WO2010002054A1 - Novel plasmid isolated from pediococcus pentosaceus and use thereof - Google Patents
Novel plasmid isolated from pediococcus pentosaceus and use thereof Download PDFInfo
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- WO2010002054A1 WO2010002054A1 PCT/KR2008/004239 KR2008004239W WO2010002054A1 WO 2010002054 A1 WO2010002054 A1 WO 2010002054A1 KR 2008004239 W KR2008004239 W KR 2008004239W WO 2010002054 A1 WO2010002054 A1 WO 2010002054A1
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- plasmid
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- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 229960005356 urokinase Drugs 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 235000013618 yogurt Nutrition 0.000 description 1
- 235000021241 α-lactalbumin Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Definitions
- the present invention relates to a novel plasmid isolated from Pediococcus pentosaceus and use thereof, and more particularly to a plasmid, having a base sequence represented by SEQ ID NO: 1, and use thereof.
- Lactic acid bacteria which are involved in the kimchi fermentation process, have been reported to have various health-promoting functions, including antimicrobial effects (Physicochemical Characteristics of Yogurt Prepared with Lactic Acid Bacteria Isolated from Kimchi. Korean J. Food Culture. 20(3):337-340(2005)), immune function enhancement, blood cholesterol-lowering effects, liver function enhancing effects, anticancer effects and antioxidative effects.
- pediocin An antimicrobial peptide secreted by the kimchi lactic bacteria Pediococcus pentosaceus is called "pediocin" and a method of producing pediocin using Pediococcus pentosaceus is disclosed in Korean Patent Laid-Open Publication No. 10-2006-0069984. However, whether any plasmid contained in Pediococcus pentosaceus shows antimicrobial activity has not been specifically reported.
- the present inventors have isolated a plasmid, which contains the antimicrobial pediocin operon, among plasmids contained in Pediococcus pentosaceus, and have determined the sequence thereof, thereby completing the present invention. [9] Therefore, it is an object of the present invention to provide a plasmid having a base sequence represented by SEQ ID NO: 1. [10] Another object of the present invention is to a cloning vector comprising the plasmid of Claim 1, and a promoter, a multiple cloning site (MSC) and a selection marker, wherein the multiple cloning site (MSC) and the selection marker are inserted in the plasmid. [11] Still another object of the present invention is to provide a shuttle vector, comprising said plasmid, the replication origin of E. coli, a promoter, a multiple cloning site
- the present invention provides a plasmid having a base sequence represented by SEQ ID NO: 1.
- the present invention provides a cloning vector comprising the plasmid of Claim 1, and a promoter, a multiple cloning site (MSC) and a selection marker, wherein the multiple cloning site (MSC) and the selection marker are inserted in the plasmid.
- the present invention provides a shuttle vector, comprising said plasmid, the replication origin of E. coli, a promoter, a multiple cloning site
- the present invention provides microorganisms transformed with said plasmid or vector.
- the present invention provides a method of producing pediocin using said plasmid.
- plasmid has the meaning common in the art, that is, refers to a circular non-chromosomal element present in bacteria. Plasmid preparation, the delivery and ligation of plasmid DNA, plasmid transformation, and the like, may be achieved using methods well known to those skilled in the art. The methods are described, for example, in Sambrook, J. et al., “Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989).
- the plasmid of the present invention has a base sequence represented by SEQ ID NO: 1
- the plasmid of the present invention is preferably isolated from, but not limited to,
- the plasmid can form a circular plasmid having a base sequence ranging from the first sequence to the last sequence of SEQ ID NO: 1.
- plasmids contained in Pediococcus pentosaceus were extracted using a plasmid DNA extraction method (large plasmid DNA extraction method, Park et al., 2008, Biotechnol. Lett. 30: 165-172), and the extracted plasmids were electrophoresed (see lane 1 in FIG. 1).
- Pediococcus pentosaceus was treated with novobiocin to obtain a strain whose antimicrobial activity has been lost, and the plasmid in the strain was electrophoresed (see lane 2 in FIG. 1).
- the electrophoresis results were compared between the plasmids extracted from the treated strain and the plasmids extracted from the wild-type strain, thus isolating the inventive plasmid having antimicrobial activity (see Example 1).
- the present inventors have analyzed the base sequence of the isolated plasmid of the present invention and, as a result, have found that the isolated plasmid has a 11762-bp base sequence of SEQ ID NO: 1 (see Example 2).
- the plasmid of the present invention can be used as a vector according to any method known in the art and can be advantageously used to produce large amounts of the antimicrobial substance pediocin.
- the plasmid of the present invention can be used as an expression vector, which is useful for the analysis of the metagenome of intestinal lactic acid bacteria or the production of enteritis- treating substances.
- the cloning vector according to the present invention comprises said plasmid, and a promoter, a multiple cloning site (MCS) and a selection marker, wherein the multiple cloning site (MSC) and the selection marker are inserted in the plasmid.
- the cloning vector of the present invention is very useful for the cloning of genes in lactic acid bacteria.
- the promoter that is used in the cloning vector of the present invention is not specifically limited, as long as it can induce expression in lactic acid bacteria.
- Examples of the promoter include, but are not limited to, a 16S rRNA promoter, a low-pH-inducible promoter, a hypoxia- inducible promoter, a ptsH (phosphotransferase system HPr (phosphocarrier protein)) promoter and the like.
- MCS Multiple cloning site
- Any MCS may be used without limitation in the cloning vector of the present invention, as long as it is MCS known in the art. It can be obtained from various vectors (e.g., pUC18, pUC19, etc.) having MCS, known in the art.
- selection marker refers to a gene which is used to confirm the cloning of a gene.
- a marker gene providing selectable phenotypes such as drug resistance, auxotropy, resistance to cytotoxic agents, or surface protein expression, may be used.
- an antibiotic-resistant gene, a color-developing enzyme gene or a luminescent/fluorescent gene may be used.
- antibiotic-resistant genes include, but are not limited to, neomycin- resistant genes, hygromycin-resistant genes, ampicillin-resistant genes, kanamycin- resistant genes, erythromycin-resistant genes and chloramphenicol acetyl transferase genes.
- color-developing enzyme gene examples include ⁇ -galactosidase genes and ⁇ -glucuronidase (GUS) genes.
- Examples of the luminescent gene include Iu- cif erase genes, and examples of the fluorescent gene include a BFP (blue fluorescent protein) gene, a CFP (cyan fluorescent protein) gene, a GFP (green fluorescent protein) gene, an YFP (yellow fluorescent protein) gene and an RFP (red fluorescent protein) gene.
- BFP blue fluorescent protein
- CFP cyan fluorescent protein
- GFP green fluorescent protein
- YFP yellow fluorescent protein
- RFP red fluorescent protein
- the shuttle vector according to the present invention comprises said plasmid, the replication origin of E. coli, a promoter, a multiple cloning site (multiple cloning site) and a selection marker.
- the shuttle vector has both the replication origin of lactic acid bacteria and the replication origin of E. coli, it can replicate in lactic acid bacteria and E. coli. Thus, the shuttle vector can replicate and express a large amount of a target gene in lactic acid bacteria and E. coli.
- the replication origin of E. coli that is used in the shuttle vector of the present invention can be obtained from E. co //-derived transformation vectors (e.g., pUC18, pUC19, etc.) well known in the art. Because the base sequence of the E. coli replication origin is well known in the art, any person skilled in the art can easily insert a known E. coli replication origin into the shuttle vector of the present invention. [36] Also, any promoter may be used without limitation in the shuttle vector of the present invention, as long as it can induce expression in lactic acid bacteria and E. coli. Examples thereof are as described above. Also, the selection marker that can be used in the shuttle vector is as described above.
- the shuttle vector of the present invention can be prepared by inserting the base sequence of the replication origin directly into the cloning vector of the present invention. Alternatively, it can also be prepared by ligating the plasmid of the present invention with the E. co //-derived transformation vector. That is, it can be prepared by digesting the inventive plasmid with, preferably, Sail or Hindlll, having one-cut site, to prepare linear plasmid DNA, and linking the prepared DNA with an E. co//-derived transformation vector, digested with the same restriction enzyme, using a genetic engineering method (e.g., ligation).
- a genetic engineering method e.g., ligation
- a target gene may be inserted into the cloning vector or the shuttle vector, which are provided in the present invention.
- target gene refers to a gene to be cloned in lactic acid bacteria or a gene to be expressed in a specific host cell.
- the target gene may be inserted into MCS present in the cloning vector or shuttle vector of the present invention.
- the target gene is amplified by PCR with primers, which comprise a restriction enzyme sequence to allow the target gene to be inserted into the MCS of each of the vectors, and then the amplified gene is digested with a restriction enzyme and inserted into the MCS of the cloning vector or shuttle vector, digested with the same restriction enzyme.
- the target gene can be operatively linked to the promoter.
- the term "operatively linked" means that one nucleic acid fragment is linked to other nucleic acid fragment so that the function or expression thereof is affected by the other nucleic acid fragment.
- the target gene can be linked such that the expression thereof can be regulated by the promoter present in the vector.
- the target gene that is used in the present invention may include all genes to be cloned in lactic acid bacteria or genes to be expressed in E. coli.
- a gene capable of increasing the probiotic activity, function and/or utility of the lactic acid bacteria may be used as a target gene.
- the plasmid of the present invention contains the pediocin operon, which makes it possible to produce antimicrobial substances.
- various genes enhancing the functions of the plasmid can be used as target genes.
- Examples of medically and industrially useful target genes which can be inserted into the MCS of the inventive vector, include genes, which encode hormones, cytokines, enzymes, clotting factors, transport proteins, receptors, regulatory proteins, structural proteins, transcription factors, antigens, antibodies and the like.
- genes which encode thrombopoietin, human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferons, interferon receptors, colony stimulating factors, glucagon-like peptides (GLP-I and so on), G-protein-coupled receptor, interleukins, interleukin receptors, enzymes, interleukin binding proteins, cytokine binding proteins, macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A, allergy inhibitor, cell necrosis glycoproteins, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressors, metastasis growth factor, alpha- 1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein-E, erythropoietin, highly glycosylated erythropoietin, angiopoietins, hemoglobin, thrombin, thrombin receptor activating
- All the vectors, which are provided in the present invention may contain, in addition to the promoter, an expression control sequence in order to control the expression of a gene.
- expression control sequence refers to a DNA sequence that regulates the expression of an operatively linked nucleic acid sequence in a specific host cell.
- the expression control sequence includes a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation.
- vectors from which some sequences, particularly sequences having structural functions, have been removed, may also be included in the scope of the vectors of the present invention, as long as they maintain the functions of the vectors of the present invention.
- microorganisms of the present invention are transformed with the vector of the present invention.
- the microorganisms preferably refer to, but are not limited to, lactic acid bacteria or
- E. coli The E. coli bacteria may be used without limitation in the present invention, as long as they are general E. coli bacteria, such as XLl-blue and JM-109.
- the microorganisms transformed as described above can be advantageously used for the mass production of proteins. Thus, large amounts of proteins can be obtained from the culture of the transformed microorganisms.
- the vector can be introduced into the cells using methods known in the art, for example, but not limited to, a heat shock method, calcium phosphate precipitation, electroporation, gene guns and other methods for introducing DNA into cells (Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988).
- the inventive method for producing pediocin comprises using the plasmid.
- pediocin may be obtained by transforming microorganisms with the plasmid of the present invention using the above-described method known in the art, and isolating and purifying pediocin from the transformed microorganisms.
- the expression of the protein can be induced by culturing the transformed microorganisms, for example, inoculating the transformed microorganisms into a suitable medium in which the transformed microorganisms can grow, subjecting the inoculated microorganisms to seed culture, inoculating the seed-cultured microorganisms in a medium for cell culture, and culturing the inoculated microorganisms in suitable conditions. After completion of the culture process, pediocin can be collected from the culture of the microorganisms.
- the collection of pediocin expressed in the culture of the microorganisms can be carried out using various isolation and purification methods known in the art. Generally, after the cell lysate is centrifuged to remove cell debris, etc., it can be subjected to precipitation, for example, salting-out (ammonium sulfate precipitation and sodium phosphate precipitation), solvent precipitation (protein fraction precipitation using acetone, ethanol, etc.), dialysis, electrophoresis and various column chromatography processes.
- salting-out ammonium sulfate precipitation and sodium phosphate precipitation
- solvent precipitation protein fraction precipitation using acetone, ethanol, etc.
- dialysis dialysis
- electrophoresis and various column chromatography processes.
- ion exchange chromatography As the chromatography processes, ion exchange chromatography, gel permeation chromatography, HPLC, reverse-HPLC, affinity column chromatography and ultrafiltration can be applied alone or in combination to purify pediocin (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1982); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press(1989); Deutscher, M., Guide to Protein Purification Methods Enzymology, vol. 182. Academic Press. Inc., San Diego, CA (1990)).
- Advantageous Effects Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1982); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press(1989); Deutscher, M., Guide to Protein Purification
- the plasmid of the present invention can be used as a vector to express target proteins in various ways and, in addition, can be advantageously used to produce large amounts of pediocin having strong antimicrobial activity against microorganisms, such as Listeria, Salmonella and Helicobacter pylori.
- FIG. 1 shows the results of the extraction of plasmids from Pediococcus pentosaceus
- lane 1 a total plasmid extracted from a wild- type strain.
- Lane 2 a total plasmid extracted from a strain, from which a pediocin-encoding plasmid has been removed.
- FIG. 2 shows the results of screening of a Pediococcus pentosaceus CBT-8 strain whose antimicrobial activity has been lost (indicated by the arrow in FIG. 2) by treating the strain with novobiocin. Best Mode for Carrying Out the Invention
- pJW79 plasmid containing an antimicrobial pediocin operon, from Pediococcus pentosaceus CBT-8.
- the plasmid of the present invention is an antimicrobial pediocin operon-containing plasmid isolated from Pediococcus pentosaceus CBT-8, and the present inventors named the isolated plasmid "pJW79".
- Example 1 a total plasmid was extracted from a wild- type strain and electrophoresed on agarose gel, and then the DNA of the inventive novel plasmid (pJW79) band found from the results of Example 1 was subjected to in-gel digestion with HindIIL The digested DNA was extracted and cloned into the cloning vector pBlueScriptll SK(+). Meanwhile, all the plasmids were digested with HindIII or CIaI, and then cloned into pBlueScriptll SK(+), thus constructing a plasmid library. The base sequences of the obtained subclones were determined using an automated DNA sequence, and the determined base sequences of the fragments were linked by alignment using the DNA analysis program DNASIS MAX (Hitachi, Japan) to construct a final base sequence.
- the inventive novel plasmid (pJW79) had a base sequence of SEQ ID NO: 1, having a length of 11762 bp.
- the plasmid of the present invention can be used as a vector to express target protein in various ways and, in addition, can be advantageously used to produce large amounts of pediocin having strong antimicrobial activity against microorganisms, including Listeria, Salmonella and Helicobacter pylori.
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Abstract
Disclosed are a novel plasmid isolated from Pediococcus pentosaceus CBT-8 and use thereof. More specifically, disclosed are a plasmid, having a base sequence represented by SEQ ID NO: 1, and use thereof. The disclosed plasmid can be used as a vector to express target protein in various ways and, in addition, can be advantageously used to produce large amounts of pediocin having strong antimicrobial activity against microorganisms, including Listeria, Salmonella and Helicobacter pylori.
Description
Description
NOVEL PLASMID ISOLATED FROM PEDIOCOCCUS PENTOSACEUS AND USE THEREOF
Technical Field
[1] This application claims the priority of Korean Patent Application No.
10-2008-0063413, filed on 1 July, 2008, the disclosure of which is incorporated herein by reference.
[2] The present invention relates to a novel plasmid isolated from Pediococcus pentosaceus and use thereof, and more particularly to a plasmid, having a base sequence represented by SEQ ID NO: 1, and use thereof. Background Art
[3] Lactic acid bacteria, which are involved in the kimchi fermentation process, have been reported to have various health-promoting functions, including antimicrobial effects (Physicochemical Characteristics of Yogurt Prepared with Lactic Acid Bacteria Isolated from Kimchi. Korean J. Food Culture. 20(3):337-340(2005)), immune function enhancement, blood cholesterol-lowering effects, liver function enhancing effects, anticancer effects and antioxidative effects.
[4] Recently, interest has been focused in probiotic functions among the characteristics of lactic acid bacteria, and thus studies on lactic bacteria having antimicrobial activity have been actively conducted. Particularly, Pediococcus pentosaceus among lactic acid bacteria contained in kimchi is known to have strong antimicrobial activities against bacteria, including Listeria, Salmonella and Helicobacter pylori, and thus studies thereon have been conducted in various ways.
[5] An antimicrobial peptide secreted by the kimchi lactic bacteria Pediococcus pentosaceus is called "pediocin" and a method of producing pediocin using Pediococcus pentosaceus is disclosed in Korean Patent Laid-Open Publication No. 10-2006-0069984. However, whether any plasmid contained in Pediococcus pentosaceus shows antimicrobial activity has not been specifically reported.
[6] To date among the Pediococcus pentosaceus strains, a strain whose entire genomic sequence is known is Pediococcus pentosaceus ATCC 27745 (GenBank accession number: NC_008525), and among plasmids contained in the strain, pMD136 (GenBank accession number: NC_001277) is the only plasmid whose entire base sequence has been determined. However, this plasmid has problems in that it contains no pediocin operon.
[7] Particularly, when a plasmid having specific antimicrobial activity, which is contained in Pediococcus pentosaceus, is found and used as a vector, various anti-
microbial substances or target proteins can be produced in large amounts in various ways. Accordingly, this plasmid is industrially highly useful, and thus studies thereon are urgently needed. Disclosure of Invention
Technical Problem
[8] The present inventors have isolated a plasmid, which contains the antimicrobial pediocin operon, among plasmids contained in Pediococcus pentosaceus, and have determined the sequence thereof, thereby completing the present invention. [9] Therefore, it is an object of the present invention to provide a plasmid having a base sequence represented by SEQ ID NO: 1. [10] Another object of the present invention is to a cloning vector comprising the plasmid of Claim 1, and a promoter, a multiple cloning site (MSC) and a selection marker, wherein the multiple cloning site (MSC) and the selection marker are inserted in the plasmid. [11] Still another object of the present invention is to provide a shuttle vector, comprising said plasmid, the replication origin of E. coli, a promoter, a multiple cloning site
(MCS) and a selection marker. [12] Still another object of the present invention is to provide microorganisms transformed with said plasmid or vector. [13] Still another object of the present invention is to provide a method of producing pediocin using said plasmid. [14] To the above objects, in one aspect, the present invention provides a plasmid having a base sequence represented by SEQ ID NO: 1. [15] In another aspect, the present invention provides a cloning vector comprising the plasmid of Claim 1, and a promoter, a multiple cloning site (MSC) and a selection marker, wherein the multiple cloning site (MSC) and the selection marker are inserted in the plasmid. [16] In still another aspect, the present invention provides a shuttle vector, comprising said plasmid, the replication origin of E. coli, a promoter, a multiple cloning site
(MCS) and a selection marker. [17] In still another object, the present invention provides microorganisms transformed with said plasmid or vector. [18] In still another aspect, the present invention provides a method of producing pediocin using said plasmid. [19]
Technical Solution [20] Hereinafter, the present invention will be described in detail.
[21] As used herein, the term "plasmid" has the meaning common in the art, that is, refers to a circular non-chromosomal element present in bacteria. Plasmid preparation, the delivery and ligation of plasmid DNA, plasmid transformation, and the like, may be achieved using methods well known to those skilled in the art. The methods are described, for example, in Sambrook, J. et al., "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989).
[22] The plasmid of the present invention has a base sequence represented by SEQ ID
NO: 1.
[23] The plasmid of the present invention is preferably isolated from, but not limited to,
Pediococcus pentosaceus CBT-8. Also, the plasmid can form a circular plasmid having a base sequence ranging from the first sequence to the last sequence of SEQ ID NO: 1.
[24] In the present invention, in order to isolate the plasmid of the present invention from wild-type Pediococcus pentosaceus, plasmids contained in Pediococcus pentosaceus were extracted using a plasmid DNA extraction method (large plasmid DNA extraction method, Park et al., 2008, Biotechnol. Lett. 30: 165-172), and the extracted plasmids were electrophoresed (see lane 1 in FIG. 1). Meanwhile, Pediococcus pentosaceus was treated with novobiocin to obtain a strain whose antimicrobial activity has been lost, and the plasmid in the strain was electrophoresed (see lane 2 in FIG. 1). The electrophoresis results were compared between the plasmids extracted from the treated strain and the plasmids extracted from the wild-type strain, thus isolating the inventive plasmid having antimicrobial activity (see Example 1).
[25] In addition, the present inventors have analyzed the base sequence of the isolated plasmid of the present invention and, as a result, have found that the isolated plasmid has a 11762-bp base sequence of SEQ ID NO: 1 (see Example 2).
[26] Furthermore, the present inventors have analyzed putative ORFs present in the plasmid of the present invention and, as a result, have found a total of ORFs and determined the locations and sizes thereof and identities with the genes of various strains (see Example 3).
[27] Accordingly, the plasmid of the present invention can be used as a vector according to any method known in the art and can be advantageously used to produce large amounts of the antimicrobial substance pediocin. In particular, the plasmid of the present invention can be used as an expression vector, which is useful for the analysis of the metagenome of intestinal lactic acid bacteria or the production of enteritis- treating substances.
[28] Thus, the cloning vector according to the present invention comprises said plasmid, and a promoter, a multiple cloning site (MCS) and a selection marker, wherein the multiple cloning site (MSC) and the selection marker are inserted in the plasmid.
[29] The cloning vector of the present invention is very useful for the cloning of genes in
lactic acid bacteria. The promoter that is used in the cloning vector of the present invention is not specifically limited, as long as it can induce expression in lactic acid bacteria. Examples of the promoter include, but are not limited to, a 16S rRNA promoter, a low-pH-inducible promoter, a hypoxia- inducible promoter, a ptsH (phosphotransferase system HPr (phosphocarrier protein)) promoter and the like.
[30] As used herein, the term "Multiple cloning site" (MCS) refers to a DNA fragment having various restriction sites. It is recognized and digested by a specific restriction enzyme, such that a target gene can be inserted into the digested MCS site. Any MCS may be used without limitation in the cloning vector of the present invention, as long as it is MCS known in the art. It can be obtained from various vectors (e.g., pUC18, pUC19, etc.) having MCS, known in the art.
[31] As used herein, the term "selection marker" refers to a gene which is used to confirm the cloning of a gene. As the selection marker, a marker gene providing selectable phenotypes such as drug resistance, auxotropy, resistance to cytotoxic agents, or surface protein expression, may be used. Preferably, an antibiotic-resistant gene, a color-developing enzyme gene or a luminescent/fluorescent gene may be used.
[32] Examples of the antibiotic-resistant genes include, but are not limited to, neomycin- resistant genes, hygromycin-resistant genes, ampicillin-resistant genes, kanamycin- resistant genes, erythromycin-resistant genes and chloramphenicol acetyl transferase genes. Also, examples of the color-developing enzyme gene include β-galactosidase genes and β-glucuronidase (GUS) genes. Examples of the luminescent gene include Iu- cif erase genes, and examples of the fluorescent gene include a BFP (blue fluorescent protein) gene, a CFP (cyan fluorescent protein) gene, a GFP (green fluorescent protein) gene, an YFP (yellow fluorescent protein) gene and an RFP (red fluorescent protein) gene. In addition, various genes known as selection markers in the art may be used in the present invention.
[33] Moreover, the shuttle vector according to the present invention comprises said plasmid, the replication origin of E. coli, a promoter, a multiple cloning site (multiple cloning site) and a selection marker.
[34] Because the shuttle vector has both the replication origin of lactic acid bacteria and the replication origin of E. coli, it can replicate in lactic acid bacteria and E. coli. Thus, the shuttle vector can replicate and express a large amount of a target gene in lactic acid bacteria and E. coli.
[35] The replication origin of E. coli that is used in the shuttle vector of the present invention can be obtained from E. co //-derived transformation vectors (e.g., pUC18, pUC19, etc.) well known in the art. Because the base sequence of the E. coli replication origin is well known in the art, any person skilled in the art can easily insert a known E. coli replication origin into the shuttle vector of the present invention.
[36] Also, any promoter may be used without limitation in the shuttle vector of the present invention, as long as it can induce expression in lactic acid bacteria and E. coli. Examples thereof are as described above. Also, the selection marker that can be used in the shuttle vector is as described above.
[37] The shuttle vector of the present invention can be prepared by inserting the base sequence of the replication origin directly into the cloning vector of the present invention. Alternatively, it can also be prepared by ligating the plasmid of the present invention with the E. co //-derived transformation vector. That is, it can be prepared by digesting the inventive plasmid with, preferably, Sail or Hindlll, having one-cut site, to prepare linear plasmid DNA, and linking the prepared DNA with an E. co//-derived transformation vector, digested with the same restriction enzyme, using a genetic engineering method (e.g., ligation).
[38] A target gene may be inserted into the cloning vector or the shuttle vector, which are provided in the present invention. As used herein, the term "target gene" refers to a gene to be cloned in lactic acid bacteria or a gene to be expressed in a specific host cell.
[39] The target gene may be inserted into MCS present in the cloning vector or shuttle vector of the present invention. For example, the target gene is amplified by PCR with primers, which comprise a restriction enzyme sequence to allow the target gene to be inserted into the MCS of each of the vectors, and then the amplified gene is digested with a restriction enzyme and inserted into the MCS of the cloning vector or shuttle vector, digested with the same restriction enzyme. The target gene can be operatively linked to the promoter. As used herein, the term "operatively linked" means that one nucleic acid fragment is linked to other nucleic acid fragment so that the function or expression thereof is affected by the other nucleic acid fragment. That is, the target gene can be linked such that the expression thereof can be regulated by the promoter present in the vector. Examples of the target gene that is used in the present invention may include all genes to be cloned in lactic acid bacteria or genes to be expressed in E. coli. For example, when the vector of the present invention is transformed into lactic acid bacteria, a gene capable of increasing the probiotic activity, function and/or utility of the lactic acid bacteria may be used as a target gene.
[40] Particularly, the plasmid of the present invention contains the pediocin operon, which makes it possible to produce antimicrobial substances. Thus, various genes enhancing the functions of the plasmid can be used as target genes.
[41] Examples of medically and industrially useful target genes, which can be inserted into the MCS of the inventive vector, include genes, which encode hormones, cytokines, enzymes, clotting factors, transport proteins, receptors, regulatory proteins, structural proteins, transcription factors, antigens, antibodies and the like. Specific
examples thereof include genes, which encode thrombopoietin, human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferons, interferon receptors, colony stimulating factors, glucagon-like peptides (GLP-I and so on), G-protein-coupled receptor, interleukins, interleukin receptors, enzymes, interleukin binding proteins, cytokine binding proteins, macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A, allergy inhibitor, cell necrosis glycoproteins, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressors, metastasis growth factor, alpha- 1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein-E, erythropoietin, highly glycosylated erythropoietin, angiopoietins, hemoglobin, thrombin, thrombin receptor activating peptide, thrombomodulin, factor VII, factor Vila, factor VIII, factor IX, factor XIII, plasminogen activating factor, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epithelial growth factor, epidermal growth factor, angiostatin, angiotensin, bone growth factor, bone stimulating protein, calcitonin, insulin, atriopeptin, cartilage inducing factor, elcatonin, connective tissue activating factor, tissue factor pathway inhibitor, follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, nerve growth factors, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, corticotropin releasing factor, thyroid stimulating hormone, autotaxin, lactoferrin, myostatin, receptors, receptor antagonists, cell surface antigens, virus derived vaccine antigens, monoclonal antibodies, polyclonal antibodies, antibody fragments and the like.
[42] All the vectors, which are provided in the present invention, may contain, in addition to the promoter, an expression control sequence in order to control the expression of a gene. As used herein, the term expression control sequence refers to a DNA sequence that regulates the expression of an operatively linked nucleic acid sequence in a specific host cell. The expression control sequence includes a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation.
[43] In addition, vectors, from which some sequences, particularly sequences having structural functions, have been removed, may also be included in the scope of the vectors of the present invention, as long as they maintain the functions of the vectors of the present invention.
[44] Moreover, the microorganisms of the present invention are transformed with the vector of the present invention.
[45] The microorganisms preferably refer to, but are not limited to, lactic acid bacteria or
E. coli. The E. coli bacteria may be used without limitation in the present invention, as long as they are general E. coli bacteria, such as XLl-blue and JM-109. The microorganisms transformed as described above can be advantageously used for the mass production of proteins. Thus, large amounts of proteins can be obtained from the culture of the transformed microorganisms.
[46] For the transformation of cells, the vector can be introduced into the cells using methods known in the art, for example, but not limited to, a heat shock method, calcium phosphate precipitation, electroporation, gene guns and other methods for introducing DNA into cells (Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988).
[47] Furthermore, the inventive method for producing pediocin comprises using the plasmid.
[48] Because the plasmid of the present invention contains the pediocin operon, pediocin may be obtained by transforming microorganisms with the plasmid of the present invention using the above-described method known in the art, and isolating and purifying pediocin from the transformed microorganisms. Specifically, the expression of the protein can be induced by culturing the transformed microorganisms, for example, inoculating the transformed microorganisms into a suitable medium in which the transformed microorganisms can grow, subjecting the inoculated microorganisms to seed culture, inoculating the seed-cultured microorganisms in a medium for cell culture, and culturing the inoculated microorganisms in suitable conditions. After completion of the culture process, pediocin can be collected from the culture of the microorganisms.
[49] The collection of pediocin expressed in the culture of the microorganisms can be carried out using various isolation and purification methods known in the art. Generally, after the cell lysate is centrifuged to remove cell debris, etc., it can be subjected to precipitation, for example, salting-out (ammonium sulfate precipitation and sodium phosphate precipitation), solvent precipitation (protein fraction precipitation using acetone, ethanol, etc.), dialysis, electrophoresis and various column chromatography processes. As the chromatography processes, ion exchange chromatography, gel permeation chromatography, HPLC, reverse-HPLC, affinity column chromatography and ultrafiltration can be applied alone or in combination to purify pediocin (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1982); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press(1989); Deutscher, M., Guide to Protein Purification Methods Enzymology, vol. 182. Academic Press. Inc., San Diego, CA (1990)).
Advantageous Effects
[50] As described above, the plasmid of the present invention can be used as a vector to express target proteins in various ways and, in addition, can be advantageously used to produce large amounts of pediocin having strong antimicrobial activity against microorganisms, such as Listeria, Salmonella and Helicobacter pylori.
[51]
Brief Description of the Drawings
[52] FIG. 1 shows the results of the extraction of plasmids from Pediococcus pentosaceus
CBT-8.
[53] In FIG. 1, lane 1: a total plasmid extracted from a wild- type strain; and
[54] Lane 2: a total plasmid extracted from a strain, from which a pediocin-encoding plasmid has been removed.
[55] FIG. 2 shows the results of screening of a Pediococcus pentosaceus CBT-8 strain whose antimicrobial activity has been lost (indicated by the arrow in FIG. 2) by treating the strain with novobiocin. Best Mode for Carrying Out the Invention
[56] Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are illustrative only, and the scope of the present invention is not limited thereto.
[57] Example 1 : Extraction and isolation of novel plasmid (^TW19)
[58] In order to extract the pediocin operon-containing plasmid from Pediococcus pentosaceus CBT-8 (Accession Number: KCTC 10297BP), a plasmid DNA extraction method (large plasmid DNA extraction method, Park et al., 2008, Biotechnol. Lett. 30: 165-172) was used to extract plasmids, and the extracted plasmids were loaded and electrophoresed on 0.8% agarose gel. The electrophoresis results are shown in FIG. 1.
[59] As shown in FIG. 1, it could be seen that at least four different plasmids were present in Pediococcus pentosaceus CBT-8 (see lane 1 in FIG. 1).
[60] Thus, the present inventors used the known method of Miller (Experiments in
Molecular Genetics, 1972, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p54) to find a plasmid (pJW79), containing an antimicrobial pediocin operon, from Pediococcus pentosaceus CBT-8.
[61] Specifically, a wild- type Pediococcus pentosaceus strain was treated with novobiocin, and then in order to find a strain whose antimicrobial activity has been lost due to the removal of a plasmid therefrom, screening was carried out on Listeria monocytogenes, the growth of which is known to be inhibited by the antimicrobial activity of the strain, using a spot-on-lawn method according to the known method of Sarkar and Banerjee (J.Food ScL Tech. 33: 231-233, 1996) (see FIG. 2). Plasmids were
extracted from the strain selected through the screening process and were subjected to agarose gel electrophoresis. The plasmids extracted from the treated strain were compared with the plasmids extracted from the wild-type strain. The electrophoresis results are shown in FIG. 1.
[62] As shown in FIG. 1, a novel plasmid, which was contained in the wild-type strain, but not contained in the strain whose antimicrobial activity has been lost, could be isolated (see lane 2 in FIG. 1). Accordingly, the plasmid of the present invention is an antimicrobial pediocin operon-containing plasmid isolated from Pediococcus pentosaceus CBT-8, and the present inventors named the isolated plasmid "pJW79".
[63] Example 2: Analysis of base sequence of novel plasmid(pJW79) according to the present invention
[64] In order to purify the inventive novel plasmid (pJW79) extracted and isolated in
Example 1, a total plasmid was extracted from a wild- type strain and electrophoresed on agarose gel, and then the DNA of the inventive novel plasmid (pJW79) band found from the results of Example 1 was subjected to in-gel digestion with HindIIL The digested DNA was extracted and cloned into the cloning vector pBlueScriptll SK(+). Meanwhile, all the plasmids were digested with HindIII or CIaI, and then cloned into pBlueScriptll SK(+), thus constructing a plasmid library. The base sequences of the obtained subclones were determined using an automated DNA sequence, and the determined base sequences of the fragments were linked by alignment using the DNA analysis program DNASIS MAX (Hitachi, Japan) to construct a final base sequence.
[65] From the above results, it could be seen that the inventive novel plasmid (pJW79) had a base sequence of SEQ ID NO: 1, having a length of 11762 bp.
[66] Example 3: Determination of putative ORFs through analysis of base sequence of inventive novel plasmid (pJW79)
[67] In order to analyze putative ORFs present in the inventive novel plasmid (pJW79) analyzed in Example 2, DNASIS MAX (Hitachi, Japan) was used, and the base sequence upstream of the analyzed ORFs was analyzed based on the promoter search program (http://www.fruitflv.org/seq tools/promoter.html). Then, whether -35 and -10 sites, which are the characteristics of the gene promoters of prokaryotes, are present in the ORFs, were examined to determine putative ORFs. Also, the identity of the putative ORFs was analyzed using a GenBank database (NCBI), and the analysis results are shown in Table 1 below.
[68] Table 1
[Table 1]
[Table ]
Characteristics of putative ORFs contained in the inventive novel plasmid (pJW79)
[69] [70]
Industrial Applicability
[71] As described above, the plasmid of the present invention can be used as a vector to express target protein in various ways and, in addition, can be advantageously used to produce large amounts of pediocin having strong antimicrobial activity against microorganisms, including Listeria, Salmonella and Helicobacter pylori.
Claims
[1] A plasmid having a base sequence represented by SEQ ID NO: 1.
[2] The plasmid of Claim 1, wherein the plasmid is isolated from Pediococcus pentosaceus CBT-8.
[3] A cloning vector comprising the plasmid of Claim 1, and a promoter, a multiple cloning site (MSC) and a selection marker, wherein the multiple cloning site (MSC) and the selection marker are inserted in the plasmid.
[4] A shuttle vector comprising the plasmid of Claim 1, the replication origin of E. coli, a multiple cloning site (MSC) and a selection marker.
[5] The vector of Claim 3 or 4, wherein the promoter is selected from the group consisting of a 16S rRNA promoter, a low-pH-inducible promoter, a hypoxia- inducible promoter and a ptsH (phosphotransferase system HPr (phosphocarrier protein)) promoter.
[6] The vector of Claim 3 or 4, wherein the selection marker is selected from the group consisting of an antibiotic-resistant gene, a color-developing enzyme gene, a luminescent gene and a fluorescent gene.
[7] The shuttle vector of Claim 4, wherein the vector is prepared by linking the plasmid of Claim 1 with an E. coli-derived transformation vector.
[8] Microorganisms transformed with any one selected from the group consisting of the plasmid of Claim 1, the cloning vector of Claim 3 and the shuttle vector of Claim 4.
[9] A method of producing pediocin using the plasmid of Claim 1.
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| DE102015111756A1 (en) | 2015-07-20 | 2017-01-26 | Eberhard Karls Universität Tübingen Medizinische Fakultät | Recombinant Orf virus vector |
| KR101915949B1 (en) * | 2018-01-09 | 2018-11-07 | 주식회사 쎌바이오텍 | Gene expression cassette and expression vector comprising the same |
| KR101915950B1 (en) * | 2018-01-09 | 2018-11-07 | 주식회사 쎌바이오텍 | Microorganisms which express and secrete cystatin for delivering drug for treating gastrointestinal diseases and pharmaceutical composition for preventing or treating gastrointestinal diseases comprising the same |
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| US6403082B1 (en) * | 1996-09-05 | 2002-06-11 | Michael E. Stiles | Bacteriocins, transport and vector system and method of use thereof |
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Non-Patent Citations (3)
| Title |
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| ALESSIO GIACOMINI ET AL., PLASMID, vol. 43, 2000, pages 111 - 122 * |
| M. DANIELSEN ET AL., JOURNAL OF APPLIED MICROBIOLOGY, vol. 102, 2007, pages 1364 - 5072 * |
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