Signal peptide for producing serratia marcescens-derived nuclease and use thereof

Abstract

The present application relates to a signal peptide for producing a Serratia marcescens-derived nuclease, and a use thereof. Specifically, the present application relates to a polypeptide comprising a signal peptide and a Serratia marcescens-derived nuclease amino acid sequence, a microorganism comprising the polypeptide, and a method for producing a Serratia marcescens-derived nuclease, comprising a step of culturing the microorganism. This application can be used for the mass production of a Serratia marcescens-derived nuclease.

Description

Signal peptide for producing nuclease from Serratia marcescens and its use

[0001] The present application relates to a signal peptide for increasing the nuclease production ability derived from Serratia marcescens and a method for producing nuclease using the same.

[0002]

[0003] Nucleases are enzymes that cleave phosphodiester bonds in nucleic acids, playing a crucial role in the replication, repair, recombination, and degradation of DNA and RNA in living organisms. Nonspecific nucleases, in particular, are crucial for medical and research applications, including the diagnosis of viral infections and cancer, the development of gene therapies and vaccines, and are also primarily used in molecular biology research. Furthermore, they are highly valuable enzymes for food and industrial applications, such as the removal of residual nucleic acids in fermented foods, the improvement of fermentation broth viscosity, and the removal of biofilms.

[0004] Nucleases are produced by various microorganisms, but among them, nuclease derived from the Gram-negative bacterium Serratia marcescens is known to have non-specific properties and high specific activity in cleaving all forms of DNA and RNA (single-stranded, double-stranded, linear, and circular), and thus much research is being conducted to produce it (US 9796994 B2).

[0005] However, when overexpressing nuclease, it can also degrade the host's nucleic acids and inhibit growth if it exists in an active state within the host cell matrix, making production using microorganisms difficult. To date, cases of nuclease production at up to 5,000 U / ml using microorganisms have been reported, but products sold using this method are still very expensive. Therefore, selecting the optimal nuclease secretion signal peptide is crucial.

[0006] Bacillus subtilis is a strain listed on the Generally Recognized as Safe (GRAS) list and has excellent protein secretion production capacity, making it a host strain for the production of various recombinant proteins. Research is particularly focused on secretory signal peptides for protein secretion production. While screening techniques and optimization methods for the production of various foreign proteins have been studied, little research has been done on the optimal signal peptide for the secretory production of Serratia marcescens-derived nuclease (He Li et al. Biochemical Engineering Journal 189 (2022) 108718).

[0007]

[0008] The present inventors completed the present application by confirming that it is possible to increase the production of nuclease derived from Serratia marcescens using a signal peptide.

[0009]

[0010] One object of the present application is to provide a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or 7; and a nuclease amino acid sequence derived from Serratia marcescens.

[0011] Another object of the present application is to provide a polynucleotide encoding the polypeptide.

[0012] Another object of the present application is to provide a microorganism comprising the polypeptide or a polynucleotide encoding the same.

[0013] Another object of the present application is to provide a method for producing a nuclease derived from Serratia marcescens, comprising the steps of culturing a microorganism containing the polypeptide or a polynucleotide encoding the polypeptide in a medium; and recovering the nuclease from the culture.

[0014] Another object of the present application is to provide 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.

[0015]

[0016] The present application provides a signal peptide for increasing the productivity of a nuclease derived from Serratia marcescens, which can be used for mass production of a nuclease derived from Serratia marcescens.

[0017]

[0018] Figure 1 is a diagram showing the halo sizes produced by each signal peptide as a result of screening for nuclease producing strains.

[0019] Figure 2 is a diagram showing the results of confirming the level of nuclease expression over time in a strain cultured through a 5L fermenter.

[0020]

[0021] 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.

[0022]

[0023] The terms used in this application are as follows.

[0024]

[0025] proteins, polypeptides

[0026] In this application, the term "protein" or "polypeptide" means a polymer or oligomer of consecutive amino acid residues. In this application, "polypeptide," "protein," and "peptide" may be used interchangeably.

[0027] In this application, amino acid sequences are described in N-terminal → C-terminal orientation unless otherwise indicated.

[0028] In the present application, with respect to an amino acid sequence, it is obvious that a polypeptide or protein “comprising” an amino acid sequence set forth in a specific sequence number, a polypeptide or protein “consisting of” an amino acid sequence set forth in a specific sequence number, or a polypeptide or protein “having” an amino acid sequence set forth in a specific sequence number may also include a polypeptide or protein in which some amino acid(s) are deleted, modified, substituted, or added, as long as it has the same or corresponding activity as a polypeptide or protein consisting of the amino acid sequence of the corresponding sequence number. For example, the polypeptide or protein may also include a polypeptide or protein having an amino acid(s) addition or deletion, a naturally occurring mutation, a silent mutation, or a conservative substitution within or before or after the polypeptide or protein (N-terminal or C-terminal), which does not alter the function of the protein, as long as it has the same or corresponding activity.

[0029]

[0030] polynucleotide

[0031] In this application, the terms "polynucleotide," "nucleic acid," or "nucleic acid molecule" refer to a polymer of nucleotides in which nucleotide units (monomers) are covalently bonded to form a long chain, and mean a strand of DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA) of a certain length or longer. In this application, "polynucleotide," "nucleic acid," and "nucleic acid molecule" may be used interchangeably.

[0032]

[0033] identity, homology

[0034] In this application, the terms "identity" or "homology" refer to the degree of similarity between two given amino acid or base sequences, which may be expressed as a percentage. In this application, "homology" and "identity" may often be used interchangeably.

[0035] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, which may be used in conjunction with default gap penalties established by the program being used.

[0036] Whether any two polynucleotide or polypeptide sequences are homologous, similar or identical 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 performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) or the GAP computer program such as the Smith-Waterman algorithm (Smith and Waterman, Adv. Appl. Math (1981) 2:482) can be determined by comparing the sequence information (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 et al.](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information database.

[0037] Additionally, whether any two polynucleotide sequences have homology, similarity or identity can be determined by a Southern hybridization experiment under appropriate hybridization conditions, which can be determined by methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual; F. M. Ausubel 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 generally hybridize along the entire sequence or at least about 50%, 60%, 70%, 80% or 90% of the entire length under stringent conditions.

[0038] In this application, the term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8). For example, it may be a condition in which polynucleotides having high homology or identity hybridize with each other, polynucleotides having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity hybridize with each other, and polynucleotides having lower homology or identity do not hybridize with each other, or a condition in which washing is performed once, specifically twice or three times, at a salt concentration and temperature equivalent to 60°C, 1ХSSC, 0.1% SDS, specifically 60°C, 0.1ХSSC, 0.1% SDS, and more specifically 68°C, 0.1ХSSC, 0.1% SDS, which is a washing condition of a typical southern hybridization.

[0039] The above hybridization can occur between nucleotides having complementary base sequences; however, the 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, adenosine is complementary to thymine, and cytosine is complementary to guanine. Accordingly, the polynucleotides of the present application may include isolated nucleic acid fragments that are complementary in their entirety, as well as substantially similar base sequences.

[0040] For example, a polynucleotide having homology or identity with the polynucleotide of the present application can be hybridized and detected at a Tm value of 55°C. 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.

[0041] 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).

[0042]

[0043] vector, transformation

[0044] The term "vector" as used in this application means a DNA preparation for delivering a desired polynucleotide into a suitable host or host cell.

[0045] For example, a vector may comprise a base 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. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences regulating the termination of transcription and translation. After being transformed into a suitable host cell (microorganism), the vector may replicate or function independently of the host genome, or may be integrated into the genome itself to replicate or function.

[0046] Additionally, as an example, the vector of the present application may include a sequence for inserting a target polynucleotide into a chromosome. Insertion of the polynucleotide into the chromosome using the vector may be accomplished by any method known in the art, such as, but not limited to, homologous recombination.

[0047] The vector used in the present application is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ series, pDC series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used as plasmid vectors. For example, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors can be used.

[0048] The above vector may further include a selection marker to determine whether the vector is transformed into a host cell or further, whether the vector is integrated into the host cell chromosome. The selection marker is used to select cells transformed with the vector or to determine whether the target polynucleotide is integrated into the chromosome. Markers that confer selectable phenotypes such as drug resistance, nutritional requirements, cytotoxic agent resistance, or expression of surface polypeptides may be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypic characteristics, thereby enabling selection of transformed cells.

[0049] In this application, the term "transformation" refers to changing the genetic characteristics of a host cell (microorganism) by introducing a target polynucleotide, or a vector containing the same, into the host cell (microorganism). The transformed polynucleotide may be inserted into the chromosome of the host cell (microorganism) or located extrachromosomally. In addition, the polynucleotide may comprise DNA or RNA. The polynucleotide may be introduced in an appropriate form depending on the purpose of introduction. For example, a polynucleotide for expressing a target polypeptide may be introduced into a host cell (microorganism) 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 a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, all of which are operably linked to the coding sequence of the target polypeptide. The expression cassette may be in the form of an expression vector capable of self-replication. Additionally, the polynucleotide may be introduced into a host cell (microorganism) in its own form and operably linked to a sequence required for expression in the host cell (microorganism), but is not limited thereto.

[0050] As used herein, the term "operably linked" refers to a configuration in which a regulatory sequence is positioned appropriately so that the regulatory sequence controls the expression of a coding sequence. Accordingly, "operably linked" includes a regulatory region of a functional domain with a known or desired activity, such as a promoter, terminator, signal sequence, or enhancer region, attached or linked to a target (gene or polypeptide) so as to regulate the expression, secretion, or function of the target according to the known or desired activity. For example, it may mean that a promoter sequence that initiates and mediates the transcription of a polynucleotide encoding a polypeptide is functionally linked to the polynucleotide sequence.

[0051] As used herein, the term “expression” includes, but is not limited to, any step involved in the production of a polypeptide, such as transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0052] As used herein, the term "expression vector" refers to a linear or circular nucleic acid molecule comprising a target polynucleotide sequence and a regulatory sequence operably linked thereto for expression thereof. For example, it may comprise the base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression regulatory region (or expression regulatory sequence) so as to enable expression of the target polypeptide in a suitable host.

[0053] In this application, the term "regulatory sequence" refers to a polynucleotide sequence necessary for regulating the expression of a target polynucleotide sequence. Each regulatory sequence may be a natural sequence (having the same origin) or a foreign sequence (derived from another gene) relative to the coding sequence, or a mutant sequence thereof, or another artificial sequence. Examples of the regulatory sequence include a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating transcription and translation termination. The minimum unit of the regulatory sequence may include a promoter, a transcription and translation termination sequence.

[0054] In this application, the term "genetic recombination" refers to a natural or artificial process in which elements that make up genes, such as DNA or RNA, are changed from their original sequence during the disassembly and reassembly process.

[0055] As used herein, the term "recombinant gene" refers to a gene with a novel genetic structure resulting from genetic recombination, such as chemical synthesis or genetic engineering techniques. The terms "recombinant gene," "recombinant DNA," and "recombinant polynucleotide" may be used interchangeably in this application. For example, the recombinant gene may include an artificial combination of nucleic acid fragments, such as regulatory sequences, that are not found together in nature.

[0056] In this application, the term “recombinant protein” means a protein produced as a result of genetic recombination.

[0057]

[0058] microorganism

[0059] In this application, the term "microorganism (or strain)" includes both wild-type microorganisms and prokaryotic or eukaryotic microorganisms that have undergone genetic modification, either naturally or artificially. It may be a microorganism that has a specific mechanism weakened or increased due to a cause such as the insertion of an external gene or the increased or inactivated activity of an endogenous gene, and may be a microorganism that includes genetic modification for the production of a desired polypeptide, protein, or product. In this application, the terms "microorganism," "strain," "host," and "host cell" may be used interchangeably.

[0060] As used herein, the term "recombinant microorganism" refers to a microorganism that has been genetically modified to exhibit a different genotype and / or phenotype compared to a naturally occurring microorganism (e.g., when the genetic modification affects the nucleic acid sequence coding of the microorganism), and may include all progeny or potential progeny of the microorganism. The terms "recombinant microorganism," "genetically modified microorganism," "recombinant host cell," "recombinant cell," and "recombinant strain" may be used interchangeably in this application. The recombinant microorganism may, for example, express a gene not found in its native (non-recombinant) form; may not express a gene expressed in its native form; or may express a native gene in a manner different from that in which it is expressed in its native form.

[0061] In this application, the term "unmodified microorganism (strain)" does not exclude a microorganism (strain) that contains a mutation that may occur naturally, and may refer to a wild-type microorganism (strain) or a natural microorganism (strain) itself, or a microorganism (strain) before its phenotype is changed by a genetic mutation caused by natural or artificial factors. In this application, the term "unmodified microorganism (strain)" may be used interchangeably with "pre-modified microorganism (strain)", "unmodified microorganism (strain)", "parent microorganism", "parent strain", "wild-type microorganism (strain)", "reference microorganism (strain)", or "reference microorganism (strain)".

[0062]

[0063] culture

[0064] In this application, the term "cultivation" refers to the growth of microorganisms under appropriately controlled environmental conditions. The cultivation process can be conducted using appropriate media and culture conditions known in the art. This cultivation process can be easily adjusted and used by those skilled in the art depending on the selected microorganism. Specifically, the cultivation process may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0065] In this application, the term "medium" refers to a material containing nutrients necessary for culturing microorganisms as its main component, and supplies nutrients and growth factors, including water, which is essential for survival and growth. Specifically, the medium and other culture conditions used for culturing the microorganisms of this application may be any medium used for culturing conventional microorganisms without particular limitation. For example, the microorganisms of this application may be cultured under aerobic conditions by controlling temperature, pH, etc. in a conventional medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins.

[0066] 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, 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 to 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.

[0067] The nitrogen source may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc.; 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.

[0068] 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.

[0069] In addition, during the cultivation of the microorganism of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. may be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during the cultivation, foaming may be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. In addition, to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, or to maintain the anaerobic and microaerobic state, nitrogen, hydrogen, or carbon dioxide gas may be injected without gas injection, but is not limited thereto.

[0070] In the cultivation of the present application, the cultivation temperature can be maintained at 20 to 45°C, specifically 25 to 40°C, and the cultivation can be performed for about 10 to 160 hours, but is not limited thereto.

[0071] In the present application, the term "culture" means a culture solution, concentrated culture solution, dried product of culture solution, culture filtrate, concentrated culture filtrate, or dried product of culture filtrate obtained by culturing a specific microorganism in a culture medium, wherein the culture solution means that it contains a specific microorganism, and the culture filtrate means that it does not substantially contain a specific microorganism (here, substantially means that a specific microorganism separated by filtration or the like is excluded, but does not mean that the filtrate is completely free of microorganisms). The culture is not limited in its formulation, and may be, for example, a liquid, an emulsion, or a solid.

[0072]

[0073] Specific description of this application

[0074] Hereinafter, specific examples of the present application will be described in more detail as follows.

[0075]

[0076] One aspect of the present application provides a polypeptide comprising an amino acid sequence of SEQ ID NO: 5 or 7; and a nuclease amino acid sequence from Serratia marcescens.

[0077]

[0078] In the present application, the amino acid sequence of SEQ ID NO: 5 or 7 refers to a signal peptide sequence. Here, the signal peptide is a type of target peptide, located at the N-terminus of a secretory protein or membrane protein, and refers to a region consisting of 15 to 30 amino acids that serves as a signal when the protein passes through the membrane.

[0079] In the present application, the signal peptide may be a peptide derived from a microorganism of the genus Bacillus, specifically a peptide derived from Bacillus subtilis, and more specifically a yqxI or yoaW signal peptide derived from Bacillus subtilis. In this case, the yqxI signal peptide has an amino acid sequence of SEQ ID NO: 5 consisting of 28 amino acids, and the yoaW signal peptide has an amino acid sequence of SEQ ID NO: 7 consisting of 24 amino acids.

[0080] In the present application, the sequence of the polynucleotide encoding the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7 can be obtained based on codon information known in the art. For example, the polynucleotide encoding the amino acid sequence of SEQ ID NO: 5 may have or include, consist of, or consist essentially 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 therewith, but is not limited thereto, and may include, without limitation, a nucleic acid molecule consisting of the polynucleotide as long as it can function as a signal peptide of the present invention identically or correspondingly. In addition, the polynucleotide encoding the amino acid sequence of SEQ ID NO: 7 may have or include, consist of, or consist essentially 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 therewith, but is not limited thereto, and may include, without limitation, a nucleic acid molecule consisting of the polynucleotide as long as it can function as a signal peptide of the present invention identically or correspondingly. In this case, the term homology or identity is as described above.

[0081] In the present application, the amino acid sequence of SEQ ID NO: 5 or 7 may be directly fused to the N-terminus of the nuclease amino acid sequence, or may be operably linked to the nuclease amino acid sequence via a linker.

[0082]

[0083] "Serratia marcescens" of the present application is a gram-negative bacterium belonging to the Enterobacteriaceae family, and is known to be widely distributed in water, soil, food, etc.

[0084] The term "nuclease" in this application refers to an enzyme that cleaves the phosphodiester bond of nucleic acids, and is known to play an important role in the replication, repair, recombination, and degradation of DNA and RNA in living organisms, and includes DNA degrading enzymes (DNase, deoxyribonuclease) and RNA degrading enzymes (RNase, ribonuclease). The nuclease in the application may be a nuclease derived from Serratia marcescens, and the nuclease derived from Serratia marcescens is known to have a non-specific property of cleaving various forms of DNA and RNA, but is not limited thereto.

[0085] In the present application, the amino acid sequence of the nuclease derived from Serratia marcescens and the polynucleotide sequence encoding the same can be obtained from known databases, such as, but not limited to, NCBI's GenBank. In the present application, the nuclease derived from Serratia marcescens can have, include, consist of, or consist essentially of the amino acid sequence of SEQ ID NO: 1.

[0086] In the present application, the Serratia marcescens-derived nuclease may include 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. In addition, it is obvious that a protein having an amino acid sequence in which a part of the sequence is deleted, modified, substituted, conservatively substituted, or added is also included within the scope of the present application, as long as it has such homology or identity and exhibits an effect corresponding to a protein including the amino acid sequence of SEQ ID NO: 1.

[0087]

[0088] In the present application, the sequence of a polynucleotide encoding the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80% or more homology or identity therewith can be obtained based on codon information known in the art. The nuclease derived from Serratia marcescens in the present application may be encoded by a polynucleotide having or including a base sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity with the base sequence of SEQ ID NO: 2, or consisting of or consisting essentially of the base sequence, but is not limited thereto. In addition, the base sequence of SEQ ID NO: 2 can be obtained from a known database, such as, but not limited to, NCBI's GenBank.

[0089] The polynucleotide of the present application may undergo various modifications in the coding region without altering the amino acid sequence of the nuclease of the present application, due to codon degeneracy or in consideration of the codons preferred by the organism to which the nuclease of the present application is to be expressed. Accordingly, it is self-evident that a polynucleotide that can be translated into a polypeptide comprising the amino acid sequence of the nuclease of the present application or a polypeptide having homology or identity therewith due to codon degeneracy may also be included in the polynucleotide of the present application.

[0090] In addition, 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 hybridizes under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence of the present application, and encodes the nuclease of the present application.

[0091]

[0092] The polypeptide of the present application may further comprise an amino acid sequence of SEQ ID NO: 5 or 7; and a nuclease amino acid sequence derived from Serratia marcescens; and an amino acid sequence of SEQ ID NO: 11.

[0093] In the present application, the amino acid sequence of SEQ ID NO: 11 refers to a propeptide sequence. Here, a propeptide refers to an inactive protein that can be converted into an active form through post-translational modification as a protein precursor, and is also used interchangeably with pre-propeptide. Many propeptides are known to be synthesized with a signal peptide at the N-terminus for secretion.

[0094] In the present application, the propeptide may be a peptide derived from a microorganism of the genus Bacillus, specifically a peptide derived from Bacillus subtilis, and more specifically an amyE propeptide derived from Bacillus subtilis. In this case, the amyE propeptide has an amino acid sequence of SEQ ID NO: 11 consisting of 8 amino acids.

[0095] In the present application, 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. For example, the polynucleotide may have or include, consist of, or consist essentially 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 therewith, but is not limited thereto, and may include, without limitation, a nucleic acid molecule consisting of the polynucleotide as long as it can function as a propeptide of the present invention identically or correspondingly. In this case, the term homology or identity is as described above.

[0096] In the present application, the amino acid sequence of SEQ ID NO: 11 may be fused to the C-terminus of the amino acid sequence of SEQ ID NO: 5 or 7 and the N-terminus of the nuclease amino acid sequence, and the amino acid sequence of SEQ ID NO: 5 or 7; the amino acid sequence of SEQ ID NO: 11; and the nuclease amino acid sequence may be operably linked to each other.

[0097]

[0098] Another aspect of the present application provides a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 5 or 7; and a nuclease amino acid sequence derived from Serratia marcescens.

[0099] The “amino acid sequence of SEQ ID NO: 5 or 7” and the “amino acid sequence of nuclease derived from Serratia marcescens” of the present application are as described above.

[0100] A polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or 7 of the present application; and a nuclease amino acid sequence derived from Serratia marcescens; can be obtained based on codon information known in the art. The polynucleotide may include the base sequence of a polynucleotide encoding a polypeptide to which the amino acid sequence of SEQ ID NO: 5 or 7 and the nuclease amino acid sequence derived from Serratia marcescens are operably linked, and may have or include, consist essentially of, or consist of a base sequence having a homology or identity of 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more thereto, but is not limited thereto.

[0101]

[0102] Another aspect of the present application provides an expression vector comprising a polynucleotide encoding a polypeptide of the present application.

[0103] The term “expression vector” in this application is as described above.

[0104]

[0105] Another aspect of the present application provides a microorganism comprising a polypeptide of the present application or a polynucleotide encoding the same.

[0106] The term "microorganism" in this application is as defined above. The microorganism comprising the polypeptide or the polynucleotide encoding it in this application may be any microorganism capable of expressing a nuclease, including the polypeptide of this application and the polynucleotide encoding it, without limitation. Specifically, the microorganism may be a microorganism of the genus Bacillus, and more specifically, Bacillus subtilis, but is not limited thereto. Furthermore, the microorganism may be a recombinant microorganism transformed with a vector comprising a polynucleotide encoding the polypeptide of this application, through which the Serratia marcescens-derived nuclease may be expressed or overexpressed in the microorganism. In this case, the method of transforming the microorganism with the vector includes any method of introducing a nucleic acid into a cell, and may be performed by selecting an appropriate standard technique known in the art depending on the host cell.

[0107]

[0108] Another aspect of the present application provides a method for producing a nuclease derived from Serratia marcescens, comprising the steps of: culturing a microorganism comprising the polypeptide of the present application or a polynucleotide encoding the same in a medium; and recovering the nuclease from the culture.

[0109] The term “cultivation” in this application is as described above.

[0110] In the method for producing a nuclease derived from Serratia marcescens of the present application, the "recovery step" may use any method known in the art for obtaining a desired product from a microorganism or culture, such as centrifugation, filtration, anion exchange chromatography, crystallization, and HPLC, but is not limited thereto. The recovery step may include a purification process, and those skilled in the art may select and utilize various known purification processes as needed.

[0111]

[0112] Another aspect of the present application provides 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.

[0113] In the composition of the present application, 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.

[0114] The composition for producing a nuclease derived from Serratia marcescens of the present application may further comprise a peptide having an amino acid sequence of SEQ ID NO: 11, wherein the amino acid sequence of SEQ ID NO: 11 is as described above.

[0115]

[0116] Another aspect of the present application provides a use for producing a nuclease having a signal peptide having an amino acid sequence of SEQ ID NO: 5 or 7.

[0117]

[0118] Hereinafter, the present application will be described in more detail through examples. These examples are intended to more specifically explain the present application, and the scope of the present application is not limited by these examples.

[0119]

[0120] Example 1. Screening of signal peptides and construction of transformed strains for the production of nuclease derived from Serratia marcescens.

[0121]

[0122] Signal peptide screening was performed using the Secretory Protein Expression System (Takara) of Bacillus subtilis strains. Nuclease was synthesized by Cosmogenetech based on the nucA sequence derived from Serratia marcescens, codon-optimized for B. subtilis (SEQ ID NO: 1), and PCR was performed using primers of SEQ ID NO: 3 and 4. The resulting product was ligated into the pBE-S vector treated with NdeI and EcoRI using the In-Fusion HD cloning kit (clontech), and then transformed into DH5α to obtain colonies. The plasmid was purified from the obtained colonies to construct the nuclease expression vector (pBE-SPaprE_nucA).

[0123] To construct a library of nuclease-producing strains for each signal peptide, SP DNA mixture (Takara) encoding 173 types of secretory signal peptides from B. subtilis and pBE-SPaprE-nucA treated with MluI and EagI were ligated using the In-FusionR HD cloning kit (clontech), and then transformed into Bacillus subtilis (BGSC 1S145) using the natural competency method (Bron, 1996), obtaining a library of approximately 400 strains.

[0124]

[0125] For screening of the nuclease-producing strain, the resulting colonies were inoculated into 300 μl LB media (10 g / l trypton, 5 g / l yeast extract, 10 g / l NaCl, 50 μg / ml kanamycin) in a 96 deep-well plate and cultured at 37°C, 800 rpm, and 24 h. Then, 3 μl of the supernatant was spotted onto a DNase Agar with Methyl Green (Kisan Bio Co., Ltd.) plate and reacted at 37°C for 2 h to confirm the resulting halo size (Fig. 1).

[0126] Through the above results, an excellent signal peptide was selected, and through sequencing, it was confirmed that the yqxI (SEQ ID NO: 5) and yoaW (SEQ ID NO: 7) signal peptide sequences were fused.

[0127]

[0128] In addition to the above-selected strains, a strain fused with the signal peptide of the B. subtilisamyE gene was constructed. Specifically, B. subtilis168 gDNA was used as a template for PCR with primers of SEQ ID NO: 9 and 10, and then ligated with pBE-SPaprE-nucA treated with MluI and EagI using the In-FusionR HD cloning kit (clontech), followed by transformation into Bacillus subtilis (BGSC 1S145), thereby constructing strain BNUC4 (SPamyE).

[0129]

[0130] Example 2. Comparative evaluation of nuclease expression by signal peptide

[0131]

[0132] The nuclease expression levels for each signal peptide were compared through flask evaluation of each strain BNUC1 (SPaprE), BNUC2 (SPyqxI), BNUC3 (SPyoaW), and BNUC4 (SPamyE) selected and produced through the above process. After culturing overnight at 37℃ in LB media, 1% was inoculated into 25 ml of titer 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, 1.5 g / l K2HPO4). Culture was performed at 37℃ and 200 rpm, and the nuclease activity of the supernatant was measured after 18 h. The nuclease activity evaluation method is as follows.

[0133]

[0134] (1) Materials

[0135] 1M Tris-HCl (Biosesang), MgCl2 (Daejeong Chemical), Albumin from bovine serum (BSA, Sigma Aldrich), Salmon sperm DNA (Invitrogen), Perchloric acid 70% (Sigma Aldrich)

[0136]

[0137] (2) Buffer solution

[0138] 1) Reagent A (1mM MgCl2, 0.1 mg / ml BSA in 50mM Tris-HCl, pH 8.0)

[0139] 2) Reagent B (0.1mg / ml Salmon sperm DNA in reagent A)

[0140] 3) 4% Perchloric acid solution

[0141]

[0142] (3) Process

[0143] 25 ul of sample appropriately diluted with Reagent A or 25 ul of Reagent A as a blank was mixed with 500 ul of Reagent B, and incubated at 37℃ for 30 minutes. 525 ul of 4% perchloric acid solution was added to terminate the reaction, and the mixture was kept on ice for 30 minutes. The mixture was centrifuged at 14,000 rpm for 6 minutes, and the supernatant was separated to measure the absorbance at 260 nm. 1 unit was defined here as the amount of enzyme required to produce a change in absorbance of 1.0 at 260 nm in optimal conditions with excess substrate for 30 minutes.

[0144]

[0145] As a result, as shown in Table 1 below, it was confirmed that the nuclease activity was significantly superior when the signal peptide of yqxI or yoaW was used compared to when the signal peptide of aprE or amyE was used.

[0146] StrainDescriptionnuclease activity (Unit / ml)BNUC1BGSC 1S145 pBE-SPaprE_nucA8946BNUC2BGSC 1S145 pBE-SPyqxI_nucA15864BNUC3BGSC 1S145 pBE-SPyoaW_nucA16238BNUC4BGSC 1S145 pBE-SPamyE_nucA3790

[0147]

[0148] Example 3. Confirmation of the effect of increasing nuclease expression when Pro-amyE peptide is fused to a signal peptide.

[0149]

[0150] To determine the effect of the amyE Pro-peptide on the nuclease secretion efficiency, eight amino acids (SEQ ID NO: 11) were additionally fused behind each yqxI or yoaW signal peptide sequence. Specifically, using the pBE-SPyqxI_nucA or pBE-SPyoaW_nucA vector constructed in Example 1 as a template and the primers of SEQ ID NO: 13, 14, 15, and 16, PCR products were ligated using the In-Fusion HD cloning kit (clontech), respectively, to construct a nuclease expression vector fused with Pro-amyE, and the vector was transformed into Bacillus subtilis (BGSC 1S145) for evaluation.

[0151] The above strain was cultured overnight at 37°C in LB media, and then inoculated at 1% in 25 ml of titer medium (Glucose 10 g / L, CSL 15 g / L, Yeast extract 20 g / L, MgCl2 2 g / L, NaSO4 2 g / L, (NH4)2SO4 2.68 g / L, K2HPO4 1.5 g / L, 50 ug / ml kanamycin). Culture was performed at 37°C and 200 rpm, and the nuclease activity of the supernatant was measured after 18 h.

[0152] As a result, as shown in Table 2 below, it was confirmed that both signal peptides, yqxI and yoaW, had a synergistic effect in which the activity was further increased by more than 20% when Pro-amyE was fused.

[0153] StrainDescriptionnuclease activity (Unit / ml)BNUC1BGSC 1S145 pBE-SPaprE_nucA9837BNUC2BGSC 1S145 pBE-SPyqxI_nucA16987BNUC3BGSC 1S145 pBE-SPyoaW_nucA18125BNUC5BGSC 1S145 pBE-SPyqxI_Pro-amyE_nucA20856BNUC6BGSC 1S145 pBE-SPyoaW_Pro-amyE_nucA22385

[0154]

[0155] Example 4. Confirmation of nuclease expression activity through 5 L fermentation tank culture

[0156]

[0157] The nuclease expression level in the strain was confirmed through a 5 L fermenter using the following method. The BNUC6 strain was cultured in 300 ml of LB media containing 50 ug / ml kanamycin, and then inoculated into 1.8 L of 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, 50 ug / ml kanamycin), and a pH of 6.7, 800 rpm, and 1 vvm of air were supplied. The temperature was maintained at 37℃ and then changed to 32.5℃ after 6 hours of culture, and culture was conducted by adding feed medium (250g / l glucose, 300g / l CSL, 20g / l Yeast extract) at a rate of 12ml / h.

[0158] After culturing, sampling was performed at 6 h, 18 h, 30 h, and 50 h, and the nuclease expression activity of the supernatant was measured. As a result, as shown in Fig. 2, it was confirmed that the strain ultimately exhibited a nuclease expression activity of 140,000 U / ml or more.

[0159]

[0160] From the above description, those skilled in the art will understand that the present application can be implemented in other specific forms without altering its technical concept 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 application should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the claims described below, and their equivalent concepts, rather than the detailed description above.

[0161]

[0162] 서열목록서열번호명칭서열1Serratia marcescens derived Nuclease AADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYTGANAALKVDRGHQAPLASLAGVSDWESLNYLSNITPQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCKN2Serratia marcescens derived NucleaseNTGATACACTGGAATCAATTGATAATTGCGCAGTTGGCTGCCCGACGGGCGGCAGCTCAAATGTTTCAATTGTGAGACACGCATACACATTGAATAATAATTCAACAACGAAGTTTGCAAATTGGGTTGCATATCATATTACAAAAGATACACCGGCAAGCGGCAAAACAAGAAATTGGAAAACAGATCCGGCACTGAATCCGGCAGATACACTGGCACCGGCAGATTATACGGGCGCAAATGCAGCACTGAAAGTTGATAGAGGCCATCAAGCACCGCTGGCATCACTGGCGGGCGTTTCAGATTGGGAATCACTGAATTATCTGTCAAATATTACACCGCAAAAATCAGATCTGAATCAAGGCGCATGGGCAAGACTGGAAGATCAAGAAAGAAAACTGATTGATAGAGCAGATATTTCATCAGTTTATACAGTTACGGGCCCGCTGTATGAAAGAGATATGGGCAAACTGCCGGGCACACAAAAAGCACATACAATTCCGTCAGCATATTGGAAAGTTATTTTTATTAATAATTCACCGGCAGTTAATCATTATGCAGCATTTCTGTTTGATCAAAATACACCGAAAGGCGCAGATTTTTGCCAATTTAGAGTTACAGTTGATGAAATTGAAAAAAGAACGGGCCTGATTATTTGGGCGGGCCTGCCGGATGATGTTCAAGCATCACTGAAATCAAAACCGGGCGTTCTGCCGGAACTGATGGGCTGCAAAAATTGA3nucA F Primer NTGCGGCCGGTGCACAT GATACACTGGAATCAATTGATAATTGCG4nucA R Primer NTTGCAGGTCGACAAGCTT TCAATTTTTGCAGCCCATCAGTTC5yqxI Signal peptide AAMFKKLLLATSALTFSLSLVLPLDGHAKA6yqxI Signal peptideNTATGTTTAAGAAATTACTTTAGCAACATCTGCATTAACATTCTCTTTATCATTAGTTCTTCCGTTGGATGGACATGCCAAAGCT7yoaW Signal peptide AAMKKMLMLAFTFLLALTIHVGEASA8yoaW Signal peptide NTATGAAAAAGATGTTGATGTTAGCTTTTACATTTCTTTTGGCTTTGACTATCCATGTAGGGGAAGCTTCGGCT9SPamyE F primer NTAGGAGAGGGACGCGT ATGTTTGCAAAACGATTCAAAACC10SPamyE R primer NTTGATTCCAGGTGTATC AGCACTCAGGGGCCG1 Propeptide AAETANKSNE12amyE Pro-peptide NTGAAACGGCGAACAAATTCGAATGAG13SPyqxI_PRO-amyE fusion F primer NTGAAACGGCGAACAAATTCGAATGAG GATACACTGGAATCAATTGATAATTGCG14SPyqxI_PRO-amyE fusion RTCGCTTCGTTCTTTCTTTCGGGG1 primer AGCTTTGGCATGTCCATCCAAC15SPyoaW_PRO-amyE fusion F primer NTGAAACGGCGAACAAATTCGAATGAG GATACACTGGAATCAATTGATAATTGCG16SPyoaW_PRO-amyE fusion R primer NTCTCATTCGATTTGTTCGCCGTTTC GGCCTCTCGAATTCGAAT

Claims

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

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

1.

3. A polypeptide according to claim 1, wherein the amino acid sequence of sequence number 5 or 7 is fused to the N-terminus of the nuclease amino acid sequence.

4. A polypeptide according to claim 1, wherein the polypeptide further comprises an amino acid sequence of SEQ ID NO: 11, 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 nuclease amino acid sequence.

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

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

7. In paragraph 6, the microorganism is a microorganism of the genus Bacillus.

8. In the 6th paragraph, the microorganism is Bacillus subtilis.

9. A method for producing a nuclease derived from Serratia marcescens, comprising: culturing a microorganism containing a polypeptide of any one of claims 1 to 4 or a polynucleotide encoding the same in a medium; and recovering the nuclease from the culture.

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. A composition for producing a nuclease derived from Serratia marcescens, wherein the composition further comprises a peptide having an amino acid sequence of SEQ ID NO:

11.

12. Use of a nuclease producing a signal peptide having an amino acid sequence of SEQ ID NO: 5 or 7.

Images