Signal peptides for producing a nuclease derived from serratia marcescens and use thereof

Abstract

The present application relates to signal peptides for producing Serratia marcescens-derived nuclease and uses thereof. In particular, the present application relates to a polypeptide comprising a signal peptide and a Serratia marcescens-derived nuclease amino acid sequence, a microorganism comprising said polypeptide, and a method of producing Serratia marcescens-derived nuclease, said method comprising the step of culturing said microorganism. The present application can be used for large-scale production of Serratia marcescens-derived nuclease.

Description

Technical Field

[0001] This disclosure relates to a signal peptide for increasing the production capacity of nucleases derived from Serratia marcescens and a method for producing nucleases using the same. Background Technology

[0002] Nucleases are enzymes that cleave phosphodiester bonds in nucleic acids and play a crucial role in the replication, repair, recombination, and degradation of DNA and RNA in living organisms. In particular, nonspecific nucleases are used for pharmaceutical and research purposes in areas such as viral infection, cancer diagnosis, gene therapy, and vaccine production, and are also widely used in molecular biology research. Furthermore, these nucleases have applications in food and industrial fields, such as removing residual nucleic acids from fermented foods, improving the viscosity of fermentation broths, and removing biofilms, thus making them enzymes of very high industrial value.

[0003] Although various microorganisms produce nucleases, those derived from the Gram-negative bacterium *Serratia marcescens* are known to possess a non-specific characteristic of cleaving all forms of DNA and RNA (including single-stranded, double-stranded, linear, and circular forms) and exhibit high specific activity. Therefore, numerous studies have been conducted to produce nucleases derived from *Serratia marcescens* (US 9,796,994 B2).

[0004] However, when nucleases are overexpressed and exist in an active state in the cytoplasm of host cells, they can also degrade nucleic acids in the host, thereby inhibiting cell growth. Therefore, the production of nucleases using microorganisms presents challenges. To date, cases of producing nucleases up to 5,000 U / mL have been reported using this method, and commercially available products produced using this method remain very expensive. Therefore, selecting the optimal secretory signal peptide for nuclease production is crucial.

[0005] Meanwhile, Bacillus subtilis is considered a GRAS (Generally Recognized As Safe) organism and possesses excellent protein secretion capabilities, thus it has been used as a host strain for the production of various recombinant proteins. In particular, research has been conducted on secretion signal peptides for protein secretion. To date, various screening techniques and optimization methods for the production of exogenous proteins have been investigated; however, the optimal signal peptide for the secretion of nucleases derived from Serratia marcescens has been rarely studied (He Li et al., Biochemical Engineering Journal, 189 (2022) 108718). Summary of the Invention

[0006] Technical issues

[0007] The inventors have demonstrated that the production capacity of nucleases derived from Serratia marcescens can be increased by using signal peptides, thereby completing this disclosure.

[0008] Technical solution

[0009] One object of this disclosure is to provide a polypeptide comprising: an amino acid sequence of SEQ ID NO: 5 or 7; and an amino acid sequence of a nuclease derived from Serratia marcescens.

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

[0011] Another object of this disclosure is to provide microorganisms containing the said polypeptide or the polynucleotide encoding it.

[0012] Another object of this disclosure is to provide a method for producing nucleases derived from Serratia marcescens, comprising: culturing microorganisms containing the polypeptide or a polynucleotide encoding the polypeptide in a culture medium; and recovering the nucleases from the culture.

[0013] Another object of this disclosure is to provide a composition for producing nucleases derived from Serratia marcescens, comprising a signal peptide having an amino acid sequence of SEQ ID NO: 5 or 7.

[0014] Beneficial effects

[0015] This disclosure provides a signal peptide for increasing the production capacity of nucleases derived from Serratia marcescens, and thus for use in the mass production of nucleases derived from Serratia marcescens. Attached Figure Description

[0016] Figure 1 This is a graph illustrating the size of the corresponding halo generated by screening nuclease-producing strains for each signal peptide.

[0017] Figure 2 This is a graph confirming the changes in nuclease expression levels over time in strains cultured in a 5 L fermenter. Detailed Implementation

[0018] This disclosure will now be described in detail. Furthermore, each description and embodiment described herein can be applied to other descriptions and embodiments. That is, all combinations of the various elements described herein fall within the scope of this disclosure. Moreover, the scope of this disclosure is not limited to the specific embodiments described below. Furthermore, those skilled in the art will be able to recognize or identify many equivalent substitutions for specific aspects of this disclosure described herein through conventional experimentation. These equivalent substitutions are intended to be included in this disclosure.

[0019] The terminology used in this article is as follows.

[0020] Proteins and polypeptides

[0021] As used herein, the terms "protein" or "peptide" refer to a polymer or oligomer of consecutive amino acid residues. In this disclosure, the terms "peptide," "protein," and "peptide" are used interchangeably.

[0022] In this disclosure, amino acid sequences are described in the direction from N-terminus to C-terminus, unless otherwise stated.

[0023] In this disclosure, with regard to the amino acid sequence, it is obvious that a polypeptide or protein "contains" the amino acid sequence shown in a specific sequence number, is "composed of" the amino acid sequence shown in a specific sequence number, or "has" the amino acid sequence shown in a specific sequence number. It may also include polypeptides or proteins in which specific amino acids are deleted, modified, substituted, or added, provided that they have the same or corresponding activity as the polypeptide or protein composed of the amino acid sequence of that sequence number. For example, a polypeptide or protein may also include polypeptides or proteins with amino acid additions or deletions, naturally occurring mutations, silent mutations, or conserved substitutions in the internal region or upstream or downstream region (N-terminus or C-terminus) of the polypeptide or protein that do not alter the function of the protein of this disclosure, provided that the polypeptide or protein exhibits the same or corresponding activity.

[0024] Polynucleotides

[0025] As used herein, the terms “polynucleotide,” “nucleic acid,” or “nucleic acid molecule” refer to a chain of DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA) of at least a certain length, which is a polymer of nucleotides in which nucleotide monomers are linked together by covalent bonds to form a long chain. In this disclosure, “polynucleotide,” “nucleic acid,” and “nucleic acid molecule” are used interchangeably.

[0026] Identity, homology

[0027] As used herein, the term “identity” or “homology” refers to the degree of similarity between two given amino acid or base sequences and may be expressed as a percentage. In this disclosure, the terms “homology” and “identity” are generally used interchangeably.

[0028] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard alignment algorithms, and a default gap penalty established by the program used can be applied together.

[0029] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined, for example, by known computer algorithms, such as the “FASTA” program with default parameters as described in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it can be performed using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), such as in the Needleman program (version 5.0.0 or later) in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, TrendsGenet. 16:276-277), or using GAP computer programs such as the Smith-Waterman algorithm (Smith and Waterman, Adv. Appl. Math (1981) 2:482) (including the GCG package (Devereux, J. et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, SF et al., J MOLEC BIOL 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994 and CARILLO et al. (1988) SIAM J Applied Math 48:1073)) compare sequence information to determine homology, similarity, or identity. For example, homology, similarity, or identity can be determined by using BLAST or ClustalW from the National Center for Biotechnology Information.

[0030] Furthermore, whether any two polynucleotide sequences are homologous, similar, or identical can be confirmed by Southern hybridization experiments under suitable hybridization conditions. Suitable hybridization conditions can be determined by methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual; FMAusubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York), but are not limited thereto. For example, homologous or identical polynucleotide sequences can typically hybridize at least about 50%, 60%, 70%, 80%, or 90% along the entire sequence or full length under stringent conditions.

[0031] As used herein, the term "strict conditions" refers to conditions that enable specific hybridization between polynucleotides. These conditions are specifically described in the literature (see Sambrook et al., above, 9.50-9.51, 11.7-11.8). For example, strict conditions may include polynucleotides with high homology or identity, i.e., polynucleotides with at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity hybridizing with each other, while polynucleotides with less homology or identity do not hybridize with each other. Alternatively, they may include typical washing conditions for Southern hybridization, i.e., washing once, particularly two or three times, at salt concentrations and temperatures corresponding to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0032] Hybridization can occur between nucleotides with complementary base sequences, but depending on the strictness of the hybridization, hybridized polynucleotides can contain some base mismatches. The term "complementary" is used to describe the relationship between the bases of nucleotides that can hybridize with each other. For example, in DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of this disclosure can comprise isolated nucleic acid fragments complementary to the entire sequence, as well as base sequences substantially similar to them.

[0033] Specifically, polynucleotides homologous to or identical with the polynucleotides disclosed herein can be subjected to a T at 55°C. m The value is detected by hybridization. In addition, T... m The value can be 60°C, 63°C or 65°C, but is not limited to these, and can be appropriately adjusted by those skilled in the art.

[0034] The appropriate stringency of hybrid polynucleotides depends on the length and complementarity of the polynucleotides, and these variables are well known in the art (e.g., J. Sambrook et al., above).

[0035] Carrier, Transformation

[0036] As used herein, the term "vector" refers to a DNA construct used to deliver desired polynucleotides into a suitable host or host cell.

[0037] In one example, the vector may contain the base sequence of a polynucleotide encoding a desired polypeptide, which is operatively linked to a suitable expression regulatory region (or expression regulatory sequence) so that the desired polypeptide can be expressed in a suitable host. The expression regulatory sequence may include a promoter capable of initiating transcription, any operator sequence regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences regulating transcription and translation termination. After transformation into a suitable host cell (microbe), the vector may replicate or function independently of the host genome, or it may integrate into the genome itself to replicate or function.

[0038] Furthermore, in one instance, the vector of this disclosure may contain a sequence for inserting the desired polynucleotide into a chromosome. Insertion of a polynucleotide into a chromosome using a vector can be performed by any method known in the art, such as homologous recombination, but is not limited thereto.

[0039] There are no particular limitations on the vectors used in this disclosure, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, granules, viruses, and bacteriophages in their native or recombinant states. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc., can be used as phage vectors or granule vectors; those based on pDZ, pDC, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc., can be used as plasmid vectors. In one example, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, etc., may be used.

[0040] The vector may also contain selection markers for confirming transformation into host cells, or further confirming insertion into the chromosome of the host cells. Selection markers are used to select cells transformed by the vector or to confirm the insertion of desired polynucleotides into the chromosome; markers that confer selectable phenotypes (such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of surface peptides) may be used. In an environment treated with a selection agent, only cells expressing the selection marker survive or exhibit a unique phenotype, thus allowing for the selection of transformed cells.

[0041] As used herein, the term "transformation" refers to the introduction of a desired polynucleotide or a vector containing it into a host cell (microorganism), thereby altering the genetic traits of the host cell (microorganism). The transformed polynucleotide can be inserted into the chromosome of the host cell (microorganism) or located extrachromosomally. Furthermore, the polynucleotide can contain DNA or RNA. Depending on the purpose of the introduction, the polynucleotide can be introduced in a suitable form. For example, a polynucleotide for expressing a desired polypeptide can be introduced into the host cell (microorganism) in the form of an expression cassette, a gene construct containing all the elements required for self-expression. Expression cassettes typically contain a promoter, transcription termination signal, ribosome binding site, and translation termination signal operably linked to the coding sequence of the desired polypeptide. Expression cassettes can be in the form of a self-replicating expression vector. Furthermore, the polynucleotide can be introduced into the host cell (microorganism) as is and can be operably linked to, but is not limited to, the desired sequence for expression in the host cell (microorganism).

[0042] As used herein, the term "operably linked" refers to an arrangement in which regulatory sequences are positioned appropriately to regulate the expression of a coding sequence. Therefore, the term "operably linked" includes the attachment or connection of a regulatory region (such as a promoter, stop codon, signal sequence, or enhancer region) with a functional domain having known or desired activity to a target (gene or polypeptide) to regulate the expression, secretion, or function of the target according to known or desired activity. For example, it could mean the functional linking of a polynucleotide sequence encoding a polypeptide to a promoter sequence that initiates and mediates polynucleotide transcription.

[0043] As used herein, the term “expression” includes, but is not limited to, any step involved in peptide production, such as transcription, post-transcriptional modification, translation, post-translational modification, secretion, etc.

[0044] As used herein, the term "expression vector" refers to a linear or circular nucleic acid molecule containing a desired polynucleotide sequence and a operatively linked regulatory sequence for its expression. For example, it may contain the base sequence of a polynucleotide encoding a desired polypeptide, which is operatively linked to a suitable expression regulatory region (or expression regulatory sequence) so that the desired polypeptide can be expressed in a suitable host.

[0045] As used herein, the term "regulatory sequence" refers to a polynucleotide sequence essential for regulating the expression of a desired polynucleotide sequence. Each regulatory sequence can be natural (from the same source) or exogenous (from a different gene) for the coding sequence, its variants, or other artificial sequences. Examples of regulatory sequences can include leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, operon sequences, sequences encoding ribosome-binding domains, and sequences regulating the termination of transcription and translation. The smallest unit of a regulatory sequence can contain a promoter and sequences that terminate transcription and translation.

[0046] As used in this article, the term “genetic recombination” refers to a natural or artificial process in which the elements that make up a gene, such as DNA or RNA, are rearranged into a sequence different from the original sequence during disassembly and recombination.

[0047] As used herein, the term "recombinant gene" refers to a gene having a novel genomic structure produced by genetic recombination, such as chemical synthesis or genetic engineering techniques. In this disclosure, the terms "recombinant gene," "recombinant DNA," and "recombinant polynucleotide" are used interchangeably. In one instance, a recombinant gene may comprise an artificial combination of nucleic acid fragments, such as regulatory sequences that cannot be found naturally.

[0048] As used in this article, the term "recombinant protein" refers to a protein produced due to genetic recombination.

[0049] microorganism

[0050] As used herein, the term "microorganism (or strain)" includes wild-type microorganisms and prokaryotic or eukaryotic microorganisms that have undergone natural or artificial genetic modification. It can be a microorganism whose specific mechanism is weakened or enhanced due to the insertion of a foreign gene or the enhancement or inactivation of an endogenous gene, and it can be a microorganism containing genetic modifications for the production of desired polypeptides, proteins, or products. In this disclosure, the terms "microorganism," "strain," "host," and "host cell" are used interchangeably.

[0051] As used herein, the term "recombinant microorganism" refers to a microorganism that has been genetically modified to exhibit a genotype and / or phenotype different from that of naturally occurring microorganisms (e.g., when the genetic modification affects how nucleic acid sequences are encoded in the microorganism), and may include all or potential offspring of that microorganism. In this disclosure, the terms "recombinant microorganism," "genetically modified microorganism," "recombinant host cell," "recombinant cell," and "recombinant strain" are used interchangeably. For example, a recombinant microorganism may express genes not found in their natural (non-recombinant) form, not express genes expressed in their natural form, or express natural genes in a manner different from their natural expression.

[0052] As used herein, the term "unmodified microorganism (strain)" does not exclude the inclusion of microorganisms (strains) that may have naturally occurring mutations, and may refer to wild-type microorganisms (strains) or natural microorganisms (strains), or microorganisms (strains) before their traits are altered by genetic variation due to natural or artificial factors. In this disclosure, the term "unmodified microorganism (strain)" may be used interchangeably with "pre-modified microorganism (strain)," "unmutated microorganism (strain)," "parental microorganism," "parental strain," "wild-type microorganism (strain)," "reference microorganism (strain)," or "standard organism (strain)."

[0053] nourish

[0054] As used herein, the term "culture" refers to the growth of microorganisms under appropriately controlled environmental conditions. The culture process can be carried out in suitable culture media and under suitable culture conditions known in the art. Such a culture process can be readily adapted and used by those skilled in the art based on the selected microorganisms. Specifically, the culture can be a batch culture, a continuous culture, and / or a fed-batch culture, but is not limited thereto.

[0055] As used herein, the term "culture medium" refers to a mixture containing nutrients essential for the cultivation of microorganisms as its main components, and the culture medium provides nutrients, growth factors, etc., including water, which is indispensable for survival and development. Specifically, any culture medium and culture conditions can be used to cultivate the microorganisms of this disclosure without particular limitation, as long as the culture medium is used for the general culture of microorganisms. For example, the microorganisms of this disclosure can be cultured under aerobic conditions in a general culture medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and / or vitamins, while controlling temperature, pH, etc.

[0056] In this disclosure, carbon sources include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; or amino acids such as glutamic acid, methionine, and lysine. Additionally, natural organic nutrients such as starch hydrolysate, molasses, molasses, rice bran, cassava, sugarcane residue, and corn steep liquor can be used. Specifically, carbohydrates such as glucose and aseptically pretreated molasses (i.e., molasses converted to reducing sugars) can be used, and various other carbon sources can be used without limitation. These carbon sources can be used alone or in combination of two or more, but are not limited thereto.

[0057] As nitrogen sources, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate can be used, or organic nitrogen sources such as amino acids (e.g., glutamic acid, methionine, and glutamine), peptone, NZ-amines, meat extracts, yeast extracts, malt extracts, corn steep liquor, casein hydrolysate, fish or its decomposition products, and defatted soybean meal or its decomposition products can be used. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0058] Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or their corresponding sodium-containing salts can be used as phosphorus sources. As for inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc., can be used, and amino acids, vitamins, and / or suitable precursors may also be included. These components or precursors can be added to the culture medium in batches or continuously. However, the culture medium is not limited to these.

[0059] During the cultivation of the microorganisms disclosed herein, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, or sulfuric acid in an appropriate manner. During cultivation, antifoaming agents such as polyethylene glycol fatty acids can be used to suppress foaming. To maintain an aerobic state in the culture medium, oxygen or oxygen-containing gas can be injected. To maintain an anaerobic or slightly aerobic state in the culture medium, no gas injection or the injection of nitrogen, hydrogen, or carbon dioxide gas is not necessary. However, the cultivation conditions are not limited to these.

[0060] In the cultivation process disclosed herein, the cultivation temperature can be maintained between 20°C and 45°C, specifically between 25°C and 40°C, and the cultivation can be carried out for approximately 10 to 160 hours, but the cultivation conditions are not limited to these.

[0061] As used herein, the term "culture" refers to a culture medium, a concentrated culture medium, a dried product of a culture medium, a culture filtrate, a concentrated culture filtrate, or a dried product of a culture filtrate obtained by culturing a specific microorganism in a culture medium. A culture medium is a solution containing a specific microorganism, while a culture filtrate is a solution that is substantially free of the specific microorganism (in particular, "substantially" means that the specific microorganism has been excluded by means of filtration, etc., but does not mean that the microorganism is completely absent from the filtrate). The form of a culture is not limited and, in one instance, may be a liquid, an emulsion, or a solid.

[0062] Detailed description of this disclosure

[0063] The specific implementation of this disclosure is described in more detail below.

[0064] One aspect of this disclosure provides a polypeptide comprising: an amino acid sequence of SEQ ID NO: 5 or 7; and an amino acid sequence of a nuclease derived from Serratia marcescens.

[0065] In this disclosure, the amino acid sequence of SEQ ID NO: 5 or 7 refers to a signal peptide sequence. Specifically, a signal peptide is a target polypeptide, referring to a region of 15 to 30 amino acids located at the N-terminus of a secretory protein or membrane protein, which acts as a signal as the protein crosses the membrane.

[0066] In this disclosure, the signal peptide may be a peptide derived from a Bacillus species, specifically a peptide derived from Bacillus subtilis, and more specifically, the yqxI or yoaW signal peptide derived from Bacillus subtilis. In particular, the yqxI signal peptide has the amino acid sequence of SEQ ID NO: 5, consisting of 28 amino acids, while the yoaW signal peptide has the amino acid sequence of SEQ ID NO: 7, consisting of 24 amino acids.

[0067] In this disclosure, the sequence of the polynucleotide encoding the amino acid sequence of SEQ ID NO: 5 or 7 can be obtained based on codon information known in the art. In one example, the polynucleotide encoding the amino acid sequence of SEQ ID NO: 5 may have, comprise, consist of, or substantially consist of the sequence of SEQ ID NO: 6 or a base sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity with the sequence of SEQ ID NO: 6, but is not limited thereto, and may include any polynucleotide without limitation, as long as it can function as the signal peptide of this disclosure in the same manner as or corresponding to the nucleic acid molecule composed of said polynucleotide. Additionally, the polynucleotide encoding the amino acid sequence of SEQ ID NO: 7 may have, contain, consist of, or substantially consist of the sequence of SEQ ID NO: 8 or a base sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity with the sequence of SEQ ID NO: 8, but is not limited thereto, and may include any polynucleotide without limitation, as long as it is capable of functioning as the signal peptide of this disclosure in the same manner as or corresponding to a nucleic acid molecule composed of said polynucleotide. In particular, “homology” or “identity” is as described above.

[0068] In this disclosure, the amino acid sequence of SEQ ID NO: 5 or 7 can be directly fused to the N-terminus of the amino acid sequence of the nuclease, or operatively linked to the amino acid sequence of the nuclease via a linker.

[0069] The "Serratia marcescens" disclosed herein refers to Gram-negative bacteria belonging to the family Enterobacteriaceae, which are known to be widely distributed in water, soil, food, etc.

[0070] As used herein, the term "nuclease" refers to an enzyme that cleaves the phosphodiester bond in nucleic acids, is known to play an important role in the replication, repair, recombination, and degradation of DNA and RNA in living organisms, and includes deoxyribonucleases (DNases) and ribonucleases (RNases). In this disclosure, the nuclease may be a nuclease derived from Serratia marcescens, and nucleases derived from Serratia marcescens are known to have nonspecific characteristics in cleaving various forms of DNA and RNA, but are not limited thereto.

[0071] In this disclosure, the amino acid sequences of nucleases derived from *Serratia marcescens* and the polynucleotide sequences encoding them are available from known databases. Examples of databases include, but are not limited to, NCBI's GenBank. In this disclosure, nucleases derived from *Serratia marcescens* may have the amino acid sequence of SEQ ID NO: 1, contain the amino acid sequence of SEQ ID NO: 1, consist of the amino acid sequence of SEQ ID NO: 1, or consist substantially of the amino acid sequence of SEQ ID NO: 1.

[0072] In this disclosure, nucleases derived from *Serratia marcescens* may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity with the amino acid sequence of SEQ ID NO: 1. Furthermore, it is apparent that any protein having an amino acid sequence in which a portion of the sequence is deleted, modified, substituted, conservedly substituted, or added may also fall within the scope of this disclosure, provided that the amino acid sequence has such homology or identity and exhibits equivalent efficacy to a protein comprising the amino acid sequence of SEQ ID NO: 1.

[0073] In this disclosure, the sequence of a polynucleotide encoding the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80% homology or identity with it, can be obtained based on codon information known in the art. In this disclosure, nucleases derived from *Serratia marcescens* can be encoded by polynucleotides having, comprising, consisting of, or substantially consisting of the following sequences: base sequences having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity with the sequence of SEQ ID NO: 2, but not limited thereto. The base sequence of SEQ ID NO: 2 can be obtained from known databases. Examples of databases include, but are not limited to, NCBI's GenBank.

[0074] Considering codon degeneracy or preferred codons in organisms expressing the nucleases of this disclosure, the polynucleotides of this disclosure can be modified in various ways in the coding region without altering the amino acid sequence of the nucleases of this disclosure. Therefore, it is evident that polynucleotides that can be translated by codon degeneracy into polypeptides consisting of the amino acid sequence of the nucleases of this disclosure, or polypeptides homologous to or identical with them, can also be included in the polynucleotides of this disclosure.

[0075] Furthermore, the polynucleotides of this disclosure may include, but are not limited to, probes that can be prepared from known gene sequences, such as any sequence that encodes a nuclease of this disclosure by hybridizing under stringent conditions with a sequence that is fully or partially complementary to the polynucleotides of this disclosure.

[0076] In addition to the amino acid sequence of SEQ ID NO: 5 or 7 and the amino acid sequence of the nuclease derived from Serratia marcescens, the polypeptide disclosed herein may also contain the amino acid sequence of SEQ ID NO: 11.

[0077] In this disclosure, the amino acid sequence of SEQ ID NO: 11 refers to the propeptide sequence. Specifically, a propeptide is a protein precursor, an inactive protein that can be converted into its active form through post-translational modifications, and is interchangeable with a precursor peptide. Many propeptides are known to be synthesized together with signal peptides for N-terminal secretion.

[0078] In this disclosure, the propeptide may be a peptide derived from a Bacillus microorganism, specifically a peptide derived from Bacillus subtilis, and more specifically, anmyE propeptide derived from Bacillus subtilis. In particular, the amyE propeptide has the amino acid sequence of SEQ ID NO: 11, consisting of 8 amino acids.

[0079] In this disclosure, the sequence of the polynucleotide encoding the amino acid sequence of SEQ ID NO: 11 can be obtained based on codon information known in the art. In one example, the polynucleotide may have, comprise, consist of, or substantially consist of the sequence of SEQ ID NO: 12 or a base sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity with the sequence of SEQ ID NO: 12, but is not limited thereto, and may include any nucleic acid molecule without limitation, as long as it can function as a propeptide of this disclosure in the same or corresponding manner as a nucleic acid molecule composed of said polynucleotide. Specifically, "homology" or "identity" is as described above.

[0080] In this disclosure, the amino acid sequence of SEQ ID NO: 11 can be directly fused to the C-terminus of the amino acid sequence of SEQ ID NO: 5 or 7 and the N-terminus of the amino acid sequence of the nuclease, and each of the amino acid sequences of SEQ ID NO: 5 or 7, the amino acid sequence of SEQ ID NO: 11 and the amino acid sequence of the nuclease can be operatively linked to each other.

[0081] Another aspect of this disclosure provides a polynucleotide encoding a polypeptide comprising: an amino acid sequence of SEQ ID NO: 5 or 7; and an amino acid sequence of a nuclease derived from Serratia marcescens.

[0082] The amino acid sequence of SEQ ID NO: 5 or 7 and the amino acid sequence of nuclease derived from Serratia marcescens are as described above.

[0083] The polynucleotides encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or 7 and the amino acid sequence of a nuclease derived from Serratia marcescens can be obtained based on codon information known in the art. The polynucleotide may comprise the base sequence of a polynucleotide encoding a polypeptide wherein the amino acid sequence of SEQ ID NO: 5 or 7 and the amino acid sequence of a nuclease derived from Serratia marcescens are operatively linked, and may have, comprise, consist of, or substantially consist of a base sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity with the base sequence, but are not limited thereto.

[0084] Another aspect of this disclosure provides an expression vector comprising a polynucleotide encoding a polypeptide of the present disclosure.

[0085] The term "expression carrier" in this disclosure is as described above.

[0086] Another aspect of this disclosure provides microorganisms that contain polypeptides of this disclosure or polynucleotides encoding them.

[0087] In this disclosure, the term "microorganism" is used as described above. In this disclosure, a microorganism comprising the polypeptide or the polynucleotide encoding it can include any microorganism, provided that the microorganism is capable of expressing a nuclease by comprising the polypeptide or the polynucleotide encoding it. Specifically, the microorganism of this disclosure can be a Bacillus microorganism, more specifically Bacillus subtilis, but is not limited thereto. Furthermore, the microorganism can be a recombinant microorganism transformed with a vector comprising a polynucleotide encoding the polypeptide of this disclosure, whereby nucleases derived from Serratia marcescens can be expressed or overexpressed in the microorganism. In particular, methods of transforming microorganisms with a vector include any method of introducing nucleic acids into cells, and can be performed by selecting suitable standard techniques known in the art according to the host cell.

[0088] Another aspect of this disclosure provides a method for producing nucleases derived from Serratia marcescens, comprising: culturing microorganisms containing polypeptides of this disclosure or polynucleotides encoding them in a culture medium; and recovering the nucleases from the culture.

[0089] The term "cultivation" in this disclosure is as described above.

[0090] In the method for producing nucleases derived from Serratia marcescens in this disclosure, "recovery" means obtaining the desired product from the microorganism or culture, and may be carried out using methods well known in the art, such as centrifugation, filtration, anion exchange chromatography, crystallization, and HPLC, but not limited thereto. Recovery may include purification processes, and those skilled in the art may select and use any of the various purification processes known in the art as needed.

[0091] Another aspect of this disclosure provides a composition for producing nucleases derived from Serratia marcescens, comprising a signal peptide having an amino acid sequence of SEQ ID NO: 5 or 7.

[0092] In the compositions disclosed herein, the amino acid sequence of SEQ ID NO: 5 or 7, the signal peptide, and the nuclease derived from Serratia marcescens are as described above.

[0093] The composition disclosed herein for producing nucleases derived from Serratia marcescens may further comprise a peptide having the amino acid sequence of SEQ ID NO: 11, and the amino acid sequence of SEQ ID NO: 11 is as described above.

[0094] Another aspect of this disclosure provides the use of a signal peptide having an amino acid sequence of SEQ ID NO: 5 or 7 for the production of nucleases.

[0095] Mode of implementing the present invention

[0096] The present disclosure is described in detail below by way of examples. The examples are provided to specifically explain the present disclosure, and the scope of the present disclosure is not limited to the examples.

[0097] Example 1. Screening of signal peptides and construction of a production strain of nuclease derived from Serratia marcescens.

[0098] Signal peptide screening was performed using the Bacillus subtilis secretory protein expression system (Takara). Based on the nucA sequence derived from Serratia marcescens, the nuclease (SEQ ID NO: 1) was synthesized from Bacillus subtilis via codon optimization using Cosmo Genetech. The synthesized sequence was amplified by PCR using primers SEQ ID NO: 3 and 4, and the PCR product was ligated into the pBE-S vector treated with NdeI and EcoRI using the In-FusionHD cloning kit (Clontech). The resulting construct was transformed into DH5α to obtain colonies. The plasmid was purified from the obtained colonies to prepare the nuclease expression vector (pBE-SPaprE_nucA).

[0099] To construct nuclease-producing strain libraries for each signal peptide, a mixture of SP DNA (Takara) encoding 173 secreted signal peptides derived from Bacillus subtilis and pBE-SPaprE-nucA treated with MluI and EagI were ligated using the In-Fusion® HD Cloning Kit (Clontech). The ligation products were then transformed into Bacillus subtilis (BGSC 1S145) using the native competent cell method (Bron, 1996), resulting in approximately 400 libraries.

[0100] To screen the constructed nuclease-producing strain, the generated colonies were inoculated into 96-well plates containing 300 μL LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 50 μg / mL kanamycin) and incubated at 37°C and 800 rpm for 24 hours. Subsequently, 3 μL of the supernatant was spotted onto DNase agar plates containing methyl green (KisanBio Co., Ltd.) and incubated at 37°C for 2 hours, and the size of the resulting halo was confirmed. Figure 1 ).

[0101] Based on the above results, a superior signal peptide was selected, and sequencing confirmed that the yqxI (SEQ ID NO: 5) and yoaW (SEQ ID NO: 7) signal peptide sequences are fused.

[0102] In addition to the selected strain, a strain incorporating the signal peptide of the Bacillus subtilis amyE gene was constructed. Specifically, PCR was performed using 168 g DNA of Bacillus subtilis as a template and primers of SEQ ID NO: 9 and 10, followed by ligation with pBE-SPaprE-nucA treated with MluI and EagI using the In-Fusion® HD Cloning Kit (Clontech). The resulting construct was transformed into Bacillus subtilis (BGSC 1S145) to prepare strain BNUC4 (SPamyE).

[0103] Example 2. Comparative evaluation of nuclease expression of each signal peptide

[0104] The nuclease expression levels of each signal peptide were compared by flask evaluation of the selected and constructed strains BNUC1 (SPaprE), BNUC2 (SPyqxI), BNUC3 (SPyoaW), and BNUC4 (SPamyE) through the above process. Each strain was incubated overnight in LB medium at 37°C, and then inoculated at a concentration of 1% into 25 mL of potency medium (10 g / L glucose, 15 g / L CSL, 20 g / L yeast extract, 2 g / L MgCl2, 2 g / L NaSO4, 2 g / L (NH4)2SO4, and 1.5 g / L K2HPO4). After incubation at 37°C and 200 rpm for 18 hours, the nuclease activity in the supernatant was measured. The method for evaluating nuclease activity is as follows.

[0105] (1) Materials

[0106] 1 M Tris-HCl (Biosesang), MgCl2 (Daejung Chemicals), bovine serum albumin (BSA, Sigma-Aldrich), salmon sperm DNA (Invitrogen), and 70% perchloric acid (Sigma-Aldrich).

[0107] (2) Buffer

[0108] 1) Reagent A (0.1 mg / ml BSA in 1 mM MgCl2, 50 mM Tris-HCl, pH 8.0)

[0109] 2) Reagent B (0.1 mg / ml salmon sperm DNA from Reagent A)

[0110] 3) 4% perchloric acid solution

[0111] (3) Procedure

[0112] Mix 25 μL of a sample appropriately diluted with reagent A, or 25 μL of reagent A (as a blank), with 500 μL of reagent B and react at 37 °C for 30 min. Then, add 525 μL of 4% perchloric acid solution to terminate the reaction and place the mixture on ice for 30 min. Centrifuge the mixture at 14,000 rpm for 6 min, separate the supernatant, and measure the absorbance at 260 nm. Specifically, one unit is defined as the amount of enzyme required to produce a 1.0 absorbance change at 260 nm within 30 min under optimal conditions in the presence of excess substrate.

[0113] The results, as shown in Table 1 below, confirm that nuclease activity was significantly higher when using the yqxI or yoaW signal peptide compared to when using the aprE or amyE signal peptide.

[0114] [Table 1]

[0115]

[0116] Example 3. Confirmation of the effect of fusion of pre-amyE peptide and signal peptide on increasing nuclease expression.

[0117] To confirm the effect of the amyE precursor on nuclease secretion efficiency, eight amino acids (SEQ ID NO: 11) were fused to the C-terminus of each of the yqxI or yoaW signal peptide sequences. Specifically, PCR products were obtained using the vectors pBE-SPyqxI_nucA or pBE-SPyoaW_nucA constructed in Example 1 as templates and primers of SEQ ID NO: 13, 14, 15, and 16. The PCR products were then ligated using the In-Fusion HD Cloning Kit (Clontech) to prepare nuclease expression vectors fused with the precursor amyE. The resulting constructs were transformed into Bacillus subtilis (BGSC 1S145) and evaluated.

[0118] The strain was incubated overnight in LB medium at 37°C, and then inoculated at a concentration of 1% into 25 mL of potency medium (10 g / L glucose, 15 g / L CSL, 20 g / L yeast extract, 2 g / L MgCl2, 2 g / L NaSO4, 2.68 g / L (NH4)2SO4, 1.5 g / L K2HPO4, and 50 μg / mL kanamycin). After incubation at 37°C and 200 rpm for 18 hours, the nuclease activity in the supernatant was measured.

[0119] The results, as shown in Table 2 below, confirm that the fusion with pre-amyE exhibits a synergistic effect for both signal peptides yqxI and yoaW, with the activity further increasing by at least 20%.

[0120] [Table 2]

[0121]

[0122] Example 4. Verification of nuclease activity in 5L fermenter cultures.

[0123] The nuclease expression levels in the strain were confirmed using a 5 L fermenter according to the following method. The BNUC6 strain was seeded in 300 mL LB medium containing 50 μg / mL kanamycin, and then inoculated into 1.8 L fermentation medium (40 g / L glucose, 50 g / L CSL, 20 g / L yeast extract, 2 g / L MgSO4, 4 g / L (NH4)2SO4, 2 g / L KH2PO4, 4 g / L CaCl2, and 50 μg / mL kanamycin). Air was supplied at pH 6.7, 800 rpm, and 1 vvm. The culture was maintained at 37 °C for 6 hours, then transitioned to 32.5 °C while fed fed medium (250 g / L glucose, 300 g / L CSL, and 20 g / L yeast extract) at a rate of 12 mL / h.

[0124] After culture, samples were taken at 6, 18, 30, and 50 hours, and the nuclease expression activity in the supernatant was measured. The results are as follows: Figure 2 The results showed that the strain ultimately exhibited nuclease expression activity of at least 140,000 U / mL.

[0125] As described above, those skilled in the art will understand that this disclosure may be implemented in other specific forms without departing from the technical spirit or essential features of this disclosure. In this regard, it should be understood that the foregoing embodiments are illustrative in all respects and should not be construed as restrictive. The scope of this disclosure should be understood to include the meaning and scope of the appended claims rather than the specific embodiments disclosed above, and all changes or variations derived from equivalent substitution concepts fall within the scope of this disclosure.

[0126] [Table 3]

[0127] [Sequence List]

[0128]

[0129]

Claims

1. A polypeptide comprising: an amino acid sequence of SEQ ID NO: 5 or 7; and an amino acid sequence of a nuclease derived from Serratia marcescens.

2. The polypeptide according to claim 1, wherein the amino acid sequence of the nuclease derived from Serratia marcescens has at least 80% homology with the amino acid sequence of SEQ ID NO:

1.

3. The polypeptide according to claim 1, wherein the amino acid sequence of SEQ ID NO: 5 or 7 is fused to the N-terminus of the amino acid sequence of the nuclease.

4. The polypeptide of claim 1, wherein the polypeptide further comprises the amino acid sequence of SEQ ID NO: 11, and wherein the amino acid sequence of SEQ ID NO: 11 is fused to the C-terminus of the amino acid sequence of SEQ ID NO: 5 or 7 and the N-terminus of the amino acid sequence of the nuclease.

5. A polynucleotide encoding a polypeptide according to any one of claims 1 to 4.

6. A microorganism comprising a polypeptide or a polynucleotide encoding the polypeptide according to any one of claims 1 to 4.

7. The microorganism according to claim 6, wherein the microorganism is a Bacillus genus microorganism.

8. The microorganism according to claim 6, wherein the microorganism is Bacillus subtilis.

9. A method for producing a nuclease derived from Serratia marcescens, comprising: Microorganisms comprising the polypeptide or the polynucleotide encoding the polypeptide according to any one of claims 1 to 4 are cultured in a culture medium; And the recovery of nucleases from cultures.

10. A composition for producing a nuclease derived from Serratia marcescens, comprising a signal peptide having an amino acid sequence of SEQ ID NO: 5 or 7.

11. The composition according to claim 10, further comprising a peptide having the amino acid sequence of SEQ ID NO:

11.

12. The use of a signal peptide having an amino acid sequence of SEQ ID NO: 5 or 7 for the production of nucleases.

Images