JP6704740B2 - Method for producing recombinant microorganism - Google Patents

Description

The present invention relates to a method for producing a recombinant microorganism.

Conventionally, in the field of genetic engineering, selectable marker genes such as drug resistance genes and auxotrophy-related genes have been used as means for selecting transformants, and drug resistance or auxotrophy deficient or acquired by these marker genes has been used. With the trait as an index, selection of transformants in which the desired gene modification has been performed has been performed. At this time, positive selection, in which only the transformant in which the desired gene modification has been performed can grow, is desired because of its good selection efficiency.

Although selectable marker genes are useful for selecting transformants, their persistence in transformed cells poses several problems. For example, when a plurality of genetic modifications are applied to one host, it is necessary to pay attention to the type of marker gene remaining in the cell. In addition, since the types of marker genes that can be used for selection of transformants are limited, the marker genes accumulated in transformants limit the number of gene modifications. Furthermore, there is a problem that the marker gene remaining in the transformant adversely affects the environment such as contamination of the wild-type microorganism with the marker gene. Therefore, development of a transformant that does not carry a selectable marker gene is desired.

A method of removing the selectable marker gene from transformants is disclosed. Patent Document 1 discloses a cassette in which a 5'terminal region of a deletion target region of a host DNA is linked to a 5'terminal region and a 3'terminal region of the deletion target region, a marker gene and a repressor gene. A method of deleting the region targeted for deletion by deleting the cassette by second homologous recombination after selecting cells in which the cassette has been integrated by homologous recombination and expression of the marker gene is selected. Have been described. In this method, a second marker gene is arranged downstream of the deletion target region, and the second marker is expressed by the deletion of the repressor in the cassette, so that the deletion target region is deleted. The desired cells are positively selected. However, with this method, the second marker gene remains intracellular.

Patent Document 2 discloses that a 5'terminal region of a deletion target region of a host DNA, a 5'terminal region and a 3'terminal region of the deletion target region (or a gene of interest located therebetween), A cassette in which a second marker gene linked to a first marker gene and a promoter is linked is integrated by homologous recombination, and a cell in which the cassette is integrated is selected by the expression of the first marker gene, followed by second homologous recombination. A method for selecting cells in which the cassette is deleted by selecting the loss of expression of the second marker gene as an index and selecting the cells in which the desired gene modification is made and the marker gene does not remain is described. .. Examples of the second marker gene include lacZ and amyE.

Non-Patent Document 1 and Patent Document 3 disclose that two types of drug resistance genes are expressed interchangeably and used as selectable marker genes when gene modification of cells is repeated. These drug resistance genes are introduced into the auxotrophy-related gene region of the host, used as a marker gene, and finally replaced again with the auxotrophy-related gene. It does not survive.

JP, 2007-11110, A JP, 2009-254350, A JP, 2013-009604, A

H. Nanamiya, et al., Microbiology, 2010, 156:2944-2952

The present invention provides a method for producing a recombinant microorganism capable of easily positively selecting a modified cell having a desired modification and suppressing the accumulation of a selectable marker gene in the modified cell.

The present inventors have previously replaced a region to be mutated in a host cell DNA with a hydrolase gene, and then replaced the hydrolase gene with a DNA of interest or a fragment for deletion to culture the cell. It is possible to easily select a recombinant cell in which the mutation target region has been replaced with the target DNA or a cell in which the mutation target region has been deleted, based on the presence or absence of the above halo formation, and in that case, a host cell On the other hand, by introducing a marker gene such as a drug resistance gene separately from the hydrolase gene, the target for determining the halo formation can be narrowed down to only cells selected by the marker. It has been found that it is easier to determine the presence or absence of. That is, by combining the selection based on the formation of halo and the selection based on the marker gene, the cells having the desired modification can be easily selected. Furthermore, even when the gene modification operation is repeated, by using the selectable marker genes by switching them interchangeably, it is possible to easily select the cells in which the desired modification has been made, and to use. The number of marker genes can be suppressed.

Therefore, in one aspect, the present invention provides a method for producing a recombinant microorganism, comprising:
(1) preparing cells of a host microorganism that co-expresses a first hydrolase expression cassette and a first selectable marker,
here,
The first hydrolase expression cassette is a DNA containing a promoter and a first hydrolase gene operably linked to the promoter, and the first hydrolase expression cassette is Replaced with the first region of interest in the DNA of the cell;
(2) The cells are cultivated in the selection medium having the first selection marker-containing strain containing the substrate for the first hydrolase, and the cells that grow on the medium and form halo are the first cells. Selecting as cells containing a hydrolase expression cassette;
(3) giving the cell selected in (2) the ability to express a second selectable marker and replacing the first hydrolase expression cassette with a first recombination cassette; and
(4) The cells prepared in (3) are cultured in the second selection marker-bearing strain selection medium containing the substrate for the first hydrolase, and the cells are grown on the medium without forming halos. Selecting cells as the cells containing the first recombination cassette,
A method of including is provided.

In a further aspect, the invention provides a method of producing a recombinant microbial host cell, the method comprising:
Introducing a DNA containing a hydrolase gene operably linked to a promoter into a microbial cell, or introducing a promoter to operably link the hydrolase gene in the cell, and a selectable marker gene And culturing the cells in the selection marker-bearing strain selection medium containing a substrate for the hydrolase, and selecting cells that have grown and formed halos on the medium.
A method of including is provided.

According to the method for producing a recombinant microorganism of the present invention, a cell into which a target DNA has been introduced or a cell in which a target DNA region has been deleted can be easily selected even when gene transfer into a host cell is repeated. can do. Furthermore, according to the method of the present invention, the number of selectable marker genes remaining in the cells after modification can be suppressed, and the selectable marker gene can be deleted from the recombinant microorganism after a series of modifications. Therefore, according to the present invention, a recombinant microorganism having no or very few selectable marker gene remains is provided.

The conceptual diagram of the manufacturing method of the recombinant microorganism of this invention. Conceptual diagram of the method for producing a recombinant microorganism of the present invention: Shows a case where two types of selectable marker genes are expressed interchangeably. Schematic diagram of the method for producing a recombinant microorganism of the present invention: shows a procedure for deleting a selectable marker gene from a recombinant cell. In the figure, the marker gene replaced with the trpBA gene region is deleted by resubstitution with trpBA.

(1. Definition)
As used herein, the term “mutation target region” refers to a region in DNA of a host microorganism that is to be mutated by substitution with the target DNA or deletion of the region. .. Regions to be mutated include unnecessary regions of host DNA, such as regions unnecessary for host substance production (spore formation-related genes, etc.), regions that inhibit host growth or substance production (phage regions, etc.), and Examples thereof include genes and gene groups (eg, two-component regulatory system genes) that have a large number of duplicates, but are not limited thereto. The region to be mutated may be a region existing in the wild-type DNA of the host microorganism, or a region artificially introduced into the host before applying the method of the present invention. Examples of the artificially introduced region include a region containing a selectable marker gene and a reporter gene. Preferably, the mutation target region is selected so as not to include a gene essential for the growth of the host microorganism (hereinafter referred to as “growth essential gene”). Here, the essential growth gene is a gene encoding a protein having a function that cannot be complemented by a method such as external addition to the medium, and is a gene that is lethal when deleted from the host genome. In other words, even if the lethal gene is known to be non-viable if the host is deleted from the host genome, that is, even if it is a lethal gene, the growth of the host can be increased by adding a complementary substance (for example, a metabolism-related substance) to the medium. As long as it can be maintained, it is not included in the “growth essential gene” in the present specification.

The size of the mutation target region used in the method for producing a recombinant microorganism of the present invention is not particularly limited, but is preferably 1 bp or more, more preferably 0.2 kb or more, further preferably 0.5 kb or more, while the mutation The size of the target region is preferably 200 kb or less, more preferably 160 kb or less, even more preferably 120 kb or less, even more preferably 80 kb or less, still more preferably 40 kb or less. When the size of the target region is 200 kb or less, there is a high probability that the growth essential gene is not included. It is known that in Bacillus subtilis, the maximum region not containing a growth-requiring gene is about 200 kb between the dltA gene and the yycH gene (for example, Proc. Natl. Acad. Sci. USA, 2003, 100). , 4678-4683).

As used herein, the “target DNA” refers to DNA to be introduced into a host microorganism. The type of the target DNA is not particularly limited, and various factors such as a DNA encoding a protein, a peptide or a polypeptide, a DNA encoding a non-coding RNA, a promoter DNA, a terminator DNA, a suppressor and an activator are bound. Examples of the DNA include the transcriptional regulatory region. The target DNA may be of a type that is naturally expressed by the host microorganism into which these DNAs are introduced, but is preferably of a type that is not naturally expressed by the host microorganism, and more preferably heterologous. It is DNA. Among the target DNAs, a DNA encoding a protein, peptide or polypeptide, or non-coding RNA is also referred to as “target gene” in the present specification. Examples of the protein, peptide or polypeptide encoded by the target gene include, but are not limited to, contractile protein, transport protein, signal protein, biological defense protein, receptor protein, structural protein, gene regulatory protein, enzyme , And functional proteins such as storage proteins, peptides or polypeptides; and proteins that act as industrially useful enzymes or bioactive factors used in the fields of foods, pharmaceuticals, cosmetics, detergents, fibers, medical tests, etc., Examples include peptides or polypeptides. The industrially useful enzymes include oxidoreductases (oxidoreductases), transferases (transferases), hydrolases (hydrolases), leaving enzymes (lyases), isomerases (isomerases), synthetic enzymes (ligases/synthases). ) And the like, and preferable examples thereof include glycoside hydrolases such as cellulase, hydrolases such as α-amylase and protease, and transferases such as chloramphenicol acetyltransferase (CAT), but are not limited thereto. Not done.

In the present specification, the term “operably linked” to a gene and a control region means that the gene and a control region (eg, promoter) involved in transcription, translation or secretion of the gene depend on the control region. It is linked so that the gene can be expressed under control. Procedures for "operable linkage" between genes and control regions are well known to those of skill in the art.

In the present specification, “replacement” of a DNA region means replacement of a part or all of the DNA region with another DNA. For example, "replacement" of a DNA region in the present specification means to replace the entire DNA region with another DNA, to leave one end of the DNA region and replace the rest with another DNA, to both ends of the DNA region. To replace with another DNA, and to insert another DNA at any position in the DNA region.

In the present specification, the term “native” used for the function or property or trait of a cell means that the function or property or trait is a wild type of the cell, or a cell before modification treatment (eg, host cell). Is used to represent the presence of

In the present specification, the term “foreign” used for the functions, properties, and traits of cells is used to represent functions, properties, and traits that are not originally present in cells but are introduced from the outside. It For example, a "foreign" gene or polynucleotide is a gene or polynucleotide that has been exogenously introduced into a cell. The foreign gene or polynucleotide may be derived from the same organism as the cell into which it was introduced, or may be derived from a different organism (ie, a heterologous gene or polynucleotide).

In the present specification, the “hydrolase gene” is an enzyme capable of forming a halo on a medium for culturing a host microorganism by hydrolyzing a substrate such as protein, starch or saccharide contained in the medium. Is a gene that encodes. Examples of the hydrolase gene include, but are not limited to, a protease gene, an amylase gene, a cellulase gene, a β-galactosidase gene, a β-glucuronidase gene, and a lipase gene.

In the present specification, "forming a halo" means that the periphery of a colony on the medium changes to transparent or translucent as compared with the original medium, or the color around the colony is different from the color of the original medium. Change to. It also includes the change of the color of colonies on the medium due to the expression of hydrolase.

In the present specification, the “substrate for hydrolase” contained in the medium is a substrate for the enzyme encoded by the above-mentioned hydrolase gene. Examples of the substrate include, but are not limited to, skim milk, peptide-pNA, peptide-MCA, peptide-AFC, etc. if the hydrolase is a protease, and starch if the hydrolase is an amylase, Amylose, amylopectin, pullulan, oligosaccharide-CNP, oligosaccharide-4NP, oligosaccharide-PNP and the like can be mentioned. If the hydrolase is cellulase, cellulose, CMC, etc. can be mentioned, and the hydrolase is β-galactosidase. If it is, ONPG, salmon GAL, X-gal, etc. are mentioned, if the hydrolase is β-glucuronidase, MUG, X-GLUC, etc. are mentioned, and if hydrolase is a lipase, tributyrin, BALB, etc. Is mentioned.

In the present specification, the “selection marker gene” is a gene that brings about a trait used as a means for selecting transformants, and examples thereof include a drug resistance gene, an auxotrophy-related gene, and a lethal gene. .. Examples of drug resistance genes include, but are not limited to, chloramphenicol resistance gene, erythromycin resistance gene, neomycin resistance gene, spectinomycin resistance gene, kanamycin resistance gene, tetracycline resistance gene, blasticidin S resistance gene, An ampicillin resistance gene etc. are mentioned. More specific examples of the drug resistance gene, chloramphenicol resistance gene derived from Streptococcus bacteria, erythromycin resistance gene derived from Lactobacillus bacteria, neomycin resistance gene derived from Streptomyces actinomycetes, corynebacterium, respectively. Examples include a spectinomycin resistance gene derived from a bacterium belonging to the genus Urum, a kanamycin resistance gene derived from an Escherichia coli, a tetracycline resistance gene derived from a bacterium belonging to the genus Salmonella, a blasticidin S resistance gene derived from a bacterium belonging to the genus Bacillus, an ampicillin resistance gene derived from Escherichia coli, and the like. Examples of auxotrophy-related genes include, but are not limited to, genes involved in amino acid biosynthetic pathway, nucleic acid biosynthetic pathway, vitamin biosynthetic pathway, and the like. For example, the auxotrophy-related gene may be a gene in which an amino acid biosynthesis-related gene naturally present in the host cell is mutated so as to show the auxotrophy of the amino acid.

In the present specification, a cell “has the ability to express a selection marker” or “expresses a selection marker” means that when the selection marker gene is a drug resistance gene, the cell expresses the drug resistance gene and the drug It means that you have acquired resistance. On the other hand, when the selectable marker gene is an auxotrophy-related gene, the cell "has the ability to express the selectable marker" or "expresses the selectable marker" means that the cell has the ability to biosynthesize nutrients essential for survival. Means to have. For example, when the selectable marker gene is an amino acid-requiring related gene, “having the expression ability of the selection marker” or “expressing the selection marker” means a gene of an amino acid that the cell does not inherently have the biosynthesis ability. The expression means that the biosynthetic ability of the amino acid is acquired.

As used herein, the term “selection marker-containing strain selection medium” refers to a medium for selecting cells that express the gene for the selection marker. For example, when the selection marker gene is a drug resistance gene, the selection marker-bearing strain selection medium is the drug-containing medium. When the selection marker gene is an auxotrophy-related gene, the selection marker-bearing strain selection medium is the nutrient-deficient medium.

(2. Method for producing recombinant microorganism)
In one aspect, the present invention provides a method for producing a recombinant microorganism. In the method for producing a recombinant microorganism of the present invention, target cells are selected with the halo forming ability of the cells as an index.

More specifically, in the method of the present invention, first, a cell in which a mutation target region to be replaced with a target DNA is replaced with an expression cassette containing a hydrolase gene (hereinafter, referred to as hydrolase expression cassette) is prepared. To do. Then, the cells are cultured in a medium containing a substrate for the hydrolase, and cells containing the hydrolase expression cassette are selected using the halo formation on the medium as an index (first selection step). ).

However, in the first selection step, when the cells grow on the medium without limitation, many colonies are generated, and adjacent colonies come into contact with each other or one halo spreads around the adjacent colonies. , It becomes difficult to distinguish the colonies formed with halos. Therefore, in the method of the present invention, it is desirable to apply selective pressure to the medium so that the cells introduced with the hydrolase gene can selectively grow in the medium. For example, in the method of the present invention, a host microorganism is allowed to co-express a first selectable marker in addition to the above hydrolytic enzyme. For example, a first selectable marker gene (M1) is introduced into a host microorganism in addition to the above hydrolase expression cassette. On the other hand, the first selection marker selection medium is used as the medium. The selection medium contains a substrate for the hydrolase. As a result, cells in which gene recombination did not occur are excluded, and colonies generated in the medium are colonies of cells expressing both the hydrolase and the first selectable marker, and only the first selectable marker. Can be any of the colonies of cells expressing. According to the method of the present invention, since excessive colony formation can be prevented, halo-forming colonies can be easily selected from the colonies on the medium. The cells selected in the first selection step that grow on a medium and have a halo-forming ability are cells that contain a hydrolase expression cassette and have a first selection marker-expressing ability (see FIG. 1). ..

Subsequently, the hydrolase expression cassette of the selected cells is replaced with a recombinant cassette containing the DNA of interest, and then the cells are cultured in a medium containing a substrate for the hydrolase. The cells into which the recombinant cassette has been introduced do not form a halo because they have lost the hydrolase expression cassette. Therefore, if cells are selected with the absence of halo formation on the medium as an index, recombinant cells in which the mutation target region is replaced with the target DNA can be obtained (second selection step).

However, even in the second selection step, if the cells grow on the medium without limitation, it becomes difficult to determine the presence or absence of halo formation. Therefore, in the method of the present invention, it is desirable to confer on the cells the ability to express the second selectable marker in parallel with the replacement of the hydrolase expression cassette with the recombinant cassette. For example, the gene (M2) of the second selection marker is introduced into the cells selected in the first selection step, in addition to the above recombination cassette. If this cell is cultured in the second selection marker selection medium containing the substrate for the hydrolase, cells in which gene recombination has not occurred are excluded. Cells that grow on the medium include the recombinant cassette and cells that express the second selectable marker but do not express hydrolase, and cells that do not contain the recombinant cassette and that contain the second selectable marker and hydrolase. Or any of the cells that co-express. Therefore, by selecting a colony that does not form a halo from the colonies formed on the medium, it is possible to obtain the target recombinant cell into which the recombination cassette containing the target DNA has been introduced. According to this method, since excessive colony formation can be prevented, colonies that do not form a halo containing the target cells can be easily selected from the colonies on the medium. The cells selected in the second selection step that grow on the medium but do not form halos are cells that contain the recombination cassette and have the second selection marker expression ability (see FIG. 1 ).

By repeating the same procedure for the recombinant cells obtained by the above procedure, double recombinant cells in which the second mutation target region is replaced with the second target DNA can be obtained. By further repeating the same steps, it is possible to obtain multiple recombinant cells in which the third or further mutation target region is replaced with the third or further target DNA (see FIG. 1).

In the method of the present invention, as the above-mentioned recombination cassette, by using a gene replacement fragment for replacing the mutation target region with the target DNA by homologous recombination, recombination in which the mutation target region is replaced with the target DNA Cells can be produced. Alternatively, in the method of the present invention, by using a gene-deleting fragment that does not contain the target DNA as the above-mentioned recombination cassette, a recombinant cell in which the mutation target region is deleted can be prepared. Therefore, according to the method for producing a recombinant microorganism of the present invention, a recombinant microorganism in which one or more genes are substituted or deleted with respect to the host DNA is produced. Preferably, the method for producing a recombinant microorganism of the present invention produces a recombinant microorganism having multiple mutations.

(2.1. Primary recombination)
In a preferred embodiment, the method for producing a recombinant microorganism of the present invention includes at least the following (1) to (4).
(1) preparing a cell of a host microorganism that co-expresses a first hydrolase expression cassette and a first selectable marker (2) comprising the cell containing a substrate for the first hydrolase Culturing in a selection medium having a first selection marker-carrying strain, and selecting cells that grow and form haloes on the medium as cells containing the first hydrolase expression cassette (3) (2) Giving the second selectable marker expression ability to the cells selected in, and replacing the first hydrolase expression cassette with the first recombination cassette (4) , Culturing in the second selection marker-bearing strain selection medium containing a substrate of the first hydrolase, and growing cells without forming a halo on the medium, the first recombinant cassette Select as cells to contain

The host microorganism used in the method for producing the recombinant microorganism of the present invention is not particularly limited, but Bacillus is preferable, and examples of the Bacillus include Bacillus subtilis, Bacillus cereus, Bacillus coagulans, and Bacillus. clausii, Bacillus circulans, Bacillus licheniformis, Paenibacillus sp. and the like, and mutants thereof. Of these, Bacillus subtilis and its mutants are more preferable as the host microorganism used in the method of the present invention.

The first hydrolase expression cassette contained in the cells of the host microorganism prepared in step (1) of the method of the present invention is a DNA containing the first hydrolase gene. The first hydrolase gene may be a gene encoding an enzyme capable of forming a halo on the medium by hydrolyzing a substrate in the medium in which the host microorganism is cultured. The first hydrolase gene is preferably selected from the group consisting of protease gene, amylase gene, cellulase gene, β-galactosidase gene, β-glucuronidase gene and lipase gene, more preferably protease gene, amylase gene, cellulase. Gene and the β-galactosidase gene. Examples of protease genes include nprE and aprE, examples of amylase genes include amyE, examples of cellulase genes include alkaline cellulase gene, examples of β-galactosidase genes include lacZ, and examples of β-glucuronidase genes include GUS and the like, lipA and the like as an example of the lipase gene, more specific examples, respectively, nprE and aprE gene from Bacillus subtilis, amyE gene from Bacillus subtilis, Bacillus sp. KSM-S237 strain derived from Alkaline cellulase gene (Japanese Unexamined Patent Publication No. 2000-210081), E. coli-derived lacZ gene, Escherichia coli-derived GUS gene, lipase genes lipA and AO60 derived from Aspergillus deficient bacteria (Japanese Unexamined Patent Publication No. 2002-186483, Japanese Unexamined Patent Publication No. 2011-2011 No. 167117) and the like, but the gene is not limited to the above-mentioned genes as long as it is a gene encoding a hydrolase whose presence or absence can be confirmed by halo formation.

In order to make the formation of the halo by the hydrolase clear to the naked eye, the hydrolase gene is preferably operably linked to a promoter and expressed under the control of the promoter. Therefore, the first hydrolase expression cassette preferably used in the present invention is a DNA containing a promoter and a first hydrolase gene operably linked to the promoter.

The first hydrolase expression cassette is replaced with the first mutation target region in the DNA of the host microbial cell. In one embodiment, the first hydrolase gene comprises an exogenous hydrolase gene. In another embodiment, the first hydrolase gene may be a hydrolase gene naturally present in the host microorganism. In this case, the hydrolase gene naturally present in the host microorganism and its control region correspond to the first mutation target region. Alternatively, by replacing the upstream regulatory region of the naturally existing hydrolase gene in the host DNA with an exogenous promoter region, a promoter and a hydrolase gene operably linked to the promoter One hydrolase expression cassette is arranged in the first mutation target region.

In the method of the present invention, the host cell is also capable of expressing the first selectable marker. In a preferred embodiment, the host cell expresses the first selectable marker by introducing a first selectable marker expression cassette. In a preferred embodiment, the first selectable marker expression cassette is DNA containing a promoter and a first selectable marker gene (M1) operably linked to the promoter.

The type of the first selection marker gene (M1) is not particularly limited as long as it can be used as a selection marker gene. In a preferred embodiment, the first selectable marker gene is a drug resistance gene. In another preferred embodiment, the first selectable marker gene is an auxotrophy related gene.

Therefore, the host microorganism cells used in the method of the present invention are cells that co-express the first hydrolase expression cassette and the first selectable marker. Preferably, in the host microbial cell, the first selectable marker expression cassette is introduced into the cell, and the first mutation target region in the DNA is replaced with the first hydrolase expression cassette. Is prepared.

In the step (2) of the method of the present invention, the cells prepared in the step (1) are cultured in the first selection marker-bearing strain selection medium containing the substrate of the first hydrolase. The cells that grow in the medium are cells that have the ability to express a first selectable marker, and those that have the first hydrolase expression cassette and the first hydrolase expression cassette. With no cells included. A cell having the first hydrolase expression cassette expresses the first hydrolase, which decomposes the substrate in the medium to form a halo. Therefore, a halo is formed around the colony of cells having the first hydrolase expression cassette on the medium. On the other hand, cells that do not have the first hydrolase expression cassette do not express the first hydrolase and therefore do not form halos.

Therefore, the target cells containing the first hydrolase expression cassette can be selected using the cell growth and halo formation on the medium as indicators (first selection step). More specifically, cells that grow on the medium and form halos are selected as target cells in which the first mutation target region has been replaced with the first hydrolase expression cassette. The halo-formed cells can be easily visually determined by the relative size of the halo around the colony. For example, among the colonies formed on the medium, a colony whose halo formation can be clearly confirmed visually as shown in FIG. 1, preferably a colony which forms a relatively larger halo, is selected as a target cell. Good. Alternatively, when the first hydrolase is β-galactosidase, β-glucuronidase or lipase, the target cells can be selected using the color development of the medium to which a reagent such as X-gal, X-GLUC or BALB is added as an index. it can.

In the procedure of the first selection step according to the present invention, when the host cell's ability to express the first selection marker is the property acquired by the introduction of the first selection marker expression cassette, the selected cell is It is a cell that has been successfully introduced with both the hydrolase expression cassette 1 and the first selection marker expression cassette. Such cells are considered to be cells in which gene recombination is extremely likely to occur even among cells that have undergone gene transfer treatment. That is, by performing a treatment for introducing both a hydrolase expression cassette and a selection marker expression cassette into cells, and then selecting cells based on cell growth and halo formation according to the above procedure, gene recombination is extremely likely to occur. A suitable competent cell can be selected as a host for the replacement cell. Therefore, the above procedure according to the present invention can be applied to a method for producing competent cells, and the application is provided as one aspect of the present invention.

In the step (3) of the method of the present invention, in the cells selected based on cell growth and halo formation in the step (2) (first selection step), the first hydrolase expression cassette is added, Replace with the first recombination cassette. The first recombination cassette may be DNA containing the first target DNA or DNA not containing the target DNA (fragment for gene deletion). When the recombination cassette contains the DNA of interest, the first mutation target region of the cell is replaced with the DNA containing the first DNA of interest, and when the recombination cassette does not contain the DNA of interest, the first region of the DNA of the cell is The mutation target region of is deleted.

When the first target DNA in the first recombination cassette is the target gene, the target gene is preferably operably linked to the control region. In other words, the first recombination cassette may contain a control region and a first gene of interest operably linked to the control region. Thereby, the target gene can be expressed under the control of the control region in the recombinant microorganism into which the recombinant cassette has been introduced. Examples of the control region include a transcription initiation control region, a translation initiation region and a secretory signal peptide. Examples of the transcription initiation control region include a promoter and a transcription initiation point. The translation initiation region includes a ribosome binding site and a start codon. The secretory signal peptide is a region involved in controlling the secretion of the expression product of the target gene.

Alternatively, by replacing the recombination cassette with the first hydrolase expression cassette so that the first gene of interest is operably linked to the control region naturally existing in the host, It can be expressed under the control of a regulatory region. Alternatively, the recombinant cassette is replaced with the first hydrolase expression cassette so that the first gene of interest is operably linked to the promoter contained in the first hydrolase expression cassette. Thus, the target gene can be expressed under the control of the control region.

In addition, in the step (3), in addition to the replacement of the first recombination cassette, the expression of a second selection marker is added to the cells selected in the step (2) (first selection step). Noh is given. The second selectable marker is a selectable marker different from the first selectable marker. In a preferred embodiment, the cell is rendered capable of expressing a second selectable marker by introducing a second selectable marker expression cassette into the cell. In step (3), the order of introducing the first recombination cassette and the second selection marker expression cassette into the cells is not particularly limited as long as they are co-expressed in the cells.

In a preferred embodiment, the second selectable marker expression cassette is DNA containing a promoter and a second selectable marker gene (M2) operably linked to the promoter. The type of the second selectable marker gene (M2) is not particularly limited as long as it can be used as a selectable marker gene, and examples thereof include the drug resistance gene and nutrition described above for the first selectable marker gene (M1). Requirement related genes are mentioned. However, the first selectable marker gene (M1) and the second selectable marker gene (M2) are different genes.

In a preferred embodiment, step (3) of the above-mentioned method of the present invention further comprises deleting the expression ability of the first selectable marker of the cells obtained in step (2). Preferably, by replacing the first selectable marker expression cassette in the cell with the second selectable marker expression cassette, the expression ability of the first selectable marker is deleted and the cell is subjected to the second selectable marker expression. It gives the expression ability of the marker. Deletion of the first selectable marker can prevent accumulation of the selectable marker gene in recombinant cells.

In a more preferred embodiment, the first selectable marker expression cassette comprises a promoter, a first selectable marker gene (M1(r)), and an inactivated second selectable marker gene (M2(s)). The second selection marker expression cassette, which comprises DNA, comprises a promoter, an inactivated first selection marker gene (M1(s)), and a second selection marker gene (M2(r)). It is DNA containing. In the cassette, the first selectable marker gene (M1(r)) and the second selectable marker gene (M2(r)) are each operably linked to the promoter in the cassette.

Examples of the inactivated first and second selection marker genes M1(s) and M2(s) include a marker gene having a modification that does not express a marker and a marker gene having a modification that loses a marker function. .. Modifications that do not express a marker include modification of the Shine-Dalgarno (SD) sequence, for example, deletion of the SD sequence and replacement of the SD sequence with an anti-SD sequence. Marker genes having a modification that loses the marker function include those in which a given drug resistance gene is point-mutated to lose drug resistance.

The order of arrangement of the first selection marker gene and the second selection marker gene in the first and second selection marker expression cassettes is not particularly limited, and either may be upstream. However, the order of arrangement of the two genes should be the same in the first selection marker expression cassette and the second selection marker expression cassette. For example, the first selectable marker expression cassette is DNA containing a promoter, M1(r) and M2(s) in that order, and the second selectable marker expression cassette is the promoter, M1(s) and M2( DNA containing r) in this order. Also, for example, the first selection marker expression cassette is DNA containing a promoter, M2(s) and M1(r) in this order, and the second selection marker expression cassette is the promoter, M2(r) and M1(r). A DNA containing (s) in this order. By configuring the first and second selection marker expression cassettes as described above, substitution between the first and second selection marker expression cassettes easily occurs, so that Replacement with a second selectable marker expression cassette is facilitated.

Then, in the step (4) of the method of the present invention, the cells in which the recombination cassette and the second selection marker have been introduced are treated with a second selection marker-bearing strain selection medium containing a substrate for the first hydrolase. Incubate with. The cells that grow in the medium are cells that have the ability to express a second selectable marker, including cells that have lost the first hydrolase expression cassette due to substitution with the first recombination cassette, Cells in which the substitution does not occur and which retains the first hydrolase expression cassette are included. Cells carrying the first recombinant cassette instead of the first hydrolase expression cassette do not express the first hydrolase and therefore do not form halos on the medium.

Therefore, it is possible to select the target cells containing the first recombination cassette with the indicators that the cells grow on the above medium and that halo formation does not occur (second selection step). More specifically, cells that grow on the medium but do not form halos are selected as cells of interest in which the first hydrolase expression cassette has been replaced by the first recombination cassette. For example, among the colonies formed on the medium, those for which clear halo formation cannot be visually confirmed may be selected as the target cells. Alternatively, when the hydrolase is β-galactosidase, β-glucuronidase or lipase, it is possible to select the target cells with the index that there is no color development in the medium to which reagents such as X-gal, X-GLUC and BALB are added. it can. Since the first hydrolase expression cassette has been arranged in the first mutation target region, the selected cells eventually have the first mutation target region in the first recombination cassette. It is a recombinant cell replaced with.

By the above procedure, a target recombinant microorganism in which the first mutation target region is replaced with the target DNA or the first mutation target region is deleted can be produced.

(2.2. Further recombination)
In one embodiment of the method for producing a recombinant microorganism of the present invention, a further recombination operation may be carried out after the above-mentioned primary recombination operation. In the further recombination, basically, the same procedure as the primary recombination is repeated for the cells obtained by the above primary recombination. More specifically, a new mutation target region in the DNA of the cells obtained by the primary recombination is replaced with a hydrolase expression cassette, cells are selected with cell growth and halo formation as indicators, and then the The hydrolase expression cassette is replaced with a recombination cassette, and the target recombinant cell is selected with an indication that halo formation is absent. By repeating the further recombination one or more times, a recombinant microorganism having multiple mutations is produced.

In a preferred embodiment, in the method for producing a recombinant microorganism of the present invention, following (1) to (4) above, the above (1′) to (4′) are repeated once or more.
(1′) Substituting an additional mutation target region in the DNA of the cell selected in (4) above with an additional hydrolase expression cassette, and imparting to the cell the ability to express an additional selectable marker;
(2′) The cells are cultured in a selection medium having the additional selection marker-containing strain containing a substrate of the additional hydrolase, and the cells that grow and form halo on the medium are added to the additional hydrolase expression cassette. Selecting as cells containing
(3′) imparting to the cells selected in (2′) the ability to express a selectable marker different from the additional selectable marker, and replacing the additional hydrolase expression cassette with an additional recombinant cassette And (4′) culturing the cell prepared in (3′) in the selective medium having the other selective marker-containing strain containing a substrate for the additional hydrolase, without forming a halo on the medium. Selecting proliferating cells as cells containing said further recombination cassette

In the step (1′), the additional mutation target region to be replaced with the additional hydrolysis expression cassette may be the same or overlapping region as the first mutation target region, but preferably the first mutation target region. It is a region different from the mutation target region. The additional hydrolase expression cassette is DNA containing an additional hydrolase gene. Preferably, the additional hydrolase expression cassette is DNA containing a promoter and an additional hydrolase gene operably linked to the promoter.

In one embodiment, the additional hydrolase gene is an exogenous hydrolase gene. In another embodiment, the additional hydrolase gene may be a hydrolase gene naturally present in the host microorganism. In this case, the hydrolase gene naturally present in the host microorganism and its control region correspond to the additional mutation target region. Alternatively, further hydrolysis comprising a promoter and a hydrolase gene operably linked to the promoter by replacing the control region upstream of the naturally occurring hydrolase gene in the host DNA with a promoter region. An enzyme expression cassette is placed in the additional region to be mutated.

Examples of the further hydrolase gene include those exemplified above for the first hydrolase gene. The first and further hydrolase genes may be the same gene or different genes, but are preferably the same gene. If the first and further hydrolase genes are the same gene, the same substrate can be used for the medium used in the primary recombination operation and the further recombination operation. Preferably, the first hydrolase expression cassette is used as the further hydrolase expression cassette.

In addition, in the step (1′), in addition to the replacement of the mutation target region with the hydrolase expression cassette, the cell obtained in (4) is provided with the expression ability of a further selectable marker. .. The further selectable marker is a selectable marker different from the second selectable marker, preferably the first selectable marker. Alternatively, the additional selectable marker can be a selectable marker that is different from both the first and second selectable markers described above. In a preferred embodiment, the cell is rendered capable of expressing the additional selectable marker by introducing into the cell an additional selectable marker expression cassette. In the step (1'), the order of introducing the hydrolase expression cassette and the selection marker expression cassette into the cells is not particularly limited as long as they are co-expressed in the cells.

In a preferred embodiment, the additional selectable marker expression cassette is DNA containing a promoter and an additional selectable marker gene (M3) operably linked to the promoter. The type of the further selectable marker gene (M3) is not particularly limited as long as it can be used as a selectable marker gene, and examples thereof include the drug resistances exemplified for the first and second selectable marker genes (M1 and M2) above. Genes and auxotrophy related genes are included. However, the further selectable marker gene (M3) is a gene different from the second selectable marker gene (M2), while it is different from the first selectable marker gene (M1) even if it is the same gene. It may be a gene. Preferably, said additional selectable marker gene (M3) is the same gene as said first selectable marker gene (M1).

In a preferred embodiment said further selectable marker expression cassette is replaced with said first or second selectable marker expression cassette. In another preferred embodiment, the additional selectable marker expression cassette is replaced with the first and second selectable marker expression cassettes. In yet another embodiment, the additional selectable marker expression cassette is located in a region of the host DNA different from the first or second selectable marker expression cassette.

In the above step (2′), the cells obtained in the step (1′) are cultured in an additional selective marker-bearing strain selection medium containing a substrate for an additional hydrolase according to the same procedure as in the above (2). , Target cells containing a further hydrolase expression cassette are selected using cell growth and halo formation on the medium as indicators. More specifically, cells that have grown on the medium and formed halos are selected as cells of interest in which additional regions for mutation have been replaced with additional hydrolase expression cassettes. Alternatively, when the hydrolase is β-galactosidase, β-glucuronidase or lipase, the desired cells can be selected using the color development of the medium containing a reagent such as X-gal, X-GLUC, BALB as an index.

In the step (3'), the additional hydrolase expression cassette is replaced with the additional recombinant cassette in the cells selected in the step (2') according to the same procedure as in the above (3). The additional recombination cassette may be DNA containing the target DNA or DNA not containing the target DNA (fragment for gene deletion). The target DNA in the additional recombination cassette may be the same as or different from the first target DNA. When the target DNA is a target gene, the target gene may be operably linked to a control region. Alternatively, the hydrolase expression cassette may be replaced with the additional recombination cassette so that the gene of interest is operably linked to a regulatory region naturally present in the host. Alternatively, the hydrolase expression cassette may be replaced with the additional recombinant cassette such that the gene of interest is operably linked to the promoter contained in the additional hydrolase expression cassette. The type of the control area is as described above.

In addition, in the step (3'), the cell selected in the step (2') is provided with the expression ability of another selectable marker in addition to the substitution of the additional recombination cassette. The alternative selectable marker is a selectable marker different from the further selectable marker, preferably the second selectable marker. In a preferred embodiment, the cell is rendered capable of expressing the other selectable marker by introducing another selectable marker expression cassette into the cell. In step (3), the order of introducing the above-mentioned additional recombination cassette and the above-mentioned another selectable marker expression cassette into cells is not particularly limited, as long as they are co-expressed in the cells.

The alternative selectable marker expression cassette is DNA containing a promoter and the alternative selectable marker gene (M4) operably linked to the promoter. The type of the other selectable marker gene (M4) is not particularly limited as long as it can be used as a selectable marker gene, and examples thereof include the drugs exemplified for the first and second selectable marker genes (M1 and M2) above. A resistance gene and an auxotrophy related gene are mentioned. However, the other selectable marker gene (M4) is a gene different from the further selectable marker gene (M3). Preferably, the other selectable marker gene (M4) is the same gene as the second selectable marker gene (M2).

In a preferred embodiment, in the step (3'), the expression ability of the additional selectable marker of the cells obtained in the step (2') is deleted. Preferably, by replacing the additional selectable marker expression cassette in the DNA of the cell with the alternative selectable marker expression cassette, the ability to express the additional selectable marker is deleted, and the expression ability of the different selectable marker is expressed in the cell. Is given. This can prevent the accumulation of the selectable marker gene in the recombinant cells.

In a preferred embodiment, the additional selectable marker expression cassette is a DNA comprising a promoter, a first selectable marker gene (M1(r)), and an inactivated second selectable marker gene (M2(s)). And the another selectable marker expression cassette is a DNA containing a promoter, an inactivated first selectable marker gene (M1(s)), and a second selectable marker gene (M2(r)). .. In a more preferred embodiment, said further selectable marker expression cassette is the first selectable marker expression cassette comprising the promoters described in (2.1) above, M1(r) and M2(s), and said alternative selectable marker expression cassette. The marker expression cassette is the second selection marker expression cassette containing the promoter, M1(s) and M2(r) described in (2.1) above. Therefore, in a more preferred embodiment of the method of the present invention, an active first selectable marker gene and an inactive second selectable marker gene are combined on the cell DNA through a primary recombination operation and a further recombination operation. A region containing a pair and a region containing a pair of an inactive first selectable marker gene and an active second selectable marker gene are constructed interchangeably to form a first selectable marker gene and a second selectable marker gene. The marker gene is repeatedly and interchangeably expressed, and cells are selected based on the expression (see FIG. 2).

Then, in the step (4′), the cells obtained in the step (3′) are treated with another selective marker-containing strain selection medium containing a substrate for further hydrolase according to the same procedure as in the above (4). The target cells containing the additional recombination cassette are selected by culturing the cells in the above manner and using the fact that cell growth and halo formation do not occur on the medium as an index. More specifically, cells that grow on the medium but do not form halos are selected as cells of interest in which an additional recombination cassette has replaced the additional hydrolase expression cassette. Alternatively, in the case where the hydrolase is β-galactosidase, β-glucuronidase or lipase, the target cells may be selected with the index that there is no color development of the medium to which reagents such as X-gal, X-GLUC and BALB are added. it can. The selected cells are double recombinant cells in which the first mutation target region has been replaced by the first recombination cassette and the further mutation target region has been replaced by the further recombination cassette.

Furthermore, by repeating the above (1′) to (4′) again for the cells obtained in the above step (4′), a plurality of mutation target regions on the DNA are respectively replaced with arbitrary target DNAs. Recombinant microorganisms that have or are deleted can be produced. In the method for producing a recombinant microorganism of the present invention, the number of times of the above-mentioned further recombination operation is not particularly limited and may be once or more, or may not be performed. Therefore, according to the method for producing a recombinant microorganism of the present invention, a recombinant microorganism in which one gene is replaced or deleted, or a recombinant microorganism having multiple mutations in which two or more genes are replaced or deleted Can be manufactured.

(2.3. Deletion of selection marker gene)
In a preferred embodiment, the method for producing a recombinant microorganism of the present invention further includes deleting the selectable marker gene used for cell selection from the recombinant microorganism obtained by the above-mentioned primary or further recombinant operation. In this embodiment, in the primary recombination operation, the first selectable marker expression cassette is replaced with a region in host microbial DNA that is involved in expression of the host trait. Using the trait and the expression of the first selectable marker as an index, cells in which the selectable marker has been introduced at a desired position can be selected. That is, a cell that does not express the trait and expresses the first selectable marker is a cell in which the first selectable marker expression cassette is replaced with a region involved in the trait expression. In this embodiment, the selectable marker expression cassette in the cell is replaced with the selectable marker expression cassette introduced later and deleted. Therefore, the selection marker expression cassette remaining in the finally obtained recombinant cells is the second selection marker expression cassette in step (4) and another selection marker expression cassette in step (4′). .. Then, the selectable marker expression cassette remaining in the recombinant cell is replaced with a region involved in expression which was first replaced with the first selectable marker expression cassette, and the selectable marker is expressed using the trait and the expression of the selectable marker as an index. It is possible to select cells that have been deleted. The cell in which the trait is expressed is a recombinant microorganism in which the selectable marker gene used for cell selection does not remain.

Examples of the region involved in the expression of a phenotype include a region of a gene which is not an essential growth gene for a host cell and whose deletion can be easily confirmed by ordinary measuring means. For example, the region involved in expression of the trait is a selectable marker gene naturally possessed by the host, for example, a drug resistance gene or an auxotrophy-related gene (amino acid biosynthetic pathway-related gene, etc.) (provided that the above-mentioned first selection Region (different from the marker gene).

Therefore, in a preferred embodiment of the method of the present invention, prior to step (1), the region involved in the phenotypic expression of the host cell is replaced with the first selectable marker expression cassette, and the phenotypic expression is absent, and Cells expressing the first selectable marker are selected. Next, the first mutation target region of the cell is replaced with the first hydrolase expression cassette to prepare a host cell (step (1)). Using the prepared cells, the above steps (2) to (4) or further steps (1') to (4') are performed. Preferably, these steps use the first selectable marker expression cassette and the second selectable marker expression cassette interchangeably. Then, the selectable marker expression cassette remaining in the obtained recombinant cells is replaced with a region involved in the expression of the trait, and then cells expressing the trait are selected (see FIG. 3). As a result, it is possible to obtain a recombinant microorganism having a desired gene modification and having no residual selectable marker gene.

(2-4. Gene modification procedure and promoter)
In the method for producing a recombinant microorganism of the present invention, preparation of a DNA fragment and replacement of a DNA region can be performed according to a method known in the art. For example, various hydrolase expression cassettes, selectable marker expression cassettes, and recombinant cassettes should be constructed according to a method such as SOE (splicing by overlap extension)-PCR method (eg, Gene, 1989, 77:61-68). You can More specifically, by preparing DNA fragments such as genes and promoters required for constructing each cassette using a conventional PCR method, etc., ligating each DNA fragment in the desired order using the SOE-PCR method. , Each cassette can be constructed. The replacement of each cassette with the intracellular DNA region can be performed by a commonly used method such as a homologous recombination method. To introduce the cassette into cells, use a general transformation method such as electroporation method, transformation method, transfection method, conjugation method, protoplast method, particle gun method, Agrobacterium method, etc. You can For example, from the upstream, construct a DNA fragment in which the 5′ side region of the host mutation target region, the promoter, the hydrolase gene, and the 3′ side region of the mutation target region are ligated in this order by the SOE-PCR method, If this is introduced into a host cell as a hydrolase expression cassette, it will be replaced with the mutation target region of the host DNA by homologous recombination. The same applies to the selection marker expression cassette and the recombination cassette.

In each recombination step of the method of the present invention, the selectable marker gene is a region (replacement with the hydrolase gene such that transformation with the hydrolase gene and transformation with the selectable marker gene occur independently. That is, it is preferable to replace with a DNA region which is not adjacent or linked to the mutation target region).

Examples of promoters that can be included in the first and further hydrolase expression cassettes, the first and second selectable marker expression cassettes, the further selectable marker expression cassette, and the other selectable marker expression cassette used in the method of the present invention include: A high expression promoter that functions in the host microorganism is preferable. Examples of high expression promoters include promoters that are expressed in the logarithmic growth phase of host microorganisms, and preferred examples include rRNA operon promoters such as PrrnO, PrrnA, PrrnB, PrrnD, PrrnE, PrrnI, PrrnJ. More preferred examples include PrrnO, PrrnA, PrrnB, PrrnD, PrrnE, PrrnI, PrrnJ and the like derived from Bacillus bacterium. The promoters that can be included in the hydrolase expression cassette and the selectable marker expression cassette may be the same promoter or different promoters.

(3. Method of producing target substance)
In a further aspect, the present invention provides a method for producing a target substance, which comprises a step of culturing the above-mentioned recombinant microorganism of the present invention. In the method for producing a target substance of the present invention, the recombinant microorganism of the present invention into which a gene encoding the target substance or a gene that promotes biosynthesis of the target substance is incorporated, is an assimilable carbon source, a nitrogen source, It is inoculated into a medium containing other essential components and cultured by a usual culture method. The target gene is expressed in the cultured cells to produce the target substance. Then, the target substance may be recovered or purified from the culture.

Regarding the composition and culture conditions of the medium used for culturing the recombinant microorganism of the present invention, and the procedures such as recovery and purification of the target substance, according to the species of the recombinant microorganism to be used, the type of the target substance, etc. Can be appropriately selected.

As the target substance produced by the method, a substance encoded by the target gene contained in the above-mentioned recombinant microorganism of the present invention, or a substance whose biosynthesis is promoted by the expression of the target gene (for example, the target gene The substance is not particularly limited as long as it is a substance biosynthesized by the enzyme encoded by. The target gene may be a gene naturally possessed by the host of the recombinant microorganism or a heterologous gene.

The following materials, manufacturing methods, uses, or methods are further disclosed herein as exemplary embodiments of the present invention. However, the present invention is not limited to these embodiments.

[1] A method for producing a recombinant microorganism,
(1) preparing cells of a host microorganism that co-expresses a first hydrolase expression cassette and a first selectable marker,
here,
The first hydrolase expression cassette is a DNA containing a promoter and a first hydrolase gene operably linked to the promoter, and the first hydrolase expression cassette is Replaced with the first region of interest in the DNA of the cell;
(2) The cells selected in (2) above are cultured in the selection medium containing the first selection marker-containing strain containing the substrate for the first hydrolase, and the cells proliferating and forming halos on the medium. Is selected as a cell containing said first hydrolase expression cassette;
(3) giving the cell the ability to express a second selectable marker and replacing the first hydrolase expression cassette with a first recombination cassette; and
(4) The cells prepared in (3) are cultured in the second selection marker-bearing strain selection medium containing the substrate for the first hydrolase, and the cells are grown on the medium without forming halos. Selecting cells as the cells containing the first recombination cassette,
Including the method.

[2] Preferably, the cell expressing the first selection marker is a cell into which the first selection marker expression cassette has been introduced, and the first selection marker expression cassette operates with a promoter and the promoter. The method according to [1], which is a DNA containing a first selectable marker gene operably linked thereto.

[3] Preferably, imparting the expression ability of the second selection marker to the cell is introduction of the second selection marker expression cassette into the cell, and the second selection marker expression cassette is a promoter. And a second selectable marker gene operably linked to the promoter, the method according to [1] or [2].

[4] The method according to any one of [1] to [3], which preferably further comprises deleting the expression ability of the first selectable marker in the cells in the above (3).

[5] The method according to [4], wherein the lack of expression ability of the first selection marker is replacement of the first selection marker expression cassette with the second selection marker expression cassette.

[6] Preferably, the first selectable marker expression cassette is a DNA containing a promoter, the first selectable marker gene, and the inactivated second selectable marker gene, and the second selectable marker gene. The selection marker expression cassette is a DNA containing a promoter, the inactivated first selection marker gene, and the second selection marker gene.
[5] The method described.

[7] Preferably, the inactivated first selectable marker gene and the inactivated second selectable marker gene have a modification in which the marker is not expressed or the marker function is lost. The method according to [6], which is a gene.

[8] Preferably, the promoter contained in the first hydrolase expression cassette, the promoter contained in the first selection marker expression cassette, and the promoter contained in the second selection marker expression cassette are the same or The method according to any one of [1] to [7], wherein the promoters are different and are expressed in the logarithmic growth phase of the host microorganism.

[9] The first hydrolase gene is
Preferably, selected from the group consisting of protease gene, amylase gene, cellulase gene, β-galactosidase gene, β-glucuronidase gene and lipase gene,
More preferably, selected from the group consisting of a protease gene, an amylase gene, a cellulase gene and a β-galactosidase gene,
The method according to any one of [1] to [8].

[10] The first recombination cassette is
Preferably, it is a DNA containing at least one selected from the group consisting of a target gene, a promoter, a terminator and a transcriptional regulatory region,
More preferably, it is a DNA containing the gene of interest operably linked to a control region,
And the target gene is a DNA encoding a protein, a peptide, a polypeptide, or a non-coding RNA,
The method according to any one of [1] to [9].

[11] The method according to any one of [1] to [9], wherein the first recombination cassette is preferably a fragment for gene deletion.

[12] The method according to any one of [1] to [11], wherein the host microorganism is preferably a bacterium of the genus Bacillus.

[13] The method according to [12], wherein the Bacillus bacterium is preferably Bacillus subtilis or a mutant strain thereof.

[14] Preferably, the method according to any one of [1] to [13], wherein the following (1′) to (4′) are further repeated once or more:
(1′) Substituting the additional mutation target region in the DNA of the cell selected in the above (4) or the following (4′) with an additional hydrolase expression cassette, and allowing the cell to express an additional selectable marker. To give,
Wherein the additional hydrolase expression cassette is a DNA containing a promoter and an additional hydrolase gene operably linked to the promoter;
(2′) The cells are cultured in a selection medium having the additional selection marker-containing strain containing a substrate of the additional hydrolase, and the cells that grow and form halo on the medium are added to the additional hydrolase expression cassette. Selecting as cells containing
(3′) imparting to the cells selected in (2′) the ability to express a selectable marker different from the additional selectable marker, and replacing the additional hydrolase expression cassette with an additional recombinant cassette And (4′) culturing the cell prepared in (3′) in the selective medium having the other selective marker-containing strain containing a substrate for the additional hydrolase, without forming a halo on the medium. Selecting proliferating cells as cells containing said additional recombination cassette.

[15] Preferably, imparting the expression ability of the further selection marker to the cell is introduction of the further selection marker expression cassette into the cell, and the further selection marker expression cassette operates with a promoter and the promoter. The method according to [14], which is a DNA containing an additional selectable marker gene that is ligated.

[16] Preferably, imparting the expression ability of the other selectable marker to the cell is introduction of another selectable marker expression cassette into the cell, and the other selectable marker expression cassette comprises a promoter, The method according to [14] or [15], which is a DNA containing another selectable marker gene operably linked to a promoter.

[17] The method according to any one of [14] to [16], which preferably further comprises, in the above (3′), further deleting the expression ability of the additional selectable marker in the cells.

[18] The method according to [17], wherein the deletion of the expression ability of the additional selectable marker is preferably replacement of the additional selectable marker expression cassette by the another selectable marker expression cassette.

[19] The method according to [18], wherein the further selection marker expression cassette is preferably the first selection marker expression cassette, and the another selection marker expression cassette is the second selection marker expression cassette.

[20] Preferably, the further selection marker expression cassette is a DNA containing a promoter, the first selection marker gene, and the inactivated second selection marker gene, and the another selection marker expression The cassette is a DNA containing a promoter, the inactivated first selection marker gene, and the second selection marker gene,
More preferably, the further selection marker expression cassette is the first selection marker expression cassette described in [6] above, and the another selection marker expression cassette is the second selection marker expression described in [6] above. Is a cassette,
[19] The method described.

[21] Preferably, the promoter contained in the additional hydrolase expression cassette, the promoter contained in the additional selectable marker expression cassette, and the promoter contained in the another selectable marker expression cassette are the same or different from each other in the host. The method according to any one of [14] to [20], which is a promoter that is expressed in the logarithmic growth phase of a microorganism.

[22] The above-mentioned further hydrolase gene is
Preferably, selected from the group consisting of protease gene, amylase gene, cellulase gene, β-galactosidase gene, β-glucuronidase gene and lipase gene,
More preferably, selected from the group consisting of a protease gene, an amylase gene, a cellulase gene and a β-galactosidase gene,
The method according to any one of [14] to [21].

[23] The method according to [22], wherein the first hydrolase gene and the further hydrolase gene are preferably the same gene.

[24] The above additional recombination cassette is
Preferably, a DNA containing a target gene, a promoter, a terminator, or a transcriptional regulatory region,
More preferably, it is a DNA containing the gene of interest operably linked to a control region,
And the target gene is a DNA encoding a protein, a peptide, a polypeptide, or a non-coding RNA,
The method according to any one of [14] to [23].

[25] The method according to any one of [14] to [23], wherein the additional recombination cassette is preferably a fragment for gene deletion.

[26] The method according to any one of [1] to [13], which preferably further comprises deleting the selectable marker expression cassette contained in the cells selected in (4) above.

[27] The method according to any one of [14] to [25], which further comprises deleting the selectable marker expression cassette contained in the cells selected in (4′) above.

[28] The method described in [26] above, preferably
The first selectable marker expression cassette is replaced with a region involved in trait expression in the DNA of the host microorganism, and deletion of the selectable marker expression cassette is included in the cells selected in (4) above. Replacing the selectable marker expression cassette with a region involved in expression of the trait,
Method.

[29] The method described in [27] above, preferably
The first selectable marker expression cassette is replaced with a region involved in phenotypic expression in the DNA of the host microorganism, and the deletion of the selectable marker expression cassette is included in the cells selected in (4′) above. The selectable marker expression cassette is replaced with a region involved in the expression of the trait,
Method.

[30] The method according to [28] or [29], wherein the region involved in expression of the trait is an auxotrophic gene region.

[31] Preferably, the genes of the first selectable marker and the second selectable marker are different genes, and each is a drug resistance gene or an auxotrophy related gene, [1] to [30] The method according to any one of 1.

[32] A method for producing a host cell of a recombinant microorganism, which comprises:
Introducing a DNA containing a hydrolase gene operably linked to a promoter into a microbial cell, or introducing a promoter to operably link the hydrolase gene in the cell, and a selectable marker gene And culturing the cells in the selection marker-bearing strain selection medium containing a substrate for the hydrolase, and selecting cells that have grown and formed halos on the medium.
Including the method.

[33] The above hydrolase gene
Preferably, selected from the group consisting of protease gene, amylase gene, cellulase gene, β-galactosidase gene, β-glucuronidase gene and lipase gene,
More preferably, selected from the group consisting of a protease gene, an amylase gene, a cellulase gene and a β-galactosidase gene,
[32] The method described.

[34] The method according to [32] or [33], wherein the selectable marker gene is preferably a drug resistance gene or an auxotrophy-related gene.

[35] The method according to any one of [32] to [34], wherein the microorganism is preferably a Bacillus bacterium, more preferably Bacillus subtilis or a mutant strain thereof.

Hereinafter, the present invention will be described more specifically with reference to examples.

The primers used in this example are shown in Table 1 below.

Reference Example 1 Construction of RIK1141 (trpB'A'::PrrnOcatpt1 erm190 hisC101) Bacillus subtilis strain using DNA extracted from Bacillus subtilis strain RIK1140 (trpB'A'::PrrnOcatpt1 erm hisC101) (JP 2013-9604A) RIK211 (trpB'A'::PrrnOkan erm190(oc) hisC101, Nanamiya et al. (2010) Microbiology 156:2977-2982) was transformed to give RIK1141 transformant showing chloramphenicol resistance and erythromycin sensitivity. It was made. hisC101 represents the Q318amber mutation in the hisC gene, which mutation shows histidine auxotrophy. Also, hisC101 is linked to the trpC2 mutation (tryptophan auxotrophy). Therefore, RIK1141 is a strain exhibiting tryptophan auxotrophy and histidine auxotrophy.

Reference Example 2 Construction of PCR DNAI fragment (trpB'A'::PrrnO-catΔSD erm190)
Using the DNA extracted from the RIK1140 strain (trpB'A'::PrrnOcatpt1 erm hisC101) as a template, PCR was performed using carΔSD1(ggagg)F primer and Em.r primer designed to delete the SD sequence of the cat gene. Then, a DNA fragment was prepared. The obtained DNA fragment was transformed into RIK1141 (Reference Example 1) to obtain a transformant RIK1143 (trpB'A'::PrrnOcatpt1ΔSD erm hisC101) which was chloramphenicol sensitive and erythromycin resistant.
Next, using the DNA of RIK202 (trpB'A'erm190) (Nanamiya et al. (2010) Microbiology 156:2944-2952) as a template, a DNA fragment containing the region from the erythromycin resistance gene to the histidine gene was empt1.f. It was prepared by PCR using the primer and the hisC-R2 primer. The DNA fragment was transformed into RIK1143 to obtain a transformant RIK1136 (trpB'A'::PrrnO-catΔSD erm190) showing erythromycin sensitivity and histidine non-requirement.
A DNA fragment PCR DNAI (trpB'A'::PrrnO-catΔSD erm190) was prepared by PCR using the DNA extracted from RIK1136 as a template and using the trpD-F primer and the hisC-R2 primer.

Reference Example 3 Preparation of competent cells CI medium (0.20% by mass ammonium sulfate, 1.40% by mass dipotassium hydrogen phosphate, 0. 0% by mass) containing tryptophan (20 μg/mL) or histidine (50 μg/mL) depending on the strain. 60% by mass potassium dihydrogen phosphate, 0.10% by mass trisodium citrate dihydrate, 0.50% by mass glucose, 0.03% by mass Amicase (manufactured by Sigma-Aldrich), 5 mM magnesium chloride) The strain was cultivated with shaking at 37° C. until the growth degree (OD600) reached about 1. After shaking culture, part of the culture solution (0.5 mL) was collected, and only the bacterial cells were collected by centrifugation, and then 1 mL of CII medium (0.20% by mass ammonium sulfate, 1.40% by mass hydrogen phosphate) was collected. Dipotassium, 0.60% by mass potassium dihydrogen phosphate, 0.10% by mass trisodium citrate dihydrate, 0.50% by mass glucose, 0.015% by mass Amicase (manufactured by Sigma-Aldrich), 5 mM By resuspending the bacterial cells in magnesium chloride, 5 μg/mL tryptophan), a competent cell suspension of Bacillus subtilis strain was prepared.

Reference Example 4 Transformation of Bacillus subtilis The obtained DNA fragment was added to 100 μL of the prepared competent cell suspension and cultured by shaking at 37° C. for 90 minutes, and then 100 μL of LB liquid medium (1 mass% tryptone, 0. 50% by mass yeast extract and 0.50% by mass NaCl) were added, and the mixture was shake-cultured at 37° C. for 1 hour. Then, depending on each transformant, one selected from chloramphenicol (5 μg/mL), erythromycin (0.5 μg/mL), skim milk (1.5%), and corn starch (1.5%). 10 μL or 100 μL of the culture solution was spread on the LB agar medium (1 mass% tryptone, 0.50 mass% yeast extract, 0.50 mass% NaCl, 1.5 mass% agar) containing the above. After stationary culture at 37° C., the grown colonies were separated as transformants.

Reference Example 5 Transformation of Bacillus subtilis Using Minimal Agar Medium The obtained DNA fragment was added to 100 μL of the prepared competent cell suspension, and the mixture was cultured at 37° C. for 90 minutes with shaking. Then, depending on each transformant, a minimal agar medium (0.20 mass% ammonium sulfate, 1.40 mass% dipotassium hydrogen phosphate, 0.60) containing tryptophan (20 μg/mL) or histidine (50 μg/mL) was used. Mass% potassium dihydrogen phosphate, 0.10 mass% trisodium citrate dihydrate, 0.03 mass% Amicase (manufactured by Sigma-Aldrich), 0.05 mass% glucose, 1 mM magnesium chloride) 5 μL or 10 μL was smeared. After stationary culture at 37° C., the grown colonies were separated as transformants.

Example 1 Selection of transformants by protease (nprE) halo (1) Construction of RIK7001 (nprE::PrrnO-nprE trpB'A'::PrrnO-catSD10 erm his101)
The promoter of the protease gene nprE of RIK1141 (Reference Example 1) was replaced with the rrnO promoter (PrrnO). Using the RIK1141 DNA as a template, the fragments of the upstream and downstream regions of the nprE gene promoter are used with the P-nprE-upF primer, P-nprE-upR primer, and P-nprE-downF primer and P-nprE-downR primer. It was prepared by PCR. The PrrnO fragment was prepared using RIK1141 as a template and the rrnO-upF primer and the PrrnO-catsd-R primer. A DNA fragment [nprE::PrrnO-nprE] was prepared from the obtained three fragments by PCR using P-nprE-upF primer and P-nprE-downR primer.
Next, the DNA of RIK1215 (ΔrrnHG1ΔrrnW2ΔrrnO1ΔrrnD1ΔrrnB2ΔrrnA1ΔrrnI2 rrnEanti-SD10 (GGAGG)trpB'A'::PrrnOcatpt1SD10 (CCTCC) erm hisC101) (JP 2013-9604 primer) was used as a template, and the DNA of JP 2013-9604F was used as a template, and DNA was used as a template. Then, a DNA fragment [trpB'A'::PrrnO-catSD10 erm his101] was prepared.
After transforming the above-mentioned [nprE::PrrnO-nprE] fragment and [trpB'A'::PrrnO-catSD10 erm his101] fragment into the RIK1141 strain, the cells were cultured on an LB plate containing erythromycin and skim milk to resist erythromycin. Cells (growth (+)) and that formed halos on the medium (halo (+)) were selected. The obtained transformant was obtained as RIK7001.

(2) Construction of RIK7011 (nprE::rrnOantiSD10 trpB'A'::PrrnO-catSD10 erm hisC101)
A downstream region fragment of the nprE gene promoter was prepared by PCR using the RIK7001 DNA prepared in (1) as a template and the P-nprE-down-F0-ver2 primer and the P-nprE-downR2 primer. The rrnO DNA fragment (rrnOantiSD10) having a modified anti-SD (anti-SD10) sequence was used as RIK1226 (ΔrrnHG1ΔrrnW2ΔrrnE1ΔrrnD1ΔrrnB2ΔrrnA1ΔrrnI2 rrnOanti-SD10 (GGAGG)trpB'A'::PrrerOCCTC1) (PRGACCTC1). Was prepared by PCR using the rrnO-upF primer and the rrnO-5R-4 primer as a template. Then, the upstream region fragment of the nprE gene promoter prepared in (1), the downstream region fragment, and the rrnOantiSD10 fragment were ligated by PCR using P-nprE-upF primer and P-nprE-downR2 primer, and [nprE ::rrnOantiSD10] fragment was generated.
Next, in order to introduce the target gene (rrnOantiSD10) into the nprE gene region of RIK7001, the prepared [nprE::rrnOantiSD10] fragment was transformed into RIK7001 strain. Cells were cultured on LB plates containing chloramphenicol and skim milk and cells that were chloramphenicol resistant (growth (+)) and did not form halos on the medium (halo (-)) were selected. Sequencing confirmed that the selected cells contained the rrnOantiSD10 sequence. The obtained transformant was obtained as RIK7011.

Example 2 Selection of transformants by protease (aprE) halo (1) Construction of RIK7002 (aprE::PrrnO-aprE trpB'A'::PrrnO-catSD10 erm his101)
The promoter of the protease gene aprE of RIK1141 (Reference Example 1) was replaced with the rrnO promoter (PrrnO). Fragments of the upstream and downstream regions of the aprE gene promoter, using RIK1141 DNA as a template, P-aprE-upF2 primer, P-aprE-upR primer, and P-aprE-downF primer and P-aprE-downR2 primer It was prepared by PCR. A DNA fragment [aprE::PrrnO-aprE] was prepared from the obtained 2 fragments and the PrrnO fragment prepared in Example 1 (1) by PCR using P-aprE-upF2 primer and P-aprE-downR2. did.
After the above-mentioned [aprE::PrrnO-aprE] fragment and the [trpB'A'::PrrnO-catSD10 erm his101] fragment prepared in Example 1 (1) were transformed into RIK1141 strain, the cells contained erythromycin and skim milk. The cells were selected from erythromycin resistant (proliferation (+)) and halo-forming (halo (+)) cells on the culture medium. The obtained transformant was obtained as RIK7002.

(2) Construction of RIK7012 (aprE::rrnOantiSD10 trpB'A'::PrrnO-catSD10 erm hisC101)
Using the RIK7002 DNA prepared in (1) as a template for the upstream and downstream fragments of the aprE gene promoter, P-aprE-upF primer, P-aprE-upR primer, and P-aprE-down-F0-ver2 primer And P-aprE-downR2 primers were used for PCR. The obtained 2 fragments and the rrnOantiSD10 fragment prepared in Example 1(2) were ligated by PCR using P-aprE-upF primer and P-aprE-downR2 primer, and the [aprE::rrnOantiSD10] fragment was obtained. It was made.
Next, in order to introduce the target gene (rrnOantiSD10) into the aprE gene region of RIK7002, the prepared [aprE::rrnOantiSD10] fragment was transformed into RIK7002 strain. Cells were cultured on LB plates containing chloramphenicol and skim milk and cells that were chloramphenicol resistant (growth (+)) and did not form halos on the medium (halo (-)) were selected. Sequencing confirmed that the selected cells contained the rrnOantiSD10 sequence. The obtained transformant was obtained as RIK7012.

Example 3 Selection of transformants using amylase (amyE) halo (1) Construction of RIK7003 (amyE::PrrnO-amyE trpB'A'::PrrnO-catSD10 erm his101)
The promoter of the amylase gene amyE of RIK1141 (Reference Example 1) was replaced with the rrnO promoter (PrrnO). Using the RIK1141 DNA as a template, the fragments of the upstream and downstream regions of the amyE gene promoter are used with the P-amyE-upF2 primer, the P-amyE-upR primer, and the P-amyE-downF primer and the P-amyE-downR2 primer. It was prepared by PCR. A DNA fragment [amyE::PrrnO-amyE] was prepared from the obtained two fragments and the PrrnO fragment prepared in Example 1 (1) by PCR using P-amyE-upF2 primer and P-amyE-downR2. ..
After the above [amyE::PrrnO-amyE] and the [trpB'A'::PrrnO-catSD10 erm his101] fragment prepared in Example 1(1) were transformed into the RIK1141 strain, the cells contained erythromycin and corn starch. Cells that were erythromycin resistant (growth (+)) and formed halos on the medium (halo (+)) were cultured on LB plates and selected. The obtained transformant was obtained as RIK7003.

(2) Construction of RIK7013 (amyE::rrnOantiSD10 trpB'A'::PrrnO-catSD10 erm hisC101)
Using the RIK7003 DNA prepared in (1) as a template for the upstream and downstream fragments of the amyE gene promoter, the P-amyE-upF primer, the P-amyE-upR primer, and the P-amyE-down-F0-ver2 primer And P-amyE-downR2 primer. The obtained 2 fragments and the rrnOantiSD10 fragment prepared in Example 1 (2) were ligated by PCR using P-amyE-upF primer and P-amyE-downR2 primer, and the [amyE::rrnOantiSD10] fragment was obtained. It was made.
Then, in order to introduce the target gene (rrnOantiSD10) into the amyE gene region of RIK7003, the prepared [amyE::rrnOantiSD10] fragment was transformed into RIK7003 strain. Cells were cultured on LB plates containing chloramphenicol and corn starch and cells were selected that were chloramphenicol resistant (growth (+)) and did not form halos on the medium (halo (-)). Sequencing confirmed that the selected cells contained the rrnOantiSD10 sequence. The obtained transformant was obtained as RIK7013.

Example 4 Selection of Transformants by Protease (nprE) Halo (1) Construction of RIK7004 (lacA::PrrnO-nprE trpB'A'::PrrnO-catSD10 erm his101)
The lacA gene of RIK1141 (Reference Example 1) was replaced with PrrnO-nprE. PCR using upstream and downstream fragments of the lacA gene, RIK1141 DNA as a template, P-lacA-upF primer and P-lacA-upR primer, and P-lacA-downF primer and P-lacA-downR primer It was made in. Next, using the DNA of RIK7001 (nprE::PrrnO-nprE trpB'A'::PrrnO-catSD10 erm his101 (Example 1(1)) as a template, the rrnO-upF primer and the PrrnO-nprE-R primer were used. A PrrnO-nprE fragment was prepared by PCR, and a DNA fragment [lacA::PrrnO-nprE] was prepared from the obtained three fragments by PCR using P-lacA-upF primer and P-lacA-downR primer. ..
After the above-mentioned [lacA::PrrnO-nprE] fragment and the [trpB'A'::PrrnO-catSD10 erm his101] fragment prepared in Example 1 (1) were transformed into RIK1141 strain, the cells contained erythromycin and skim milk. The cells were selected from erythromycin resistant (proliferation (+)) and halo-forming (halo (+)) cells on the culture medium. The obtained transformant was obtained as RIK7004.

(2) Construction of RIK7014 (lacA::rrnOantiSD10 trpB'A'::PrrnO-catSD10 erm hisC101)
By using RIK1226 (Example 1 (2)) as a template and rrnO-upF primer and rrnO-5R-4+XbaI primer, PCR was performed to prepare a rrnOantiSD10 fragment having an XbaI cleavage site added to the 3'end. Next, using the DNA of RIK1141 (Reference Example 1) as a template, PCR was performed using the P-nprE-down-F0+XbaI primer and the P-lacA-downR primer, and the lacA gene with an XbaI cleavage site added to the 5'end. A downstream region fragment was prepared. The obtained two fragments were treated with a restriction enzyme (XbaI) and then ligated by a ligation reaction to prepare a DNA fragment [lacA::rrnOantiSD10].
Next, in order to introduce the target gene (rrnOantiSD10) into the lacA gene region of RIK7004, the prepared [lacA::rrnOantiSD10] fragment was transformed into RIK7004 strain. Cells were cultured on LB plates containing chloramphenicol and skim milk and cells that were chloramphenicol resistant (growth (+)) and did not form halos on the medium (halo (-)) were selected. Sequencing confirmed that the selected cells contained the rrnOantiSD10 sequence. The obtained transformant was obtained as RIK7014.

Example 5 Selection of transformants by protease (nprE) halo (1) Construction of RIK7005 strain (nprB::PrrnO-nprE trpB'A'::PrrnO-catSD10 erm his101)
The nprB gene of RIK1141 (Reference Example 1) was replaced with PrrnO-nprE. PCR using the upstream region and downstream region fragment of the nprB gene, RI-K1141 DNA as a template, P-nprB-upF primer and P-nprB-upR primer, and P-nprB-downF primer and P-nprB-downR primer It was made in. A DNA fragment [nprB::PrrnO-nprE] was prepared using the obtained two fragments, the PrrnO fragment prepared in Example 1 (1), the P-nprB-upF primer and the P-nprB-downR primer.
After the above [nprB::PrrnO-nprE] fragment and the [trpB'A'::PrrnO-catSD10 erm his101] fragment prepared in Example 1 (1) were transformed into RIK1141 strain, the cells contained erythromycin and skim milk. The cells were selected from erythromycin resistant (proliferation (+)) and halo-forming (halo (+)) cells on the culture medium. The obtained transformant was obtained as RIK7005.

(2) Construction of RIK7015 (nprB::rrnOantiSD10 trpB'A'::PrrnO-catSD10 erm hisC101)
A downstream region fragment of the nprB gene in which an XbaI cleavage site was added to the 5'end by PCR using RIK1141 (Reference Example 1) DNA as a template and P-nprE-down-F0+XbaI primer and P-nprB-downR primer Was produced. This fragment and the rrnOantiSD10 fragment prepared in Example 4(2) with an XbaI cleavage site added at the 3'end were treated with a restriction enzyme (XbaI) and ligated by a ligation reaction to give a DNA fragment [nprB::rrnOantiSD10]. It was made.
Next, in order to introduce the target gene (rrnOantiSD10) into the nprB gene region of RIK7005, the prepared [nprB::rrnOantiSD10] fragment was transformed into RIK7005 strain. Cells were cultured on LB plates containing chloramphenicol and skim milk and cells that were chloramphenicol resistant (growth (+)) and did not form halos on the medium (halo (-)) were selected. Sequencing confirmed that the selected cells contained the rrnOantiSD10 sequence. The resulting transformant was obtained as RIK7015.

Example 6 Deletion of marker gene from recombinant cells
Construction of RIK7213 (amyE::rrnOantiSD10 trpC2) Tryptophan synthetic gene (including trpC2 mutation) by PCR using DNA extracted from Bacillus subtilis strain 168 (trpC2) as a template and trpD-F primer and hisC-R2 primer To a histidine synthetic gene were prepared. RIK7013 (Example 3(2)) was transformed with the prepared DNA fragment, and the cells were cultured in a minimal medium containing tryptophan to select transformants showing tryptophan auxotrophy and histidine non-auxotrophy. The selected transformant was obtained as RIK7213. This is a recombinant cell having the gene of interest (rrnOantiSD10) and no drug resistance marker.

Example 7 Preparation of double recombinant cells (1) Construction of RIK7021 strain (amyE::rrnOantiSD10 trpB'A'::PrrnO-catΔSD erm190)
After the PCR DNAI (Reference Example 2) was transformed into the RIK7013 strain (Example 3(2)), the cells were cultured in a minimal medium containing tryptophan to obtain a transformant showing tryptophan auxotrophy and histidine non-auxotrophy. Selected. It was confirmed that the obtained transformant was sensitive to chloramphenicol and sensitive to erythromycin, and RIK7021 was obtained.

(2) Construction of RIK7032 (nprE::PrrnO-nprE amyE::rrnOantiSD10 trpB'A'::PrrnO-cat ΔSD erm hisC101)
Using the DNA extracted from RIK7001 (Example 1(1)), the DNA fragments [nprE::PrrnO-nprE] and [erm his101] were transformed into RIK7021 prepared in (1) above. Cells were cultured on LB plates containing erythromycin and skim milk, and cells that formed halos (halo (+)) on the medium and proliferated (proliferation (+)) were selected. The selected cells were further selected by culturing on LB plates containing erythromycin, LB plates containing chloramphenicol, minimal agar medium containing tryptophan, minimal agar medium containing tryptophan and histidine, and selecting chloramphenic acid. Cells showing call sensitivity, erythromycin resistance and histidine requirement were selected. The obtained transformant was obtained as RIK7032.

(3) Construction of RIK7051 (amyE::rrnOantiSD10 nprE::rrnOantiSD10 trpB'A'::PrrnO-catSD10 erm hisC101)
Using the DNA extracted from RIK7011 (Example 1(2)), the DNA fragments [nprE::rrnOantiSD10] and [trpB'A'::PrrnO-catSD10 erm hisC101] were transformed into RIK7032 prepared in (1) above. Converted. Cells were cultivated on LB plates containing chloramphenicol and skim milk and cells that did not form halos on the medium (halo (-)) and grew (growth (+)) were selected. The selected cells were further selected by culturing on LB plates containing erythromycin, LB plates containing chloramphenicol, minimal agar medium containing tryptophan, minimal agar medium containing tryptophan and histidine, and selecting chloramphenic acid. Transformants showing call resistance, erythromycin resistance, and histidine requirement were selected. The obtained transformant was obtained as RIK7051.

Example 8 Deletion of Marker Gene from Recombinant Cell (1) Construction of RIK7221 (amyE::rrnOantiSD10 nprE::rrnOantiSD10 trpC2) In the same procedure as in Example 6, the region from the tryptophan synthetic gene to the histidine synthetic gene was included. A DNA fragment was prepared. RIK7051 (Example 7(3)) was transformed with the prepared DNA fragment, and a transformant showing tryptophan auxotrophy and histidine non-auxotrophy was selected. The selected transformant was obtained as RIK7221. This is a double recombinant cell having a target gene (rrnOantiSD10) at two positions on DNA and having no drug resistance marker.

The flow of the procedure for producing a recombinant in this example is shown below.

Claims (28)

A method for producing a recombinant microorganism, comprising:
(1) preparing cells of a host microorganism that co-expresses a first hydrolase expression cassette and a first selectable marker,
here,
The first hydrolase expression cassette is a DNA containing a promoter and a first hydrolase gene operably linked to the promoter, and the first hydrolase expression cassette is Replaced with the first region of interest in the DNA of the cell;
(2) The cells are cultivated in the selection medium having the first selection marker-containing strain containing the substrate for the first hydrolase, and the cells that grow on the medium and form halo are the first cells. Selecting as cells containing a hydrolase expression cassette;
(3) giving the cell selected in (2) the ability to express a second selectable marker and replacing the first hydrolase expression cassette with a first recombination cassette; and
(4) The cells prepared in (3) are cultured in the second selection marker-bearing strain selection medium containing the substrate for the first hydrolase, and the cells are grown on the medium without forming halos. Selecting cells as the cells containing the first recombination cassette,
Including the method. The cell expressing the first selectable marker is a cell into which the first selectable marker expression cassette has been introduced, and the first selectable marker expression cassette is a promoter and a first operably linked to the promoter. The method according to claim 1, which is a DNA containing one selection marker gene. The imparting of the expression ability of the second selectable marker to the cells is the introduction of the second selectable marker expression cassette into the cells, and the second selectable marker expression cassette operates with a promoter and the promoter. The method according to claim 1 or 2, which is a DNA containing a second selectable marker gene operably linked to the DNA. The method according to any one of claims 1 to 3, further comprising deleting the expression ability of the first selectable marker in the cell in the step (3). The method according to claim 4, wherein the lack of expression ability of the first selectable marker is replacement of the first selectable marker expression cassette with the second selectable marker expression cassette. The first selection marker expression cassette is a DNA containing a promoter, the first selection marker gene, and the inactivated second selection marker gene, and the second selection marker expression cassette, A DNA containing a promoter, the inactivated first selectable marker gene, and the second selectable marker gene,
The method according to claim 5. The host microorganism wherein the promoter contained in the first hydrolase expression cassette, the promoter contained in the first selection marker expression cassette, and the promoter contained in the second selection marker expression cassette are the same or different. The method according to any one of claims 1 to 6, which is a promoter that is expressed in the logarithmic growth phase. 8. The first hydrolase gene is selected from the group consisting of a protease gene, an amylase gene, a cellulase gene, a β-galactosidase gene, a β-glucuronidase gene, and a lipase gene. the method of. The method according to claim 1, wherein the first recombination cassette is a DNA containing at least one selected from the group consisting of a target gene, a promoter, a terminator, and a transcriptional regulatory region. The method according to any one of claims 1 to 8, wherein the first recombination cassette is a DNA containing a gene of interest operably linked to a control region. The method according to claim 1, wherein the first recombination cassette is a fragment for gene deletion. The method according to claim 1, wherein the host microorganism is a bacterium of the genus Bacillus. The method according to claim 12, wherein the Bacillus is Bacillus subtilis or a mutant strain thereof. The method according to any one of claims 1 to 13, further comprising repeating the following (1') to (4') one or more times:
(1′) Substituting the additional mutation target region in the DNA of the cell selected in the above (4) or the following (4′) with an additional hydrolase expression cassette, and allowing the cell to express an additional selectable marker. To give,
Wherein the additional hydrolase expression cassette is a DNA containing a promoter and an additional hydrolase gene operably linked to the promoter;
(2′) The cells are cultured in a selection medium having the additional selection marker-containing strain containing a substrate of the additional hydrolase, and the cells that grow and form halo on the medium are added to the additional hydrolase expression cassette. Selecting as cells containing
(3′) imparting to the cells selected in (2′) the ability to express a selectable marker different from the additional selectable marker, and replacing the additional hydrolase expression cassette with an additional recombinant cassette And (4′) culturing the cell prepared in (3′) in the selective medium having the other selective marker-containing strain containing a substrate for the additional hydrolase, without forming a halo on the medium. Selecting proliferating cells as cells containing said additional recombination cassette. Giving the cell the ability to express the additional selectable marker is the introduction of the additional selectable marker expression cassette into the cell, the additional selectable marker expression cassette being a promoter and a further operably linked to the promoter. The method according to claim 14, which is a DNA containing a selectable marker gene. Giving the cell the ability to express the other selectable marker is introduction of the other selectable marker expression cassette into the cell, and the other selectable marker expression cassette is operably linked to a promoter. 16. The method according to claim 14 or 15, which is a DNA containing the selected another selectable marker gene. The method according to any one of claims 14 to 16, further comprising, in (3′), deleting the ability of the cell to express the additional selectable marker. 18. The method of claim 17, wherein the lack of expression ability of said additional selectable marker is the replacement of said additional selectable marker expression cassette by said another selectable marker expression cassette. 19. The method of claim 18, wherein the additional selectable marker expression cassette is the first selectable marker expression cassette and the alternative selectable marker expression cassette is the second selectable marker expression cassette. The further selection marker expression cassette is a DNA containing a promoter, the first selection marker gene, and the inactivated second selection marker gene, and the another selection marker expression cassette is a promoter, A DNA containing the activated first selection marker gene and the second selection marker gene,
The method according to claim 19. The promoter contained in the further hydrolase expression cassette, the promoter contained in the further selectable marker expression cassette, and the promoter contained in the another selectable marker expression cassette are the same or different in the logarithmic growth phase of the host microorganism. The method according to any one of claims 14 to 20, which is a promoter that expresses. The method according to any one of claims 14 to 21, wherein the further hydrolase gene is selected from the group consisting of a protease gene, an amylase gene, a cellulase gene, a β-galactosidase gene, a β-glucuronidase gene and a lipase gene. .. 23. The method of claim 22, wherein the first hydrolase gene and the additional hydrolase gene are the same gene. The method according to any one of claims 14 to 23, wherein the additional recombination cassette is a DNA containing at least one selected from the group consisting of a gene of interest, a promoter, a terminator, and a transcriptional regulatory region. 24. The method according to any one of claims 14 to 23, wherein the additional recombination cassette is a DNA containing a gene of interest operably linked to a control region. 24. The method of any of claims 14-23, wherein the additional recombination cassette is a gene deletion fragment. The method according to any one of claims 1 to 13, further comprising deleting the selectable marker expression cassette contained in the cells selected in (4). 27. The method of any of claims 14-26, further comprising deleting the selectable marker expression cassette contained in the cells selected in (4').