JP2008199963A - Negative selection marker gene and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a marker gene enabling the negative selection in a target cell. <P>SOLUTION: The gene encoding a polypeptide having levansucrase activities and not having cleaving activities of a signal sequence, or a polypeptide having the levansucrase activities and not having the signal sequence is used as the negative selection marker. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a gene that can be used as a negative selectable marker and use thereof, and more particularly to a gene that inhibits the growth of a host cell in the presence of sucrose when expressed in the host cell and use thereof. Is.

  For the production of useful substances using metabolic engineering techniques, a technique for destroying a plurality of genes on the genome and a technique for introducing a plurality of foreign genes onto the genome are used. A drug resistance gene is used as a selection marker gene for efficiently selecting cell lines in which gene disruption and foreign gene introduction have been performed. Such a selection marker is said to be a positive selection marker that selects (survives) cells containing the selection marker.

  In recent years, as technology has advanced, there has been a need to perform a plurality of gene modifications on one genome. When a plurality of genetic modifications are performed on a single target, it is necessary to use a combination of a plurality of selectable markers. However, the types of drug resistance genes that can be used as selectable markers are limited. For this reason, development of a new selectable marker that can be used is desired.

  As another type of selectable marker, a negative selectable marker that eliminates (kills) cells containing the selectable marker is known. As a negative selection marker gene, SacB gene derived from Bacillus, tetAR gene, rpsL gene and the like are known (Non-patent Document 1). The negative selection marker may be used alone or in combination with a positive selection marker as a double selection method such as the positive-negative selection (PNS) method.

  The SacB gene is a gene encoding a SacB protein, which is one of enzymes having levansucrase activity. SacB protein breaks down sucrose and produces levan. Gram-negative bacteria cannot grow in the presence of sucrose when SacB protein is expressed in cells. Thus, by excluding (negative selection) the cell line introduced with the SacB gene in the presence of sucrose, it is possible to efficiently select the target genetically modified cell line (Non-patent Document 2).

  The reason why the expression of the SacB gene in Gram-negative bacteria confers lethality to cells in the presence of sucrose has not been clarified. In gram-negative bacteria, levansucrase activity is known to be localized in the periplasm, and the accumulation of high-molecular-weight levan produced by levansucrase activity in the periplasm imparts lethality to cells. This is considered as one of the factors.

  Gram-positive bacteria do not have an outer membrane, so there is no periplasmic region. In Gram-positive bacteria that do not have an outer membrane, it is thought that SacB protein is released outside the cell, and as a result, levan cannot be produced or accumulated in the cell. However, even in the case of Gram-positive bacteria, in some bacteria such as Corynebacterium and Mycobacterium, lethality is imparted to the cells by the expression of the SacB gene (Non-patent Document 3). The results shown in Non-Patent Document 3 are due to the outer membrane-like structure formed due to mycolic acid in Gram-positive bacteria containing mycolic acids in the cell wall, such as Corynebacterium and Mycobacterium. This is probably because the SacB protein stayed in the cell and levan accumulated in the periplasm-like region similarly formed.

  Since ordinary gram-positive bacteria do not have mycolic acid in the cell wall, levan is not produced or accumulated in the cells. Therefore, even if the SacB gene is expressed in the cell, the cell is not lethal in the presence of sucrose. This indicates that the SacB gene cannot be used as a negative selection marker in normal gram-positive bacteria.

  By inserting the gene to be introduced between the promoter and the marker gene, the positive selection marker gene can be used as a negative selection marker. Similarly, a negative selection marker gene can be used as a positive selection marker by inserting the gene to be introduced between the promoter and the marker gene. Of course, the insertion site of the gene to be introduced is not limited to between the promoter and the marker gene, and even if the target gene is inserted into the marker gene, the expression of the marker gene can be suppressed.

  A technique using the SacB gene as a positive selection marker by introducing multiple copies of the SacB gene into Escherichia coli and overexpressing it in cells using this viewpoint has been reported (Patent Document 1).

In addition, a mutant SacB gene lacking signal sequence cleavage activity is introduced into a Gram-positive bacterium having no mycolic acid in the cell wall and overexpressed in the cell, thereby positively mutating the SacB gene. A technique used as a selection marker has been reported (Patent Document 2).
WO92 / 14819 (publication date: September 3, 1992) Special table 11-1151427 gazette (publication date: December 14, 1999) Reyrat J M et al. , Infection and Immunity, Sept. 1998; 4011-4017 Jagar W et al. , Journal of Bacteriology 1992; 5462-5465 Pelic V et al. , Journal of Bacteriology 1996; 1971-1199. Borchert TV et al. , Journal of Bacteriology 1991; 276-282. Bramucci MG et al. , Applied and Environmental Microbiology 1996; 3948-3953.

  If SacB protein lacks its signal sequence cleavage activity, the signal sequence is not cleaved from the SacB precursor protein and is not secreted outside the cell (Non-patent Document 4). Of course, such a mutant SacB protein does not have periplasmic localization as in the wild type, but the mutant SacB protein exists in the vicinity of the cytoplasmic membrane by overexpressing the gene in the cell. It is considered that levan was generated and accumulated near the cytoplasmic membrane and cell wall, and the mutant SacB gene functioned as a positive selection marker (Non-patent Document 5).

  However, in order to use the mutant SacB gene as a positive selection marker, as described in Patent Document 2, a multicopy mutant SacB gene must be present in the host cell. When a single-copy mutant SacB gene is introduced into Gram-positive bacterial cells, sucrose-induced lethality is not observed in the host cells (Non-patent Document 5).

  Of the selectable marker genes used for the double selection marker, the marker gene used for the second selection preferably has a single copy number introduced into the host cell. In particular, when a plurality of genetic modifications are performed on a single target, it is very important that only a single copy gene exists and can function as a marker. In the methods described in Patent Document 2 and Non-Patent Document 5, since a host cell into which a single-copy mutant SacB gene has been introduced is not lethal in the presence of sucrose, the mutant SacB gene is used as a negative selection marker gene. I can't do it.

  Furthermore, in recent years, development of a technique that does not leave a selection marker gene as a heterologous gene in a transformant is eagerly desired. However, a selection marker having a configuration such as Patent Documents 1 and 2 may be applied to such a technique. I can't.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a negative selection marker gene that can be used in Gram-positive bacteria and a gene modification technique using the same.

  The selectable marker fusion gene according to the present invention is a fusion gene comprising a negative selectable marker gene and a positive selectable marker gene, and the negative selectable marker gene and the positive selectable marker gene can operate on the first and second promoters, respectively. The polypeptide encoded by the negative selectable marker gene has a levansucrase activity and does not have a signal sequence cleavage activity, or has a levansucrase activity and has a signal sequence. It is characterized by not being a polypeptide.

  In the selection marker fusion gene according to the present invention, the first and second promoters are preferably promoters derived from Gram-positive bacteria.

  In the selectable marker fusion gene according to the present invention, the negative selectable marker gene may be a gene derived from a gene selected from the group consisting of a SacB gene, a fefA gene, a lev gene, an mlft gene, an lsc gene, and an lsxA gene. Good.

  In the selection marker fusion gene according to the present invention, also the negative selectable marker gene, Bacillus subtilis, Bacillus amyloliquefaciens, Lactobacillus sanfranciscensis, Lactobacillus johnsonii, Lactobacillus reuteri, Leuconostoc mesenteroides, Oenococcus oeni, derived cells selected from the group consisting of Paenibacillus polymyxa It may be a gene derived from the wild type SacB gene.

  In the selectable marker fusion gene according to the present invention, the negative selectable marker gene may also be a gene encoding any of the following polypeptides (A) to (C): (A) wild type A polypeptide comprising an amino acid sequence in which at least one Ala of Ala-X-Ala present at positions 20 to 44 in the amino acid sequence encoded by the SacB gene is substituted; or (B) above (A) A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the polypeptide shown in the above, having a levansucrase activity and a signal sequence cleavage activity. Or (C) a fragment of the polypeptide shown in (A) or (B) above A, a polypeptide having no cleavage activity has and signal sequence levansucrase activity is.

  In the selectable marker fusion gene according to the present invention, the negative selectable marker gene may also be a gene encoding any of the following polypeptides (A) to (C): (A) wild type A polypeptide comprising an amino acid sequence in which the amino acid at the N-terminal side of Ala-X-Ala existing at positions 20 to 44 in the amino acid sequence encoded by the SacB gene is deleted; or (B) above (A) A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the polypeptide shown in the above, and having a levansucrase activity and not having a signal sequence Or (C) a fragment of the polypeptide shown in (A) or (B) above, wherein Polypeptide having no have and signal sequence activity.

  In the selectable marker fusion gene according to the present invention, the negative selectable marker gene may also be a gene encoding any of the following polypeptides (A) to (C): (A) SEQ ID NO: A polypeptide comprising an amino acid sequence in which at least one Ala of Ala-X-Ala present at positions 20 to 44 in the amino acid sequence represented by any one of 1 to 8 is substituted; or (B) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of the polypeptide shown in (A) above, having a levansucrase activity and a signal sequence cleavage activity Or (C) a fragment of the polypeptide shown in (A) or (B) above. A preparative has a levansucrase activity and have no polypeptide having the cleavage activity of the signal sequence.

  In the selectable marker fusion gene according to the present invention, the negative selectable marker gene may also be a gene encoding any of the following polypeptides (A) to (C): (A) SEQ ID NO: A polypeptide comprising an amino acid sequence in which the amino acid at the N-terminal side of Ala-X-Ala existing at positions 20 to 44 in the amino acid sequence shown in any of 1 to 8 is deleted; or (B) A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the polypeptide shown in (A) above, having levansucrase activity and having a signal sequence Or (C) a fragment of the polypeptide shown in (A) or (B) above, comprising a levan Have cyclase activity and a polypeptide having no signal sequences.

  In the selectable marker fusion gene according to the present invention, the positive selectable marker gene may be a drug resistance gene.

  In the selectable marker fusion gene according to the present invention, the positive selectable marker gene may also be a drug resistance gene derived from a Gram-positive bacterium.

  In the selectable marker fusion gene according to the present invention, the positive selectable marker gene may also include an erythromycin resistance gene, a chloramphenicol resistance gene, an alpha-galactosidase gene, a phosphobeta-glatosidase gene, an alanine racemase gene, a thymylate synapse gene, an oxidase synapse gene, A gene selected from the group consisting of: a gene, an amber suppressor gene, an aspartate aminotransferase gene, a nisin immunity gene, a lactacin immunity gene, a levanase gene, a crythromycin-resistant gene, and a lincomycin-resistant gene It may be a child.

  The selectable marker fusion gene according to the present invention may be a gene used as a double selection marker in Gram-positive bacterial cells.

  In the selection marker fusion gene according to the present invention, the cells also, Leuconostoc, Pediococcus, Streptococcus, Lactobacillus, Melisscoccus, Enterococcus, Lactococcus, Carnobacterium, Vagococcus, Tetragenococcus, Atopobium, Weissella, Lactosphaera, Oenococcus, Abiotrophia, Paralactobacillus, Granulicatella, Atopobacter Or a cell selected from the group consisting of Alkabacterium and Olsenella.

  The selectable marker fusion gene according to the present invention may be a gene for modifying the genomic sequence of a target cell without leaving a heterologous sequence in the genome.

  The double selection kit for Gram-positive bacterial cells according to the present invention is characterized by comprising the selection marker fusion gene according to the present invention.

  The method of double-selecting a target cell according to the present invention comprises a step of introducing the fusion gene according to the present invention into a Gram-positive bacterium cell; a step of culturing the cell under conditions for positive selection; Removing the marker gene; and culturing the cell under conditions for negative selection. The step of removing the negative selection marker gene may be a step of removing the entire fusion gene.

  The negative selectable marker gene according to the present invention is such that the polypeptide encoded by the gene has a levansucrase activity and does not have a signal sequence cleavage activity, or has a levansucrase activity and a signal sequence. It is characterized by being a polypeptide that it does not have.

  A kit for negatively selecting a target cell according to the present invention is characterized by comprising the negative selection marker gene according to the present invention.

  In the kit according to the present invention, the negative selection marker gene may be a gene having the above-described characteristics, and is preferably operably linked to a promoter derived from a Gram-positive bacterium. Moreover, the said cell should just be a cell which has the characteristic mentioned above.

  Using the present invention, it is possible to negatively select Gram-positive bacterial cells. Moreover, if this invention is used, it is possible to double-select Gram positive bacteria cells. Furthermore, the present invention can be used as a targeting technique that does not leave a selection marker gene as a heterologous gene in a transformant.

[1. Negative selectable marker gene)
The present inventors have found that a specific variant of a gene encoding a polypeptide having levansucrase activity functions as a selection marker gene for negative selection of cells.

  That is, the present invention provides a negative selection marker gene for negative selection of cells. The negative selection marker gene is a gene that functions as a selection marker for eliminating (killing) cells containing this gene, and selecting a target cell using this gene is called negative selection. Specifically, in negative selection, cell lines in which the negative selection marker gene has been introduced are eliminated by culturing the cells under predetermined conditions that allow only cells that have not been introduced with the negative selection marker gene to grow. The target cell into which the negative selection marker gene has not been introduced can be selected.

  Examples of the negative selection marker gene used in Gram-positive bacterial cells include uraylphosphophosphoyltransferase (upp) gene, beta-lactamase repressor (blaI) gene, mazF gene, and the like (“mazF, a novel counter molecule-select”). unmarked chromosomal manipulation in Bacillus subtilis. ", Nucleic acids research, vol34 (2006), and" New integrated method to generate bilites substitut. s free of selection markers. ", Applied environmental microbiology, 7241-7250 (2004) see).

  The negative selectable marker gene according to the present invention encodes a mutant of levansucrase. As used herein, unless otherwise specified, “levansucrase” is intended to be “a polypeptide having levansucrase activity” and is used interchangeably with “levansucrase polypeptide” or “levansucrase protein”. Is done.

  As used herein, “levansucrase activity” intends the activity of degrading sucrose to produce levan. “Levansucrase variant” is used interchangeably with “mutant levansucrase”. The mutant levansucrase is specifically a polypeptide having levansucrase activity and no signal sequence cleavage activity, or a polypeptide having levansucrase activity and no signal sequence. .

  “Signal sequence” is intended as a “polypeptide consisting of a signal sequence”, and is a sequence necessary when a secreted protein or the like moves outside a cell. The precursor of levansucrase synthesized in the cell is converted into a mature sequence by cleaving the processing site of the signal sequence and secreted outside the cell. As used herein, the “signal sequence” of levansucrase has the function of transferring levansucrase expressed in cells to the periplasm or periplasm-like region in gram-negative or some gram-positive bacteria. In other Gram-positive bacteria, it is intended to be a polypeptide having a function of transferring levansucrase expressed intracellularly to the outside of the cell.

  One of the “mutant levansucrases”, “polypeptide having levansucrase activity and not having signal sequence cleavage activity”, is such that the signal sequence is not removed from the levansucrase precursor. Variant levansucrase polypeptides with mutated processing sites are contemplated. In such mutants, the signal sequence cannot be removed from the levansucrase precursor, so the mature sequence of levansucrase polypeptide cannot be transferred as described above.

  Another “mutant levansucrase”, “polypeptide having levansucrase activity and no signal sequence”, is a mutant levansucrase mutated so that the signal sequence is missing from the levansucrase precursor. A polypeptide is contemplated. Since such a mutant levansucrase polypeptide does not have a signal sequence, the mature sequence of levansucrase polypeptide cannot be transferred as described above.

  Thus, a “mutant levansucrase polypeptide” is a polypeptide that retains the levansucrase activity of a wild-type polypeptide and does not have a signal sequence cleavage activity or has a signal sequence. Polypeptides not intended are contemplated. Further, the “mutant levansucrase gene” is intended to be a gene encoding the mutant levansucrase polypeptide.

  In addition, as used herein, “polypeptide” is used interchangeably with “peptide” or “protein”. “Gene” is used interchangeably with “polynucleotide”, “nucleic acid” or “nucleic acid molecule”. As used herein, “base sequence” is used interchangeably with “nucleic acid sequence” or “nucleotide sequence” and as a sequence of deoxyribonucleotides (abbreviated as A, G, C, and T). Indicated.

  As described above, a mutant gene of a gene encoding a polypeptide having levansucrase activity can be employed as the negative selectable marker gene according to the present invention. Preferred genes encoding a polypeptide having levansucrase activity include the SacB gene, the fefA gene (accession number: AJ508391, levantocase from Lactobacillus sanfranciscensis), and the lev gene (accession number: Q8Gvcil4, Lc). Gene (accession number: AAT81165, levansucrose derived from Leuconostoc mesenteroides), lsc gene (accession number: O68609, levansuc from Pseudomonas syringae) race), lsxA gene (accession number: BAA93720, levansucrose derived from Gluconacetobacter xylinus), but is not limited thereto.

  As a negative selection marker gene of the present invention, a gene derived from the SacB gene will be described as an example.

  As described above, the SacB gene is a gene encoding a SacB protein that is one of the polypeptides having levansucrase activity. The SacB protein is produced by cleaving the signal sequence after being produced as a SacB precursor protein. SacB protein breaks down sucrose and produces levan. The wild-type SacB gene, Bachillus subtilis, Bacillus amyloliquefaciens, Lactobacillus sanfranciscensis, Lactobacillus johnsonii, Lactobacillus reuteri, Leuconostoc mesenteroides, Oenococcus oeni, or there may be mentioned the wild-type SacB gene from Paenibacillus polymyxa, but are not limited to.

  The SacB precursor proteins derived from the eight cells listed as examples are provided as amino acid sequences shown in SEQ ID NOs: 1 to 8, and the nucleotide sequence constituting the polynucleotide encoding the polypeptide is SEQ ID NO: 9 It is provided as the base sequence shown in -16. Among the SacB genes derived from the above eight cells, the Bacillus subtilis-derived SacB gene is synthesized in the cell as a SacB precursor protein having a signal sequence of 29 amino acids.

  In the negative selection marker gene of the present invention, (A) in the amino acid sequence encoded by the wild-type SacB gene, Ala at least one of Ala-X-Ala present at positions 20 to 44 is substituted. It may be a gene encoding a polypeptide consisting of an amino acid sequence. That is, since the site involved in the cleavage activity of the signal sequence of the wild-type SacB precursor protein provided as an amino acid sequence as shown in SEQ ID NOs: 1 to 8 is mutated, it has a cleavage activity of the signal sequence. It may be a gene encoding a mutant SacB protein that is not present.

  The negative selectable marker gene of the present invention includes (B) a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the polypeptide shown in (A) above. Further, it may be a gene encoding a polypeptide having levansucrase activity and not having signal sequence cleavage activity.

  Furthermore, the negative selectable marker gene of the present invention is (C) a fragment of the polypeptide shown in (A) or (B) above, which has levansucrase activity and does not have signal sequence cleavage activity. It may be a gene encoding a polypeptide.

  In addition, the negative selection marker gene of the present invention has (A) an amino acid sequence encoded by the wild-type SacB gene that lacks an amino acid on the N-terminal side from Ala-X-Ala present at positions 20 to 44. It can be a gene encoding a polypeptide consisting of a certain amino acid sequence. That is, a gene encoding a mutant SacB protein that does not have a signal sequence because the signal sequence of the wild-type SacB precursor protein provided as an amino acid sequence as shown in SEQ ID NOs: 1 to 8 is mutated It can be.

  Furthermore, the negative selectable marker gene of the present invention comprises (B) a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the polypeptide shown in (A) above. Further, it may be a gene encoding a polypeptide having levansucrase activity and no signal sequence.

  Furthermore, the negative selectable marker gene of the present invention is a polypeptide that is (C) a fragment of the polypeptide shown in (A) or (B) above and has levansucrase activity and no signal sequence. It may be a gene encoding.

  It is well known in the art that some amino acids in the amino acid sequence of a polypeptide can be easily modified without significantly affecting the structure or function of the polypeptide. Furthermore, it is also well known that there are variants that not only artificially modify, but also do not significantly alter the structure or function of the natural protein. One skilled in the art can readily mutate one or several amino acids in the amino acid sequence of a polypeptide using well-known techniques.

In another embodiment, the mutant form of the gene according to the present invention is a gene encoding the polypeptide of (A) to ( C ) above, and (I) one or several in the base sequence of the gene (II) a gene that hybridizes under stringent conditions with a gene consisting of the base sequence of the gene or its complementary sequence; or (III) the gene It may be a gene consisting of a base sequence that is at least 80% identical to the base sequence.

  Hybridization is described in “Molecular Cloning: A Laboratory Manual 3rd edition, J. Sambrook and D. W. Russll, Cold Spring Harbor Laboratory, NY (2001)” (incorporated herein by reference). It can be carried out by a known method such as a known method.

  As used herein, “stringent hybridization conditions” include hybridization solution (50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate ( pH 7.6) in 5 × Denhart solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA) after overnight incubation at 42 ° C., then 0.1 × SSC at about 65 ° C. It is intended to be filter washed in.

  The present invention also provides a method for negatively selecting a target cell and a kit for negatively selecting a target cell.

  The method of negatively selecting a target cell according to the present invention is characterized by using the negative selectable marker gene according to the present invention. Specifically, a step of introducing a negative selectable marker gene into a cell, and negative selection Preferably, the method includes the step of culturing the cells under conditions for. The method for negatively selecting a target cell according to the present invention is preferably used for negatively selecting a Gram-positive bacterial cell.

  In addition, a kit for negative selection of a target cell according to the present invention is characterized by comprising the negative selection marker gene according to the present invention. The kit for negatively selecting a target cell according to the present invention is preferably used for negatively selecting a Gram-positive bacterial cell. As used herein, a “kit” is intended to be a form in which at least one of various components is contained in another substance (eg, a container).

  In order to specifically describe the negative selectable marker gene according to the present invention, the mutant SacB gene derived from the Bacillus subtilis-derived SacB gene has been described as an example. In the present invention, the negative selectable marker gene is the above mutation. Those of ordinary skill in the art who have read this specification will readily appreciate that they are not limited to type SacB genes.

  Thus, the target cell can be negatively selected by using the negative selection marker gene according to the present invention.

[2. Selectable marker fusion gene]
The present invention also provides a selectable marker fusion gene for selecting cells of interest. As used herein, “selection marker fusion gene” is intended to be a marker gene for selecting a target cell multiple times, and includes a positive selection marker gene and a negative selection marker gene. “Selectable marker fusion gene” is used interchangeably with “double selectable marker fusion gene” or “positive-negative selectable marker fusion gene”. As used herein, “double selection” is not limited to a selection consisting of only a single positive selection and a single negative selection, but at least one positive selection and at least one selection. Selection including negative selection is contemplated.

  The selection marker fusion gene according to the present invention includes the negative selection marker gene described above, and further includes a positive selection marker gene. The positive selection marker gene is a gene that functions as a selection marker for selecting (surviving) cells containing this gene, and selecting a target cell using this gene is called positive selection. Specifically, in the positive selection, by culturing the cells under a predetermined condition capable of growing only the cells into which the positive selection marker gene has been introduced, the cell line into which the positive selection marker gene has not been introduced is excluded, A target cell into which a positive selection marker gene has been introduced can be selected.

  The positive selection marker gene contained in the selection marker fusion gene according to the present invention is preferably a drug resistance gene, and more preferably a drug resistance gene derived from a Gram-positive bacterium. As the positive selection marker gene of the present invention, preferably, an erythromycin resistance gene, a chloramphenicol resistance gene, an alpha-galactosidase gene, a phospho beta-gluctosidase gene, an alanine racemase gene, a thymylate synthase gene, an ochresuprap gene, , Aspartate aminotransferase gene, nisin immunity gene, lactacin immunity gene, levanase gene, crymyrmycin resistance gene, or lincomycin resistance gene, but is not limited thereto.

  The positive selection marker gene and the negative selection marker gene contained in the selection marker fusion gene according to the present invention are operably linked to the first and second promoters, respectively, in order to select cells multiple times.

  As used herein, “operably linked (linked)” means that the sequence of interest is placed under the control of specific transcriptional translational regulatory sequences (eg, promoters, enhancers, etc.). By doing so, it is intended that the expression can be regulated. By placing a promoter upstream of the gene of interest, the gene of interest is operably linked to the promoter. Note that the promoter need not be located adjacent to the target gene.

  As used herein, a “promoter” is intended to be a gene involved in initiating transcription of an operably linked gene. Since the negative selection marker gene and the positive selection marker gene contained in the selection marker fusion gene according to the present invention preferably independently express the negative selection marker protein and the positive selection marker protein, each operates on a separate promoter. It is preferable that they are connected.

  Thus, each promoter independently controls transcription of the linked gene. In the selectable marker fusion gene according to the present invention, the first promoter linked to the negative selectable marker gene is preferably a promoter derived from Gram-positive bacteria (promoter derived from Bacillus subtilis), and the first promoter linked to the positive selectable marker gene. One promoter is preferably a gram-positive bacterium-derived promoter (Lactococcus-derived promoter), but is not limited thereto.

  The selectable marker fusion gene according to the present invention can be used as a double selection marker in Gram-positive bacterial cells. As used herein, a “dual selectable marker” is intended to be a bifunctional marker gene that is used to negatively and positively select cells. In “double selection”, for example, cells in which the target gene and marker gene are inserted into the genome are selected under positive selection conditions, and then cells in which the marker gene is excluded from the genome by homologous recombination are negative. A technique for selecting only a target cell (that is, a targeted cell without leaving a selectable marker gene in the genome) by growing under selection conditions can be mentioned, but is not limited thereto.

  A cell preferable as an application target of the present invention is preferably a Gram-positive bacterial cell. Gram positive bacteria refer to bacteria that are stained purple by Gram staining. Representative gram-positive bacteria include cocci (Micrococcus genus, Staphylococcus genus, Streptococcus genus), spore-forming rods (Bacillus genus, Clostridium genus), lactic acid bacteria (Lactobacillus genus etc.), coryneform bacteria (Corynebacterium genus, etc.) (Streptomyces genus).

  The cells preferred as the application target of the present invention are more preferably Gram-positive bacterial cells that do not contain mycolic acid in the cell wall. As used herein, “a Gram-positive bacterium that does not contain mycolic acid in the cell wall” does not include the genus Corynebacterium, the genus Mycobacterium, the genus Nocardia, and the like.

  It is even more preferable that the cells preferred as the application target of the present invention are so-called lactic acid bacteria cells. As used herein, “lactic acid bacteria” are intended to belong to bacteria among microorganisms that acquire energy by decomposing carbohydrates to produce lactic acid. Preferred lactic acid bacteria include Lactobacillus genus, Leuconostoc genus, Pediococcus genus, Streptococcus genus, Melissococcus genus, Enterococcus genus, Lactococcus genus, Carnobacterium genus, Vacococcus genus, Tetragenus genus, Examples include, but are not limited to, the genus Paralactobacillus, Granulacatella, Atopobacter, Alkabacterium, and Olsenella.

  The selectable marker fusion gene according to the present invention can be used to modify the genomic sequence of a target cell without leaving a heterologous sequence in the genome. As used herein, “heterologous sequence” is used interchangeably with “heterologous gene sequence” or “foreign gene sequence” and intends a gene sequence derived from a cell different from the cell into which the gene is to be introduced. Is done. Further, “modifying the genomic sequence of the target cell without leaving the heterologous sequence in the genome” is intended to perform gene targeting without leaving the selectable marker gene as a heterologous gene in the transformant.

  Thus, the target cell can be selected with higher accuracy by using the selection marker fusion gene according to the present invention.

[3. Vector containing selection marker fusion gene)
The present invention provides a vector comprising the gene described above. The vector according to the present invention is not particularly limited as long as it contains the above-described gene. For example, an expression vector into which cDNA of a negative selectable marker gene or a selectable marker fusion gene is inserted can be mentioned. Examples of the method for producing a vector according to the present invention include, but are not limited to, a method using a plasmid, phage, cosmid or the like.

  In the case of an expression vector, the specific type of the vector is not particularly limited, and a vector that can be expressed in a host cell can be appropriately selected. That is, a vector in which the gene of interest is incorporated into various plasmids or the like according to the type of host cell may be used as an expression vector so that the gene can be expressed reliably. If such an expression vector is used, the polypeptide encoded by the selectable marker fusion gene can be expressed in the cell into which the gene has been introduced.

[4. Method and kit for double selection of Gram-positive bacterial cells]
The present invention provides methods and kits for double selection of cells of interest.

  The method for double-selecting cells according to the present invention is characterized by using the above-described selection marker fusion gene, specifically, the step of introducing the above-mentioned fusion gene into the cell; conditions for positive selection Preferably, the method comprises culturing the cell under; removing the negative selectable marker gene; and culturing the cell under conditions for negative selection. The method of negatively selecting a target cell according to the present invention is preferably used for double selection of Gram-positive bacterial cells. For the step of introducing the selectable marker fusion gene into Gram-positive bacterial cells, gene transfer techniques well known in the art (eg, electroporation, calcium phosphate method, liposome method, DEAE dextran method, etc.) can be employed. In addition, techniques for inserting a target gene into the genome of a target cell are also well known in the art.

  The “conditions for positive selection” are conditions under which a cell expressing a polypeptide encoded by the positive selection marker gene contained in the selection marker fusion gene of the present invention can be grown, and the polypeptide is expressed. Conditions where non-viable cells cannot grow are contemplated. For example, when an erythromycin resistance gene is used as the positive selection marker gene, the "condition for positive selection" is that the medium contains an amount of erythromycin so that cells not having the erythromycin resistance gene cannot grow. Is intended.

  In addition, in the step of removing the negative selection marker gene, only the negative selection marker gene is removed from the fusion gene inserted into the genome of the Gram-positive bacterial cell in order to enable negative selection of the cell. However, all of the above fusion genes may be removed. Removal of the negative selectable marker gene can be realized by replacing the negative selectable marker gene sequence integrated on the genome with another base sequence (for example, by homologous recombination).

  Specifically, a negative selection marker gene is amplified by amplifying the base sequence in the peripheral region of the negative selectable marker gene and using the amplified base sequence to homologously recombine the region where the negative selectable marker gene is inserted on the genome. Remove the gene from the genome. In addition, by using the gene fragment connecting the amplified base sequence and the foreign gene, homologous recombination of the region where the negative selection marker gene was inserted on the genome, the negative selection marker gene was removed from the genome. It is also possible (see FIG. 5).

  Furthermore, “conditions for negative selection” are conditions under which cells expressing a polypeptide encoded by the negative selection marker gene contained in the selection marker fusion gene of the present invention cannot grow, and the polypeptide is expressed. Conditions under which cells that are not able to grow are intended. For example, when a mutant SacB gene derived from a Bacillus subtilis-derived SacB gene is used as the negative selection marker gene, the “condition for negative selection” is an amount that prevents the cells having the mutant SacB gene from growing. It is contemplated that sucrose is included in the medium.

  As described above, the polypeptide encoded by the mutant SacB gene has a levansucrase activity and does not have a signal sequence cleavage activity, or has a levansucrase activity and a signal sequence. The polypeptide is present in the cell because it is not a polypeptide. Thereby, when the cells are cultured in the presence of a predetermined concentration of sucrose, the cells in which the polypeptide is expressed can be excluded, and the cells can be negatively selected.

  The double selection kit for Gram-positive bacterial cells according to the present invention is characterized by comprising the above-described selection marker fusion gene. The kit for double-selecting cells of interest according to the present invention is preferably used for double-selecting Gram-positive bacteria cells. Thus, the Gram-positive bacterial cell is transformed using the selection marker fusion gene of the present invention, and the Gram-positive bacterial cell is positively selected by culturing under the conditions for positive selection. Negative selection can be performed by culturing under conditions for negative selection. The double selection kit according to the present invention may further include cells to be subjected to double selection.

  If the method and kit for double-selecting Gram-positive bacteria cells according to the present invention are used, the above-mentioned Gram-positive bacteria cells into which the selection marker gene has been introduced are positively selected and then further selected negatively. It is possible to double select cells. The present invention can also be applied to a technique for selecting only targeted cells without leaving a selectable marker gene in the genome.

  Note that the method and kit for double-selecting Gram-positive bacteria cells need not be limited to the above-described embodiment, and a specific method for double-selecting cells other than those described above and a kit for double-selection of cells. The embodiments will be readily understood by those of ordinary skill in the art who have read this specification.

  Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention is not limited to these examples, and various modifications are possible within the scope shown in the claims and the above embodiments. Embodiments obtained by appropriately combining technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[1. Materials and methods]
[1-1. Construction of mutant SacB gene]
Based on the SacB gene sequence of Bacillus subtilis registered in GenBank (GenBank Accession # NP_391325), a primer (SacB_PstI) for amplifying a region including the promoter region of the SacB gene was synthesized. Next, a primer (SacB (W29) _R) in which alanine immediately before the signal peptide processing site of the SacB gene was replaced with tryptophan was synthesized. Fragment 1 was amplified by PCR amplification using Bacillus subtilis genomic DNA as a template.

  PCR amplification is performed on SacB_PstI_F primers (5′-ggggctgcaaaaaaaggcaactttagtccccatgcaacaagaacac-3 ′ (SEQ ID NO: 17)) and SacB (W29) _R primers (5′-ttttggttcgtcgtgttgttgtgttgttgttgtgtt After the reaction at 94 ° C for 2 minutes, 30 cycles of denaturation at 94 ° C for 30 seconds, annealing at 53 ° C for 30 seconds, and extension at 74 ° C for 1 minute are performed, and the reaction solution is stored at 4 ° C. did.

  The PCR reaction solution was applied to a 1% agarose gel and electrophoresed at 100 V for 40 minutes. Only the amplification product of interest was excised from the agarose gel and purified using QIAquick Gel Extraction Kit (QIAGEN).

  In addition, SacB (W29) _F, which is a reverse complementary sequence of SacB (W29) _R, and SacB_BamHI that amplifies up to the termination codon of the SacB gene were synthesized. Fragment 2 was amplified by PCR amplification using Bacillus subtilis genomic DNA as a template.

  PCR amplification is included in the SacB (W29) _F primer (5′-aactcaagcgtttttggaaaaagaacagaaccaaaaa-3 ′ (SEQ ID NO: 19)) and the SacB_BamHI_R primer (5′-cccggatccggtatctgtttttttttt) After the reaction at 94 ° C for 2 minutes, 30 cycles of denaturation at 94 ° C for 30 seconds, annealing at 53 ° C for 30 seconds, and extension at 74 ° C for 1 minute are performed, and the reaction solution is stored at 4 ° C. did.

  The PCR reaction solution was applied to a 1% agarose gel and electrophoresed at 100 V for 40 minutes. Only the amplification product of interest was excised from the agarose gel and purified using a QIAquick Gel Extraction Kit (manufactured by QIAGEN).

  Next, PCR amplification was performed using SacB_PstI_F and SacB_BamHI_R using the mixed solution containing the purified fragment 1 and fragment 2 as a template, and a mutant SacB gene fragment was prepared.

  PCR amplification was carried out using the reaction solution shown in Table 3 at 94 ° C for 2 minutes, followed by 30 cycles of denaturation at 94 ° C for 30 seconds, annealing at 53 ° C for 30 seconds, and extension at 74 ° C for 1 minute. And the reaction was stored at 4 ° C.

  The PCR reaction solution was applied to a 1% agarose gel and electrophoresed at 100 V for 40 minutes. Only the target amplification product was excised from the agarose gel and purified using a QIAquick Gel Extraction Kit (manufactured by QIAGEN) to construct a mutant SacB gene fragment.

[1-2. Construction of mutant SacB gene transfer vector]
The mutant SacB gene fragment was treated with restriction enzymes (PstI and BamHI), applied to a 1% agarose gel, and electrophoresed at 100 V for 40 minutes. Only the fragment of interest was excised from the agarose gel and purified using a QIAquick Gel Extraction Kit (QIAGEN). Further, pUC18 (manufactured by Takara Bio Inc.) was treated with restriction enzymes (PstI and BamHI), applied to a 1% agarose gel, and electrophoresed at 100 V for 40 minutes. Only pUC18 cleaved with restriction enzymes (PstI and BamHI) was excised from the agarose gel and purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).

  The mutated SacB gene and pUC18 cleaved with restriction enzymes (PstI and BamHI) were ligated using Ligation-convenience kit (Nippon Gene), and the resulting plasmid was used to produce Escherichia coli Chemical competent cell-TOP10 (Invitrogen). Transformed. The transformed somatic cells were inoculated into an LB agar medium containing 100 ppm of ampicillin to construct mutant SacB / pUC18.

[1-3. Construction of positive-negative selection marker fusion gene]
As a positive selection marker used for double selection of cells together with the mutant SacB gene, an erythromycin resistance gene derived from Lactococcus lactis, which is one of so-called lactococci, was used. As shown in the schematic diagram of FIG. 2, a positive-negative selection marker fusion gene was constructed in which a mutant SacB gene and an erythromycin resistance gene were linked to a SacB promoter derived from Bacillus subtilis and an EmR promoter derived from Lactococcus, respectively.

  The erythromycin resistance gene and promoter as a positive selection marker were constructed as follows. Based on the plasmid pIL253 sequence derived from Lactococcus lactis (GenBank Accession # AF041239), an erythromycin resistance gene containing the promoter region shown in SEQ ID NO: 21 was synthesized. BamHI was added to the 5 'end of the synthetic fragment, and a PstI site and a SacI site were added to the 3' end.

  The synthesized fragment was treated with restriction enzymes (BamHI and SacI), applied to a 1% agarose gel, and electrophoresed at 100 V for 40 minutes. Only the fragment of interest was excised from the agarose gel and purified using QIAquick Gel Extraction Kit (QIAGEN). The mutant SacB / pUC18 was treated with restriction enzymes (BamHI and SacI), then applied to a 1% agarose gel and electrophoresed at 100 V for 40 minutes. Only the mutant SacB / pUC18 cleaved with restriction enzymes (BamHI and SacI) was excised from the agarose gel and purified using QIAquick Gel Extraction Kit (QIAGEN).

  The erythromycin resistance gene cleaved with restriction enzymes (BamHI and SacI) and the mutant SacB / pUC18 were ligated using Ligation-convenience kit (Nippon Gene), and the resulting plasmid was used to produce Escherichia coli Chemical competent cell-TOP10 (Invitrogen). ) Was transformed. The transformant cells were inoculated into an LB agar medium containing 100 ppm ampicillin and 500 ppm erythromycin to construct a mutant SacB_EmR / pUC18.

[1-4. Cloning of mutant SacB_EmR fragment into E. coli-Lactobacillus reuteri shuttle vector]
The mutant SacB_EmR fragment was cloned into the E. coli-Lactobacillus reuteri (L. reuteri) shuttle vector (pUC18_pGT232) represented by the plasmid map shown in FIG. pGT232 (GenBank Accession # U21859) It is a plasmid of unknown function (cryptic plasmid) retained by the reuteri 100-23 strain.

  Cloning was performed as follows. Mutant SacB_EmR / pUC18 was treated with a restriction enzyme (PstI), applied to a 1% agarose gel, and electrophoresed at 100 V for 40 minutes. Only the mutant SacB_EmR fragment was excised from the agarose gel and purified using the QIAquick Gel Extraction Kit (QIAGEN).

  In addition, pUC18_pGT232 was treated with a restriction enzyme (PstI), and then E. coli was treated. Dephosphorylation was carried out using E. coli alkaliline phosphatase (manufactured by TOYOBO). The dephosphorylation reaction solution was applied to a 1% agarose gel and electrophoresed at 100 V for 40 minutes. pUC18_pGT232 was excised from the agarose gel and purified using the QIAquick Gel Extraction Kit (QIAGEN).

  The mutated SacB_EmR fragment and pUC18_pGT232 cleaved with a restriction enzyme (PstI) and ligation-convenience kit (Nippon Gene) were ligated, and the resulting plasmid was used to transform Escherichia coli Chemical competent cell-TOP10 (Invitrogen). . Transformant cells were inoculated into an LB agar medium containing 100 ppm ampicillin and 500 ppm erythromycin to construct mutant SacB_EmR / pUC18_pGT232.

[1-5. Mutant SacB_EmR / pUC18_pGT232 L. Introduction to reuteri]
Mutant SacB_EmR / pUC18_pGT232 was transferred to L. A clone introduced into reuteri was prepared as follows.

Plasmids were extracted from the mutant SacB_EmR / pUC18_pGT232-introduced Escherichia coli, and after ethanol precipitation, the DNA pellet was dissolved in sterile water. In an MRS liquid medium (Difco), L. reuteri was inoculated and static culture was performed at 37 ° C. until OD ₆₀₀ = 0.8. After removing the medium by centrifugation, the cell pellet was suspended in sterilized water and centrifuged again. This operation was repeated 5 times.

  The cell pellet was suspended in 250 μl of 30% (wt / vol) PEG 1500, and 100 μl was dispensed. Mutant SacB_EmR / pUC18_pGT232 2 μg and L. reuteri recipient bacteria (manufactured by Japan collection of microorganisms) were transferred to a 0.2 cm electroporation cuvette (manufactured by Bio-rad), and a gene pulser (manufactured by Bio-rad) set at 2.5 kV, 25 μF, 200Ω was used. An electric pulse was applied. Then, L. Reuteri recipient bacteria were inoculated into 1 ml of MRS liquid medium (Difco) and statically cultured at 37 ° C. for 2 hours. In addition, L. reuteri recipient bacteria were inoculated into MRS agar medium containing 8 ppm of erythromycin, cultured at 37 ° C. overnight, reuteri (mutant SacB_EmR / pUC18_pGT232) was prepared.

[1-6. L. of mutant SacB_EmR fragment. Introduction into reuteri cells]
In order to produce a gene disruption strain in which the lactate dehydrogenase gene was replaced with a mutant SacB_EmR fragment using homologous recombination, first, a gene disruption fragment necessary for gene disruption by homologous recombination was constructed as follows.

  Two homologous regions 3 kb required for homologous recombination were PCR amplified. PCR amplification, Lr_ldh1511_nes_F (5'-tcctggagctcttgcttatgtattgagtgg-3 '(SEQ ID NO: 22)), and ldh1511_FR4 (5'-catttcgtgaatgcctcgagttcataaaaaccgc-3' (SEQ ID NO: 23)), and ldh1511_SF4 (5'-gcggtttttatgaactcgaggcattcacgaaatg-3 '(SEQ ID NO: 24)) and Lr_ldh1511_nes_R (5′-ttgggtgactccttttctcattggcaatggc-3 ′ (SEQ ID NO: 25)), respectively, using the reaction solution shown in Table 4 for 30 seconds at 98 ° C., followed by denaturation at 98 ° C. for 10 seconds, Annealing at 48 ° C for 30 seconds and 72 ° C Extension for 45 seconds is performed for 30 cycles, after further reaction for 90 seconds at 72 ° C., and stored the reaction solution at 4 ° C..

  Each PCR reaction solution was purified using Wizard SV PCR & gel clean-up system (manufactured by Progema), and 0.1 μl of each purified PCR reaction solution was mixed and used as a template for the second PCR reaction. The second PCR reaction was performed using Lr_ldh1511_nes_F (5′-tcctggagctctttgctttagtgtattgattgg-3 ′ (SEQ ID NO: 26)) and Lr_ldh1511_nes_R (5′-tttgtgctctgtgtgctgtgctgtg) , After 2 minutes of reaction at 94 ° C., 30 cycles of denaturation at 94 ° C. for 10 seconds, annealing at 60 ° C. for 30 seconds, and extension at 72 ° C. for 90 seconds, and further reaction at 72 ° C. for 180 seconds, The solution was stored at 4 ° C.

  The PCR reaction solution was applied to an agarose gel, and only a PCR amplified fragment of about 6 kb was excised from the agarose gel and purified using Wizard SV PCR & gel clean-up system (Progema). The purified PCR amplified fragment of about 6 kb was treated with a restriction enzyme (SacI), mixed with charomid 9-36 (manufactured by Nippon Gene) treated separately with restriction enzyme (SacI), and then ethanol precipitation was performed.

  After the DNA pellet was dissolved in sterilized water, a ligation reaction was performed at 16 ° C. for 30 minutes using Ligation convenient kit (manufactured by Nippon Gene). This ligation product was obtained from E. coli using LAMBDA INN (Nippon Gene). It was introduced into E. coli DH5α (manufactured by TOYOBO). The introduced cells were selected on LBAp 100 ppm agar medium to construct ldh_fragment / charomid.

  Next, the mutant SacB_EmR fragment was inserted into the XhoI site in the 6 kb amplified fragment as follows. PCR amplification of the mutant SacB_EmR fragment was performed using the mutant SacB_EmR / pUC18 as a template. PCR amplification includes SacB_F (XhoI) (5′-atgcctcgaaaaaaaggcaactttagtccccatgc-3 ′ (SEQ ID NO: 28)), and EmR_R (XhoI) (5′-gctcctctgagggtggtgcggtgcggtgcggtgcggtggtgcggtggg3-3)) , Followed by 30 cycles of reaction at 98 ° C for 30 seconds, denaturation at 98 ° C for 10 seconds, annealing at 48 ° C for 30 seconds, and extension at 72 ° C for 45 seconds, followed by reaction at 72 ° C for 45 seconds Thereafter, the reaction solution was stored at 4 ° C. The XhoI site was added to the primer used.

  The PCR reaction solution was purified using Wizard SV PCR & gel clean-up system (manufactured by Polymer), and then XhoI treatment was performed at 37 ° C. overnight. The XhoI reaction solution was applied to an agarose gel, and only the target fragment was excised from the gel and purified using Wizard SV PCR & gel clean-up system (manufactured by Polymer). The purified fragment was mixed with XhoI-treated ldh_fragment / charomid, followed by ethanol precipitation. After the DNA pellet was dissolved in sterilized water, a ligation reaction was performed at 16 ° C. for 30 minutes using a Ligation convenience kit (manufactured by Nippon Gene). The ligation product was obtained from E. coli using LAMBDA INN (Nippon Gene). It was introduced into E. coli DH5α (manufactured by TOYOBO). The introduced cells were selected on LBAp100ppmEm500ppm agar medium to construct ldh_mutant SacB_EmR / charomid.

  ldh_mutation SacB_EmR / charomid It was introduced into reuteri JCM1112 to produce a lactate dehydrogenase gene disruption strain in which the mutant SacB_EmR fragment was inserted into the genome.

[2. result]
[2-1. L. mutated SacB_EmR / pUC18_pGT232 introduced. reuteri's lethal effect in the presence of sucrose]
L. reuteri (mutant SacB_EmR / pUC18_pGT232) was inoculated into sucrose-added MRS agar medium (manufactured by Difco), and the lethal effect of sucrose was verified as follows.

L. reuteri (mutant SacB_EmR / pUC18_pGT232) was precultured in 11 ml of MRS liquid medium (manufactured by Difco) containing 8 ppm of erythromycin. The next day, 100 μl of the culture solution was transferred to 11 ml of MRS liquid medium (Difco) containing 8 ppm of erythromycin, cultured to OD ₆₀₀ = 2.0, and serial dilution series from 10 ⁻² to 10 ⁻⁵ using sterilized water. Produced. 100 μl of this was inoculated into MRS agar medium (manufactured by Difco) containing 8 ppm of erythromycin supplemented with 0%, 1%, 5%, 10% and 15% by weight of sucrose, and the number of colonies formed the next day Counted.

In the sucrose-added medium, the effect of reducing the number of cells grown to 1/10 ⁴ or less in the sucrose-added 5 wt% medium was confirmed. This can be said to be a lethal effect sufficient to use the mutant SacB gene as a negative selection marker in Gram-positive bacterial cells. Therefore, the mutant SacB_EmR can be used as a selection marker gene for positive-negative selection (PNS) in Gram-positive bacterial cells.

  If the present invention is used, negative selection of Gram-positive bacteria, which could not be achieved before, becomes possible, which can contribute to the development of a new genetic modification technique. Moreover, since the genome sequence of the target cell can be altered without leaving the heterologous gene in the genome, it can contribute to the development of a food that does not contain a recombinant gene.

FIG. 1 is a schematic diagram showing a method for constructing a mutant SacB gene. FIG. 2 is a schematic diagram showing a gene fragment containing a mutant SacB gene and an erythromycin resistance gene. FIG. 3 shows E. coli-L. It is a figure which shows the plasmid map of a reuteri shuttle vector. FIG. 4 is a schematic diagram showing a method for constructing a mutant SacB_EmR fragment. FIG. 5 is a schematic diagram showing a method for destroying a mutant SacB_EmR fragment.

Claims (23)

A fusion gene comprising a negative selectable marker gene and a positive selectable marker gene,
The negative selectable marker gene and the positive selectable marker gene are operably linked to first and second promoters, respectively;
The polypeptide encoded by the negative selectable marker gene is:
A selectable marker fusion gene, which is a polypeptide having levansucrase activity and no signal sequence cleavage activity, or a polypeptide having levansucrase activity and no signal sequence.   The fusion gene according to claim 1, wherein the first and second promoters are promoters derived from Gram-positive bacteria.   The fusion gene according to claim 1, wherein the negative selection marker gene is derived from a gene selected from the group consisting of a SacB gene, a fefA gene, a lev gene, an mlft gene, an lsc gene, and an lsxA gene.   The negative selection marker gene is derived from Bacillus subtilis, Bacillus amyloliquefaciens, Lactobacillus sanfranciscensis, Lactobacillus johnsonii, Lactobacillus reuteri, Leuconostoc mesenteroides, Oenococcus oeni, the wild-type SacB gene derived from a cell selected from the group consisting of Paenibacillus polymyxa, claim 2. The fusion gene according to 1. The negative selectable marker gene is
(A) a polypeptide comprising an amino acid sequence in which at least one of Ala-X-Ala present at positions 20 to 44 in the amino acid sequence encoded by the wild-type SacB gene is substituted; or (B ) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of the polypeptide shown in (A) above, having levansucrase activity and cleavage of the signal sequence A polypeptide having no activity; or (C) a fragment of the polypeptide shown in (A) or (B) above, having a levansucrase activity and not having a signal sequence cleavage activity The fusion gene of claim 1 which encodes a polypeptide. The negative selectable marker gene is
(A) a polypeptide comprising an amino acid sequence in which the amino acid at the N-terminal side of Ala-X-Ala existing at positions 20 to 44 in the amino acid sequence encoded by the wild-type SacB gene is deleted; or (B ) A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the polypeptide shown in (A) above, having levansucrase activity and having a signal sequence. Or (C) a fragment of the polypeptide shown in (A) or (B) above, which encodes a polypeptide having levansucrase activity and no signal sequence The fusion gene according to claim 1. The negative selectable marker gene is
(A) A polypeptide comprising an amino acid sequence in which at least one Ala of Ala-X-Ala present at positions 20 to 44 in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 8 is substituted Or (B) a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the polypeptide shown in (A) above, and having levansucrase activity; A polypeptide not having a signal sequence cleavage activity; or (C) a fragment of the polypeptide shown in (A) or (B) above, having levansucrase activity and having a signal sequence cleavage activity. The fusion gene of claim 1, which encodes a polypeptide that does not have. The negative selectable marker gene is
(A) a polypeptide comprising an amino acid sequence in which the amino acid at the N-terminal side of Ala-X-Ala existing at positions 20 to 44 is deleted in the amino acid sequence shown in any of SEQ ID NOs: 1 to 8 Or (B) a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the polypeptide shown in (A) above, and having levansucrase activity; A polypeptide not having a signal sequence; or (C) a fragment of the polypeptide shown in (A) or (B) above, which has a levansucrase activity and does not have a signal sequence. The fusion gene of claim 1 which encodes a peptide.   The fusion gene according to claim 1, wherein the positive selection marker gene is a drug resistance gene.   The fusion gene according to claim 1, wherein the positive selection marker gene is a drug resistance gene derived from a Gram-positive bacterium.   The positive selection marker gene is an erythromycin resistance gene, a chloramphenicol resistance gene, an alpha-galactosidase gene, a phosphobeta-lactosidase gene, an alanine racemase gene, a thymylase synthase gene, an ochresupraspase gene, an ochresuprastor gene The fusion gene according to claim 1, wherein the fusion gene is selected from the group consisting of an immunoglobulin gene, a lactacin Immunity gene, a levanase gene, a crythyrmycin resistance gene, and a lincomycin resistance gene.   The fusion gene according to claim 1 for use as a marker for double selection in Gram-positive bacterial cells.   Wherein the cell is selected Leuconostoc, Pediococcus, Streptococcus, Lactobacillus, Melisscoccus, Enterococcus, Lactococcus, Carnobacterium, Vagococcus, Tetragenococcus, Atopobium, Weissella, Lactosphaera, Oenococcus, Abiotrophia, Paralactobacillus, Granulicatella, Atopobacter, Alkalibacterium, from the group consisting of Olsenella The fusion gene according to claim 12.   The fusion gene according to claim 1, for modifying the genomic sequence of the target cell without leaving the heterologous sequence in the genome.   A kit for double selection of cells comprising the gene according to claim 1.   The kit according to claim 15, wherein the cell is a Gram-positive bacterial cell.   Said cell is selected Leuconostoc, Pediococcus, Streptococcus, Lactobacillus, Melisscoccus, Enterococcus, Lactococcus, Carnobacterium, Vagococcus, Tetragenococcus, Atopobium, Weissella, Lactosphaera, Oenococcus, Abiotrophia, Paralactobacillus, Granulicatella, Atopobacter, Alkalibacterium, from the group consisting of Olsenella The kit according to claim 15. A method of double-selecting cells of interest comprising:
Introducing the fusion gene of claim 1 into a cell;
Culturing the cells under conditions for positive selection;
Removing the negative selectable marker gene; and culturing the cell under conditions for negative selection.   19. The method of claim 18, wherein the cell is a gram positive bacterial cell.   Wherein the cell is selected Leuconostoc, Pediococcus, Streptococcus, Lactobacillus, Melisscoccus, Enterococcus, Lactococcus, Carnobacterium, Vagococcus, Tetragenococcus, Atopobium, Weissella, Lactosphaera, Oenococcus, Abiotrophia, Paralactobacillus, Granulicatella, Atopobacter, Alkalibacterium, from the group consisting of Olsenella The method of claim 18. A kit for negative selection of a target cell comprising a negative selectable marker gene,
A kit wherein the polypeptide encoded by the gene is a polypeptide having levansucrase activity and not having a signal sequence cleavage activity, or a polypeptide having levansucrase activity and not having a signal sequence.   The kit according to claim 21, wherein the cells are Gram-positive bacteria cells.   Wherein the cell is selected Leuconostoc, Pediococcus, Streptococcus, Lactobacillus, Melisscoccus, Enterococcus, Lactococcus, Carnobacterium, Vagococcus, Tetragenococcus, Atopobium, Weissella, Lactosphaera, Oenococcus, Abiotrophia, Paralactobacillus, Granulicatella, Atopobacter, Alkalibacterium, from the group consisting of Olsenella The kit according to claim 21.

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