sequence of SEQ ID NO. 75 or the amino acid sequence of SEQ ID NO. 76, or may consist of the said amino acid sequence.

[0026] If the polypeptide encoded by the above gene exhibits protease activity, it is obvious that a gene encoding a polypeptide in which some amino acid sequences are deleted, modified, substituted, conservatively substituted, or added may also be included in the genes of the present application. Furthermore, if the above polypeptide exhibits protease activity, it is obvious that a polypeptide in which some amino acid sequences are deleted, modified, substituted, conservatively substituted, or added may also be included in the polypeptides of the present application.

[0027] For example, this may involve the addition or deletion of amino acid sequences at the N-terminus, C-terminus, and / or within the aforementioned amino acid sequence, natural mutations, latent mutations, or conservative substitutions that do not affect protease activity. The term “conservative substitution” refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions may have little to no effect on the activity of the protein or polypeptide.

[0028] In the present application, the phrase “a polynucleotide or polypeptide has, includes, consists of, or is essentially composed of a specific nucleic acid sequence (base sequence) or amino acid sequence” may mean that the polynucleotide or polypeptide essentially includes the specific nucleic acid sequence (base sequence) or amino acid sequence, and may be interpreted as including (or not excluding) a “substantially equivalent sequence” in which a variation (deletion, substitution, modification, and / or addition) is applied to the specific nucleic acid sequence (base sequence) or amino acid sequence to maintain the original function and / or intended function of the polynucleotide or polypeptide. In one example, the phrase “a polynucleotide or polypeptide has, includes, is composed of, or is essentially composed of a specific nucleic acid sequence (base sequence) or amino acid sequence” means that the polynucleotide or polypeptide (i) essentially includes the specific nucleic acid sequence (base sequence) or amino acid sequence, or (ii) has 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 90.5% or more, 91% or more, 91.5% or more, 92% or more, 92.5% or more, 93% or more, 93.5% or more, 94% or more, 94.5% or more, 95% or more, 95.5% or more, 96% or more, 96.5% or more, 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, 99.5% or more, or 99.It may mean that it consists of or essentially includes a nucleic acid sequence or amino acid sequence having 9% or more homology or identity and maintains its original function and / or intended function. In one example, the intended function may mean a function that increases (enhances) or imparts the recombinant protein production capacity of a microorganism.

[0029] In this application, 'homology' or 'identity' refers to the degree of similarity between two given amino acid sequences or base sequences and may be expressed as a percentage. The terms homology and identity may often be used interchangeably.

[0030] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard arrangement algorithms, and a default gap penalty established by the program used may be utilized. Practically, homologous or identical sequences can generally be hybridized with the entire sequence or a part thereof under moderate or high stringent conditions. It is evident that hybridization also includes hybridization with polynucleotides containing common codons or codons that account for codon degeneracy.

[0031] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using a known computer algorithm, such as the “FASTA” program, using default parameters as in, for example, Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] (Including Academic Press, San Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST from the National Biotechnology Information Database Center or ClustalW.

[0032] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as that described in, for example, Smith and Waterman, Adv. Appl. Math (1981) 2:482, or Needleman et al. (1970), J Mol Biol. 48:443. In summary, a GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). The default parameters for a GAP program are (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), or Gribskov et al. (1986) Nucl. Acids Res. 14: A weighted comparison matrix of 6745 (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0034] In this application, the term “microorganism (or strain)” includes both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and may be microorganisms in which specific mechanisms are weakened or strengthened due to causes such as the insertion of external genes or the weakening of the activity of endogenous genes, and may be microorganisms that include genetic modification for the production of a desired polypeptide, protein, or product.

[0035] In this application, the term “weakening” of a gene or polypeptide encompasses the concept of either reduced activity or lack of activity relative to its intrinsic activity. Such weakening may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation. The “intrinsic activity” refers to the activity of a specific gene or polypeptide originally possessed by the parent strain, wild-type, or non-modified microorganism prior to phenotypic change, in cases where phenotypic changes occur due to genetic mutations caused by natural or artificial factors. This may be used interchangeably with “pre-modification activity.” The statement that the activity of a gene or polypeptide is “inactivated, deficient, reduced, down-regulated, reduced, or attenuated” relative to its intrinsic activity may mean that the expression of said gene or polypeptide has decreased compared to the parent strain or non-modified microorganism prior to phenotypic change.

[0036] In one example, the attenuation of the activity of a gene having 75% or more sequence homology with the nucleic acid sequence of SEQ ID NO. 1 or a polypeptide encoded by said gene may include cases where the expression of said gene is inhibited (e.g., inhibition of transcription into mRNA or translation of mRNA), and the concentration (expression level) of the polypeptide encoded by said gene within the cell is low compared to a control group (wild type or a microorganism in which the activity of said gene is not attenuated), or cases where the polypeptide encoded by said gene is not expressed at all. Such attenuation of the activity of the gene or polypeptide may be performed by any method known in the art, but is not limited thereto, and may be achieved by the application of various 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.).

[0037] In one example, the weakening of the gene or polypeptide is

[0038] 1) Deletion of all or part of the gene encoding a polypeptide;

[0039] 2) Modification of the expression regulatory region (or expression regulatory sequence) to reduce the expression of the gene encoding the polypeptide;

[0040] 3) Modification of the amino acid sequence constituting the polypeptide so as to remove or weaken the activity of the polypeptide (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence);

[0041] 4) Modification of the gene sequence encoding the polypeptide so as to remove or weaken the activity of the polypeptide (e.g., deletion / substitution / addition of one or more nucleotide bases on the nucleotide base sequence of the polypeptide gene to code for a polypeptide modified so as to remove or weaken the activity of the polypeptide);

[0042] 5) A modification of the nucleotide sequence encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide;

[0043] 6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide;

[0044] 7) Addition of a sequence complementary to the Shine-Dalgarno sequence to the upstream end of the Shine-Dalgarno sequence of a polypeptide-coding gene to form a secondary structure incapable of ribosome attachment;

[0045] 8) Addition of a reverse-transcribed promoter to the 3' end of the ORF (open reading frame) of a gene sequence encoding a polypeptide (Reverse transcription engineering, RTE);

[0046] 9) Regulation of the cellular localization of polypeptides; or

[0047] 10) It may be based on two or more combinations selected from 1) to 9) above, but is not specifically limited thereto.

[0048] For example, the deletion of part or all of the gene encoding the above 1) polypeptide may be the removal of the entire polynucleotide encoding the intrinsic target polypeptide within the chromosome, replacement with a polynucleotide in which some nucleotides have been deleted, or replacement with a marker gene.

[0049] Additionally, modification of the expression regulatory region (or expression regulatory sequence) described in 2) above may be a deletion, insertion, non-conservative or conservative substitution, or a combination thereof, resulting in a mutation on the expression regulatory region (or expression regulatory sequence), or replacement with a sequence having weaker activity. The expression regulatory region includes, 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.

[0050] In addition, the above 3) modification of the nucleotide sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide may, for example, be a substitution with a nucleotide sequence encoding another start codon that has a lower polypeptide expression rate compared to the intrinsic start codon, but is not limited thereto.

[0051] In addition, modifications to the amino acid sequences or polynucleotide sequences of 4) and 5) above may involve the occurrence of sequence variations in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to weaken the activity of the polypeptide, or may involve replacement with an amino acid sequence or polynucleotide sequence modified to have weaker activity or an amino acid sequence or polynucleotide sequence modified to have no activity, but are not limited thereto. For example, gene expression may be inhibited or weakened by introducing a variation within the polynucleotide sequence to form a stop codon, but are not limited thereto. The "stop codon" is a codon on the mRNA that does not specify an amino acid and acts as a signal indicating that the protein synthesis process has ended; generally, three types, UAA, UAG, and UGA, may be used as stop codons.

[0052] For the introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide 6) above, refer to the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

[0053] 7) In order to form a secondary structure in which ribosome attachment is impossible, the addition of a sequence complementary to the Shine-Dalgarno sequence to the front of the Shine-Dalgarno sequence of a gene encoding a polypeptide may make mRNA translation impossible or slow it down.

[0054] Reverse transcription engineering (RTE) of a promoter that is transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide above may weaken the activity by creating an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide.

[0055] The above 9) regulation of the intracellular localization of the polypeptide may involve targeting the polypeptide to a specific intracellular organelle or a specific intracellular space. For example, it may involve targeting to the periplasm or cytoplasm through the addition or removal of a leader sequence that functions for the targeting of the polypeptide, but is not limited thereto.

[0056] Modification of part or all of a gene in the microorganism of the present application may be induced by (a) homologous recombination using a vector for chromosome insertion within the microorganism or genome editing using engineered nucleases (e.g., CRISPR-Cas9) and / or (b) treatment by light and / or chemicals such as ultraviolet rays and radiation, but is not limited thereto. The method of modifying part or all of the gene may include methods using DNA recombination technology. For example, deletion of part or all of the gene may be achieved by inducing homologous recombination by injecting a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism. The injected nucleotide sequence or vector may include a dominant selection marker, but is not limited thereto.

[0057] In one example, the attenuation of the gene may be caused by a recombination method. The recombination method may include homologous recombination. The homologous recombination method may induce homologous recombination between the sequence of the gene and the endogenous gene in the microorganism when a vector containing a partial sequence of a gene encoding a polypeptide is transformed into the microorganism and cultured in the presence of a selection marker product.

[0059] The microorganism provided in the present application may be a microorganism with weakened gene activity having 75% or more sequence homology with the nucleic acid sequence of SEQ ID NO. 1, specifically a microorganism genetically modified through a vector to weaken the gene activity (e.g., a recombinant microorganism), but is not limited thereto.

[0060] The above microorganism may be a microorganism that has the ability to produce recombinant proteins or has an enhanced ability to produce recombinant proteins. That the above microorganism has the ability to produce recombinant proteins or has an enhanced ability to produce recombinant proteins means that the ability to produce recombinant proteins is newly conferred compared to a control group (non-modified microorganism without the ability to produce recombinant proteins, cells prior to recombination, parent strains, and / or wild-type strains, etc.), or that the ability to produce recombinant proteins is enhanced compared to a control group that has the ability to produce recombinant proteins (cells prior to recombination, parent strains, etc.).

[0061] In this application, "non-modified microorganism" does not exclude strains containing mutations that may naturally occur in microorganisms, and may refer to wild-type strains or natural-type strains themselves, or strains before their traits are altered by genetic mutations caused by natural or artificial factors. For example, the non-modified microorganism may refer to a strain in which the activity of the gene is not weakened or is not weakened, depending on one example. The term "non-modified microorganism" may be used interchangeably with "pre-modification strain," "pre-modification microorganism," "non-variant strain," "non-modified strain," "non-variant microorganism," or "reference microorganism." In one example, the non-modified microorganism, which is the target strain for comparing whether the recombinant protein production capacity increases, may be the Corynebacterium glutamicum ATCC13032 strain, the Corynebacterium glutamicum ATCC13869 strain, etc., but is not limited thereto.

[0062] The above microorganism (or strain, recombinant cell) may include a mutation that further increases the production of recombinant protein, and the location of the mutation and / or the type of gene and / or protein subject to the mutation may be included without limitation as long as it increases the production of recombinant protein. The above recombinant cell may be used without limitation as long as it is a cell capable of transformation.

[0063] The above recombinant protein may refer to any type of protein having biologically useful activity intended to be produced using the protein expression, secretion, and production system of microorganisms, and may refer to any protein that can be expressed in a host cell by inserting a polynucleotide encoding the said protein into a recombinant vector as a protein intended to be produced in large quantities by a person skilled in the art. For example, the said target protein may be collagen, collagen-derived polypeptide, hormone, hormone analog, cytokine, antigen, antigen-binding fragment, antibody, cell receptor, enzyme, transport protein, structural protein, serum, cell protein, antimicrobial peptide, antioxidant peptide, etc.

[0064] In one example, the recombinant protein is a human insulin precursor, human FGF2 (Fibroblast Growth Factor), human EGF (Epidermal Growth Factor), collagen (collagen derived from, e.g., humans, pigs, cattle, chickens, fish, etc.), collagen-derived polypeptide (a polypeptide containing a collagen-derived amino acid sequence), etanercept, epoetin alpha, infliximab, interferon alpha, insulin lispro, filgrastim, imiglucerase, glatiramer acetate, rituximab, pegfilgrastim, insulin grastim, adalimumab, trastuzumab, Bevacizumab, ranibizumab, insulin, growth hormone, tumor necrosis factor-alpha, interleukin-7, insulin-like growth factor 2, interferon gamma, interferon alpha, interleukin-2, osteogenic protein, recombinant plasminogen-activator, bone morphogenetic protein 2, antifungal peptide,Tissue plasminogen activator, immunoglobulin G, erythropoietin, granulocyte-macrophage stimulating factor, granulocyte-colony stimulating factor, muromomab, abciximab, daclizumab, basiliximab, palivizumab, ibritumomab, omalizumab, epalizumab, tositumomab, cetuximab, natalizumab, alkaline phosphatase (alkaline It may be one or more selected from the group consisting of phosphatase (PhoA), levan fructotransferase (LFT), cellulose binding domain, cholera toxin B, organophosphohydrolase, calcitonin, blood coagulation factors, hirudin, and monoclonal antibody 5T4, but is not limited thereto.

[0065] In one example, the recombinant protein may further include a tag for protein purification, expression, dissolution, or detection enhancement of the target protein. Various tags that can be used for these purposes are known in the industry and may be, for example, GST tags, FLAG tags, polyarginine tags, polyhistidine tags, for example, 6His-tags, MBP tags, S-tags, influenza virus HA tags, thioredoxin tags, or Staphylococcus protein A tags.

[0066] In one example, the above human insulin precursor may be used by obtaining an amino acid sequence from a known database (UniProtKB / Swiss-Prot: P01308.1), or by substituting the protein sequence from 25 to 110, excluding the protein secretion signal sequence from 1 to 24 in the UniProtKB / Swiss-Prot: P01308.1 sequence, with the protein sequence from 55 to 89 specified as C-peptide, with AAK (T Kjeldsen et al. 1996, doi: 10.1016 / 0378-1119(95)00822-5.) (e.g., the amino acid sequence of SEQ ID NO. 51), but is not limited thereto.

[0067] In one example, the amino acid sequence of the above-mentioned human FGF2 (Human FGF2) may be obtained and used from a known database (UniProtKB / Swiss-Prot: P09038.3), or the protein sequence from 143 to 288 may be used excluding the amino acid sequence from 1 to 142 specified as Propeptide in the above-mentioned UniProtKB / Swiss-Prot: P09038.3 sequence (e.g., the amino acid sequence of SEQ ID NO. 59), but is not limited thereto.

[0068] In one example, the above human EGF (Human EGF) may be used by obtaining an amino acid sequence from a known database (UniProtKB / Swiss-Prot: P01133.2), or the protein sequence from 971 to 1023 specified as PRO_0000007541 in the above UniProtKB / Swiss-Prot: P01133.2 sequence may be used (e.g., the amino acid sequence of SEQ ID NO. 65), but is not limited thereto.

[0069] In one example, the recombinant protein may be a protein and / or peptide having a desired activity in vivo (e.g., prevention, alleviation, and / or therapeutic activity of a specific disease or symptom, or activity to replace a substance required by the body), and may be one or more selected from the group consisting of, for example, proteins or peptides of enzymatic activity (e.g., protease, kinase, phosphatase, etc.), receptor proteins or peptides, transporter proteins or peptides, bactericidal and / or endotoxin-binding polypeptides, structural proteins or peptides, immunopeptides, toxins, antibiotics, hormones, growth factors, vaccines, etc. In one example, the desired polypeptide may be one or more selected from the group consisting of hormones, cytokines, tissue plasminogen activators, immunoglobulins (e.g., antibodies or their antigen-binding fragments or variants). The above immunoglobulin may be of any isotype (e.g., IgA, IgD, IgG, IgM, or IgE) and may be, for example, an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4). The above antigen-binding fragment is a fragment possessing the antigen-binding ability of the original antibody and may be any fragment of an antibody containing about 20 or more amino acids, for example, about 100 or more amino acids. The above antigen-binding fragment may be one or more selected from the group consisting of the antigen-binding site of the antibody, e.g., CDS, Fab fragment, F(ab)2 fragment, Fv, scFv, multibody containing multiple binding domains (e.g., diabody, triabody, tetrabody, etc.), single-domain antibody, affibody, etc. The above antibody variant is a derivative of an antibody or antibody fragment having the same binding function as the antibody but having an amino acid sequence modified from the original antibody.The antibody and / or antigen-binding fragment may be, for example, a mouse antibody, a human antibody, a chimeric antibody, a humanized antibody, or a human antibody. The antibody and / or antigen-binding fragment may be isolated from living organisms or may be of non-naturally occurring origin. The antibody and / or antigen-binding fragment may be produced synthetically or recombinantly. The antibody may be a monoclonal antibody. In another embodiment, the target polypeptide may be one or more selected from the group consisting of insulin, human growth hormone (hGH), insulin-like growth factor, EGF, VERF and various growth factors, various receptors, tissue plasminogen activator (tPA), erythropoietin (EPO), cytokines (e.g., interleukins such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18), interferon (IFN)-alpha, -beta, -gamma, -omega or -tau, tumor necrosis factor (TNF) such as TNF-alpha, beta or gamma, TRAIL, G-CSF, GM-CSF, M-CSF, MCP-1, etc.

[0070] The recombinant vector may additionally include a gene encoding a secretion signal peptide. For the expression and secretion of the recombinant protein through the transport system of microorganisms, the recombinant protein may be linked to a secretion signal peptide at its N' end. The recombinant vector may include a gene encoding the recombinant protein and a gene encoding the secretion signal peptide in an appropriate order so as to express a form in which a secretion signal peptide is linked to the N' end of the recombinant protein.

[0071] The above secretion signal peptides may be used without limitation as long as they are secretion signal peptides capable of inducing the secretion of recombinant proteins in microorganisms, and in one example, Cg0955 secretion signal peptide, CgR0079 secretion signal peptide, CgR0120 secretion signal peptide, CgR0124 secretion signal peptide, CgR0900 secretion signal peptide, CgR0949 secretion signal peptide, CgR1023 secretion signal peptide, CgR1448 secretion signal peptide, CgR2137 secretion signal peptide, CgR2627 secretion signal peptide, CgR2926 secretion signal peptide, E. coli ( E. coli ) derived TorA secretory signaling peptide and Atrobacter globiformis ( Arthrobacter globiformis It may be one or more selected from the group consisting of ) derived IMD secretion signal peptides, but is not limited thereto. That is, any secretion signal peptide capable of inducing the secretion of recombinant proteins in microorganisms may be used without limitation.

[0072] The recombinant vector may include a promoter operably linked to the gene encoding the recombinant protein and / or the gene encoding the secretion signal peptide for the expression of the gene encoding the recombinant protein and / or the gene encoding the secretion signal peptide.

[0073] In this specification, "promoter" may mean an untranscribed nucleotide sequence upstream or downstream of a coding region that includes a binding site for polymerase and has transcription initiation activity for a promoter target gene (e.g., a gene encoding a recombinant protein or a tween-arginine transposase, etc.), such as a DNA region to which polymerase binds to initiate transcription of the gene.

[0074] The above promoter may use any promoter sequence commonly used for gene expression, and examples of known promoters include, but are not limited to, CJ1 to CJ7 promoters (US Patent 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13(sm3) promoter (US Patent 10584338 B2), O2 promoter (US Patent 10273491 B2), tkt promoter, yccA promoter, etc.

[0075] In one example, the promoter may be used without limitation as long as it can regulate transcription initiation in a cell, e.g., a viral cell, a bacterial cell, a eukaryotic cell, an insect cell, a plant cell, or an animal cell. For example, the promoter may be one or more selected from the group consisting of, but not limited to, promoters of prokaryotic or mammalian viruses such as the CMV promoter (cytomegalovirus promoter), SV40 promoter, adenovirus promoter (major late promoter), pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, vaccinia virus 7.5K promoter, and HSV tk promoter, and animal cell promoters such as the metallothionin promoter and the beta-actin promoter.

[0076] In one example, the microorganism with increased recombinant protein production capacity may have increased recombinant protein production capacity by about 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 80% or more, 90% or more, 100% or more (there is no specific upper limit, and it may be, for example, about 1000% or less). In another example, the microorganism with increased recombinant protein production capacity may have a recombinant protein production capacity that is increased by about 1.1 times or more, 1.15 times or more, 1.2 times or more, 1.25 times or more, 1.3 times or more, 1.35 times or more, 1.4 times or more, 1.45 times or more, 1.5 times or more, 1.55 times or more, 1.6 times or more, 1.65 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, or 2 times or more compared to the parent strain before mutation or the non-mutated microorganism.

[0077] The above term “about” refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes, but is not limited to, all numerical values ​​within a range equivalent to or similar to the numerical value following the term “about.”

[0079] The above microorganism may be a microorganism of the genus Corynebacterium.

[0080] The above-mentioned microorganism of the genus Corynebacterium is Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium crudilactis ( Corynebacterium crudilactis ), Corynebacterium deserti ( Corynebacterium deserti ), Corynebacterium epiphysiens ( Corynebacterium efficiens ), Corynebacterium calunae ( Corynebacterium callunae ), Corynebacterium stationaryis ( Corynebacterium stationis ), Corynebacterium singulare ( Corynebacterium singular ), Corynebacterium halotolerans ( Corynebacterium halotolerans ), Corynebacterium striatum ( Corynebacterium striatum ), Corynebacterium pollutisoli ( Corynebacterium pollutisoli ), Corynebacterium imitans ( Corynebacterium imitans ), Corynebacterium testudinoris ( Corynebacterium testudinoris ), and Corynebacterium flavescens ( Corynebacterium flavescens It may be one or more microorganisms selected from a group consisting of ), but is not limited thereto.

[0081] In one example, the microorganism of the genus Corynebacterium may be Corynebacterium glutamicum.

[0083] Another aspect provides a method for producing (or manufacturing) a recombinant protein comprising the step of culturing the microorganism in a culture medium.

[0084] The method for producing a recombinant protein of the present application may include the step of culturing the microorganism in a culture medium.

[0085] In this application, "culture" means growing the microorganism, such as a strain of Corynebacterium glutamicum, under appropriately controlled environmental conditions. The culture process may be carried out according to suitable media and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culture may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0086] In this application, "medium" refers to a substance mixed with nutrients as the main component required to culture the microorganism, such as a strain of Corynebacterium glutamicum, and supplies nutrients and growth factors, including water, which is indispensable for survival and growth. Specifically, the medium and other culture conditions used for culturing the microorganism of this application may be any medium used for culturing ordinary microorganisms without special limitations, but the microorganism of this application may be cultured under aerobic conditions while controlling the temperature, pH, etc., in a conventional medium containing a suitable carbon source, nitrogen source, phosphorus, inorganic compounds, amino acids, and / or vitamins.

[0087] Specifically, culture media for the above-mentioned microorganisms, such as strains of the genus Corynebacterium, can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D. Corynebacterium, USA, 1981)].

[0088] In the present application, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvate, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. Additionally, natural organic nutrient sources such as starch hydrolysate, molasses (e.g., blackstrap molasses), rice husk, cassava, sugarcane residue, and corn steeping liquid may be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted into reducing sugars) may be used, and other carbon sources in appropriate amounts may be used without limitation. These carbon sources may be used individually or in combination of two or more types, but are not limited thereto.

[0089] The above nitrogen sources may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc., amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0090] The above ingredients may include monopotassium phosphate, dipotassium phosphate, or corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc., and may also include amino acids, vitamins, and / or suitable precursors. These components or precursors may be added to the culture medium in a batch or continuous manner. However, they are not limited thereto.

[0091] In addition, during the cultivation of the microorganisms, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc., may be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during cultivation, foam generation may be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, to maintain an aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen, or carbon dioxide gas may be injected without gas injection to maintain an anaerobic and microaerobic state, but is not limited thereto.

[0092] In the culture of the present application, the culture temperature can be maintained at 20 to 45°C, specifically 25 to 40°C, and culture can be performed for about 10 to 160 hours, but is not limited thereto.

[0093] The recombinant protein produced by the culture of the present application may be secreted into the culture medium or remain within the cell.

[0094] The method for producing a recombinant protein according to the present application may additionally include, for example, the step of preparing the microorganism, the step of preparing a medium for culturing the microorganism, or a combination thereof (in any order), prior to the culturing step.

[0095] The method for producing a recombinant protein according to the present application may further include a step of recovering the recombinant protein from a culture medium (a culture medium in which the culture is performed) or a microorganism (e.g., a strain of the genus Corynebacterium) according to the culture. The recovery step may be additionally included after the culture step.

[0096] The above recovery may involve collecting the desired recombinant protein using a suitable method known in the art according to the culture method of the microorganism of the present application, such as a batch, continuous, or fed-batch culture method. For example, various chromatographs such as centrifugation, filtration, treatment with a crystallizing protein precipitating agent (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof may be used, and the desired recombinant protein may be recovered from the culture medium or microorganism using a suitable method known in the art.

[0097] In addition, the method for producing a recombinant protein according to the present application may additionally include a purification step. The purification may be performed using a suitable method known in the art. In one example, where the method for producing a recombinant protein according to the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the order, or simultaneously or integrated into a single step, but are not limited thereto.

[0099] Another aspect provides a composition for producing recombinant proteins comprising one or more selected from the group consisting of the microorganism and the culture medium in which the microorganism is cultured.

[0100] Another aspect provides a use for producing recombinant proteins using one or more selected from the group consisting of the microorganism and the culture medium in which the microorganism is cultured.

[0101] The composition of the present application may further include any suitable excipients commonly used in compositions for producing recombinant proteins, and such excipients may be, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents, but are not limited thereto. Effects of the invention

[0103] The present application provides a microorganism of the genus Corynebacterium with weakened gene activity having 75% or more sequence homology with the nucleic acid sequence of SEQ ID NO. 1, and said microorganism has excellent recombinant protein expression ability. Brief explanation of the drawing

[0105] Figure 1 shows the analysis of proteins present in the supernatant of a culture medium in which the Corynebacterium glutamicum ATCC13032 strain was cultured. Specific details for implementing the invention

[0106] The present invention will be explained in more detail below through the following examples. However, these are merely illustrative of the invention, and the scope of the invention is not limited by these examples.

[0108] Example 1. Protein analysis using LC-MS and selection of candidate candidates

[0109] To analyze the proteins present in the culture medium of the wild-type Corynebacterium glutamicum ATCC13032 strain, one colony of the strain was plated onto the compound agar plate below and solid-state cultured at 30°C for 16 hours. This was then inoculated with 1 blue loop (SPL, Cat: SPL90010) into a 250 ml flask containing 25 ml of culture medium and cultured with shaking at 200 rpm for 48 hours at 30°C.

[0110] Compound Plate Medium (pH 7.0)

[0111] Glucose 10 g, Peptone 10 g, Beef extract 5 g, Yeast extract 5 g, Brain Heart Infusion 18.5 g, NaCl 2.5 g, Urea 2 g, Sorbitol 91 g, Agar 20 g (based on 1 liter of distilled water)

[0112] <Culture Medium (pH 7.0)>

[0113] Glucose 63 g, ammonium sulfate 28 g, MgSO4.7H2O 1.2 g, KH2PO4 1.1 g, yeast extract 1 g, d-biotin 1 mg, thiamine-HCl 25 mg, calcium-pantothenic acid 25 mg, MnSO4.5H2O 10 mg, ZnSO4.5H2O 2.5 mg, CuSO4.5H2O 2.5 mg, FeSO4.5H2O 10 mg, CaCO3 30 g (based on 1 liter of distilled water)

[0114] After the fermentation was completed, proteins present in the supernatant of the culture medium were analyzed as follows. The supernatant of the culture medium, from which the cells had settled by centrifugation, was separated using a 4–20% Tris-glycine polyacrylamide gel via the SDS-PAGE (Laemmli, 1970) method and stained using DIRECTBLUE(TM) gel staining solution (SCGBIOMAX, Cat: BDS-1000).

[0115] The stained results and protein expression patterns are shown in Figure 1. The bands present on the gel were divided into eight major groups, and proteins were identified using the in-gel digestion method (Rosenfeld, 1992) and LC-MS / MS (LC: ACQUITY UPLC H-Class, MS: Vion IMS QTof MS (Waters Co., Milford, MA, USA). The whole genome sequence of Corynebacterium glutamicum ATCC13032 was referenced from a known database (NCBI, GenBank: BA000036.3), and the signal peptide sequence prediction and signal peptide possession potential of the detected proteins were confirmed by referring to a known database (SignalP 5.0). The detected proteins were named proteins 01 to 08 as shown in Table 1 below.

[0116] Band Gene Annotation NCBI reference coding (Protein sequence) SignalP (5.0) Size (kDa) 명명 01 NCgl2525 (cg2896, Cgl2614) WP_011265980 0.7506 78.8 protein 01 02 NCgl1480 (cg1735, Cgl1538) WP_011014435 0.8577 63.0 protein 02 03 NCgl2107 (Cg2401, Cgl2187) WP_011014944 0.8075 38.1 protein 03 04 NCgl0757 (Cg0901, Cgl0791) WP_011013891 0.9103 36.8 protein 04 05 NCgl1337 (cg1577, Cgl1391) WP_003858702 0.7237 33.5 Protein 05 06 NCgl0339 (cg0339, Cgl0282) WP_011013586 - 31.4 Protein 06 07 NCgl1048 (cg1243, Cgl1093) WP_011014118 0.5306 28.2 Protein 07 08 NCgl0508 (cg0620, Cgl0530) WP_011013710 0.8899 21.8 Protein 08

[0117] Example 2. Preparation of a strain having a deletion in the gene encoding the protein selected in Example 1.

[0118] In order to increase the secretory production capacity of the recombinant protein, a vector was constructed as follows to delete the protein in the culture medium analyzed in Example 1.

[0119] A deletion vector to delete the NCgl2525 gene encoding protein O1 in Table 1 above was constructed as follows. First, using the genome of wild-type Corynebacterium glutamicum ATCC13032 as a template, the homologous recombinant A arm was amplified using the primer pair of SEQ ID NO. 3 and SEQ ID NO. 4, and the homologous recombinant B arm was amplified using the primer pair of SEQ ID NO. 5 and SEQ ID NO. 6 by the PCR method. The obtained gene fragments and the vector pDC24 (SEQ ID NO. 77), which was cleaved with SalI and BamHI restriction enzymes, were cloned using the Gibson assembly method, transformed into E. coli DH5α, and plated on LB solid medium containing 25 mg / L kanamycin. PCR was performed using the primer pair of SEQ ID NO. 39 and SEQ ID NO. 40 to select transformed colonies. Subsequently, a plasmid was obtained from a selected colony using a commonly known plasmid extraction method and named pDC24-ΔNCgl2525. Deletion vectors of genes encoding proteins O2 to O8 were constructed using substantially the same method as above, and the primers used to construct each deletion vector are shown in Table 2 below.

[0120] vector Homologous arm Sequence number nucleotide sequence pDC24-ΔNCgl2525 A Sequence No. 03 atgcctgcaggtcgacCTGCCTACAAGGATCTCTT Sequence No. 04 GTTATCCATCTTGAAAAAAGCATTCCTTGTGGGCGCTTTGTC B Sequence number 05 GACAAAGCGCCCACAAGGAATGCTTTTTCAAGATGGATAAC Sequence number 06 cggtacccggggatccCAAACGCTACAATACCTTC pDC24-ΔNCgl1480 A Sequence number 07 atgcctgcaggtcgacGCCATGGTGGAGACAAAAC Sequence number 08 CCAGGTAAAAGGTGTCACGTTTCCTCCTATGAATCTTGA B Sequence number 09 TCAAGATTCATAGGAGGAAACGTGACACCTTTTACCTGG Sequence number 10 cggtacccggggatccTTATAGCACTTTCGGTAGG pDC24-ΔNCgl2107 A Sequence number 15 atgcctgcaggtcgacCCTTTCCTCGGCACTAAAC Sequence number 16 GTTAGAGTAGTGGAGTTGCGAAATAGTTCGTCAGGAGAATC B Sequence number 17 GATTCTCCTGACGAACTATTTCGCAACTCCACTACTCTAAC Sequence number 18 cggtacccggggatccCCAGGTCTTAACAACGTTC pDC24-ΔNCgl0757 A Sequence No. 19 atgcctgcaggtcgacGAGAGAATACCCCAGGGAG Sequence number 20 AGATATCTTTAGTTCACTTTCGAACGGCGGATTTTCTAAATT B Sequence number 21 GAAAGTGAACTAAAGATATCTAATTTAGAAAATCCGCCGTTC Sequence number 22 cggtacccggggatccACCCTTCAAAATCAGCATC pDC24-ΔNCgl1337 A Sequence number 23 atgcctgcaggtcgacTCGGCGAGAACAAGAAATC Sequence No. 24 TAATGCTTTTCGACGTCATCTGTTTTGCTTCTCCTTTTCCC B Sequence number 25 GGGAAAAGGAGAAGCAAACAGATGACGTCGAAAAGCATTA Sequence number 26 cggtacccggggatccTTCTCAATCCCCTTTGGTC pDC24-ΔNCgl0339 A Sequence number 27 atgcctgcaggtcgacAGCTTCACTATTCTCACTGT Sequence number 28 CTGTCCTGCTTTACCAATGCGACAATAACAAGAATGCTTA B Sequence number 29 TAAGCATTCTTGTTATTGTCGCATTGGTAAAGCAGGACAG Sequence number 30 cggtacccggggatccTTATTAGGCGGTATCTTGGAC pDC24-ΔNCgl1048 A Sequence No. 31 atgcctgcaggtcgacGATCTTTATGGGGCGTTTTC Sequence No. 32 CAACCATCTAGACTGTTCTTTAATATGCTGATCTCCCTGC B Sequence number 33 GCAGGGAGATCAGCATATTAAAGAACAGTCTAGATGGTTG Sequence No. 34 cggtacccggggatccCAAAATGATCAACGCCATC pDC24-ΔNCgl0508 A Sequence number 35 atgcctgcaggtcgacGCTCAGGCCAAGCACAACCA Sequence number 36 GAAGCTTTAGGTTAGGTTGCGGCTTTGAACCTTTCATTTTC B Sequence number 37 GAAAATGAAAGGTTCAAGCCGCAACCTAACCTAAAGCTTC Sequence number 38 cggtacccggggatccTCATCCTCGGCAACACCGGA

[0121] To construct a strain deficient in the NCgl2525 gene encoding the above protein 01, the above pDC24-ΔNCgl2525 vector was transformed into wild-type Corynebacterium glutamicum ATCC13032 by electroporation, and a strain deficient in the NCgl2525 gene was obtained through a secondary crossover process. PCR was performed using the primer pair of SEQ ID NOs. 3 and 6, which can amplify adjacent regions including the site where the gene was inserted, to confirm the genetic modification. The microorganism obtained in this manner was named ATCC13032::ΔNCgl2525.

[0122] In order to produce strains with a deletion of the gene encoding proteins O2 to O8 in substantially the same manner as above, strains were produced using each deletion vector, and the produced strains are shown in Table 3 below.

[0123] vector strain pDC24-ΔNCgl2525 ATCC13032::ΔNCgl2525 pDC24-ΔNCgl1480 ATCC13032::ΔNCgl1480 pDC24-ΔNCgl2107 ATCC13032::ΔNCgl2107 pDC24-ΔNCgl0757 ATCC13032::ΔNCgl0757 pDC24-ΔNCgl1337 ATCC13032::ΔNCgl1337 pDC24-ΔNCgl0339 ATCC13032::ΔNCgl0339 pDC24-ΔNCgl1048 ATCC13032::ΔNCgl1048 pDC24-ΔNCgl0508 ATCC13032::ΔNCgl0508

[0124] Example 3. Preparation of a strain expressing GFP (green fluorescent protein) protein

[0125] To construct a GFP-expressing strain, a vector for expressing the GFP expression cassette on the genome was constructed as follows. The GFP protein sequence used was QKN22541.1 from NCBI (GenBank: QKN22541.1, SEQ ID NO. 41). The nucleotide sequence for expressing the GFP protein of SEQ ID NO. 41 in Corynebacterium glutamicum ATCC13032 is as shown in SEQ ID NO. 42.

[0126] order GFP protein (Sequence No. 41) MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIEL KGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGNSITSYSIHYTKL Gene encoding GFP protein (Sequence No. 42) ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGGAATTCGATAACTTCGTATAGCATACATTATACGAAGTTA

[0127] Using the genome of wild-type Corynebacterium glutamicum ATCC13032 as a template, the homologous recombinant A arm was amplified using the primer pair of SEQ ID NO. 43 and SEQ ID NO. 44, and the homologous recombinant B arm was amplified using the primer pair of SEQ ID NO. 45 and SEQ ID NO. 46 by the PCR method. Using the genome of wild-type Corynebacterium glutamicum ATCC13032 as a template, the promoter sequence of the gapA gene (NCgl1526) was amplified using the primer pair of SEQ ID NO. 47 and SEQ ID NO. 48 by the PCR method. To express the effluxing GFP, the protein sequence of NCgl2185 analyzed as the TAT pathway secretion signal sequence via SignalP 5.0 was used. Using the primer pair of SEQ ID NO. 49 and SEQ ID NO. 50, the gene fragment of the protein secretion signal sequence of the NCgl2185 (cg2485 and Cgl2265) gene was amplified by the PCR method. The obtained gene fragment, the GFP gene fragment, and the vector pDC24, which was cleaved with SalI and BamHI restriction enzymes, were cloned using the Gibson assembly method, transformed into E. coli DH5α, and plated on LB solid medium containing 25 mg / L of kanamycin. PCR was performed using the primer pair of SEQ ID NO. 39 and SEQ ID NO. 40 to select transformed colonies. Subsequently, a plasmid was obtained from the selected colonies using a commonly known plasmid extraction method and named pDC24-PgapA_SPn2185_GFP.

[0128] 프라이머 서열 서열번호 39 TATTACGCCAGCTGGCGAAA 서열번호 40 GCTTTACACTTTATGCTTCC 서열번호 43 ATGCCTGCAGGTCGACAGATCATGGTGCCGACAAAG 서열번호 44 TCAATCATCTAAATTTCTTCTAGGGTTTGATGCAAAAATT 서열번호 45 TACATTATACGAAGTTATAGCTAGGTTGGATGCAAAAATC 서열번호 46 CGGTACCCGGGGATCCGAAGCTCAATGCCAGATACT Sequence number 47 AATTTTTGCATCAAACCCTAGAAGAAATTTAGATGATTGA Sequence number 48 CGTCTGCTTAACTGTGGCATGTTGTGTCTCCTCTAAAGAT Sequence number 49 ATCTTTAGAGGAGACACAACATGCCACAGTTAAGCAGACG Sequence number 50 AGCTCCTCGCCCTTGCTCACAGCGCGTGCAGGTGTGCCCG

[0129] To construct a strain expressing GFP, the above pDC24-PgapA_SPn2185_GFP vector was transformed into wild-type Corynebacterium glutamicum ATCC13032 and ATCC13032::ΔNCgl2525 using the electro-pulse method (Appl. Microbiol. Biothcenol.(1999) 52:541-545), and a strain with the inserted GFP expression cassette was obtained through a secondary crossover process. PCR was performed using the primer pair SEQ ID NO. 43 and SEQ ID NO. 46, which can amplify adjacent regions including the site where the gene was inserted, to confirm the genetic modification. The microorganism obtained in this way was named ATCC13032::ΔNCgl02525::PgapA_SPn2185_GFP.

[0130] In order to construct strains that express GFP protein and have genes encoding proteins O2 to O8 deleted in substantially the same manner as above, strains were constructed using each deletion vector, and the constructed strains are shown in Table 6 below.

[0131] base strain GFP-expressing strain Corynebacterium glutamicum ATCC13032 ATCC13032::PgapA_SPn2185_GFP ATCC13032::ΔNCgl2525 ATCC13032::ΔNCgl2525::PgapA_SPn2185_GFP ATCC13032::ΔNCgl1480 ATCC13032::ΔNCgl1480::PgapA_SPn2185_GFP ATCC13032::ΔNCgl2107 ATCC13032::ΔNCgl2107::PgapA_SPn2185_GFP ATCC13032::ΔNCgl0757 ATCC13032::ΔNCgl0757::PgapA_SPn2185_GFP ATCC13032::ΔNCgl1337 ATCC13032::ΔNCgl1337::PgapA_SPn2185_GFP ATCC13032::ΔNCgl0339 ATCC13032::ΔNCgl0339::PgapA_SPn2185_GFP ATCC13032::ΔNCgl1048 ATCC13032::ΔNCgl1048::PgapA_SPn2185_GFP ATCC13032::ΔNCgl0508 ATCC13032::ΔNCgl0508::PgapA_SPn2185_GFP

[0132] Example 4. Screening of strains with enhanced GFP protein expression

[0133] To screen for strains with increased GFP expression among the nine strains prepared in Example 3, they were cultured as follows. To culture the ATCC13032::ΔNCgl2525::PgapA_SPn2185_GFP strain, one colony was plated onto the compound agar plate below and solid-state cultured at 30°C for 16 hours. One blue loop (SPL, Cat: SPL90010) was inoculated into a 250 ml flask containing 25 ml of culture medium and cultured with shaking at 200 rpm for 48 hours at 30°C.

[0134] The other 8 strains of Table 6 were each cultured using substantially the same method as above.

[0135] Compound Plate Medium (pH 7.0)

[0136] Glucose 10 g, Peptone 10 g, Beef extract 5 g, Yeast extract 5 g, Brain Heart Infusion 18.5 g, NaCl 2.5 g, Urea 2 g, Sorbitol 91 g, Agar 20 g (based on 1 liter of distilled water)

[0137] <Culture Medium (pH 7.0)>

[0138] Glucose 63 g, ammonium sulfate 28 g, MgSO4.7H2O 1.2 g, KH2PO4 1.1 g, yeast extract 1 g, d-biotin 1 mg, thiamine-HCl 25 mg, calcium-pantothenic acid 25 mg, MnSO4.5H2O 10 mg, ZnSO4.5H2O 2.5 mg, CuSO4.5H2O 2.5 mg, FeSO4.5H2O 10 mg, CaCO3 30 g (based on 1 liter of distilled water)

[0140] To analyze the fluorescence intensity of the GFP protein expressed in the culture medium, the following procedure was performed. First, the GFP fluorescence intensity of the culture supernatant, after settling the bacterial cells using a centrifuge, was measured using a 96-well black plate (SPL, cat: 33396) under conditions of an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The fluorescence intensity was [calculated] at the OD of the culture. 600 The values ​​were standardized based on the values. The average values ​​of the 3 replicate data for each strain are shown in Table 7 below.

[0141] GFP-expressing strain (GFP 485 / 528 / OD 600 ) / 1,000 Increase rate (%) compared to the control group ATCC13032::PgapA_SPn2185_GFP (Control) 192.0 - ATCC13032::ΔNCgl2525::PgapA_SPn2185_GFP 200.1 4.22 ATCC13032::ΔNCgl1480::PgapA_SPn2185_GFP 195.8 1.90 ATCC13032::ΔNCgl2107::PgapA_SPn2185_GFP 188.3 -1.70 ATCC13032::ΔNCgl0757::PgapA_SPn2185_GFP 198.0 3.19 ATCC13032::ΔNCgl1337::PgapA_SPn2185_GFP 193.7 0.86 ATCC13032::ΔNCgl0339::PgapA_SPn2185_GFP 190.1 -0.98 ATCC13032::ΔNCgl1048::PgapA_SPn2185_GFP 262.7 37.19 ATCC13032::ΔNCgl0508::PgapA_SPn2185_GFP 203.9 4.53

[0142] As can be seen in Table 7 above, the fluorescence intensity of the ATCC13032::ΔNCgl1048::PgapA_SPn2185_GFP strain was improved by approximately 37% compared to the control group (ATCC13032::PgapA_SPn2185_GFP). From the above results, it was confirmed that deletion of the NCgl1048 gene can improve protein secretion ability.

[0144] Example 5. Preparation of strains expressing human insulin precursor, human FGF2, and human EGF

[0145] We aimed to investigate changes in the expression levels of human insulin precursor, human FGF2, and human EGF following the deletion of the NCgl1048 gene.

[0147] 1) Human Insulin Precursor

[0148] First, the human insulin precursor sequence was used by substituting the protein sequence from 55 to 89, specified as C-peptide, with AAK (T Kjeldsen et al. 1996, doi: 10.1016 / 0378-1119(95)00822-5.) from the protein secretion signal sequence from 1 to 24 on the P01308 sequence specified on UNIPROT (UniProtKB / Swiss-Prot: P01308.1) (Sequence No. 51). Codon optimization was performed to express the protein of Sequence No. 51 in Corynebacterium glutamicum ATCC13032, and the nucleic acid sequence of the gene encoding the protein of Sequence No. 51 is the same as the nucleic acid sequence of Sequence No. 52.

[0149] order Human insulin precursor (sequence number 51) FVNQHLCGSHLVEALYLVCGERGFFYTPKTAAKGIVEQCCTSICSLYQLENYCN Gene encoding human insulin precursor (Sequence No. 52) tttgttaatcaacacttatgtggttcccacttagtggaagccctttatctagtgtgcggagagcgtggctttttctatacccctaagACCgcagcaaagggtatcgtggagcaatgttgcAcctctATCtgcagtctttaccaacttgaaaattactgcaat

[0150] To construct an expression vector for expressing a human insulin precursor, the promoter sequence of the gapA gene (NCgl1526) was amplified by PCR using the primer pair of SEQ ID NO. 53 and SEQ ID NO. 54, with the genome of wild-type Corynebacterium glutamicum ATCC13032 as a template. To express effluxing GFP, the protein sequence of NCgl0059 analyzed as the TAT pathway secretion signal sequence via SignalP 5.0 was used. The gene fragment of the protein secretion signal sequence of the NCgl0059 (cg0079, Cgl0060) gene was PCR-coated using the primer pair of SEQ ID NO. 55 and SEQ ID NO. 56. The above-mentioned obtained gene fragment, human insulin precursor, and the vector pCES208 (Korean Registered Patent Publication KR 10-1673080 B1, J.Microbiol. Biotechnol., 18:639-647, 2008), which was cleaved with SalI and BamHI restriction enzymes, were cloned using the Gibson assembly method, transformed into E. coli DH5α, and plated on LB solid medium containing 25 mg / L of kanamycin. PCR was performed using the primer pair of SEQ ID NO. 57 and SEQ ID NO. 58 to select transformed colonies. Subsequently, a plasmid was obtained from the selected colonies using a commonly known plasmid extraction method and named pCES208-PgapA_SPn0059_hINS.

[0151] Primer sequence Sequence number 53 GGCCCCCCTCGAGGTCGACGAAGAAATTTAGATGATTGA Sequence number 54 GCAGATTTAAAGCTAGGCATGTTGTGTCTCCTCTAAAGAT Sequence number 55 ATCTTTAGAGGAGACACAACATGCCTAGCTTTAAATCTGC Sequence number 56 CataagtgttgattaacaaaCGATGATGCTGACGTTTGGA Sequence number 57 AGTACTGATCCTCCGGCGTT Sequence number 58 GTGATATGGGGCAAATGGTG

[0152] To produce a strain expressing a human insulin precursor, the above pCES208-PgapA_SPn0059_hINS vector was transformed into wild-type Corynebacterium glutamicum ATCC13032 and the ATCC13032::ΔNCgl1048 strain produced in Example 2 using the electro-pulse method (Appl. Microbiol. Biothcenol.(1999) 52:541-545) to obtain a strain into which the vector was inserted. The microorganisms obtained in this way were named ATCC13032 / pCES208-PgapA_SPn0059_hINS and ATCC13032::ΔNCgl1048 / pCES208-PgapA_SPn0059_hINS, respectively.

[0154] 2) Human FGF2

[0155] For the amino acid sequence of Human FGF2, the protein sequence from 143 to 288 was used, excluding the amino acid sequence from 1 to 142 specified as Propeptide in the P09038 sequence (UniProtKB / Swiss-Prot: P09038.3) specified in UNIPROT. (Sequence No. 59) Codon optimization was performed to express the protein of Sequence No. 59 in Corynebacterium glutamicum ATCC13032, and the nucleic acid sequence of the gene encoding the protein of Sequence No. 51 is the same as the nucleic acid sequence of Sequence No. 60.

[0156] order Human FGF2 (sequence number 59) PALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS Gene encoding human FGF2 (Sequence No. 60) CCTGCGCTCCCCGAAGATGGCGGATCTGGAGCGTTTCCTCCGGGACATTTCAAGGATCCAAAGCGGCTCTATTGTAAAAACGGTGGCTTCTTTCTCCGCATTCATCCTGACGGACGTGTCGATGGAGTACGCGAAAAATCTGACCCTCATATCAAACTCCAGCTCCAGGCCGAGGAACGGGGAGTTGTTTCTATCAAGGGTGTTTGCGCTAACCGTTAT TTGGCAATGAAAGAAGATGGTCGCCTTCTGGCCTCAAAGTGCGTTACTGATGAGTGCTTTTTTTTTTCGAGCGCCTTGAGTCGAACAATTATAATACTTATCGGAGCCGTAAATACAcCTCCTGGTATGTCGCTCTGAAACGCACCGGACAGTATAAATTGGGAtCCAAGACTGGCCCAGGTCAGAAGGCCATCCTGTTCTTGCCAATGAGCGCCAAATCC

[0157] To construct an expression vector for expressing Human FGF2, the promoter sequences of the gapA genes (NCgl1526, cg1791, Cgl1588) were amplified by PCR using the primer pair of SEQ ID NO. 61 and SEQ ID NO. 62, with the genome of wild-type Corynebacterium glutamicum ATCC13032 as a template. To express efflux-type GFP, the protein sequence of NCgl0801 analyzed as the TAT pathway secretion signal sequence via SignalP 5.0 was used. The gene fragment of the protein secretion signal sequence of the NCgl0801 gene was PCR-treated using the primer pair of SEQ ID NO. 63 and SEQ ID NO. 64. The above-mentioned obtained gene fragment, human FGF2, and the vector pCES208, which was cleaved with SalI and BamHI restriction enzymes, were cloned using the Gibson assembly method, transformed into E. coli DH5α, and plated on LB solid medium containing 25 mg / L of kanamycin. PCR was performed using the primer pair of SEQ ID NO. 57 and SEQ ID NO. 58 to select transformed colonies. Subsequently, a plasmid was obtained from the selected colonies using a commonly known plasmid extraction method and named pCES208-PgapA_SPn0801_hFGF2.

[0158] Primer sequence Sequence number 61 GGCCCCCCTCGAGGTCGACGAAGAAATTTAGATGATTGA Sequence number 62 CCTCGGCGGTTTATTTGCATGTTGTGTCTCCTCTAAAGAT Sequence number 63 ATCTTTAGAGGAGACACAACATGCAAATAAACCGCCGAGG Sequence number 64 CCATCTTCGGGGAGCGCAGGGGCGTTGGCCTTTGGCATAA

[0159] To produce a strain expressing Human FGF2, the above pCES208-PgapA_SPn0801_hFGF2 vector was transformed into wild-type Corynebacterium glutamicum ATCC13032 and the ATCC13032::ΔNCgl1048 strain produced in Example 2 using the electro-pulse method (Appl. Microbiol. Biothcenol.(1999) 52:541-545) to obtain a strain into which the vector was inserted. The microorganisms obtained in this way were named ATCC13032 / pCES208-PgapA_SPn0801_hFGF2 and ATCC13032::ΔNCgl1048 / pCES208-PgapA_SPn0801_hFGF2.

[0161] 3) Human EGF

[0162] The sequence of Human EGF was the protein sequence from 971 to 1023 specified as PRO_0000007541 on the P01133 sequence specified on UNIPROT (UniProtKB / Swiss-Prot: P01133.2) (Sequence No. 65). Codon optimization was performed to express the protein of Sequence No. 65 in Corynebacterium glutamicum ATCC13032, and the nucleic acid sequence of the gene encoding the protein of Sequence No. 65 is the same as the nucleic acid sequence of Sequence No. 66.

[0163] order Human EGF (Sequence No. 65) NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR Gene encoding human EGF (Sequence No. 66) ACGAAGCTCCCACCACTTCAAGTCACGATACTGACAACGCTCTCCAATATATCCTACCACACAATTACAGGCGTATTTGTCCAGTGCTTCAATATACATGCACACGCCGTCGTGCAAGCAGTAACCGTCGTGGCTGAGCGGACATTCGGAATCAGAATT

[0164] To construct an expression vector for expressing Human EGF, the promoter sequence of the gapA gene (NCgl1526) was amplified by PCR using the primer pair of SEQ ID NOs. 67 and 68, with the genome of wild-type Corynebacterium glutamicum ATCC13032 as a template. To express efflux GFP, the protein sequence of NCgl0334 analyzed as the SEC pathway secretion signal sequence via SignalP 5.0 was used. The protein secretion signal sequence gene fragment of the NCgl0334 (cg0411, Cgl0341) gene was PCR-treated using the primer pair of SEQ ID NOs. 69 and 70. The above-mentioned obtained gene fragment, human EGF, and the pCES208 vector, which was cleaved with SalI and BamHI restriction enzymes, were cloned using the Gibson assembly method, transformed into E. coli DH5α, and plated on LB solid medium containing 25 mg / L of kanamycin. PCR was performed using the primer pair of SEQ ID NO. 55 and SEQ ID NO. 56 to select transformed colonies. Subsequently, a plasmid was obtained from the selected colonies using a commonly known plasmid extraction method and named pCES208-PgapA_SPn0334_hEGF.

[0165] Primer sequence Sequence number 67 GGCCCCCCTCGAGGTCGACGAAGAAATTTAGATGATTGA Sequence number 68 GTTTTACGCATGCCAATCATGTTGTGTCTCCTCTAAAGAT Sequence number 69 ATCTTTAGAGGAGACACAACATGATTGGCATGCGTAAAAC Sequence number 70 GGACATTCGGAATCAGAATTCGCCTGCACTGGTGAGATGG

[0166] To produce a strain expressing human EGF, the above pCES208-PgapA_SPn0334_hEGF vector was transformed into wild-type Corynebacterium glutamicum ATCC13032 and the ATCC13032::ΔNCgl1048 strain produced in Example 2 using the electro-pulse method (Appl. Microbiol. Biothcenol.(1999) 52:541-545) to obtain a strain into which the vector was inserted. The microorganisms obtained in this way were named ATCC13032 / pCES208-PgapA_SPn0334_hEGF and ATCC13032::ΔNCgl1048 / pCES208-PgapA_SPn0334_hEGF.

[0168] Example 6. Evaluation of expression of Human Insulin Precursor, Human FGF2, and Human EGF

[0169] The six strains prepared in Example 5 were evaluated as follows. For the culture of the ATCC13032::ΔNCgl1048 / pCES208-PgapA_SPn0059_hINS strain expressing a human insulin precursor, one colony was plated onto the compound agar plate below and solid-state cultured at 30°C for 16 hours. One blue loop (SPL, Cat: SPL90010) was inoculated into a 250 ml flask containing 25 ml of culture medium and cultured with shaking at 200 rpm for 48 hours at 30°C.

[0170] Five other strains prepared in Example 5 were each cultured in substantially the same manner as above.

[0171] Compound Plate Medium (pH 7.0)

[0172] Glucose 10 g, Peptone 10 g, Beef extract 5 g, Yeast extract 5 g, Brain Heart Infusion 18.5 g, NaCl 2.5 g, Urea 2 g, Sorbitol 91 g, Agar 20 g (based on 1 liter of distilled water)

[0173] <Culture Medium (pH 7.0)>

[0174] Glucose 63 g, ammonium sulfate 28 g, MgSO4.7H2O 1.2 g, KH2PO4 1.1 g, yeast extract 1 g, d-biotin 1 mg, thiamine-HCl 25 mg, calcium-pantothenic acid 25 mg, MnSO4.5H2O 10 mg, ZnSO4.5H2O 2.5 mg, CuSO4.5H2O 2.5 mg, FeSO4.5H2O 10 mg, CaCO3 30 g (based on 1 liter of distilled water)

[0176] After the completion of fermentation, the supernatant of the culture medium was analyzed as follows. The supernatant of the culture medium, from which the bacterial cells had settled via centrifugation, was separated using a 4–20% Tris-glycine polyacrylamide gel via SDS-PAGE (Laemmli, 1970) and stained using DIRECTBLUE(™) gel staining solution (SCGBIOMAX, Cat: BDS-1000). The concentration of the band corresponding to the human insulin precursor on the gel was analyzed using a Calibrated Densitometer (BIO-RAD, GS-900). Additionally, to further confirm whether the band of that size was indeed the human insulin precursor, it was verified by LC-MS / MS analysis using the in-gel digestion method (Rosenfeld, 1992).

[0177] Culture and analysis of strains expressing human FGF2 and human EGF were performed using substantially the same method as above. The average values ​​of the 3 replicate data for each strain are shown in Tables 14 to 16 below.

[0178] strain Human insulin precursor expression level (OD) Increase (%) compared to the control group ATCC13032 / pCES208-PgapA_SPn0059_hINS (Control group) 43.73 - ATCC13032::ΔNCgl1048 / pCES208-PgapA_SPn0059_hINS 69.97 60.00%

[0179] strain Human FGF2 expression level (OD) Increase (%) compared to the control group ATCC13032 / pCES208-PgapA_SPn0801_hFGF2 (control group) 103.2 - ATCC13032::ΔNCgl1048 / pCES208-PgapA_SPn0801_hFGF2 148.7 44.09%

[0180] strain Human EGF expression level (OD) Increase (%) compared to the control group ATCC13032 / pCES208-PgapA_SPn0334_hEGF (control group) 150.9 - ATCC13032::ΔNCgl1048 / pCES208-PgapA_SPn0334_hEGF 208.2 37.97%

[0181] As can be seen in Tables 14 to 16 above, it was confirmed that the ATCC13032::ΔNCgl1048 / pCES208-PgapA_SPn0059_hINS strain, which expresses a human insulin precursor with a deletion of the NCgl1048 gene, showed an increase in the expression of the human insulin precursor by approximately 60% compared to the control group (ATCC13032 / pCES208-PgapA_SPn0059_hINS). In strains expressing other target proteins, human FGF2 and human EGF, the expression of the target proteins increased by 44% and 38%, respectively, thus confirming that the effect of this NCgl1048 gene deletion is observed for various target proteins.

[0183] Example 7. Preparation of a gene-deleted strain having the nucleic acid sequence of SEQ ID NO. 2

[0184] To determine whether the same effect is observed when a deletion trait of a gene having a nucleic acid sequence having 98.6% homology with the NCgl1048 gene having the nucleic acid sequence of SEQ ID NO. 1 is introduced into Corynebacterium glutamicum ATCC13869, another subtype of Corynebacterium glutamicum ATCC13032 (nucleic acid sequence code: BBD29_05870, amino acid sequence code: ANU33319.1 in the whole genome of Corynebacterium glutamicum ATCC13869 (GenBank: CP016335.1); nucleic acid sequence of SEQ ID NO. 2), a strain with a deletion of the gene having the nucleic acid sequence of SEQ ID NO. 2 was constructed.

[0185] To delete the gene having the nucleic acid sequence of SEQ ID NO. 2, a vector was constructed as follows. First, using the genome of wild-type Corynebacterium glutamicum ATCC13869 as a template, the homologous recombinant A arm was amplified using the primer pair of SEQ ID NO. 71 and SEQ ID NO. 72, and the homologous recombinant B arm was amplified using the primer pair of SEQ ID NO. 73 and SEQ ID NO. 74 by the PCR method. The obtained gene fragment and the vector pDC24, which was cleaved with SalI and BamHI restriction enzymes, were cloned using the Gibson assembly method, transformed into E. coli DH5α, and plated on LB solid medium containing 25 mg / L kanamycin. PCR was performed using the primer pair of SEQ ID NO. 39 and SEQ ID NO. 40 to select transformed colonies. Subsequently, a plasmid was obtained from a selected colony using a commonly known plasmid extraction method and named pDC24-ΔBDD29_05870.

[0186] Primer sequence Sequence number 71 atgcctgcaggtcgacACTCGCTGAGGCGAAGTAC Sequence number 72 GACCATCTAGACTGTTCTTTAATATGCTGATCTCCCTGC Sequence number 73 GCAGGGAGATCAGCATATTAAAGAACAGTCTAGATGGTC Sequence number 74 cggtacccggggatccGAAGGCCACAAAAATATTGC Sequence number 39 TATTACGCCAGCTGGCGAAA Sequence number 40 GCTTTACACTTTATGCTTCC

[0187] To construct a strain with a gene deletion having the nucleic acid sequence of SEQ ID NO. 2, the above pDC24-ΔBDD29_05870 vector was transformed into wild-type Corynebacterium glutamicum ATCC13869 by the electro-pulse method (Appl. Microbiol. Biothcenol.(1999) 52:541-545), and a strain with a GFP expression cassette inserted was obtained through a secondary crossover process. PCR was performed using the primer pair of SEQ ID NO. 71 and SEQ ID NO. 74, which can amplify adjacent regions including the site where the gene was inserted, to confirm the genetic modification. The microorganism obtained in this way was named ATCC13869::ΔBDD29_05870.

[0189] Example 8. Preparation of strains expressing human insulin precursor, human FGF2, and human EGF

[0190] To produce a strain expressing a human insulin precursor, the above pCES208-PgapA_SPn0059_hINS vector was transformed into wild-type Corynebacterium glutamicum ATCC13869 and the ATCC13869::ΔBDD29_05870 strain produced in Example 7 using the electro-pulse method (Appl. Microbiol. Biothcenol.(1999) 52:541-545) to obtain a strain into which the vector was inserted. The microorganisms thus obtained were named ATCC13689 / pCES208-PgapA_SPn0059_hINS and ATCC13869::ΔBDD29_05870 / pCES208-PgapA_SPn0059_hINS.

[0191] In order to construct strains expressing Human FGF2 in substantially the same manner as above, the ATCC13869 / pCES208-PgapA_SPn0801_hFGF2 and ATCC13869::ΔBDD29_05870 / pCES208-PgapA_SPn0801_hFGF2 strains were obtained using the pCES208-PgapA_SPn0801_hFGF2 vector.

[0192] In order to construct strains expressing Human EGF in substantially the same manner as above, ATCC13869 / pCES208-PgapA_SPn0334_hEGF and ATCC13869 / ΔBDD29_05870 / pCES208-PgapA_SPn0334_hEGF were obtained using the above pCES208-PgapA_SPn0334_hEGF vector.

[0194] Example 9. Evaluation of expression of Human Insulin Precursor, Human FGF2, and Human EGF

[0195] The six strains prepared in Example 8 were evaluated as follows. For the culture of the ATCC13869::ΔBDD29_05870 / pCES208-PgapA_SPn0059_hINS strain expressing a human insulin precursor, one colony was plated onto the compound agar plate below and solid-state cultured at 30°C for 16 hours. One blue loop (SPL, Cat: SPL90010) was inoculated into a 250 ml flask containing 25 ml of culture medium and cultured with shaking at 200 rpm for 48 hours at 30°C. In the same manner, culture was performed for the other strains.

[0196] Five other strains prepared in Example 8 were each cultured in substantially the same manner as above.

[0197] Compound Plate Medium (pH 7.0)

[0198] Glucose 10 g, Peptone 10 g, Beef extract 5 g, Yeast extract 5 g, Brain Heart Infusion 18.5 g, NaCl 2.5 g, Urea 2 g, Sorbitol 91 g, Agar 20 g (based on 1 liter of distilled water)

[0199] <Culture Medium (pH 7.0)>

[0200] Glucose 63 g, ammonium sulfate 28 g, MgSO4.7H2O 1.2 g, KH2PO4 1.1 g, yeast extract 1 g, d-biotin 1 mg, thiamine-HCl 25 mg, calcium-pantothenic acid 25 mg, MnSO4.5H2O 10 mg, ZnSO4.5H2O 2.5 mg, CuSO4.5H2O 2.5 mg, FeSO4.5H2O 10 mg, CaCO3 30 g (based on 1 liter of distilled water)

[0202] After the completion of fermentation, the culture supernatant was analyzed as follows. The culture supernatant, from which the bacterial cells had settled via centrifugation, was separated using a 4–20% Tris-glycine polyacrylamide gel via SDS-PAGE (Laemmli, 1970) and stained using DIRECTBLUE(™) gel staining solution (SCGBIOMAX, Cat: BDS-1000). The concentration of the band corresponding to the human insulin precursor on the gel was analyzed using a Calibrated Densitometer (BIO-RAD, GS-900). Additionally, to further confirm whether the band of that size was indeed the human insulin precursor, it was verified by LC-MS / MS analysis using the in-gel digestion method (Rosenfeld, 1992).

[0203] Culture and analysis of strains expressing human FGF2 and human EGF were performed using substantially the same method as above. The average values ​​of the 3 replicate data for each strain are shown in Tables 18 to 20 below.

[0204] strain Human insulin precursor expression level (OD) Increase (%) compared to the control group ATCC13869 / pCES208-PgapA_SPn0059_hINS 31.33 - ATCC13869::ΔBDD29_05870 / pCES208-PgapA_SPn0059_hINS 48.56 55.00%

[0205] strain Human FGF2 expression level (OD) Increase (%) compared to the control group ATCC13869 / pCES208-PgapA_SPn0801_hFGF2 78.86 - ATCC13869::ΔBDD29_05870 / pCES208-PgapA_SPn0801_hFGF2 110.1 39.62%

[0206] strain Human EGF expression level (OD) Increase (%) compared to the control group ATCC13869 / pCES208-PgapA_SPn0334_hEGF 109.4 - ATCC13869::ΔBDD29_05870 / pCES208-PgapA_SPn0334_hEGF 154.3 41.04%

[0207] As can be seen in Tables 18 to 20 above, it was confirmed that in the case of deletion of the NCgl1048 gene (Sequence No. 1) as well as deletion of the BBD29_05870 gene (Sequence No. 2), which has 98.6% homology with the NCgl1048 gene, the ATCC13869::ΔBDD29_05870 / pCES208-PgapA_SPn0059_hINS strain expressing a human insulin precursor showed an increase in the expression of the human insulin precursor by about 55% compared to the control group (ATCC13869 / pCES208-PgapA_SPn0059_hINS). In strains expressing other target proteins, human FGF2 and human EGF, the expression levels of the target proteins increased by 40% and 41%, respectively, indicating that deletion of genes with high homology to the NCgl1048 gene can increase the expression and secretion capabilities of various target proteins.

[0209] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

Claim 1 A Corynebacterium glutamicum microorganism having reduced activity of a gene containing the nucleic acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2, and increased ability to produce recombinant protein compared to a control group in which the activity of said gene is not reduced. Claim 2 A microorganism according to claim 1, wherein the gene codes for a protein having proteolytic enzyme activity. Claim 3 A microorganism according to claim 1, having proteolytic enzyme activity and having weakened activity of a polypeptide comprising the amino acid sequence of SEQ ID NO. 75 or SEQ ID NO.

76. Claim 4 delete Claim 5 In paragraph 1, the microorganism is a microorganism having all or part of the gene missing. Claim 6 In claim 1, the recombinant protein is one or more selected from the group consisting of collagen, collagen-derived polypeptides, hormones, cytokines, antibodies, antibiotic peptides, and antioxidant peptides, a microorganism. Claim 7 In claim 1, the microorganism is one or more types selected from the group consisting of human insulin precursor, human FGF2 (Human Fibroblast Growth Factor 2), and human EGF (Human Epidermal Growth Factor). Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 A method for producing a recombinant protein comprising the step of culturing a microorganism of any one of claims 1 to 3 and claims 5 to 7 in a culture medium. Claim 12 A method for producing a recombinant protein according to claim 11, further comprising, after the culturing step, a step of recovering the recombinant protein from the cultured microorganism, the medium, or both. Claim 13 A composition for producing recombinant protein comprising one or more selected from the group consisting of a microorganism of any one of claims 1 to 3 and claims 5 to 7 and a culture medium in which said microorganism is cultured.

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