KR20250147854A - Microorganisms of Corynebacterium genus with enhanced productivity of recombinant protein - Google Patents

Abstract

The present application provides a microorganism of the genus Corynebacterium having a weakened activity of a gene having a sequence homology of 75% or more with the nucleic acid sequence of sequence number 1, wherein the microorganism has an excellent ability to express a recombinant protein.

Description

Microorganisms of the genus Corynebacterium with enhanced productivity of recombinant protein

The present application relates to a Corynebacterium genus microorganism having increased recombinant protein production capacity and a method for producing a recombinant protein comprising a step of culturing the microorganism.

Corynebacterium glutamicum possesses industrial advantages as an expression host for recombinant protein production due to its lack of endotoxin and low protease activity. These recombinant proteins are widely used in the food, pharmaceutical, and chemical industries, including in enzymes and biological products. Corynebacterium glutamicum can secrete proteins produced within its cells into the culture supernatant via a unique excretion mechanism, allowing for efficient recombinant protein production by recovering the proteins from the culture medium.

Korean Patent Registration No. 10-2501259 B1

The purpose of the present application is to provide a microorganism having increased production capacity of a recombinant protein and having weakened activity of a gene having a sequence homology of 75% or more with the nucleic acid sequence of SEQ ID NO: 1.

Another object of the present application relates to a method for producing a recombinant protein, comprising a step of culturing the microorganism in a medium.

Another object of the present application is to provide a composition for producing a recombinant protein, comprising at least one selected from the group consisting of the above microorganism and a medium in which the above microorganism is cultured.

This is specifically explained as follows. Meanwhile, each description and embodiment disclosed in this application can also 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 limited by the specific descriptions described below. Furthermore, those skilled in the art will recognize or ascertain numerous equivalents to the specific embodiments of this application described in this application using only routine experimentation. Furthermore, such equivalents are intended to be encompassed by this application.

One aspect provides a microorganism having increased production capacity of a recombinant protein, wherein the activity of a gene having at least 75% sequence homology or identity with the nucleic acid sequence of SEQ ID NO: 1 is weakened.

The gene having the nucleic acid sequence of the above SEQ ID NO: 1 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).

The inventors of the present application discovered that a microorganism in which the activity of a gene having a sequence homology of 75% or more with the nucleic acid sequence of the above sequence number 1 is weakened has an increased ability to produce recombinant proteins.

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

In one example, the gene is (i) at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, It may encode a protein having a sequence homology or identity of at least 99.5% or at least 99.9%; and (ii) having a peptidase activity.

The protein having protease activity encoded by the above gene may be a protease 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.

In one example, the gene may be derived from a microorganism of the genus Corynebacterium. The above-mentioned Corynebacterium genus microorganisms are Corynebacterium glutamicum, Corynebacterium crudilactis , Corynebacterium deserti , Corynebacterium efficiens , Corynebacterium callunae, Corynebacterium stationis , Corynebacterium singulare , Corynebacterium halotolerans , Corynebacterium striatum , Corynebacterium pollutisoli, It may be at least one microorganism selected from the group consisting of, but is not limited to, Corynebacterium imitans , Corynebacterium testudinoris, and Corynebacterium flavescens .

In one example, the gene may be from Corynebacterium glutamicum, and in one example, the gene may be from Corynebacterium glutamicum ATCC13032 or Corynebacterium glutamicum ATCC13869.

In one example, the gene may be the NCgl1048 gene (SEQ ID NO: 1) from Corynebacterium glutamicum ATCC13032 (genome sequence, GenBank: CP025533.1 or GenBank: BA000036.3) or the BBD29_05870 gene (SEQ ID NO: 2) from Corynebacterium glutamicum ATCC13869 (genome sequence, GenBank: CP016335.1).

In one specific embodiment, the gene may comprise or consist of a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

The above microorganism has a nucleic acid sequence of sequence number 1, a nucleic acid sequence of sequence number 2, or a nucleic acid sequence of sequence number 1 of 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, A microorganism may be a microorganism having an attenuated activity of a polypeptide encoded by a gene comprising a nucleic acid sequence having a sequence homology or identity of 99% or more, 99.5% or more, or 99.9% or more.

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

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

The above polypeptide may comprise or consist of the amino acid sequence of SEQ ID NO: 75 or the amino acid sequence of SEQ ID NO: 76.

If the polypeptide encoded by the above gene exhibits protease activity, it is self-evident 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 gene of the present application. Furthermore, if the above polypeptide exhibits protease activity, it is self-evident that a polypeptide in which some amino acid sequences are deleted, modified, substituted, conservatively substituted, or added may also be included in the polypeptide of the present application.

For example, it may have additions or deletions, spontaneous mutations, silent mutations, or conservative substitutions of amino acid sequences that do not affect protease activity at the N-terminus, C-terminus, and/or within the amino acid sequence. The term “conservative substitution” refers to the replacement of one amino acid with another amino acid having similar structural and/or chemical properties. Such amino acid substitutions 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 or no effect on the activity of a protein or polypeptide.

In the present application, the phrase “a polynucleotide or polypeptide has, includes, consists of, or consists essentially 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 mutation (deletion, substitution, modification, and/or addition) is added to the specific nucleic acid sequence (base sequence) or amino acid sequence within the scope of maintaining the original function and/or desired function of the polynucleotide or polypeptide. In one example, a polynucleotide or polypeptide "has, comprises, consists of, or consists essentially of a particular nucleic acid sequence (base sequence) or amino acid sequence" means that the polynucleotide or polypeptide (i) essentially comprises the particular nucleic acid sequence (base sequence) or amino acid sequence, or (ii) is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, It may mean that it consists of or essentially contains a nucleic acid sequence or amino acid sequence having a homology or identity of 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 99.9% or more, and maintains the original function and/or the desired function. In one example, the desired function may mean the function of increasing (improving) or imparting the recombinant protein production ability of a microorganism.

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

Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and may be combined with default gap penalties established by the program being used. In practice, homologous or identical sequences can generally hybridize with all or part of the sequence under moderate or high stringency conditions. It should be appreciated that hybridization also includes hybridization with polynucleotides containing common codons or codons that take codon degeneracy into account.

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

Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information, for example, using a GAP computer program such as that of Needleman et al. (1970), J Mol Biol. 48:443, as disclosed, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In brief, the 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). Default parameters for the GAP program include (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and (2) a comparison matrix as disclosed by Gribskov et al. (1986) Nucl. Acids Res. 48:443, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979). 14: 6745 weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) permutation 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.

In this application, the term "microorganism (or strain)" includes both wild-type microorganisms and microorganisms that have undergone genetic modification naturally or artificially, and may be a microorganism that has a specific mechanism weakened or strengthened due to causes such as the insertion of an external gene or the weakening of the activity of an endogenous gene, and may be a microorganism that includes genetic modification for the production of a desired polypeptide, protein or product.

In this application, the term “attenuation” of a gene or polypeptide includes both a decrease in activity or absence of activity compared to the intrinsic activity. The term “attenuation” may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation. The term “intrinsic activity” refers to the activity of a specific gene or polypeptide that a parent strain, wild type, or unmodified microorganism originally had before the phenotype change, when the phenotype changes due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activity before modification.” The fact that the activity of a gene or polypeptide is “inactivated, deficient, reduced, down-regulated, reduced, or attenuated” compared to the intrinsic activity may mean that the expression of the gene or polypeptide is reduced compared to the parent strain or unmodified microorganism before the phenotype change.

In one example, the weakening 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 the gene may include a case where the expression of the gene is inhibited (e.g., inhibition of transcription into mRNA or translation of mRNA), such that the concentration (expression amount) of the polypeptide encoded by the gene in a cell is lower than that of a control (wild type or a microorganism in which the activity of the gene is not weakened), or a case where the polypeptide encoded by the gene is not expressed at all. Such weakening 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 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.).

In one example, weakening of the gene or polypeptide

1) Deletion of all or part of a gene encoding a polypeptide;

2) Modification of the expression control region (or expression control sequence) so as to reduce the expression of the gene encoding the polypeptide;

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

4) Modification of the gene sequence encoding the polypeptide such that the activity of the polypeptide is eliminated or weakened (e.g., deletion/substitution/addition of one or more nucleic acid bases in the nucleic acid sequence of the polypeptide gene such that the polypeptide is modified such that the activity of the polypeptide is eliminated or weakened);

5) Modification of the base sequence encoding the initiation codon or 5'-UTR region of a gene transcript encoding a polypeptide;

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

7) Addition of a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of a gene encoding a polypeptide to form a secondary structure to which ribosome attachment is impossible;

8) Addition 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 (Reverse transcription engineering, RTE);

9) Controlling the cellular localization of polypeptides; or

10) It may be a combination of two or more selected from 1) to 9), but is not particularly limited thereto.

For example, the deletion of part or all of the gene encoding the polypeptide described above 1) may be the removal of the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, replacement with a polynucleotide having some nucleotides deleted, or replacement with a marker gene.

In addition, the above 2) modification of the expression control region (or expression control sequence) may be a mutation in the expression control region (or expression control sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or replacement with a sequence having weaker activity. The expression control 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.

In addition, the above 3) modification of the base sequence encoding the initiation codon or 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, a substitution with a base sequence encoding another initiation codon having a lower polypeptide expression rate than the endogenous initiation codon, but is not limited thereto.

In addition, the modification of the amino acid sequence or polynucleotide sequence of the above 4) and 5) may be a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to weaken the activity of the polypeptide, or replacement with an amino acid sequence or polynucleotide sequence improved to have weaker activity, or an amino acid sequence or polynucleotide sequence improved to have no activity, but is not limited thereto. For example, by introducing a mutation in the polynucleotide sequence to form a stop codon, the expression of the gene may be inhibited or weakened, but is not limited thereto. The "stop codon" is a codon that does not specify an amino acid among the codons on mRNA and serves as a signal to indicate the end of the protein synthesis process, and generally, three types of UAA, UAG, and UGA can be used as stop codons.

6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that complementarily binds to the transcript of the gene encoding the polypeptide may be described, 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].

7) Addition of a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of a gene encoding a polypeptide to form a secondary structure to which ribosome attachment is impossible may render mRNA translation impossible or slow it down.

8) The addition of a promoter transcribed 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) may weaken the activity by creating an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide.

The above 9) regulation of the intracellular location of a polypeptide may target the polypeptide to a specific organelle or specific intracellular space within the cell. For example, targeting to the periplasm or cytoplasm may be achieved by adding or removing a leader sequence that functions in targeting the polypeptide, but is not limited thereto.

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

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 involve transforming a microorganism with a vector containing a portion of a sequence of a gene encoding a polypeptide and culturing the microorganism in the presence of a selectable marker product, thereby causing homologous recombination between the portion of the gene and an endogenous gene within the microorganism.

The microorganism provided in the present application may be a microorganism in which the activity of a gene having a sequence homology of 75% or more with the nucleic acid sequence of the above sequence number 1 is weakened, and specifically, a microorganism (e.g., a recombinant microorganism) genetically modified through a vector so that the activity of the gene is weakened, but is not limited thereto.

The above microorganism may be a microorganism having recombinant protein production ability or an improved recombinant protein production ability. The microorganism having recombinant protein production ability or an improved recombinant protein production ability may mean that the recombinant protein production ability is newly granted compared to a control group (such as an unmodified microorganism without recombinant protein production ability, a cell before recombination, a parent strain, and/or a wild-type strain), or that the recombinant protein production ability is improved compared to a control group (such as a cell before recombination, a parent strain, etc.) having recombinant protein production ability.

In the present application, the term "unmodified microorganism" does not exclude a strain that contains a mutation that can occur naturally in a microorganism, and may refer to a wild-type strain or a natural strain itself, or a strain before its characteristics are changed by a genetic mutation caused by natural or artificial factors. For example, the unmodified microorganism may refer to a strain in which the activity of the gene is not weakened or before it is weakened, according to an example. The term "unmodified microorganism" may be used interchangeably with "pre-modified strain," "pre-modified microorganism," "unmutated strain," "unmodified strain," "unmutated microorganism," or "reference microorganism." In one example, the unmodified microorganism, which is a target strain for comparing whether the recombinant protein production ability increases, may be, but is not limited to, the Corynebacterium glutamicum ATCC13032 strain, the Corynebacterium glutamicum ATCC13869 strain, etc.

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

The above recombinant protein may refer to any kind of protein having biologically useful activity that is to be produced using a protein expression, secretion and production system of a microorganism, and may refer to any protein that a person skilled in the art wishes to mass-produce, and that can be expressed in a host cell by inserting a polynucleotide encoding the protein into a recombinant vector. For example, the target protein may be collagen, a collagen-derived polypeptide, a hormone, a hormone analog, a cytokine, an antigen, an antigen-binding fragment, an antibody, a cell receptor, an enzyme, a transport protein, a structural protein, a serum, a cellular protein, an antibiotic peptide, an antioxidant peptide, etc.

In one example, the recombinant protein is human insulin precursor, human FGF2 (Human FGF2, Fibroblast Growth Factor), human EGF (Human EGF, Epidermal Growth Factor), collagen (collagen, such as collagen derived from human, pig, cow, chicken, fish, etc.), collagen-derived polypeptide (polypeptide comprising a collagen-derived amino acid sequence), etanercept, epoetin alpha, infliximab, interferon beta, insulin lispro, filgrastim, imiglucerase, glatiramer acetate, rituximab, pegfilgrastim, insulin grastim, adalimumab, trastuzumab (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 receptor, granulocyte-colony stimulating factor, muromomab, abciximab, daclizumab, basiliximab, palivizumab, ibritumomab, omalizumab, efalizumab, tositumomab, cetuximab, natalizumab, alkaline phosphatase (PhoA), levan fructotransferase (LFT), cellulose binding domain, cholera toxin B (cholera toxin B), organophosphohydrolase, calcitonin, blood coagulation factors, hirudin, and monoclonal antibody 5T4, but is not limited thereto.

In one example, the recombinant protein may further comprise a tag for protein purification, expression of the target protein, solubilization, or enhanced detection. Various tags are known in the art for use for this purpose, including, but not limited to, a GST tag, a FLAG tag, a polyarginine tag, a polyhistidine tag, such as a 6His tag, an MBP tag, an S-tag, an influenza virus HA tag, a thioredoxin tag, or a Staphylococcus aureus protein A tag.

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

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

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

In one example, the recombinant protein may be a protein and/or peptide having a desired activity in a living body (e.g., an activity for preventing, alleviating, and/or treating a specific disease or symptom, or an activity for replacing a biologically necessary substance), and may be, for example, at least one selected from the group consisting of a protein or peptide having enzymatic activity (e.g., a protease, a kinase, a phosphatase, etc.), a receptor protein or peptide, a transporter protein or peptide, a bactericidal and/or endotoxin-binding polypeptide, a structural protein or peptide, an immune polypeptide, a toxin, an antibiotic, a hormone, a growth factor, a vaccine, etc. In one example, the desired polypeptide may be at least one selected from the group consisting of a hormone, a cytokine, a tissue plasminogen activator, an immunoglobulin (e.g., an antibody or an antigen-binding fragment or variant thereof), etc. The immunoglobulin may be of any isotype (e.g., IgA, IgD, IgG, IgM, or IgE), and may be, for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4) molecule. The antigen-binding fragment may be any fragment of an antibody that retains the antigen-binding ability of the original antibody and comprises at least about 20 amino acids, for example, at least about 100 amino acids. The antigen-binding fragment may be at least one selected from the group consisting of an antigen-binding site of an antibody, for example, a CDS, a Fab fragment, an F(ab)2 fragment, an Fv, an scFv, a multibody comprising multiple binding domains (e.g., a diabody, a triabody, a tetrabody, etc.), a single domain antibody, an affibody, etc. The antibody variant is a derivative of an antibody or antibody fragment that has the same binding function as an antibody but has an amino acid sequence that is altered from that of 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 a living organism or non-naturally occurring. The antibody and/or antigen-binding fragment may be synthetically or recombinantly produced. The antibody may be a monoclonal antibody. In another specific embodiment, the target polypeptide is at least one selected from the group consisting of various growth factors such as insulin, human growth hormone (hGH), insulin-like growth factor, EGF, VERF, etc., 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. Can be.

The recombinant vector may further comprise a gene encoding a secretory signal peptide. For expression and secretion of the recombinant protein through the transport system of a microorganism, the recombinant protein may be linked to a secretory signal peptide at the N'-terminus. The recombinant vector may comprise a gene encoding a recombinant protein and a gene encoding a secretory signal peptide in an appropriate order so as to enable expression of the recombinant protein in a form in which the secretory signal peptide is linked to the N'-terminus.

The above secretion signal peptide can be used without limitation as long as it is a secretion signal peptide that can induce secretion of a recombinant protein in a microorganism, and in one example, one selected from the group consisting of 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, TorA secretion signal peptide derived from E. coli , and IMD secretion signal peptide derived from Arthrobacter globiformis This may be, but is not limited to, any secretory signal peptide capable of inducing secretion of a recombinant protein in a microorganism may be used without limitation.

The above recombinant vector may include a promoter operably linked to a gene encoding the recombinant protein and/or a gene encoding the secretory signal peptide, for expression of the gene encoding the recombinant protein and/or the gene encoding the secretory signal peptide.

As used herein, "promoter" may mean an untranslated nucleotide sequence upstream or downstream of a coding region, which comprises a binding site for polymerase and has transcription initiation activity of a promoter target gene (e.g., a gene encoding a recombinant protein or twin-arginine transposase, etc.), such as a DNA region to which a polymerase binds to initiate transcription of the gene.

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 No. US 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 No. US 10584338 B2), O2 promoter (US Patent No. US 10273491 B2), tkt promoter, yccA promoter, etc.

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

In one example, the microorganism with increased recombinant protein production ability may have an increased recombinant protein production ability of 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, or 100% or more (there is no particular upper limit, and for example, it may be about 1000% or less) compared to the parent strain before mutation or the unmodified microorganism. In another example, the microorganism with increased recombinant protein production ability may have a recombinant protein production ability 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 unmodified microorganism.

The term “about” above includes all ranges including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all ranges of values equal to or similar to the value following the term “about,” but is not limited thereto.

The above microorganism may be a microorganism of the genus Corynebacterium.

The above-mentioned Corynebacterium genus microorganisms include Corynebacterium glutamicum, Corynebacterium crudilactis , Corynebacterium deserti , Corynebacterium efficiens , Corynebacterium callunae, Corynebacterium stationis , Corynebacterium singulare , Corynebacterium halotolerans , Corynebacterium striatum , Corynebacterium pollutisoli, It may be at least one microorganism selected from the group consisting of, but is not limited to, Corynebacterium imitans , Corynebacterium testudinoris, and Corynebacterium flavescens .

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

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

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

In this application, "cultivation" refers to growing the microorganism, such as a Corynebacterium glutamicum strain, under appropriately controlled environmental conditions. The culturing process can be performed using any suitable medium and culture conditions known in the art. This culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing process may be batch, continuous, and/or fed-batch, but is not limited thereto.

In the present application, "medium" means a material containing nutrients as a main component necessary for culturing the microorganism, for example, a Corynebacterium glutamicum strain, and supplies nutrients and growth factors, including water essential for survival and growth. Specifically, the medium and other culture conditions used for culturing the microorganism of the present application may be any medium used for culturing general microorganisms without particular limitation, but the microorganism of the present application may be cultured under aerobic conditions while controlling temperature, pH, etc. in a general medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and/or vitamin.

Specifically, culture media for the above 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)].

In the present application, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrolysate, molasses (e.g., blackstrap molasses), rice winter, cassava, sugarcane bagasse, and corn steep liquor may be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugar) may be used, and other appropriate amounts of carbon sources may be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

The nitrogen source 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.; organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

The above-mentioned components may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and/or suitable precursors may be included. These components or precursors may be added to the medium in batch or continuous manner, but are not limited thereto.

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

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

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

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

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

The above recovery may be performed by 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 chromatographies such as centrifugation, filtration, treatment with a crystallized protein precipitant (salting out method), 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 medium or microorganism using a suitable method known in the art.

Additionally, the recombinant protein production method of the present application may additionally include a purification step. The purification may be performed using any suitable method known in the art. In one example, if the recombinant protein production method of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed sequentially or discontinuously, regardless of order, or may be performed simultaneously or integrated into a single step, but is not limited thereto.

Another aspect provides a composition for producing a recombinant protein comprising at least one selected from the group consisting of the above microorganism and a medium in which the above microorganism is cultured.

Another aspect provides a use for producing a recombinant protein, comprising at least one selected from the group consisting of the above microorganism and a medium in which the above microorganism is cultured.

The composition of the present application may further comprise any suitable excipient commonly used in compositions for producing recombinant proteins, and such excipients may be, for example, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, or isotonic agents.

The present application provides a microorganism of the genus Corynebacterium having a weakened activity of a gene having a sequence homology of 75% or more with the nucleic acid sequence of sequence number 1, wherein the microorganism has an excellent ability to express a recombinant protein.

Figure 1 shows an analysis of proteins present in the supernatant of a culture medium in which Corynebacterium glutamicum ATCC13032 strain was cultured.

The present invention will be described in more detail below with reference to the following examples. However, these examples are provided solely to illustrate the present invention, and the scope of the present invention is not limited by these examples.

Example 1. Analysis of proteins in culture medium using LC-MS and selection of defective candidates.

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

<Composite plate (pH 7.0)>

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 (per 1 liter of distilled water)

<Culture medium (pH 7.0)>

Glucose 63 g, ammonium sulfate 28 g, MgSO ₄ . 7H ₂ O 1.2 g, KH ₂ PO ₄ 1.1 g, yeast extract 1 g, d-biotin 1 mg, thiamine-HCl 25 mg, calcium-pantothenic acid 25 mg, MnSO ₄ . 5H ₂ O 10 mg, ZnSO ₄ . 5H ₂ O 2.5 mg, CuSO ₄ . 5H ₂ O 2.5 mg, FeSO ₄ . 5H ₂ O 10 mg, CaCO ₃ 30 g (per 1 liter of distilled water)

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

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

Band Gene Annotation NCBI reference coding (Protein sequence) SignalP (5.0) Size (kDa) denomination 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

Example 2. Production of a strain lacking the gene encoding the protein selected in Example 1.

In order to increase the secretion production capacity of the recombinant protein, a vector was constructed as follows to delete the protein in the culture solution analyzed in Example 1.

In Table 1 above, a deletion vector for deleting the NCgl2525 gene encoding protein 01 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 using the PCR method. The obtained gene fragment and the vector pDC24 (SEQ ID NO: 77) digested with SalI and BamHI restriction enzymes were cloned using the Gibson assembly method, and then transformed into Escherichia coli DH5α and plated on LB solid medium containing 25 mg/L kanamycin. To select the transformed colonies, PCR was performed using the primer pair of SEQ ID NO: 39 and SEQ ID NO: 40. Subsequently, a plasmid was obtained from the selected colony using a commonly known plasmid extraction method, and this was designated pDC24-ΔNCgl2525. Deletion vectors encoding genes for proteins 02 to 08 were constructed using substantially the same method as above, and the primers used to construct each deletion vector are as shown in Table 2 below.

vector Homologous arm Sequence number base sequence pDC24-ΔNCgl2525 A Sequence number 03 atgcctgcaggtcgacCTGCCTACAAGGATCTCTT Sequence number 04 GTTATCCATCTTGAAAAAGCATTCCTTGTGGGCGCTTTGTC 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 number 19 atgcctgcaggtcgacGAGAGAATACCCCAGGGAG Sequence number 20 AGATATCTTTAGTTCACTTTCGAACGGCGGATTTTCTAAATT B Sequence number 21 GAAAGTGAACTAAAGATATCTAATTTAGAAAATCCGCCGTTC Sequence number 22 cggtacccggggatccACCCTTCAAAATCAGCATC pDC24-ΔNCgl1337 A Sequence number 23 atgcctgcaggtcgacTCGGCGAGAACAAGAAATC Sequence number 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 number 31 atgcctgcaggtcgacGATCTTTATGGGGCGTTTTC Sequence number 32 CAACCATCTAGACTGTTCTTTAATATGCTGATCTCCCTGC B Sequence number 33 GCAGGGAGATCAGCATATTAAAGAACAGTCTAGATGGTTG Sequence number 34 cggtacccggggatccCAAAATGATCAACGCCATC pDC24-ΔNCgl0508 A Sequence number 35 atgcctgcaggtcgacGCTCAGGCCAAGCACAACCA Sequence number 36 GAAGCTTTAGGTTAGGTTGCGGCTTTGAACCTTTCATTTTC B Sequence number 37 GAAAATGAAAGGTTCAAGCCGCAACCTAACCTAAAGCTTC Sequence number 38 cggtacccggggatccTCATCCTCGGCAACACCGGA

To produce a strain lacking the NCgl2525 gene encoding the above protein 01, the pDC24-ΔNCgl2525 vector was transformed into wild-type Corynebacterium glutamicum ATCC13032 by electroporation, and a strain lacking the NCgl2525 gene was obtained through a second crossing process. The genetic manipulation was confirmed by performing PCR using a pair of primers of SEQ ID NOs: 3 and 6 capable of amplifying adjacent regions including the position where the gene was inserted. The microorganism thus obtained was named ATCC13032::ΔNCgl2525.

In order to produce strains lacking the genes encoding proteins 02 to 08 in substantially the same manner as above, strains were produced using each deletion vector, and the produced strains are as shown in Table 3 below.

vector strains 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

Example 3. Production of a strain expressing GFP (green fluorescent protein) protein

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

order GFP protein (SEQ ID NO: 41) MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIEL KGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGNSITSYSIHYTKL Gene encoding GFP protein (SEQ ID NO: 42) ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACC ACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGA AGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGG CGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGGAATTCGATAACTTCGTATAGCATACATTATACGAAGTTA

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 using the PCR method. The promoter sequence of the gapA gene (NCgl1526) was amplified using the genome of wild-type Corynebacterium glutamicum ATCC13032 as a template and the primer pair of SEQ ID NO: 47 and SEQ ID NO: 48 using the PCR method. To express the exudate GFP, the protein sequence analyzed as a TAT pathway secretion signal sequence using SignalP 5.0 among the protein sequences of NCgl2185 was used. A gene fragment of the protein secretion signal sequence of the NCgl2185 (cg2485 and Cgl2265) gene was amplified using the PCR method using the primer pair of SEQ ID NO: 49 and SEQ ID NO: 50. The obtained gene fragment, the GFP gene fragment, and the vector pDC24 digested with SalI and BamHI restriction enzymes were cloned using the Gibson assembly method, and then transformed into E. coli DH5α and plated on LB solid medium containing 25 mg/L kanamycin. To select the transformed colonies, PCR was performed using the primer pair of SEQ ID NO: 39 and SEQ ID NO: 40. Thereafter, a plasmid was obtained from the selected colony using a conventionally known plasmid extraction method, and this was designated pDC24-PgapA_SPn2185_GFP.

Primer sequence Sequence number 39 TATTACGCCAGCTGGCGAAA Sequence number 40 GCTTTACACTTTATGCTTCC Sequence number 43 ATGCCTGCAGGTCGACAGATCATGGTGCCGACAAAG Sequence number 44 TCAATCATCTAAATTTCTTCTAGGGTTTGATGCAAAAATT Sequence number 45 TACATTATACGAAGTTATAGCTAGGTTGGATGCAAAAATC Sequence number 46 CGGTACCCGGGGATCCGAAGCTCAATGCCAGATACT Sequence number 47 AATTTTTGCATCAAACCCTAGAAGAAATTTAGATGATTGA Sequence number 48 CGTCTGCTTAACTGTGGCATGTTGTGTCTCCTCTAAAGAT Sequence number 49 ATCTTTAGAGGAGACACAACATGCCACAGTTAAGCAGACG Sequence number 50 AGCTCCTCGCCCTTGCTCACAGCGCGTGCAGGTGTGCCCG

To construct a strain expressing GFP, the pDC24-PgapA_SPn2185_GFP vector was transformed into wild-type Corynebacterium glutamicum ATCC13032 and ATCC13032::ΔNCgl2525 by the electric pulse method (Appl. Microbiol. Biothcenol. (1999) 52:541-545), and a strain with an inserted GFP expression cassette was obtained through a second crossing process. The genetic manipulation was confirmed by performing PCR using the primer pair of SEQ ID NOs: 43 and 46 that can amplify the adjacent region including the position where the gene was inserted. The microorganism thus obtained was named ATCC13032::ΔNCgl02525::PgapA_SPn2185_GFP.

In order to produce a strain in which the genes encoding proteins 02 to 08 are deleted and which expresses the GFP protein in substantially the same manner as above, strains were produced using each deletion vector, and the produced strains are as shown in Table 6 below.

Base strain GFP expression 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

Example 4. Screening for strains with improved GFP protein expression

In order to screen for strains with increased GFP expression among the 9 strains produced in Example 3, they were cultured as follows. To culture the ATCC13032::ΔNCgl2525::PgapA_SPn2185_GFP strain, one colony was spread on the complex plate medium below and cultured on solid 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 at 30°C for 48 hours.

Each of the eight other strains in Table 6 was cultured in substantially the same manner as above.

<Composite plate (pH 7.0)>

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 (per 1 liter of distilled water)

<Culture medium (pH 7.0)>

Glucose 63 g, ammonium sulfate 28 g, MgSO ₄ . 7H ₂ O 1.2 g, KH ₂ PO ₄ 1.1 g, yeast extract 1 g, d-biotin 1 mg, thiamine-HCl 25 mg, calcium-pantothenic acid 25 mg, MnSO ₄ . 5H ₂ O 10 mg, ZnSO ₄ . 5H ₂ O 2.5 mg, CuSO ₄ . 5H ₂ O 2.5 mg, FeSO ₄ . 5H ₂ O 10 mg, CaCO ₃ 30 g (per 1 liter of distilled water)

To analyze the fluorescence intensity of the GFP protein expressed in the culture medium, the following procedure was followed. First, the GFP fluorescence intensity of the culture supernatant, from which the cells had been settled by centrifugation, was measured under conditions of an excitation wavelength of 485 nm and an emission wavelength of 528 nm using a 96-well black plate (SPL, cat: 33396). The fluorescence intensity was normalized to the OD ₆₀₀ value of the culture. The average value of the data from three replicates for each strain is shown in Table 7 below.

GFP expression strain (GFP _485/528 /OD ₆₀₀ )/1,000 Increase rate compared to 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 March 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

As can be seen in Table 7 above, the fluorescence intensity of the ATCC13032::ΔNCgl1048::PgapA_SPn2185_GFP strain was enhanced 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 enhance protein secretion ability.

Example 5. Production of strains expressing human insulin precursor, human FGF2, and human EGF.

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

1) Human insulin precursor

First, the human insulin precursor sequence was used by replacing the protein sequence from 55-89, which is designated as C-peptide, with AAK among the protein sequence from 25-110, excluding the protein secretion signal sequence from 1-24, in the P01308 sequence specified in UNIPROT (UniProtKB/Swiss-Prot: P01308.1) (SEQ ID NO: 51). Codon optimization was performed to express the protein of SEQ ID NO: 51 in Corynebacterium glutamicum ATCC13032, and the nucleic acid sequence of the gene encoding the protein of SEQ ID NO: 51 is the same as the nucleic acid sequence of SEQ ID NO: 52.

order human insulin precursor (SEQ ID NO: 51) FVNQHLCGSHLVEALYLVCGERGFFYTPKTAAKGIVEQCCTSICSLYQLENYCN Gene encoding human insulin precursor (SEQ ID NO: 52) tttgttaatcaacacttatgtggttcccacttagtggaagccctttatctagtgtgcggagagcgtggctttttctatacccctaagACCgcagcaaagggtatcgtggagcaatgttgcAcctctATCtgcagtctttaccaacttgaaaattactgcaat

To construct an expression vector expressing 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 using the genome of wild-type Corynebacterium glutamicum ATCC13032 as a template. To express the efflux-type GFP, the protein sequence of NCgl0059, which was analyzed as a TAT pathway secretion signal sequence using SignalP 5.0, was used. The gene fragment of the protein secretion signal sequence of the NCgl0059 (cg0079, Cgl0060) gene was PCR-amplified using the primer pair of SEQ ID NO: 55 and SEQ ID NO: 56. The obtained gene fragment, human insulin precursor, and pCES208 vector (Korean Patent Publication No. KR 10-1673080 B1, J. Microbiol. Biotechnol., 18:639-647, 2008) digested 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. To select the transformed colonies, PCR was performed using the primer pair of SEQ ID NO: 57 and SEQ ID NO: 58. Thereafter, the plasmid was obtained from the selected colony using a conventionally known plasmid extraction method, and this was named pCES208-PgapA_SPn0059_hINS.

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

To construct a strain expressing human insulin precursor, the pCES208-PgapA_SPn0059_hINS vector was transformed into wild-type Corynebacterium glutamicum ATCC13032 and the ATCC13032::ΔNCgl1048 strain constructed in Example 2 by the electric 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 ATCC13032/pCES208-PgapA_SPn0059_hINS and ATCC13032::ΔNCgl1048/pCES208-PgapA_SPn0059_hINS, respectively.

2) Human FGF2

The amino acid sequence of human FGF2 (Human FGF2) was the protein sequence from 143 to 288, excluding the amino acid sequence from 1 to 142, which is designated as a propeptide in the P09038 sequence specified in UNIPROT (UniProtKB/Swiss-Prot: P09038.3). (SEQ ID NO: 59) Codon optimization was performed to express the protein of SEQ ID NO: 59 in Corynebacterium glutamicum ATCC13032, and the nucleic acid sequence of the gene encoding the protein of SEQ ID NO: 51 is the same as the nucleic acid sequence of SEQ ID NO: 60.

order human FGF2 (SEQ ID NO: 59) PALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS Gene encoding human FGF2 (SEQ ID NO: 60) CCTGCGCTCCCCGAAGATGGCGGATCTGGAGCGTTTCCTCCGGGACATTTCAAGGATCCAAAGCGGCTCTATTGTAAAAACGGTGGCTTCTTTCTCCGCATTCATCCTGACGGACGTGTCGATGGAGTACGCGAAAAATCTGACCCTCATATCAAACTCCAGCTCCAGGCCGAGGAACGGGGAGTTGTTTCTATCAAGGGTGTTTGCGCTAACCGTTAT TTGGCAATGAAAGAAGATGGTCGCCTTCTGGCCTCAAAGTGCGTTACTGATGAGTGCTTTTTTTTTTCGAGCGCCTTGAGTCGAACAATTATAATACTTATCGGAGCCGTAAATACAcCTCCTGGTATGTCGCTCTGAAACGCACCGGACAGTATAAATTGGGAtCCAAGACTGGCCCAGGTCAGAAGGCCATCCTGTTCTTGCCAATGAGCGCCAAATCC

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

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

To construct a strain expressing human FGF2, the pCES208-PgapA_SPn0801_hFGF2 vector was transformed into wild-type Corynebacterium glutamicum ATCC13032 and the ATCC13032::ΔNCgl1048 strain constructed in Example 2 by the electric pulse method (Appl. Microbiol. Biothcenol. (1999) 52:541-545), thereby obtaining a strain into which the vector was inserted. The microorganisms thus obtained were named ATCC13032/pCES208-PgapA_SPn0801_hFGF2 and ATCC13032::ΔNCgl1048/pCES208-PgapA_SPn0801_hFGF2.

3) Human EGF

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

order human EGF (SEQ ID NO: 65) NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR Gene encoding human EGF (SEQ ID NO: 66) ACGAAGCTCCCACCACTTCAAGTCACGATACTGACAACGCTCTCCAATATATCCTACCACACAATTACAGGCGTATTTGTCCAGTGCTTCAATATACATGCACACGCCGTCGTGCAAGCAGTAACCGTCGTGGCTGAGCGGACATTCGGAATCAGAATT

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

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

To construct a strain expressing human EGF, the pCES208-PgapA_SPn0334_hEGF vector was transformed into wild-type Corynebacterium glutamicum ATCC13032 and the ATCC13032::ΔNCgl1048 strain constructed in Example 2 by the electric 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 ATCC13032/pCES208-PgapA_SPn0334_hEGF and ATCC13032::ΔNCgl1048/pCES208-PgapA_SPn0334_hEGF.

Example 6. Evaluation of expression of human insulin precursor, human FGF2, and human EGF.

The six strains produced in Example 5 were evaluated as follows. For the culture of ATCC13032::ΔNCgl1048/pCES208-PgapA_SPn0059_hINS strain expressing human insulin precursor, one colony was plated on the complex plate medium below and cultured on solid 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 at 30°C for 48 hours.

In substantially the same manner as above, five other strains produced in Example 5 were each cultured.

<Composite plate (pH 7.0)>

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 (per 1 liter of distilled water)

<Culture medium (pH 7.0)>

Glucose 63 g, ammonium sulfate 28 g, MgSO ₄ . 7H ₂ O 1.2 g, KH ₂ PO ₄ 1.1 g, yeast extract 1 g, d-biotin 1 mg, thiamine-HCl 25 mg, calcium-pantothenic acid 25 mg, MnSO ₄ . 5H ₂ O 10 mg, ZnSO ₄ . 5H ₂ O 2.5 mg, CuSO ₄ . 5H ₂ O 2.5 mg, FeSO ₄ . 5H ₂ O 10 mg, CaCO ₃ 30 g (per 1 liter of distilled water)

After fermentation, the supernatant of the culture medium was 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 by SDS-PAGE (Laemmli, 1970) and stained using DIRECTBLUE(TM) gel staining solution (SCGBIOMAX, Cat: BDS-1000). The concentration of the band corresponding to the human insulin precursor on the gel was compared using a Calibrated Densitometer (BIO-RAD, GS-900). In addition, to further confirm whether the band of the corresponding size was the human insulin precursor, LC-MS/MS analysis was performed using the in-gel digestion method (Rosenfeld, 1992).

Strains expressing human FGF2 and human EGF were cultured and analyzed using substantially the same method as described above. The average values of three replicates of data for each strain are shown in Tables 14 to 16 below.

strains Human insulin precursor expression level (O.D) Increase (%) compared to the control group ATCC13032/pCES208-PgapA_SPn0059_hINS (control) 43.73 - ATCC13032::ΔNCgl1048/pCES208-PgapA_SPn0059_hINS 69.97 60.00%

strains Human FGF2 expression level (O.D) Increase (%) compared to control group ATCC13032/pCES208-PgapA_SPn0801_hFGF2 (control) 103.2 - ATCC13032::ΔNCgl1048/pCES208-PgapA_SPn0801_hFGF2 148.7 44.09%

strains Human EGF expression level (O.D) Increase (%) compared to control group ATCC13032/pCES208-PgapA_SPn0334_hEGF (control) 150.9 - ATCC13032::ΔNCgl1048/pCES208-PgapA_SPn0334_hEGF 208.2 37.97%

As can be confirmed in Tables 14 to 16 above, the ATCC13032::ΔNCgl1048/pCES208-PgapA_SPn0059_hINS strain expressing human insulin precursor with the NCgl1048 gene deletion showed an approximately 60% increase in the expression level of human insulin precursor compared to the control group (ATCC13032/pCES208-PgapA_SPn0059_hINS). In strains expressing other target proteins, human FGF2 and human EGF, the expression levels of the target proteins also increased by 44% and 38%, respectively, confirming that the effect of the NCgl1048 gene deletion appears on various target proteins.

Example 7. Production of a strain lacking a gene having the nucleic acid sequence of sequence number 2.

To confirm whether the same effect is exhibited when a deletion trait of a gene having a nucleic acid sequence that is 98.6% homologous to the NCgl1048 gene having a nucleic acid sequence of SEQ ID NO: 1 in Corynebacterium glutamicum ATCC13869, a subtype of Corynebacterium glutamicum ATCC13032 (nucleic acid sequence code: BBD29_05870, amino acid sequence code: ANU33319.1; nucleic acid sequence of SEQ ID NO: 2 in the complete genome of Corynebacterium glutamicum ATCC13869 (GenBank: CP016335.1)) was introduced, a strain was constructed in which the gene having a nucleic acid sequence of SEQ ID NO: 2 was deleted.

In order to delete the gene having the nucleic acid sequence of the above 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 using the PCR method. The obtained gene fragment and the vector pDC24 digested with SalI and BamHI restriction enzymes were cloned using the Gibson assembly method, and then transformed into Escherichia coli DH5α and plated on LB solid medium containing 25 mg/L kanamycin. To select the transformed colonies, PCR was performed using the primer pair of SEQ ID NO: 39 and SEQ ID NO: 40. Afterwards, a plasmid was obtained from the selected colony using a commonly known plasmid extraction method and named pDC24-ΔBDD29_05870.

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

To produce a strain lacking the gene having the nucleic acid sequence of SEQ ID NO: 2, the pDC24-ΔBDD29_05870 vector was transformed into wild-type Corynebacterium glutamicum ATCC13869 by the electric pulse method (Appl. Microbiol. Biothcenol. (1999) 52:541-545), and a strain with an inserted GFP expression cassette was obtained through a second crossing process. The genetic manipulation was confirmed by performing PCR using the primer pair of SEQ ID NO: 71 and SEQ ID NO: 74 capable of amplifying the adjacent region including the position where the gene was inserted. The microorganism thus obtained was named ATCC13869::ΔBDD29_05870.

Example 8. Production of strains expressing human insulin precursor, human FGF2, and human EGF

To construct a strain expressing human insulin precursor, the pCES208-PgapA_SPn0059_hINS vector was transformed into wild-type Corynebacterium glutamicum ATCC13869 and the ATCC13869::ΔBDD29_05870 strain constructed in Example 7 by the electric 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.

In order to produce a strain expressing human FGF2 (Human FGF2) in substantially the same manner as above, ATCC13869/pCES208-PgapA_SPn0801_hFGF2 and ATCC13869::ΔBDD29_05870/pCES208-PgapA_SPn0801_hFGF2 strains were obtained using the pCES208-PgapA_SPn0801_hFGF2 vector.

In order to produce a strain 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 pCES208-PgapA_SPn0334_hEGF vector.

Example 9. Evaluation of expression of human insulin precursor, human FGF2, and human EGF.

The six strains produced in Example 8 were evaluated as follows. For the culture of ATCC13869::ΔBDD29_05870/pCES208-PgapA_SPn0059_hINS strain expressing human insulin precursor, one colony was plated on the complex plate medium below and cultured on solid 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 at 30°C for 48 hours. Similarly, culture was performed in the same manner for other strains.

In substantially the same manner as above, five other strains produced in Example 8 were each cultured.

<Composite plate (pH 7.0)>

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 (per 1 liter of distilled water)

<Culture medium (pH 7.0)>

Glucose 63 g, ammonium sulfate 28 g, MgSO ₄ . 7H ₂ O 1.2 g, KH ₂ PO ₄ 1.1 g, yeast extract 1 g, d-biotin 1 mg, thiamine-HCl 25 mg, calcium-pantothenic acid 25 mg, MnSO ₄ . 5H ₂ O 10 mg, ZnSO ₄ . 5H ₂ O 2.5 mg, CuSO ₄ . 5H ₂ O 2.5 mg, FeSO ₄ . 5H ₂ O 10 mg, CaCO ₃ 30 g (per 1 liter of distilled water)

After fermentation, the culture supernatant was analyzed as follows. The culture supernatant, from which the cells had been sedimented by centrifugation, was separated on a 4–20% Tris-glycine polyacrylamide gel using SDS-PAGE (Laemmli, 1970) and stained with DIRECTBLUE(TM) gel staining solution (SCGBIOMAX, Cat: BDS-1000). The concentration of the band corresponding to the human insulin precursor on the gel was compared using a Calibrated Densitometer (BIO-RAD, GS-900). In addition, to further confirm whether the band of the corresponding size was the human insulin precursor, LC-MS/MS analysis was performed using the in-gel digestion method (Rosenfeld, 1992).

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

strains Human insulin precursor expression level (O.D) Increase (%) compared to control group ATCC13869/pCES208-PgapA_SPn0059_hINS 31.33 - ATCC13869::ΔBDD29_05870/pCES208-PgapA_SPn0059_hINS 48.56 55.00%

strains Human FGF2 expression level (O.D) Increase (%) compared to control group ATCC13869/pCES208-PgapA_SPn0801_hFGF2 78.86 - ATCC13869::ΔBDD29_05870/pCES208-PgapA_SPn0801_hFGF2 110.1 39.62%

strains Human EGF expression level (O.D) Increase (%) compared to control group ATCC13869/pCES208-PgapA_SPn0334_hEGF 109.4 - ATCC13869::ΔBDD29_05870/pCES208-PgapA_SPn0334_hEGF 154.3 41.04%

As can be seen in Tables 18 to 20 above, even when the BBD29_05870 gene (SEQ ID NO: 2), which has 98.6% homology with the NCgl1048 gene, is deleted in addition to the deletion of the NCgl1048 gene (SEQ ID NO: 1), the ATCC13869::ΔBDD29_05870/pCES208-PgapA_SPn0059_hINS strain, which expresses human insulin precursor, was confirmed to have an increase in the expression amount of human insulin precursor by approximately 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 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 capacity of various target proteins.

From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Attach an electronic file of the sequence list

Claims (13)

A microorganism having increased recombinant protein production ability and having weakened activity of a gene having a sequence homology of 75% or more with the nucleic acid sequence of sequence number 1. A microorganism according to claim 1, wherein the gene encodes a protein having protein-decomposing enzyme activity. A microorganism having a weakened activity of a polypeptide having a protein-decomposing enzyme activity having a sequence homology of 75% or more with the amino acid sequence of SEQ ID NO: 75 in claim 1. A microorganism in claim 1, wherein the gene comprises a nucleic acid sequence of sequence number 1 or sequence number 2. In claim 1, the microorganism is a microorganism in which all or part of the gene is missing. A microorganism in claim 1, wherein the recombinant protein is at least one selected from the group consisting of collagen, collagen-derived polypeptides, hormones, cytokines, antibodies, antibiotic peptides, and antioxidant peptides. A microorganism in claim 1, wherein the recombinant protein is at least one selected from the group consisting of human insulin precursor, human FGF2, and human EGF. In the first paragraph, the microorganism has an increased ability to produce a recombinant protein compared to a control group in which the activity of the gene is not weakened. In claim 1, the microorganism is a microorganism of the genus Corynebacterium. In claim 1, the microorganism is Corynebacterium glutamicum. A method for producing a recombinant protein, comprising the step of culturing a microorganism according to any one of claims 1 to 10 in a medium. A method for producing a recombinant protein, further comprising, after the culturing step, a step of recovering the recombinant protein from the cultured microorganism, the medium, or both. A composition for producing a recombinant protein, comprising at least one selected from the group consisting of a microorganism according to any one of claims 1 to 10 and a medium in which the microorganism is cultured.