Variant of twin-arginine translocase and method for producing target protein using same

Abstract

The present application relates to a twin-arginine translocase subunit TatC variant, a microorganism comprising the TatC variant, a composition for producing a target protein, using the microorganism, and a method for producing the target protein. The microorganism comprising the TatC variant has the effect of improving the secretion and productivity of a target protein.

Description

Mutant of twin-arginine transport protein and method for producing target protein using same

[0001] Cross-citation with related application(s)

[0002] This application claims the benefit of priority from Republic of Korea Patent Application No. 10-2023-0197062, dated December 29, 2023, the entire contents of which are incorporated herein by reference.

[0003] The present application relates to a variant of a twin-arginine transport protein and a method for producing a target protein using the same.

[0004]

[0005] Microorganisms of the genus Corynebacterium have long been used in industrial fields for the production of amino acids and nucleic acids, and their safety has been recognized. While Corynebacterium possesses a transport system for secreting intracellular proteins, they are known to have a very limited number of endogenous proteins, including proteases, that can be secreted extracellularly. Therefore, utilizing Corynebacterium microorganisms to build an extracellular secretion production system for target proteins facilitates the recovery and purification of target proteins, making them highly applicable to industrial applications.

[0006] Among the previous literature that attempted to utilize microorganisms of the genus Corynebacterium for the secretion and production of target proteins, there were also studies that attempted to enhance the secretion of target proteins [Korean Patent Publication No. 10-2022-0049827], but they were limited to enhancing the expression of some of the protein transport system genes inherently possessed by microorganisms of the genus Corynebacterium, and therefore, there are clear limitations in the effect of enhancing the secretion and production capacity of target proteins.

[0007]

[0008] [Prior Art Literature]

[0009] [Patent Document]

[0010] (Patent Document 1) Republic of Korea Patent Publication KR 10-2022-0049827 A

[0011]

[0012] The purpose of this application is to include an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence of SEQ ID NO. 1,

[0013] (1) Substitution of amino acids corresponding to the 106th and 107th residues from the N-terminus in the amino acid sequence of sequence number 1 with other amino acids; or

[0014] (2) It provides a polypeptide in which the amino acids corresponding to the 106th, 107th, and 108th residues from the N-terminus in the amino acid sequence of sequence number 1 are replaced with other amino acids.

[0015] Another object of the present application is to provide a polynucleotide encoding the polypeptide.

[0016] Another object of the present application is to provide a recombinant vector comprising the polynucleotide.

[0017] Another object of the present application is to provide a polypeptide and a target protein; or

[0018] A microorganism producing a target protein is provided, comprising a polynucleotide encoding the polypeptide and a gene encoding the target protein.

[0019] Another object of the present application is to provide a composition for producing a target protein, comprising the microorganism.

[0020] Another object of the present application is to provide a method for producing a target protein, comprising a step of culturing the microorganism in a medium.

[0021]

[0022] 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.

[0023]

[0024] The present application is described in more detail below.

[0025]

[0026] polypeptide

[0027] The present application comprises an amino acid sequence having at least 90% sequence homology with the amino acid sequence of SEQ ID NO: 1; or an amino acid sequence of SEQ ID NO: 1;

[0028] A polypeptide is provided in which the amino acid residues corresponding to the 106th and 107th residues from the N-terminus in the amino acid sequence of SEQ ID NO: 1, or the 106th, 107th and 108th residues, are replaced with amino acids different from the original amino acid.

[0029] The above polypeptide may be a polypeptide having twin-arginine translocase activity.

[0030] The above polypeptide may be a twin-arginine translocase subunit TatC protein.

[0031] In one example, the twin-arginine translocase subunit TatC protein may be derived from Corynebacterium glutamicum (WP_011014400.1) and may be represented by the amino acid sequence of SEQ ID NO: 1.

[0032]

[0033] In one example, the polypeptide comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 1,

[0034] (1) Substitution of amino acids corresponding to the 106th and 107th residues from the N-terminus in the amino acid sequence of sequence number 1 with other amino acids; or

[0035] (2) It may be a polypeptide in which the amino acids corresponding to the 106th, 107th, and 108th residues from the N-terminus in the amino acid sequence of sequence number 1 are replaced with other amino acids.

[0036] In one example, the polypeptide comprises the amino acid sequence of SEQ ID NO: 1,

[0037] (1) In the amino acid sequence of sequence number 1, the 106th and 107th amino acids from the N-terminus are replaced with other amino acids; or

[0038] (2) It may be a polypeptide in which the 106th, 107th, and 108th amino acids from the N-terminus in the amino acid sequence of sequence number 1 are substituted with other amino acids.

[0039] The amino acid corresponding to the 106th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 1 or the 106th amino acid from the N-terminus in the amino acid sequence of SEQ ID NO: 1 may be threonine.

[0040] Accordingly, the amino acid corresponding to the 106th residue or the 106th amino acid may be substituted with alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan. In one specific example, the amino acid corresponding to the 106th residue or the 106th amino acid may be substituted with alanine.

[0041] The amino acid corresponding to the 107th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 1 or the 107th amino acid from the N-terminus in the amino acid sequence of SEQ ID NO: 1 may be proline.

[0042] Accordingly, the amino acid corresponding to the 107th residue or the 107th amino acid may be substituted with serine, arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan. In one specific example, the amino acid corresponding to the 107th residue or the 107th amino acid may be substituted with serine.

[0043] The amino acid corresponding to the 108th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 1 or the 108th amino acid from the N-terminus in the amino acid sequence of SEQ ID NO: 1 may be glycine.

[0044] Accordingly, the amino acid corresponding to the 108th residue or the 108th amino acid may be substituted with alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan. In one specific example, the amino acid corresponding to the 108th residue or the 108th amino acid may be substituted with alanine.

[0045] In one example, the polypeptide may be:

[0046] A sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 1,

[0047] (1) In the amino acid sequence of SEQ ID NO: 1, the amino acid corresponding to the 106th residue from the N-terminus is replaced with alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan, and the amino acid corresponding to the 107th residue is replaced with serine, arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; or

[0048] (2) In the amino acid sequence of sequence number 1, the amino acid corresponding to the 106th residue from the N-terminus is replaced with alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan, the amino acid corresponding to the 107th residue is replaced with serine, arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan, and the amino acid corresponding to the 108th residue is replaced with alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, It may be a polypeptide substituted with threonine, asparagine, glutamine, cysteine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

[0049] In one example, the polypeptide may be:

[0050] A sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 1,

[0051] (1) In the amino acid sequence of sequence number 1, the amino acid corresponding to the 106th residue from the N-terminus is replaced with alanine and the amino acid corresponding to the 107th residue is replaced with serine; or

[0052] (2) It may be a polypeptide in which the amino acid corresponding to the 106th residue from the N-terminus in the amino acid sequence of sequence number 1 is substituted with alanine, the amino acid corresponding to the 107th residue is substituted with serine, and the amino acid corresponding to the 108th residue is substituted with alanine.

[0053] In one example, the polypeptide comprises:

[0054] (1) In the amino acid sequence of sequence number 1, the 106th amino acid is replaced with alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan, and the 107th amino acid is replaced with serine, arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; or

[0055] (2) In the amino acid sequence of sequence number 1, the 106th amino acid from the N-terminus is substituted with alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan, the 107th amino acid is substituted with serine, arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan, and the 108th amino acid is substituted with alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, It may be a polypeptide substituted with glutamine, cysteine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

[0056] In one example, the polypeptide may be:

[0057] (1) In the amino acid sequence of sequence number 1, the 106th amino acid from the N-terminus is substituted with alanine and the 107th amino acid is substituted with serine; or

[0058] (2) It may be a polypeptide in which the 106th amino acid from the N-terminus in the amino acid sequence of sequence number 1 is substituted with alanine, the 107th amino acid is substituted with serine, and the 108th amino acid is substituted with alanine.

[0059] In one specific embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 1,

[0060] (1) A polypeptide in which the 106th amino acid from the N-terminus in the amino acid sequence of sequence number 1 is substituted with alanine and the 107th amino acid is substituted with serine; or

[0061] (2) It may be a polypeptide in which the 106th amino acid from the N-terminus in the amino acid sequence of sequence number 1 is substituted with alanine, the 107th amino acid is substituted with serine, and the 108th amino acid is substituted with alanine.

[0062] In one specific embodiment, the polypeptide may comprise or consist of the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 24.

[0063] If the polypeptide contains a mutation in which the amino acid residues corresponding to the 106th and 107th residues from the N-terminus in the amino acid sequence of SEQ ID NO: 1, or the amino acid residues corresponding to the 106th, 107th, and 108th residues, are substituted with amino acids other than the original amino acids, it is obvious that the polypeptide of the present application can be included even if amino acid residues other than the above amino acid residues are deleted, modified, substituted, or added, as long as it exhibits twin-arginine translocase activity. For example, it may be a case in which the polypeptide has a sequence addition or deletion that does not alter the activity of the polypeptide, a naturally occurring mutation, a silent mutation, or a conservative substitution at the N-terminus, C-terminus, and / or within the amino acid sequence of the polypeptide.

[0064] The term "conservative substitution" refers to the replacement of one amino acid with another amino acid with similar structural and / or chemical properties. Such amino acid substitutions may generally be 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.

[0065] In one example, the polypeptide comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 1,

[0066] (1) Substitution of amino acids corresponding to the 106th and 107th residues from the N-terminus in the amino acid sequence of sequence number 1 with other amino acids; or

[0067] (2) In the amino acid sequence of sequence number 1, the amino acids corresponding to the 106th, 107th and 108th residues from the N-terminus are replaced with other amino acids,

[0068] It may be a polypeptide having twin-arginine translocase activity.

[0069] In one example, the polypeptide having the twin-arginine translocase activity may be a twin-arginine translocase subunit TatC protein.

[0070] In one example, the polypeptide (e.g., a polypeptide having twin-arginine translocase activity) or twin-arginine translocase subunit TatC protein 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, and Corynebacterium imitans. The microorganism may be one or more species selected from the group consisting of, but is not limited to, Corynebacterium imitans, Corynebacterium testudinoris, and Corynebacterium flavescens.

[0071] As used herein, the term "variant polypeptide" refers to a polypeptide in which one or more amino acids are conservatively substituted and / or modified, thereby differing from the amino acid sequence of the variant polypeptide before the mutation, but retaining functions or properties. Such variant polypeptides can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant polypeptide may be increased, unchanged, or decreased compared to the polypeptide before the mutation. Additionally, some variant polypeptides may include variant polypeptides in which one or more portions, such as the N-terminal leader sequence or the transmembrane domain, are deleted. Other variant polypeptides may include variant polypeptides in which portions are deleted from the N- and / or C-terminus of the mature protein. The above term "variant polypeptide" may be used interchangeably with terms such as variant, modification, mutated protein, mutation, and variant (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, variant, etc.), and is not limited thereto as long as the term is used in the meaning of mutation. In addition, the variant polypeptide may include deletion or addition of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence involved in protein translocation co-translationally or post-translationally may be conjugated to the N-terminus of the variant polypeptide.Additionally, the above mutant polypeptides may be conjugated with other sequences or linkers to enable identification, purification, or synthesis.

[0072] For the purpose of the present application, the mutant polypeptide comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9% sequence homology with the amino acid sequence of SEQ ID NO: 1,

[0073] (1) Substitution of amino acids corresponding to the 106th and 107th residues from the N-terminus in the amino acid sequence of sequence number 1 with other amino acids; or

[0074] (2) It may be a polypeptide in which the amino acids corresponding to the 106th, 107th, and 108th residues from the N-terminus in the amino acid sequence of sequence number 1 are replaced with other amino acids.

[0075] The above mutant polypeptide may have increased twin-arginine translocase activity and / or activity that increases the secretion and production ability of a target protein of a microorganism compared to a polypeptide before mutation (e.g., a polypeptide comprising the amino acid sequence of SEQ ID NO: 1).

[0076]

[0077] polynucleotide

[0078] Another aspect provides a polynucleotide encoding the polypeptide.

[0079] In this application, the term "polynucleotide" may mean a DNA or RNA strand of a certain length or longer, which is a polymer of nucleotides in which nucleotide units (monomers) are linked in a long chain shape by covalent bonds.

[0080] The polynucleotide comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9% sequence homology with the nucleic acid sequence of SEQ ID NO: 2,

[0081] In the amino acid sequence of sequence number 1, the codon encoding the amino acid residue corresponding to the 106th and 107th residues from the N-terminus is replaced with a codon encoding another amino acid, or

[0082] It may be a polynucleotide in which the codons encoding amino acid residues corresponding to the 106th, 107th, and 108th residues from the N-terminus in the amino acid sequence of SEQ ID NO: 1 are replaced with codons encoding other amino acids.

[0083] It was explained that the amino acid corresponding to the 106th residue from the N-terminus in the amino acid sequence of SEQ ID NO. 1 or the 106th amino acid from the N-terminus in the amino acid sequence of SEQ ID NO. 1 is threonine, the amino acid corresponding to the 107th residue or the 107th amino acid is proline, and the amino acid corresponding to the 108th residue or the 108th amino acid is glycine.

[0084] Accordingly, the codon encoding the amino acid corresponding to the 106th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 1 or the 106th amino acid from the N-terminus in the amino acid sequence of SEQ ID NO: 1 may be ACU, ACC, ACA or ACG, the amino acid corresponding to the 107th residue or the codon encoding the 107th amino acid may be CCU, CCC, CCA or CCG, the amino acid corresponding to the 108th residue or the codon encoding the 108th amino acid may be GGU, GGC, GGA or GGG.

[0085] It is well known in the art that the codon encoding each amino acid comprises a polynucleotide sequence. Various modifications can be made to the codon without altering the amino acid sequence of the polypeptide, taking into account codon degeneracy or preferred codons in the organism intended to express the polypeptide.

[0086] In one specific embodiment, the polynucleotide may comprise or consist of a nucleic acid sequence of SEQ ID NO: 19 or SEQ ID NO: 25.

[0087]

[0088] The polynucleotide of the present application may include, without limitation, a probe that can be prepared from a known genetic sequence, for example, a sequence that can hybridize under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence of the present application. The term “stringent conditions” refers to conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, polynucleotides having high homology or identity, at least 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 99.9% or more of homology or identity are hybridized with each other, and more than that Conditions under which polynucleotides with low homology or identity do not hybridize with each other, or washing conditions of typical southern hybridization, such as 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.Conditions for washing once, specifically two to three times, can be listed, at a salt concentration and temperature equivalent to 1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0089] Hybridization requires that two nucleotide sequences be complementary, but hybridized polynucleotides may contain some mismatches between bases, depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Accordingly, the polynucleotides of the present application may also include isolated nucleic acid fragments that are complementary in their entirety, as well as substantially similar nucleic acid sequences.

[0090] Specifically, a polynucleotide having homology or identity with the polynucleotide of the present application can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the conditions described above. In addition, the Tm value may be, but is not limited to, 60°C, 63°C, or 65°C, and can be appropriately adjusted by a person skilled in the art depending on the purpose.

[0091] The appropriate stringency for hybridizing the polynucleotides depends on the length and degree of complementarity of the polynucleotides, variables which are well known in the art (e.g., J. Sambrook et al., supra).

[0092] In this specification, “consisting of a sequence,” “consisting essentially of a sequence,” or “comprising a sequence” may mean all instances of including the sequence, but is not intended to exclude instances of including sequences other than the sequence.

[0093] In this specification, the phrase "a polynucleotide (which may be used interchangeably with a "gene") or a polypeptide (which may be used interchangeably with a "protein") "comprises or consists of or is represented by a specific nucleic acid sequence or amino acid sequence" may mean that the polynucleotide or polypeptide essentially includes the specific nucleic acid sequence or amino acid sequence, and may be interpreted as including (or not excluding) a "substantially equivalent sequence" in which a non-significant mutation (deletion, substitution, modification, and / or addition) is added to the specific nucleic acid sequence or amino acid sequence to the extent that the original function and / or the desired function of the polynucleotide or polypeptide is maintained.

[0094] As used herein, 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.

[0095] 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.

[0096] 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.

[0097] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as that disclosed, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482, or in, for example, Needleman et al. (1970), J Mol Biol. 48:443. 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 coding sequence matrix, as disclosed by Gribskov et al. (1986) Nucl. Acids Res. 48:443, and (3) a binary comparison matrix, containing values ​​of 1 for identity and 0 for non-identity, 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.

[0098] As used herein, the term "corresponding to" refers to an amino acid residue at a position listed in a polypeptide, or an amino acid residue that is similar, identical, or homologous to the residue listed in the polypeptide. Identifying an amino acid at a corresponding position may determine a specific amino acid in a sequence that references a particular sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0099] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, and based on this, each amino acid residue of the amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, a sequence alignment algorithm such as that described in the present application can identify the position of an amino acid, or the position at which a modification such as a substitution, insertion, or deletion occurs, by comparing it with a query sequence (also referred to as a “reference sequence”).

[0100] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277) can be used, but is not limited thereto, and any sequence alignment program known in the art, pairwise sequence comparison algorithm, etc. can be appropriately used.

[0101]

[0102] recombinant vector

[0103] Another aspect provides a recombinant vector comprising the polynucleotide.

[0104] As used herein, the term "vector" refers to a DNA construct for delivering a target polynucleotide into a suitable host or host cell. For example, it may include, but is not limited to, a nucleic acid sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the target polypeptide in a suitable host cell. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and / or a sequence for regulating the termination of transcription and / or translation. After being transformed into a suitable host cell, the vector may be maintained independently of the genome (genome) of the host cell, or may be integrated into the genome of the host cell. For example, the target polynucleotide may be integrated into a chromosome via an insertion vector. Insertion of the polynucleotide into a chromosome can be accomplished by any method known in the art, for example, but not limited to, homologous recombination.

[0105] The vector usable in this specification is not particularly limited as long as it is replicable in a host cell, and may be selected from all commonly used vectors. Examples of commonly used vectors include plasmids, cosmids, viruses, bacteriophages, etc. in a natural or recombinant state. For example, as the vector, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc. can be used as a phage vector or a cosmid vector, and pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series, etc. can be used as a plasmid vector. Specifically, examples include, but are not limited to, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors.

[0106] The above vector may further comprise a selection marker to determine whether the vector has been introduced into a transformed cell or has been integrated into the genome of the transformed cell. The selection marker is used to determine whether the vector-transformed cell or the polynucleotide has been integrated, and may be selected from genes that confer a selectable phenotype, such as drug resistance, nutrient requirement, cytotoxic agent resistance, or surface protein expression. In an environment treated with a selective agent, only cells expressing the selection marker will survive or exhibit other phenotypic characteristics, thereby enabling the selection of transformed cells.

[0107] Expression of the above polypeptide in a microorganism can be performed by introducing a polynucleotide encoding the above polypeptide, or a vector containing the polynucleotide, into a host cell and culturing a recombinant cell (e.g., a microorganism) containing the same.

[0108] The introduction of a polynucleotide encoding the polypeptide or a vector containing the polynucleotide into a microorganism can be performed by a person skilled in the art by appropriately selecting a known transformation method. As used herein, the term "transformation" refers to changing the genetic characteristics of a host cell (microorganism) by introducing a target polynucleotide or a vector containing the polynucleotide into the host cell (microorganism). The transformed polynucleotide may be positioned by insertion into the chromosome of the host cell or may be positioned extrachromosomally. The polynucleotide may be introduced in an appropriate form depending on the purpose of introduction. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for autonomous expression. The expression cassette may typically include expression control elements such as a promoter, a transcription termination signal, a ribosome binding site, and / or a translation termination signal that are operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of autonomous replication. Additionally, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence necessary for expression in the host cell. The term "operably linked" as used herein may mean that the polynucleotide is functionally linked to an expression control element (e.g., a promoter) so that transcriptional regulation (e.g., transcription initiation) of the polynucleotide can be performed. Operable linkage can be performed using genetic recombination techniques known in the art.

[0109] The method for transforming the above polynucleotide into a host cell can be performed by any method for introducing a nucleic acid into a cell (microorganism), and can be performed by appropriately selecting a transformation technique known in the art depending on the host cell. Examples of the known transformation methods include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) precipitation, DEAE-dextran, cationic liposome, lipofection, and lithium acetate-DMSO.

[0110] The above recombinant vector may include the polynucleotide and a gene encoding the target protein.

[0111] The above target 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 above 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.

[0112] In one example, the target protein is collagen (e.g., collagen derived from human, porcine, bovine, 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, 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 The target protein may be at least one selected from the group consisting of, but is not limited to, cholera toxin B, organophosphohydrolase, calcitonin, blood coagulation factors, hirudin, and monoclonal antibody 5T4. In one example, the target protein may further include a tag for improving protein purification, expression, solubilization, or detection of the target protein. Various tags that can be used for this purpose are known in the art, and may be, for example, GST tag, FLAG tag, polyarginine tag, polyhistidine tag, for example, 6His-tag, MBP tag, S-tag, influenza virus HA tag, thioredoxin tag, or staphylococcal protein A tag.

[0113] In one example, the target protein may be, but is not limited to, bovine collagen protein (bovine COL6A1, NP_001137337.1; SEQ ID NO: 5) or a polypeptide derived therefrom (SEQ ID NO: 6 or SEQ ID NO: 32) or YGK peptide (SEQ ID NO: 34), which is an antioxidant functional protein derived from Saccharomyces cerevisiae.

[0114] In one example, the target protein may be a protein and / or peptide having a desired activity in a living body (e.g., activity for preventing, alleviating, and / or treating a specific disease or symptom, or 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., protease, kinase, 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 target 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.

[0115] The recombinant vector may further comprise a gene encoding a secretory signal peptide. In order to express and secrete the target protein through the transport system of a microorganism, the target protein may be linked to a secretory signal peptide at the N'-terminus. The recombinant vector may comprise a gene encoding the target protein and a gene encoding the secretory signal peptide in an appropriate order so as to enable expression of the target protein in a form in which the secretory signal peptide is linked to the N'-terminus.

[0116] 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 target protein in a microorganism, and in one example, it is at least 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. However, this is not limited to the above. That is, any secretory signal peptide capable of inducing secretion of a target protein from a microorganism can be used without limitation.

[0117] The above recombinant vector may include a promoter operably linked to a gene encoding a target protein and / or a gene encoding a secretory signal peptide, for the expression of the gene encoding the target protein and / or the gene encoding a secretory signal peptide.

[0118] 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 target protein or twin-arginine transposase, etc.), such as a DNA region to which a polymerase binds to initiate transcription of the gene.

[0119] 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.

[0120] 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.

[0121] The above recombinant vector may include at least one selected from the group consisting of a polynucleotide encoding a polypeptide provided in the present application, a gene encoding a target protein, and a gene encoding a secretory signal peptide.

[0122] The above recombinant vector may include the above polynucleotide, a gene encoding a target protein, and a gene encoding a secretory signal peptide.

[0123] The above polynucleotide, the gene encoding the target protein and the gene encoding the secretory signal peptide may each be operably linked to the above promoter, and the promoters may be the same or different.

[0124]

[0125] The polypeptide provided in the present application may have enhanced twin-arginine translocase activity. The polypeptide may have enhanced expression, secretion, and / or production capabilities of a target protein.

[0126] As used herein, the term “enhancement” of polypeptide activity means that the activity of a polypeptide is increased compared to the intrinsic activity within a host cell (microorganism). The term “enhancement” may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Here, activation, enhancement, up-regulation, overexpression, and increase may all include exhibiting an activity that was not originally present, or exhibiting an enhanced activity compared to the intrinsic activity or the activity before modification. The term “intrinsic activity” refers to the activity of a specific polypeptide that a parent strain or unmodified microorganism originally possessed before the trait change, when the trait change is caused by genetic mutation due to natural or artificial factors. This may be used interchangeably with “activity before modification.” “Enhanced,” “upregulated,” “overexpressed,” or “increased” the activity of a polypeptide relative to its intrinsic activity means that the activity and / or concentration (expression amount) of a particular polypeptide is improved compared to the activity and / or concentration (expression amount) that the parent strain or unmodified microorganism originally had prior to the transformation.

[0127] The above enhancement can be achieved by introducing an exogenous polypeptide, or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. Whether the activity of the polypeptide is enhanced can be confirmed by an increase in the activity level of the polypeptide, the expression level, or the secretion and / or production amount of the target protein linked to the secretory signal peptide recognized by the polypeptide.

[0128] Enhancement of the activity of the above polypeptide can be achieved by applying various methods well known in the art, and is not limited as long as the activity of the target polypeptide can be enhanced compared to the microorganism before modification. Specifically, it may be achieved by using genetic engineering and / or protein engineering, which are routine methods of molecular biology and are well known to those skilled in the art, but are not limited thereto (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0129] Specifically, the enhancement of the polypeptide of the present application is

[0130] 1) Increase in the intracellular copy number of a polynucleotide encoding a polypeptide;

[0131] 2) Replacing the gene expression control region on the chromosome that codes for a polypeptide with a highly active sequence;

[0132] 3) Modification of the base sequence encoding the initiation codon or 5'-UTR region of a gene transcript encoding a polypeptide;

[0133] 4) Modification of the amino acid sequence of the polypeptide so as to enhance polypeptide activity;

[0134] 5) Modification of the polynucleotide sequence encoding the polypeptide so as to enhance the activity of the polypeptide (e.g., modification of the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the activity of the polypeptide);

[0135] 6) Introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same;

[0136] 7) Codon optimization of polynucleotides encoding polypeptides;

[0137] 8) Analyzing the tertiary structure of the polypeptide and selecting the exposed portion to modify or chemically modify; or

[0138] 9) It may be one or a combination of two or more selected from 1) to 8), but is not particularly limited thereto.

[0139] More specifically,

[0140] The increase in the intracellular copy number of the polynucleotide encoding the polypeptide described above may be achieved by introducing into the host cell a vector capable of replicating and functioning independently of the host, to which the polynucleotide encoding the polypeptide is operably linked. Alternatively, the polynucleotide encoding the polypeptide may be achieved by introducing one copy or two or more copies into the chromosome of the host cell. The introduction into the chromosome may be performed by introducing into the host cell a vector capable of inserting the polynucleotide into the chromosome of the host cell, but is not limited thereto. The vector is as described above.

[0141] 2) Replacing the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide with a sequence having strong activity may be, for example, a mutation in the sequence such as deletion, insertion, non-conservative or conservative substitution, or a combination thereof to further enhance the activity of the expression control region, or replacement with a sequence having stronger activity. The expression control region may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. As an example, it may be, but is not limited to, replacing the original promoter with a strong promoter.

[0142] Examples of known strong promoters include, but are not limited to, the CJ1 to CJ7 promoters (US Patent No. US 7662943 B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (US Patent No. US 10584338 B2), the O2 promoter (US Patent No. US 10273491 B2), the tkt promoter, and the yccA promoter.

[0143] 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 higher polypeptide expression rate than the endogenous initiation codon, but is not limited thereto.

[0144] The modification of the amino acid sequence or polynucleotide sequence of the above 4) and 5) may be, but is not limited to, 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 enhance the activity of the polypeptide, or replacement with an amino acid sequence or polynucleotide sequence improved to have stronger activity, or an amino acid sequence or polynucleotide sequence improved to have increased activity. The replacement may be specifically performed by inserting the polynucleotide into a chromosome by homologous recombination, but is not limited thereto. The vector used at this time may additionally include a selection marker to confirm whether or not chromosomal insertion has occurred. The selection marker is as described above.

[0145] The introduction of the foreign polynucleotide exhibiting the activity of the polypeptide as described above 6) may be the introduction into the host cell of a foreign polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide. The foreign polynucleotide is not limited in its origin or sequence as long as it exhibits the same / similar activity as the polypeptide. The method used for the introduction may be performed by a person skilled in the art appropriately selecting a known transformation method, and the polypeptide may be produced by expressing the introduced polynucleotide in the host cell, thereby increasing its activity.

[0146] The above 7) codon optimization of a polynucleotide encoding a polypeptide may be codon optimization of an endogenous polynucleotide to increase transcription or translation within a host cell, or codon optimization of a foreign polynucleotide to achieve optimized transcription or translation within a host cell.

[0147] The above 8) analyzing the tertiary structure of a polypeptide and selecting an exposed portion to modify or chemically modify may be done by, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing the sequence information of known proteins, determining a template protein candidate based on the degree of sequence similarity, confirming the structure based on this, and selecting an exposed portion to modify or chemically modify, and modifying or modifying it.

[0148] Such enhancement of polypeptide activity may be, but is not limited to, an increase in the activity or concentration of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild type or pre-transformed microorganism, or an increase in the amount of a product produced from the polypeptide.

[0149]

[0150] microorganism

[0151] Another aspect provides a microorganism comprising at least one member selected from the group consisting of the polypeptide, the polynucleotide, and a vector comprising the polynucleotide.

[0152] In this application, the term "microorganism (or strain)" may encompass both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification. The microorganism may be a microorganism whose specific mechanism has been strengthened or weakened, such as by the insertion of an external gene or the enhancement or weakening of the activity of an endogenous gene, and may be a microorganism that includes genetic modification for the production of a target protein. In this application, the terms "microorganism," "strain," "host," and "host cell" may be used interchangeably.

[0153] The microorganism (or strain, recombinant cell) of the present application may be a microorganism having twin-arginine translocase activity, or having enhanced twin-arginine translocase activity, or having the ability to express, secrete, and / or produce a target protein, or having increased ability to express, secrete, and / or produce a target protein.

[0154] As described above, in the above microorganism, the target protein may be linked to a secretory signal peptide.

[0155] 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 genetic mutation caused by natural or artificial factors. For example, the unmodified microorganism may refer to a strain into which the mutant polypeptide of the present application or a polynucleotide encoding the mutant polypeptide is not introduced, or before it is introduced. The term "unmodified microorganism" may be used interchangeably with "pre-modified strain," "pre-modified microorganism," "unmutated strain," "unmodified microorganism," or "reference microorganism."

[0156] The above microorganism may be a microorganism of the genus Corynebacterium (Corynebacterium sp.). 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, and Corynebacterium imitans. The microorganism may be one or more species selected from the group consisting of, but is not limited to, Corynebacterium imitans, Corynebacterium testudinoris, and Corynebacterium flavescens.

[0157] In one example, the target strain for comparing whether the secretion and production ability of the target protein increases may be a wild-type Corynebacterium genus microorganism, such as Corynebacterium glutamicum ATCC13032 strain, comprising a wild-type twin-arginine translocase subunit TatC protein (e.g., amino acid sequence of SEQ ID NO: 1).

[0158] In one example, the secretion and production ability of the target protein may be increased by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, 200% or more, 210% or more, 220% or more, 230% or more, 240% or more, 250% or more, 260% or more, 270% or more, 280% or more, 290% or more, or 300% or more in the secretion and production ability of the target protein compared to the parent strain before mutation or the unmodified microorganism.

[0159] In one example, the microorganism with improved secretion and production ability of the target protein has a slightly higher secretion and production ability of the target protein compared to the parent strain or the unmodified microorganism before mutation.

[0160] 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.

[0161]

[0162] Composition and production method for target protein production

[0163] Another aspect is to provide a composition for producing a target protein comprising at least one selected from the group consisting of the above microorganism and a medium in which the above microorganism is cultured.

[0164] Another aspect provides for the use of the microorganism for producing a desired protein.

[0165] The composition of the present application may further comprise any suitable excipient commonly used in compositions for producing target 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.

[0166]

[0167] Another aspect provides a method for producing (or manufacturing) a target protein, comprising a step of culturing the microorganism in a medium.

[0168] The method for producing the target protein of the present application may include a step of culturing the microorganism in a medium.

[0169] 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.

[0170] 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.

[0171] 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)].

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] The target protein produced by the culture of the present application may be secreted into the medium or remain within the cell.

[0178] The method for producing the target 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.

[0179] The method for producing the target protein of the present application may additionally include a step of recovering the target 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.

[0180] The above recovery may be performed by collecting the target 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 target protein may be recovered from the medium or microorganism using a suitable method known in the art.

[0181] Additionally, the method for producing the target protein 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 method for producing the target protein 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.

[0182]

[0183] The present application provides a twin-arginine translocase subunit TatC mutant, and a microorganism comprising the mutant has an excellent ability to secrete a target protein.

[0184]

[0185] Figure 1 shows the production concentration of the target protein (bCOL6_1) in a Corynebacterium glutamicum strain expressing the wild-type TatC protein or a TatC mutant containing an amino acid single substitution mutation (lane 1: wild-type TatC protein; lane 2: TatC T106A mutant; lane 3: TatC P107S mutant; lane 4: TatC G108A mutant).

[0186] Figure 2 shows the production concentration of the target protein (bCOL6_1) in a Corynebacterium glutamicum strain expressing the wild-type TatC protein or a TatC mutant containing an amino acid combination substitution mutation (lane 1: wild-type TatC protein; lane 2: TatC T106A / P107S mutant; lane 3: TatC P107S / G108A mutant; lane 4: TatC T106A / G108A mutant; lane 5: TatC T106A / P107S / G108A mutant).

[0187] Figure 3 shows the production concentration of the target protein (bCOL6_1) in Corynebacterium strains expressing Corynebacterium glutamicum TatC protein, wild-type TatC protein from Escherichia coli, TatC P97S mutant from Escherichia coli, wild-type TatC protein from Bacillus subtilis, and TatC S95A / P96S / G97A mutant from Bacillus subtilis (lane 1: Corynebacterium glutamicum TatC protein; lane 2: wild-type TatC protein from E. coli; lane 3: TatC P97S mutant from E. coli; lane 4: wild-type TatC protein from Bacillus subtilis; lane 5: TatC S95A / P96S / G97A mutant from Bacillus subtilis).

[0188] Figure 4 shows the production concentration of the target protein (bCOL6_1) in a Corynebacterium glutamicum strain in which a gene encoding the TatC T106A / P107S mutant or the TatC T106A / P107S / G108A mutant has been introduced into the chromosome (lane 1: wild type; lane 2: TatC T106A / P107S / G108A mutant; lane 3: TatC T106A / P107S / G108A mutant).

[0189] Figure 5 shows the production concentration of the target protein (bCOL6_2) in a Corynebacterium glutamicum strain in which a gene encoding the TatC T106A / P107S mutant or the TatC T106A / P107S / G108A mutant has been introduced into the chromosome (lane 1: wild type; lane 2: TatC T106A / P107S / G108A mutant; lane 3: TatC T106A / P107S / G108A mutant).

[0190] Figure 6 shows the production concentration of the target protein (YGK peptide) in a Corynebacterium glutamicum strain in which a gene encoding the TatC T106A / P107S mutant or the TatC T106A / P107S / G108A mutant has been introduced into the chromosome (lane 1: wild type; lane 2: TatC T106A / P107S / G108A mutant; lane 3: TatC T106A / P107S / G108A mutant).

[0191]

[0192] 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.

[0193]

[0194] Example 1. Production of a target protein using a Corynebacterium glutamicum strain expressing a TatC gene containing a single or combined amino acid mutation.

[0195] The twin-arginine translocase (Tat) system, a protein transport system inherent in Corynebacterium glutamicum strains, functions to secrete target proteins with a secretory signal peptide sequence recognized by this system to the outside of the cell membrane. In order to increase the production of target proteins using Corynebacterium glutamicum strains, we explored strategies to enhance the secretion capacity of the Tat system. It is known that the TatC protein, a component of the Tat system, plays a proofreading role by recognizing the folding state of the target protein to be secreted and selecting only proteins with the correct folding state for secretion.

[0196] In the present application, a TatC mutant in which the amino acid at residues 106, 107, and / or 108 of the Corynebacterium glutamicum TatC protein was substituted with another amino acid was produced, and the effect on the secretion ability of a Corynebacterium glutamicum strain expressing the mutant was confirmed.

[0197]

[0198] Example 1-1. Construction of a simultaneous expression vector of the TatC gene and a model target protein containing a single amino acid mutation.

[0199] In order to compare the secretion and production ability of target proteins of Corynebacterium glutamicum strains expressing Corynebacterium glutamicum TatC mutants, an expression vector was constructed by simultaneously introducing a partial sequence of bovine collagen type 6 (bovine COL6A1) as a model target protein and Corynebacterium glutamicum TatC. Specifically, the bovine COL6A1 sequence information [SEQ ID NO: 5] (Accession No. NP_001137337.1) was obtained from the National Institutes of Health (NIH GenBank) in the United States, and a C-terminal HisX6 tag (HHHHHH; SEQ ID NO: 75) for protein purification was added to a portion of the amino acid sequence, and named bCOL6_1 [SEQ ID NO: 6]. Based on the bCOL6_1 amino acid sequence, codon optimization was performed to synthesize a gene having a nucleotide sequence of [SEQ ID NO: 7]. Using the synthesized gene DNA as a template, PCR was performed using the primers [SEQ ID NO: 36] and [SEQ ID NO: 37] to obtain a bCOL6_1 gene fragment. The PCR reaction was performed using Pfu-X DNA Polymerase (Solgent, Cat. No. SPX16-R500) and the manufacturer's protocol was followed.

[0200] Genomic DNA of the wild-type Corynebacterium glutamicum ATCC13032 strain was extracted using a G-spin Total DNA extraction mini kit (Intron, Cat. No. 17045) according to the protocol provided in the kit, and PCR was performed using the genomic DNA as a template and the primers of [SEQ ID NO: 38] and [SEQ ID NO: 39] to obtain a TatC gene fragment. The amino acid sequence of the TatC protein of the wild-type Corynebacterium glutamicum ATCC13032 strain is the amino acid sequence of SEQ ID NO: 1 (WP_011014400.1), and the gene sequence encoding the amino acid sequence is the nucleic acid sequence of SEQ ID NO: 2.

[0201] In order to obtain the CgR0949 secretion signal peptide sequence [SEQ ID NO: 8] recognized by the Tat system, PCR was performed using the ATCC13032 genomic DNA as a template and the primers [SEQ ID NO: 40] and [SEQ ID NO: 41], and a CgR0949 secretion signal peptide fragment was obtained. In order to obtain the promoter sequence, PCR was performed using the CJ7 promoter (Pcj7) [SEQ ID NO: 10] (US Patent No. US 7662943 B2) as a template and the primers [SEQ ID NO: 42] and [SEQ ID NO: 43], and a Pcj7 fragment was obtained. In addition, in order to secure the pyk promoter (Ppyk) sequence [SEQ ID NO: 11], PCR was performed using the ATCC13032 genomic DNA as a template and the primers [SEQ ID NO: 44] and [SEQ ID NO: 45], and a Ppyk fragment was obtained.

[0202] The obtained Pcj7 fragment, CgR0949 secretion signal peptide fragment, bCOL6_1 gene fragment, Ppyk fragment, TatC gene fragment and pCES208 vector digested with XbaI restriction enzyme ("Construction of heat-inducible expression vector of Corynebacterium glutamicum and C. ammoniagenes: fusion of lambda operator with promoters isolated from C. ammoniagenes." Journal of microbiology and biotechnology 18.4 (2008): 639-647.) were cloned using In-fusion HD Cloning Kit (Takara Bio Inc, Cat. No. 638933) to obtain a recombinant vector, which was named “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(WT)”.

[0203] In order to secure a TatC gene fragment containing a single amino acid mutation, PCR was performed using the ATCC13032 genomic DNA as a template, primers of [SEQ ID NO: 38] and [SEQ ID NO: 46], and primers of [SEQ ID NO: 47] and [SEQ ID NO: 39], to obtain gene fragments for the T106A mutant in which threonine, the 106th amino acid of the TatC protein, was substituted with alanine. From the same template, PCR was performed using primers of [SEQ ID NO: 38] and [SEQ ID NO: 48], and primers of [SEQ ID NO: 49] and [SEQ ID NO: 39], and gene fragments for the P107S mutant in which proline, the 107th amino acid of the TatC protein, was substituted with serine were obtained, and PCR was performed using primers of [SEQ ID NO: 38] and [SEQ ID NO: 50], and primers of [SEQ ID NO: 51] and [SEQ ID NO: 39], and gene fragments for the G108A mutant in which glycine, the 108th amino acid of the TatC protein, was substituted with alanine were obtained. Recombinant vectors were obtained by cloning each amino acid single mutant gene fragment of TatC, Pcj7 fragment, CgR0949 secretion signal peptide fragment, bCOL6_1 gene fragment, Ppyk fragment, and pCES208 vector digested with XbaI restriction enzyme using In-fusion HD Cloning Kit, and were named “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(T106A)”, “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(P107S)”, and “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(G108A)”, respectively.

[0204] The amino acid sequences of the above TatC T106A mutant, TatC P107S mutant, and TatC G108A mutant are amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, and the gene sequences encoding the above amino acid sequences are nucleic acid sequences of SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 17, respectively.

[0205]

[0206] Example 1-2. Production of a target protein using a Corynebacterium glutamicum strain expressing a TatC gene containing a single amino acid mutation.

[0207] The recombinant vectors produced in the above Example 1-1 were transformed into the Corynebacterium glutamicum wild type ATCC13032 strain by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), and the strains into which the recombinant vectors were introduced were named “ATCC13032-bCOL6_1-tatC(WT)”, “ATCC13032-bCOL6_1-tatC(T106A)”, “ATCC13032-bCOL6_1-tatC(P107S)”, and “ATCC13032-bCOL6_1-tatC(G108A)”, respectively.

[0208] Culture evaluations were conducted to confirm the target protein secretion and production capacity of the four Corynebacterium glutamicum strains described above. Specifically, colonies of each strain were subcultured in nutrient medium, and then each strain was inoculated into a 250-ml corner-baffle flask containing 25 ml of production medium, followed by shaking culture at 200 rpm for 48 hours at 30°C. Each medium contained 50 mg / L of kanamycin antibiotic to maintain the recombinant vector.

[0209] [Nutrient medium (pH 7.2)]

[0210] Glucose 10g, meat extract 5g, polypeptone 10g, sodium chloride 2.5g, yeast extract 5g, agar 20g, urea 2g, kanamycin 50mg (per 1 liter of distilled water)

[0211] [Production medium (pH 7.0)]

[0212] Glucose 60g, ammonium sulfate 30g, yeast extract 10g, potassium phosphate dibasic 1g, magnesium sulfate heptahydrate 0.4g, iron sulfate heptahydrate 20mg, manganese sulfate pentahydrate 1.8mg, biotin 1.8mg, thiamine-HCl 9mg, calcium carbonate 50g, kanamycin 50mg (per 1 liter of distilled water)

[0213]

[0214] After the culture was completed under the above conditions, the production concentration of the bCOL6-1 target protein was confirmed through analysis of the culture supernatant. Specifically, the culture medium was centrifuged at 5000 × g for 10 minutes to sediment the cells, and only the culture supernatant was separated and electrophoresed on a 4-20% Tris-Glycine SDS Precast Gel (Labis Koma, Cat. No. KG75355) using the SDS-PAGE (Laemmli, 1970) method. The gel was stained using DIRECTBLUE™ gel staining solution (SCGBIOMAX, Cat. No. BDS-1000), and the image of the stained gel was acquired using a Calibrated Densitometer (BIO-RAD, GS-900) and the concentration of the target protein in the culture medium was analyzed. In addition, LC-MS analysis using the in gel digestion method (Rosenfeld, 1992) was performed to confirm whether the amino acid sequence included in the protein band of the corresponding size matched the target protein. The concentration of bCOL6-1 target protein production of the above four strains is shown in Figure 1 and Table 1 below.

[0215] Number Classification Strain Name Purpose Protein Concentration (mg / L) Change compared to control group (%) 1 Control group ATCC13032-bCOL6_1-tatC (WT) 279.8-2 Experimental group ATCC13032-bCOL6_1-tatC (T106A) 165.7-40.78% 3 ATCC13032-bCOL6_1-tatC (P107S) 202.2-27.73% 4 ATCC13032-bCOL6_1-tatC (G108A) 228.9-18.19%

[0216] As shown in Table 1 above, the strain expressing the TatC mutant containing the amino acid single substitution mutation did not show an increase in the concentration of the target protein (bCOL6_1) compared to the strain expressing the wild-type TatC protein (ATCC13032-bCOL6_1-tatC(WT)), which is the control.

[0217]

[0218] Example 1-3. Production of a target protein using a Corynebacterium glutamicum strain expressing a TatC gene containing an amino acid integration mutation.

[0219] Recombinant vectors were constructed to evaluate the effect of combining the amino acid single substitution mutations of Example 1-1 on the secretion and production ability of the target protein of a Corynebacterium glutamicum strain.

[0220] Specifically, PCR was performed using the genomic DNA of the above-mentioned Corynebacterium glutamicum ATCC13032 strain as a template, primers of [SEQ ID NO: 38] and [SEQ ID NO: 52], and primers of [SEQ ID NO: 53] and [SEQ ID NO: 39], to obtain gene fragments for the T106A and P107S integrated mutants. From the same template, PCR was performed using the primers [SEQ ID NO: 38] and [SEQ ID NO: 54], and the primers [SEQ ID NO: 55] and [SEQ ID NO: 39], and gene fragments for the P107S, G108A integration mutants were obtained, and from the same template, PCR was performed using the primers [SEQ ID NO: 38] and [SEQ ID NO: 56], and the primers [SEQ ID NO: 57] and [SEQ ID NO: 39], and gene fragments for the T106A, G108A integration mutants were obtained. Similarly, from the same template, PCR was performed using the primers [SEQ ID NO: 38] and [SEQ ID NO: 58], and the primers [SEQ ID NO: 59] and [SEQ ID NO: 39], and gene fragments for the T106A, P107S, G108A integration mutants were obtained.

[0221] The amino acid integration mutant gene fragments of the above TatC and the Pcj7 fragment, CgR0949 secretion signal peptide fragment, bCOL6_1 gene fragment, Ppyk fragment obtained in Example 1-1, and the pCES208 vector digested with XbaI restriction enzyme were cloned using an In-fusion HD Cloning Kit to obtain recombinant vectors, and “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(T106A / P107S)”, “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(P107S / G108A)”, “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(T106A / G108A)”, It was named “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(T106A / P107S / G108A)”.

[0222] The amino acid sequences of the above TatC T106A / P107S mutant, TatC P107S / G108A mutant, TatC T106A / G108A mutant, and TatC T106A / P107S / G108A mutant are amino acid sequences of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24, respectively, and the gene sequences encoding the above amino acid sequences are nucleic acid sequences of SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 25, respectively.

[0223]

[0224] The above-mentioned recombinant vectors were transformed into the wild-type Corynebacterium glutamicum ATCC13032 strain by electroporation, and the strains into which the recombinant vectors were introduced were named “ATCC13032-bCOL6_1-tatC(T106A / P107S)”, “ATCC13032-bCOL6_1-tatC(P107S / G108A)”, “ATCC13032-bCOL6_1-tatC(T106A / G108A)”, and “ATCC13032-bCOL6_1-tatC(T106A / P107S / G108A)”, respectively. In order to compare the secretion and production ability of the target protein of the above four types of Corynebacterium glutamicum strains and the ATCC13032-bCOL6_1-tatC (WT) strain obtained in Example 1-2, the strains were cultured in the same manner as in Example 1-2, and the concentration of the target protein in the culture solution was analyzed, which is shown in Figure 2 and Table 2 below.

[0225] Number Classification Strain Name Purpose Protein Concentration (mg / L) Change compared to control group (%) 1 Control group ATCC13032-bCOL6_1-tatC (WT) 278.6-2 Experimental group ATCC13032-bCOL6_1-tatC (T106A / P107S / G108A) 771.31 76.85% 3 ATCC13032-bCOL6_1-tatC (T106A / P107S) 781.21 80.4% 4 ATCC13032-bCOL6_1-tatC (P107S / G108A) 201-27.85% 5 ATCC13032-bCOL6_1-tatC (T106A / G108A) 267.2-4.09%

[0226] As shown in Table 2 above, among the strains expressing TatC variants including amino acid combination substitution mutations, the ATCC13032-bCOL6_1-tatC(T106A / P107S) strain and the ATCC13032-bCOL6_1-tatC(T106A / P107S / G108A) strain showed a target protein concentration approximately 2.8 times higher than the strain expressing the wild-type TatC protein (ATCC13032-bCOL6_1-tatC(WT)), which is the control group.

[0227]

[0228] Example 2. Production of target protein using a Corynebacterium glutamicum strain expressing a TatC gene derived from a foreign microorganism containing an amino acid integration mutation.

[0229] The results of Example 1 above showed that the T106A / P107S mutant and the T106A / P107S / G108A mutant of Corynebacterium glutamicum TatC improved the secretion and production ability of the target protein of the Corynebacterium glutamicum strain. To expand on this, it was confirmed whether the same effect of improving the secretion and production ability of the target protein was exhibited when the equivalent mutation was applied to the TatC gene derived from a foreign microorganism. Specifically, the amino acid sequence information [SEQ ID NO: 3; WP_000109943.1] encoded by the TatC gene of Escherichia coli [SEQ ID NO: 4] and the amino acid sequence information [SEQ ID NO: 26; WP_000109943.1] encoded by the TatC gene of Bacillus subtilis [SEQ ID NO: 27] were obtained from the GenBank of the National Institutes of Health. WP_010886426.1] was obtained and homology analysis was performed with the amino acid sequence information [SEQ ID NO: 1; WP_011014400.1] encoded by the TatC gene [SEQ ID NO: 2] of Corynebacterium glutamicum using BLAST of the National Institutes of Health. As a result, it was confirmed that the 97th amino acid, proline, of the TatC protein derived from E. coli and the 96th amino acid, proline, of the TatC protein derived from Bacillus subtilis correspond to the 107th amino acid, proline, of the TatC derived from Corynebacterium glutamicum. Since the 96th and 98th amino acids of the TatC protein derived from E. coli are alanine, a TatC P97S mutant was constructed in which the 97th amino acid, proline, was substituted solely with serine, and a S95A / P96S / G97A mutant was constructed in which the 95th amino acid, serine, the 96th amino acid, proline, and the 97th amino acid, glycine, of the TatC protein derived from Bacillus subtilis were substituted with alanine. Specifically, genomic DNA of E. coli K-12 KCTC 2223 strain and Bacillus subtilis KCTC 3135 strain were extracted using a G-spin Total DNA extraction mini kit according to the protocol provided in the kit.Using the genomic DNA of the E. coli K-12 KCTC 2223 strain extracted above as a template, PCR was performed using the primers [SEQ ID NO: 60] and [SEQ ID NO: 61] to obtain a wild-type E. coli TatC gene fragment, and using the primers [SEQ ID NO: 60] and [SEQ ID NO: 62] from the same template, and the primers [SEQ ID NO: 63] and [SEQ ID NO: 61] to perform PCR, and gene fragments for the E. coli TatC P97S mutant were obtained. Similarly, PCR was performed using the genomic DNA of the Bacillus subtilis KCTC 3135 strain extracted above as a template and the primers [SEQ ID NO: 64] and [SEQ ID NO: 65] to obtain a wild-type Bacillus subtilis TatC gene fragment, and from the same template, PCR was performed using the primers [SEQ ID NO: 64] and [SEQ ID NO: 66], and the primers [SEQ ID NO: 67] and [SEQ ID NO: 65] to obtain gene fragments for the Bacillus subtilis TatC S95A / P96S / G97A mutant.

[0230] The wild type gene fragment and P97S mutant gene fragment of the above E. coli TatC, and the wild type gene fragment and S95A / P96S / G97A mutant gene fragment of Bacillus subtilis TatC, the Pcj7 fragment, the CgR0949 secretion signal peptide fragment, the bCOL6_1 gene fragment, the Ppyk fragment obtained in Example 1-1, and the pCES208 vector digested with XbaI restriction enzyme were cloned using an In-fusion HD Cloning Kit to obtain recombinant vectors, and “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_EctatC(WT)”, “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_EctatC(P97S)”, They were named “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_BstatC(WT)” and “pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_BstatC(S95A / P96S / G97A).”

[0231] The amino acid sequences of the above-mentioned E. coli-derived TatC P97S mutant and Bacillus subtilis-derived TatC S95A / P96S / G97A mutant are amino acid sequences of SEQ ID NO: 28 and SEQ ID NO: 30, respectively, and the gene sequences encoding the above-mentioned amino acid sequences are nucleic acid sequences of SEQ ID NO: 29 and SEQ ID NO: 31, respectively.

[0232]

[0233] The above-mentioned recombinant vectors were transformed into the Corynebacterium glutamicum wild-type ATCC13032 strain by electroporation, and the strains into which the recombinant vectors were introduced were named “ATCC13032-bCOL6_1-EctatC(WT)”, “ATCC13032-bCOL6_1-EctatC(P97S)”, “ATCC13032-bCOL6_1-BstatC(WT)”, and “ATCC13032-bCOL6_1-BstatC(S95A / P96S / G97A)”, respectively. In order to compare the secretion and production ability of the target protein of the above four types of Corynebacterium glutamicum strains and the ATCC13032-bCOL6_1-tatC (WT) strain obtained in Example 1-2, the strains were cultured in the same manner as in Example 1-2, and the concentration of the target protein in the culture solution was analyzed, which is shown in Figure 3 and Table 3 below.

[0234] Number Classification Strain Name Purpose Protein Concentration (mg / L) Change compared to control group (%) 1 Control group ATCC13032-bCOL6_1-tatC (WT) 266.3-2 Experimental group ATCC13032-bCOL6_1-EctatC (WT) 116.1-56.40% 3 ATCC13032-bCOL6_1-EctatC (P97S) 116.5-56.25% 4 ATCC13032-bCOL6_1-BstatC (WT) 164.5-38.23% 5 ATCC13032-bCOL6_1-BstatC (S95A / P96S / G97A) 179.3-32.67%

[0235] As shown in Table 3 above, it was confirmed that the Corynebacterium glutamicum strain expressing E. coli TatC (ATCC13032-bCOL6_1-EctatC(WT)) or the Corynebacterium glutamicum strain expressing Bacillus subtilis TatC (ATCC13032-bCOL6_1-BstatC(WT)) showed a lower target protein concentration than the control strain expressing Corynebacterium glutamicum TatC (ATCC13032-bCOL6_1-tatC(WT)). In addition, a Corynebacterium glutamicum strain (ATCC13032-bCOL6_1-EctatC(P97S)) into which an E. coli TatC P97S mutant, which has an amino acid substitution mutation corresponding to the P107S mutation of Corynebacterium glutamicum TatC, and a Corynebacterium glutamicum strain (ATCC13032-bCOL6_1-BstatC(S95A / P96S / G97A)) into which an Bacillus subtilis TatC S95A / P96S / G97A mutant, which has an amino acid substitution mutation corresponding to the T106A / 9107S / G108A mutation of Corynebacterium glutamicum TatC, were introduced, respectively, and a Corynebacterium glutamicum strain expressing wild-type E. coli TatC and a wild-type Bacillus subtilis TatC were introduced. No significant increase in target protein concentration was observed compared to the expressing Corynebacterium glutamicum strain.

[0236] These results indicate that the effect of the Corynebacterium glutamicum TatC mutant confirmed in Examples 1-3 on improving secretion and production of target proteins is not universally observed for TatC derived from foreign microorganisms.

[0237]

[0238] Example 3. Production of an improved strain of Corynebacterium glutamicum in which a TatC gene containing an amino acid integration mutation has been introduced into the chromosome and production of various target proteins using the strain.

[0239] From the results of Example 1 above, an improved strain was produced by introducing the Corynebacterium glutamicum TatC T106A / P107S mutant or the TatC T106A / P107S / G108A mutant, which was confirmed to improve the secretion and production ability of the target protein of the Corynebacterium glutamicum strain, into the chromosome. Specifically, PCR was performed using the primers of [SEQ ID NO: 68] and [SEQ ID NO: 69] using the pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(T106A / P107S) or pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(T106A / P107S / G108A) vector DNA as a template, and gene fragments for the TatC T106A / P107S mutant and the TatC T106A / P107S / G108A mutant were obtained, respectively. The above TatC integrated mutant gene fragment and the pDZ vector (US Patent No. US 9109242 B2) digested with XbaI restriction enzyme were cloned using the In-fusion HD Cloning Kit to obtain recombinant vectors, and each of these was introduced into the chromosome of wild-type Corynebacterium glutamicum ATCC13032 strain by electroporation to cause homologous recombination. Afterwards, the improved strains in which the wild-type TatC gene on the chromosome was replaced with the integrated mutant gene were confirmed through PCR using the primers of [SEQ ID NO: 68] and [SEQ ID NO: 69] for the colonies that went through the second crossing process, and these were named “ATCC13032::tatC(T106A / P107S)” and “ATCC13032::tatC(T106A / P107S / G108A)”, respectively.

[0240] In order to compare the secretion and production ability of the target protein of Corynebacterium glutamicum strains into which the TatC mutant was introduced, an expression vector for the bCOL6_1 target protein was constructed. Specifically, PCR was performed using the primers of [SEQ ID NO: 42] and [SEQ ID NO: 70] using the pCES208-Pcj7_CgR0949_bCOL6_1-Ppyk_tatC(WT) vector DNA as a template, and a gene fragment for the Pcj7_CgR0949_bCOL6_1 expression cassette was obtained. The gene fragment and the pCES208 vector digested with XbaI restriction enzyme were cloned using the In-fusion HD Cloning Kit to obtain a recombinant vector, which was named “pCES208-Pcj7_CgR0949_bCOL6_1”. The pCES208-Pcj7_CgR0949_bCOL6_1 recombinant vector was transformed into the Corynebacterium glutamicum wild-type ATCC13032 strain and the ATCC13032::tatC(T106A / P107S) and ATCC13032::tatC(T106A / P107S / G108A) improved strains by electroporation, and the strains into which the recombinant vector was introduced were named “ATCC13032-bCOL6_1”, “ATCC13032::tatC(T106A / P107S)-bCOL6_1”, and “ATCC13032::tatC(T106A / P107S / G108A)-bCOL6_1”, respectively. In order to compare the secretion and production ability of the target protein of the three Corynebacterium glutamicum strains mentioned above, they were cultured in the same manner as in Example 1-2, and the concentration of the target protein in the culture solution was analyzed, which is shown in Figure 4 and Table 4 below.

[0241] Number Classification Strain Name Purpose Protein Concentration (mg / L) Change compared to control group (%) 1 Control group ATCC13032-bCOL6_185.3-2 Experimental group ATCC13032::tatC(T106A / P107S)-bCOL6_1193.1126.38% 3 ATCC13032::tatC(T106A / P107S / G108A)-bCOL6_1188.4120.87%

[0242] As shown in Table 4 above, it was confirmed that the Corynebacterium glutamicum strain (ATCC13032::tatC(T106A / P107S)-bCOL6_1) in which the Corynebacterium glutamicum TatC T106A / P107S variant was introduced into the chromosome and the Corynebacterium glutamicum strain (ATCC13032::tatC(T106A / P107S / G108A)-bCOL6_1) in which the Corynebacterium glutamicum TatC T106A / P107S / G108A variant was introduced into the chromosome showed a target protein concentration more than twice as high as the control strain (ATCC13032-bCOL6_1).

[0243] From these results, it was confirmed that a Corynebacterium glutamicum strain expressing the TatC T106A / P107S mutant or the T106A / P107S / G108A mutant by introducing it into the chromosome exhibited improved target protein secretion and production ability.

[0244]

[0245] In order to confirm whether the effect of enhancing the secretion and production of target proteins exhibited by the Corynebacterium glutamicum strain with the TatC mutant introduced into the chromosome is the same for other target proteins, expression vectors for target proteins other than bCOL6_1 were constructed. Specifically, among the bovine COL6A1 sequence information, a C-terminal HisX6 tag (HHHHHH; SEQ ID NO: 75) for protein purification was added to some amino acid sequences different from those of bCOL6_1, and named bCOL6_2 [SEQ ID NO: 32], and after codon optimization, a gene having a nucleic acid sequence of [SEQ ID NO: 33] was synthesized. Using the synthesized DNA as a template, PCR was performed using the primers of [SEQ ID NO: 71] and [SEQ ID NO: 72], to obtain a bCOL6_2 gene fragment. Meanwhile, in order to produce an expression vector for YGK peptide (including C-terminal HisX6 tag) [SEQ ID NO: 34] [Mirzaei et al., Journal of Functional Foods, 2015, Journal of Functional Foods Volume 19, Part A, December 2015, Pages 259-268], an antioxidant functional protein derived from Saccharomyces cerevisiae, codon optimization was performed to synthesize a gene having a nucleic acid sequence of [SEQ ID NO: 35], and using the synthesized DNA as a template, PCR was performed using primers of [SEQ ID NO: 73] and [SEQ ID NO: 74] to obtain a YGK gene fragment. Recombinant vectors were obtained by cloning the above-mentioned gene fragments, the Pcj7 fragment obtained in Example 1-1, the CgR0949 secretion signal peptide fragment, and the pCES208 vector cut with XbaI restriction enzyme using the In-fusion HD Cloning Kit, and were named “pCES208-Pcj7_CgR0949_bCOL6_2” and “pCES208-Pcj7_CgR0949_YGK,” respectively.

[0246] The above recombinant vectors were transformed into the wild type Corynebacterium glutamicum ATCC13032 strain and the improved strains ATCC13032::tatC(T106A / P107S) and ATCC13032::tatC(T106A / P107S / G108A) by electroporation, and the strains into which the recombinant vectors were introduced were named “ATCC13032-bCOL6_2”, “ATCC13032::tatC(T106A / P107S)-bCOL6_2”, “ATCC13032::tatC(T106A / P107S / G108A)-bCOL6_2”, “ATCC13032-YGK”, They were named “ATCC13032::tatC(T106A / P107S)-YGK” and “ATCC13032::tatC(T106A / P107S / G108A)-YGK”.

[0247] First, in order to compare the secretion and production ability of the target protein of the three Corynebacterium glutamicum strains into which the bCOL6_2 expression vector was introduced, the strains were cultured in the same manner as in Example 1-2, and the concentration of the target protein in the culture solution was analyzed, which is shown in Fig. 5 and Table 5 below.

[0248] Number Classification Strain Name Purpose Protein Concentration (mg / L) Change compared to control group (%) 1 Control group ATCC13032-bCOL6_2110.6-2 Experimental group ATCC13032::tatC(T106A / P107S)-bCOL6_2176.559.59% 3 ATCC13032::tatC(T106A / P107S / G108A)-bCOL6_2165.849.91%

[0249] As shown in Table 5 above, it was confirmed that the Corynebacterium glutamicum strain (ATCC13032::tatC(T106A / P107S)-bCOL6_2) in which the TatC T106A / P107S mutant was introduced into the chromosome and the Corynebacterium glutamicum strain (ATCC13032::tatC(T106A / P107S / G108A)-bCOL6_2) in which the TatC T106A / P107S / G108A mutant was introduced into the chromosome showed a target protein concentration that was about 1.5 times higher for the bCOL6_2 target protein compared to the control strain (ATCC13032-bCOL6_2).

[0250]

[0251] Next, in order to compare the secretion and production ability of the target protein of the three Corynebacterium glutamicum strains into which the YGK expression vector was introduced, they were cultured in the same manner as in Example 1-2, and the concentration of the target protein in the culture solution was analyzed, which is shown in Fig. 6 and Table 6 below.

[0252] Number Classification Strain Name Purpose Protein Concentration (mg / L) Change compared to control group (%) 1 Control group ATCC13032-YGK49.4-2 Experimental group ATCC13032::tatC(T106A / P107S)-YGK134.0171.26% 3 ATCC13032::tatC(T106A / P107S / G108A)-YGK148.6200.81%

[0253] As shown in Table 6 above, it was confirmed that the Corynebacterium glutamicum strain (ATCC13032::tatC(T106A / P107S)-bCOL6_2) in which the TatC T106A / P107S mutant was introduced into the chromosome and the Corynebacterium glutamicum strain (ATCC13032::tatC(T106A / P107S / G108A)-bCOL6_2) in which the TatC T106A / P107S / G108A mutant was introduced into the chromosome showed 2.7-fold and 3-fold higher target protein concentrations for the YGK target protein, respectively, compared to the control strain (ATCC13032-bCOL6_2).

[0254] From the above results, it was confirmed that the Corynebacterium glutamicum strain in which the TatC T106A / P107S mutant or the TatC T106A / P107S / G108A mutant was introduced into the chromosome exhibited improved target protein secretion and production ability for various sequences and types of target proteins.

[0255]

[0256] 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.

Claims

1. Contains an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence of sequence number 1; (1) Substitution of amino acids corresponding to the 106th and 107th residues from the N-terminus in the amino acid sequence of sequence number 1 with other amino acids; or (2) A polypeptide in which the amino acids corresponding to the 106th, 107th, and 108th residues from the N-terminus in the amino acid sequence of sequence number 1 are replaced with other amino acids.

2. In paragraph 1, (1) The amino acid corresponding to the 106th residue from the N-terminus in the amino acid sequence of sequence number 1 is replaced with alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; and The amino acid corresponding to residue 107 is replaced with serine, arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; or (2) The amino acid corresponding to the 106th residue from the N-terminus in the amino acid sequence of sequence number 1 is replaced with alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; The amino acid corresponding to residue 107 is replaced with serine, arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; and A polypeptide wherein the amino acid corresponding to residue 108 is replaced with alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

3. In paragraph 1, (1) In the amino acid sequence of sequence number 1, the amino acid corresponding to the 106th residue from the N-terminus is replaced with alanine and the amino acid corresponding to the 107th residue is replaced with serine; or (2) A polypeptide in which the amino acid corresponding to the 106th residue from the N-terminus in the amino acid sequence of sequence number 1 is substituted with alanine, the amino acid corresponding to the 107th residue is substituted with serine, and the amino acid corresponding to the 108th residue is substituted with alanine.

4. A polypeptide according to claim 1, wherein the polypeptide has twin-arginine translocase activity.

5. A polypeptide according to claim 1, wherein the polypeptide is a twin-arginine translocase subunit TatC protein.

6. A polypeptide in the first paragraph, wherein the polypeptide is derived from a microorganism of the genus Corynebacterium.

7. A polypeptide according to claim 6, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.

8. In the first paragraph, comprising an amino acid sequence of sequence number 1, (1) Substitution of the 106th and 107th residues in the amino acid sequence of sequence number 1 with other amino acids; or (2) A polypeptide in which the 106th, 107th, and 108th residues in the amino acid sequence of sequence number 1 are substituted with other amino acids.

9. In paragraph 8, (1) In the amino acid sequence of sequence number 1, the 106th residue from the N-terminus is replaced with alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; and A replacement of residue 107 with serine, arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; or (2) Substitution of the 106th residue from the N-terminus in the amino acid sequence of sequence number 1 with alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; A replacement of residue 107 with serine, arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; and A polypeptide wherein residue 108 is substituted with alanine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

10. In paragraph 8, (1) In the amino acid sequence of sequence number 1, the 106th residue from the N-terminus is substituted with alanine and the 107th residue is substituted with serine; or (2) A polypeptide in which the 106th residue from the N-terminus in the amino acid sequence of sequence number 1 is substituted with alanine, the 107th residue is substituted with serine, and the 108th residue is substituted with alanine.

11. A polypeptide comprising an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 24 in claim 8.

12. A polynucleotide encoding a polypeptide according to any one of claims 1 to 11.

13. A recombinant vector comprising the polynucleotide of clause 12.

14. A recombinant vector further comprising a gene encoding a target protein according to claim 13.

15. A recombinant vector according to claim 14, wherein the target protein is at least one selected from the group consisting of collagen, collagen-derived polypeptides, hormones, cytokines, antibodies, antibiotic peptides, and antioxidant peptides.

16. The polypeptide and target protein of paragraph 1; or A microorganism producing a target protein, comprising a polynucleotide encoding the above polypeptide and a gene encoding the target protein.

17. In paragraph 16, the microorganism is a microorganism with enhanced secretion and production ability of the target protein.

18. In paragraph 16, the microorganism is a microorganism of the genus Corynebacterium.

19. In claim 18, the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.

20. A composition for producing a target protein, comprising at least one selected from the group consisting of a microorganism according to any one of claims 16 to 19 and a medium in which the microorganism is cultured.

21. A method for producing a target protein, comprising a step of culturing a microorganism according to any one of claims 16 to 19 in a medium.

22. A method for producing a target protein, comprising, in claim 21, additionally, after the culturing step, a step of recovering the target protein from the cultured microorganism, the medium, or both.

Images