Method for producing target substance

Abstract

The present invention provides a method for producing a target protein by culturing cells, the method being capable of stably maintaining a target plasmid without using an antibiotic. The present invention relates to a method for producing a target substance, the method including a step for culturing cells that are transformed with a target plasmid (A), wherein: each of the transformed cells includes the target plasmid (A) and a temperature-sensitive plasmid (B) and are obtained by a method that includes a step for culturing cells in each of which the function of an essential gene on the chromosome has been reduced at a temperature at which the temperature-sensitive plasmid (B) is eliminated; the target plasmid (A) includes the essential gene and a target substance gene and does not include an antibiotic-resistant gene; and the temperature-sensitive plasmid (B) includes the essential gene and the antibiotic-resistant gene.

Description

Method for producing the target substance

[0001] This invention relates to a method for producing a target substance.

[0002] In nature, organisms possessing plasmid DNA in addition to chromosomal DNA are known. Techniques for modifying plasmid DNA to serve as vectors for artificially expressing target substances, thereby conferring new functions to host cells, are commonly known. This genetically engineered expression of a target substance is achieved by introducing a vector containing the gene for that substance into host cells and then culturing the host cells. However, since the vector is merely an unwanted foreign factor to the host cells, there is a problem in that the vector is easily lost during continuous culture. To avoid this problem, various methods have been developed to stably maintain the vector within host cells.

[0003] The most widely used method for maintaining a vector stably is to introduce an antibiotic resistance gene into the vector in addition to the gene for the target substance, and then culture host cells in the presence of the antibiotic. Since host cells cannot survive without the antibiotic resistance gene, the vector containing the antibiotic resistance gene is also maintained stably. However, there is a risk of the antibiotic resistance gene contaminating the product. Furthermore, there are concerns that the release of the antibiotic resistance gene or the antibiotic itself into the environment could lead to the emergence of antibiotic-resistant bacteria.

[0004] Therefore, a method has been proposed to transform cells using a vector that contains the gene for the target protein but does not contain the antibiotic resistance gene (Patent Document 1). Furthermore, in order to maintain the vector without relying on antibiotics, it has also been proposed to introduce a vector containing the gene encoding frr (ribosome release factor) into a host in which the chromosomal gene encoding frr has been inactivated (Patent Document 2).

[0005] Japanese Patent Publication No. 2021-193932, Japanese Patent Publication No. 2017-500042

[0006] The method described in Patent Document 1 can produce cells containing a vector that does not contain antibiotic resistance genes, but the efficiency of vector introduction is insufficient because cell selection is not possible. Furthermore, the vector is lost when the resulting cells are cultured, so there is room for improvement in the vector retention rate. The frr (ribosome release factor) gene used in the method described in Patent Document 2 is a protein-coding gene, so introducing a plasmid containing the frr gene into cells may affect the productivity of the target protein. The object of the present invention is to provide a method for producing a target protein by cell culture that can stably maintain the target plasmid in cells without using antibiotics.

[0007] As a result of the inventors' investigations, they discovered that by culturing cells containing the target plasmid (A) and a temperature-sensitive plasmid (B), in which the function of essential genes on the chromosome is reduced, at a temperature at which the temperature-sensitive plasmid (B) is removed, it is possible to introduce the target plasmid (A) with high efficiency and stably maintain it while producing the target protein, even though the target plasmid (A) does not contain antibiotic resistance genes. This led to the completion of the present invention.

[0008] This disclosure includes the following forms: <1> A method for producing a target substance, comprising the step of culturing cells transformed with a target plasmid (A), wherein the transformed cells comprise the target plasmid (A) and a temperature-sensitive plasmid (B), and the cells have reduced function of essential genes on the chromosome, and are obtained by a method comprising the step of culturing the cells at a temperature at which the temperature-sensitive plasmid (B) is removed, wherein the target plasmid (A) comprises the essential gene and the target substance gene, but does not comprise an antibiotic resistance gene, and the temperature-sensitive plasmid (B) comprises the essential gene and the antibiotic resistance gene. <2> The method for producing a target substance according to claim 1, further comprising the steps of obtaining cells comprising the temperature-sensitive plasmid (B) and having reduced function of essential genes on the chromosome, and introducing the target plasmid (A) into the cells, prior to the step of culturing at a temperature at which the temperature-sensitive plasmid (B) is removed. <3> The method for producing a target substance according to claim 1 or 2, wherein the essential gene is a gene that does not encode a protein. <4> The method for producing a target substance according to claim 3, wherein the essential gene is a tRNA gene. <5> A method for producing the target substance according to any one of claims 1 to 4, wherein the total length of the target plasmid (A) is 2.5 kbp or more. <6> A method for producing the target substance according to any one of claims 1 to 5, wherein the cells are actinomycetes. <7> A method for producing the target substance according to any one of claims 1 to 6, wherein the target substance is a protein. <8> A method for producing cells transformed with the target plasmid (A), comprising the step of culturing cells comprising the target plasmid (A) and a temperature-sensitive plasmid (B), wherein the function of essential genes on the chromosome is reduced, at a temperature at which the temperature-sensitive plasmid (B) is removed, wherein the target plasmid (A) comprises the essential gene and the target substance gene, but does not contain an antibiotic resistance gene, and the temperature-sensitive plasmid (B) comprises the essential gene and the antibiotic resistance gene. <9> Cells comprising a target plasmid (A) which contains essential genes and a target substance gene but does not contain an antibiotic resistance gene, wherein the function of the essential gene on the chromosome is reduced. <10> Cells according to claim 9, wherein the essential gene is a tRNA gene.<11> A cell comprising a target plasmid (A) containing an essential gene and a target substance gene but not an antibiotic resistance gene, and a temperature-sensitive plasmid (B) containing the essential gene and the antibiotic resistance gene, wherein the function of the essential gene on the chromosome is reduced. <12> The cell according to item 11, wherein the essential gene is a tRNA gene.

[0009] In this invention, a method for producing a target protein by cell culture allows for the stable maintenance of the target plasmid within cells without the use of antibiotics.

[0010] This is a schematic diagram showing the process of obtaining transformed cells in the method for producing the target substance of the present invention. This is the gene map of plasmid pDNKO3::tRNA-Trp. This is the gene map of plasmid pDNX2421. This shows the selection results by replica method of cells transformed with the target plasmid (A). This shows the relative activity of PLA2 produced by the method of the present invention. This shows the relative activity of glucanase produced by the method of the present invention. This shows the relative activity of protease produced by the method of the present invention.

[0011] <Method for Producing the Target Substance> The method for producing the target substance of the present invention includes a step of culturing cells transformed with a target plasmid (A), wherein the transformed cells include the target plasmid (A) and a temperature-sensitive plasmid (B), and the cells, in which the function of essential genes on the chromosome is reduced, are obtained by a method including a step of culturing the cells at a temperature at which the temperature-sensitive plasmid (B) is removed, wherein the target plasmid (A) includes the essential genes and the target substance gene, but does not include antibiotic resistance genes, and the temperature-sensitive plasmid (B) includes the essential genes and the antibiotic resistance genes.

[0012] In the cell culture process, cells transformed with the target plasmid (A), as described later, are cultured. The culture medium can be either a natural or synthetic medium, as long as it contains a carbon source, nitrogen source, inorganic salts, etc., that the cells can utilize, and allows for efficient cell culture. Examples of carbon sources include carbohydrates such as glycerol, glucose, galactose, fructose, xylose, sucrose, raffinose, and starch; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol. Examples of nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other inorganic acids or ammonium salts of organic acids or other nitrogen-containing compounds. Other substances that may be used include peptone, meat extract, fish extract, corn steep liquor, yeast extract, and various amino acids. Examples of inorganic substances include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate. Additionally, vegetable oil, surfactants, and defoaming agents such as silicone may be added as needed.

[0013] Among those listed above, a culture medium mainly composed of peptone, fish extract, corn steep liquor, or yeast extract is preferred, and a culture medium containing yeast extract is more preferred. The concentration of yeast extract is preferably 0.5 to 10% by weight in the culture medium, and more preferably 1 to 5% by weight.

[0014] The culture conditions can be appropriately selected depending on the type of culture medium and culture method, and there are no particular restrictions as long as the conditions allow cells to proliferate and produce the target substance. Typically, culture is carried out under aerobic conditions such as shaking culture or aerated stirring culture in a liquid medium. The shaking speed during shaking culture is preferably 50 to 300 rpm, and more preferably 100 to 200 rpm. The culture temperature is preferably 25 to 35°C, and more preferably 27 to 30°C. The pH of the culture medium is preferably pH 3 to 9, and more preferably pH 6 to 8. The culture time is preferably 24 to 96 hours, and more preferably 48 to 72 hours. Subculturing may also be performed by diluting the culture solution 10 to 250 times. The number of subculturing cycles is preferably 2 to 4 to maintain high purity of the target substance.

[0015] If the target substance is a target protein, it is preferable to include a step of purifying the target protein after the cell culture step. In the step of purifying the target protein, if the target protein accumulates inside the cells, the cells are recovered by centrifugation or filter filtration, the recovered cells are disrupted by sonication or the like, and then a cell-free extract is obtained by centrifugation or solid-liquid separation using a filter aid such as diatomaceous earth or cellulose powder. Using this as a starting material, the target protein can be purified by general protein purification methods such as salting out, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, and affinity chromatography. If the target protein is secreted outside the cells, the target protein can be purified in the same way, except that the culture supernatant is used instead of the cell-free extract.

[0016] <Cells> The cells used to produce the target substance are transformed with the target plasmid (A). These cells are obtained by a method that includes culturing cells containing the target plasmid (A) and a temperature-sensitive plasmid (B), in which the function of essential genes on the chromosome is reduced, at a temperature at which the temperature-sensitive plasmid (B) is removed.

[0017] The cells used are not particularly limited as long as they are capable of expressing the target substance; microbial cells, animal cells, and plant cells can all be used. From the viewpoint of productivity of the target substance and ease of culture, microbial cells are preferred, and bacteria are more preferred.

[0018] Examples of bacteria include actinomycetes such as the genera Streptomyces and Rhodococcus for which host-vector systems have been developed; and bacteria such as the genera Escherichia, Bacillus, Pseudomonas, Serratia, Brevibacterium, Corynebacterium, Streptococcus, and Lactobacillus for which host-vector systems have been developed. Other genera include Saccharomyces, Kluyveromyces, Schizosaccharomyces, Zygosaccharomyces, Yarrowia, Trichosporon, and Rhodosporidium. Examples include yeasts for which host-vector systems have been developed, such as those belonging to the genera Ridium, Pichia, and Candida; and fungi for which host-vector systems have been developed, such as those belonging to the genera Neurospora, Aspergillus, Cephalosporium, and Trichoderma. Among these, actinomycetes are preferred, and Streptomyces is more preferred.

[0019] The genus Streptomyces includes Streptomyces lividans, Streptomyces violaceoruber, Streptomyces cinnamoneus, Streptomyces avermetilis, Streptomyces mirabilis, Streptomyces thermoviolaceus, and Streptomyces halsteady. Examples include *Streptomyces halstedii*, *Streptomyces griseus*, *Streptomyces coelicor*, *Streptomyces venezuelae*, and *Streptomyces thermoviolaceus*.

[0020] A specific strain of Streptomyces lividans is Streptomyces lividans strain 1326. This strain is also known as Streptomyces violaceoruber strain 1326, and its culture and purified products have been confirmed to be safe for use in food.

[0021] An example of the genus Escherichia is Escherichia coli.

[0022] Examples of animal cells include those derived from humans, mice, rats, dogs, monkeys, Chinese hamsters, fruit flies, armyworms, and nettle moths. Examples of plant cells include those derived from tobacco, corn, and rice.

[0023] The cells described above may be cells that naturally express the target substance, or they may not naturally express the target substance. Even in the case of cells that naturally express the target substance, the productivity of the target substance can be improved by transforming them with a vector.

[0024] Figure 1 is a schematic diagram illustrating the process of obtaining transformed cells in the method for producing the target substance of the present invention.

[0025] <Essential Genes> Cells have reduced function of essential genes on their chromosomes. In Figure 1, cell 4 has chromosome 3 in which the function of essential genes is reduced. In this invention, essential genes refer to genes whose reduced function would prevent the cell from surviving. Depending on the type of cell, the genes essential for the cell's survival will differ, and the essential genes can be determined according to the type of cell used. In this invention, cells with reduced function of essential genes on their chromosomes refer to cells that cannot survive without supplementation of the function of those essential genes from an external source. Examples of supplementation of essential gene function include transformation using extrachromosomal DNA such as plasmids. In the case of essential genes being metabolic enzymes, cell culture in a culture medium containing their metabolites is also an option.

[0026] Essential genes can include both protein-coding and non-protein-coding genes. Examples of non-protein-coding genes include tRNA and rRNA genes. Examples of protein-coding genes include genes encoding ribosome recycling factors, genes encoding translation initiation factors, and genes encoding toxin-antitoxin systems.

[0027] If the essential gene is a protein-coding gene, when the target plasmid (A) is introduced into cells, the production of the protein encoded by the essential gene contained in the target plasmid (A) may occupy factors necessary for protein production in the cells, such as ribosomes, potentially inhibiting the production of the target substance. Furthermore, depending on the type of protein encoded by the essential gene, its overexpression may be toxic to cells. When the essential gene is a non-protein-coding gene, these risks can be reduced. Therefore, it is preferable for the essential gene to be a non-protein-coding gene, and a tRNA gene is even more preferable because its function on bacterial chromosomes is easily reduced.

[0028] As tRNA genes, tRNA genes corresponding to common amino acids such as Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val can be used. Among these, Trp-tRNA genes (also called tRNA-Trp genes) and Phe-tRNA genes (also called tRNA-Phe genes) are preferred.

[0029] Methods for reducing the function of essential genes on cell chromosomes will be discussed later.

[0030] <Target Plasmid (A)> Target plasmid (A) contains essential genes and target substance genes, but does not contain antibiotic resistance genes.

[0031] The essential gene contained in target plasmid (A) is a gene that has the same function as the essential gene whose function is reduced on the cell's chromosome. However, the nucleotide sequence of the essential gene contained in target plasmid (A) does not need to be the same as the nucleotide sequence of the essential gene whose function is reduced on the cell's chromosome. The essential gene contained in target plasmid (A) may have a different sequence from the essential gene on the chromosome, as long as it is within a range that allows the cell with reduced function of the essential gene on the chromosome to survive.

[0032] <Target Substance Gene> The target substance encoded by the target substance gene is not particularly limited as long as it can be expressed in cells, but may be a target protein, a physiologically active peptide, a non-coding RNA, etc., and is preferably a target protein. Examples of target proteins include enzymes, hormones, receptors, structural proteins such as collagen, transport proteins such as hemoglobin, and contractile proteins such as myosin. Among these, enzymes are preferred because they are easy to apply to food products.

[0033] Examples of enzymes include phospholipase, glucanase, protease, α-amylase, β-amylase, maltogenic amylase, glucan 1,4-α-maltotriohydrolase, glucan 1,4-α-maltohexaohydrolase, cellulase, hemicellulase, galactolipase, glucose oxidase, ascorbic acid oxidase, peroxidase, lipoxygenase, catalase, glutathione dehydrogenase, peptidase, transglutaminase, cyclodextrin glucanotransferase, triacylglycerol lipase, phosphodiesterase, esterase, muramidase, phosphatase, glutaminase, chitosanase, and chitinase. Examples of phospholipases include phospholipase D and phospholipase A2. Among these, phospholipase, glucanase, and protease are preferred, phospholipase is more preferred, and phospholipase A2 is even more preferred.

[0034] Examples of DNA encoding phospholipase include the following (a), (b), or (c): (a) DNA containing the nucleotide sequence shown in Sequence ID No. 1 of the sequence listing; (b) DNA encoding a polypeptide having phospholipase activity and exhibiting 85% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 1 of the sequence listing; (c) DNA encoding a polypeptide having phospholipase activity, consisting of a nucleotide sequence in which one or more nucleotides are deleted, inserted, substituted, and / or added to the nucleotide sequence shown in Sequence ID No. 1 of the sequence listing.

[0035] The sequence identity with the base sequence shown in Sequence ID No. 1 is preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more.

[0036] In the nucleotide sequence shown in Sequence ID No. 1 of the sequence listing, the number of deletions, insertions, substitutions and / or additions is preferably 243 or less, more preferably 162 or less, even more preferably 81 or less, even more preferably 32 or less, and particularly preferably 20, 10, 5, 4, 3, or 2 or less.

[0037] Examples of the DNA encoding glucanase include the following DNAs (a), (b), or (c). (a) DNA containing the base sequence shown in SEQ ID NO: 2 of the Sequence Listing; (b) DNA showing 85% or more sequence identity with the base sequence shown in SEQ ID NO: 2 of the Sequence Listing and encoding a polypeptide having glucanase activity; (c) DNA consisting of a base sequence in which one or more bases in the base sequence shown in SEQ ID NO: 2 of the Sequence Listing are deleted, inserted, substituted and / or added and encoding a polypeptide having glucanase activity.

[0038] The sequence identity with the base sequence shown in SEQ ID NO: 2 is preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more.

[0039] In the base sequence shown in SEQ ID NO: 2 of the Sequence Listing, the number of bases to be deleted, inserted, substituted and / or added is preferably 179 or less, more preferably 119 or less, even more preferably 59 or less, even more preferably 23 or less, and particularly preferably 20, 10, 5, 4, 3, or 2 or less.

[0040] Examples of the hormone include growth hormone, follicle-stimulating hormone, insulin, calcitonin and the like.

[0041] <Antibiotic Resistance Gene> The target plasmid (A) does not contain an antibiotic resistance gene. The use of an antibiotic resistance gene can lead to the generation of resistant bacteria due to the outflow of the gene into the environment or the outflow of the antibiotic added to the medium as a selection pressure into the environment. However, since the target plasmid (A) does not contain an antibiotic resistance gene, these risks can be eliminated. An antibiotic resistance gene refers to a gene that maintains the activity of decomposing an antibiotic or inhibiting the action of an antibiotic. The antibiotics mentioned here include thiostrepton, penicillin, kanamycin, vancomycin, erythromycin, viomycin, neomycin, streptomycin, tetracycline, chloramphenicol, hygromycin B, tubercactinomycin N, rifampicin, spectinomycin, lincomycin, clarithromycin, apramycin, ribostamycin, etc.

[0042] <Replication Origin Sequence> The target plasmid (A) contains a replication origin sequence in order to be maintained and amplified in the cell. The replication origin sequence is not particularly limited as long as it can promote the replication of the plasmid in the host cell. For example, the replication origin sequence derived from plasmid vector pIJ101, the replication origin sequence derived from plasmid vector pSG5, the replication origin sequence derived from plasmid vector SLP2, etc. can be mentioned. Among these, the replication origin sequence derived from pIJ101 is preferred. The length of the replication origin sequence is preferably 500 - 2000 bp, and more preferably 1000 - 1500 bp.

[0043] The target plasmid (A) can be either a low-copy plasmid or a high-copy plasmid, and can be appropriately selected considering the yield of the target substance and the influence on the host cell. For example, using a low-copy plasmid can reduce the load on the host cell. Here, a low-copy plasmid refers to a plasmid containing 1 - 99 copies in one host cell, and a high-copy plasmid refers to a plasmid containing 100 or more copies in one host cell.

[0044] <Promoter Sequence> The target plasmid (A) contains a promoter sequence within the gene encoding the target substance. The promoter sequence may be the promoter inherent to the target substance, or it may be a heterologous promoter. When a heterologous promoter is used, examples of genes from which the promoter originates include metalloendopeptidase genes, phospholipase D (PLD) genes, xylose isomerase genes, xylanase genes, amylase genes, and protease genes. Among these, promoter sequences derived from metalloendopeptidase genes and phospholipase D (PLD) genes are preferred. The length of the promoter sequence is preferably 50 to 2000 bp, and more preferably 60 to 400 bp.

[0045] Examples of DNA containing a promoter sequence derived from a metalloendopeptidase gene include the following DNAs: (a) DNA containing the nucleotide sequence shown in Sequence ID No. 3 of the sequence listing; (b) DNA exhibiting 85% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 3 of the sequence listing and inducing the expression of the target protein in cells; (c) DNA consisting of a nucleotide sequence in which one or more nucleotides are deleted, inserted, substituted and / or added to the nucleotide sequence shown in Sequence ID No. 3 of the sequence listing and inducing the expression of the target protein in cells.

[0046] The sequence identity with the base sequence shown in Sequence ID No. 3 is preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more.

[0047] In the nucleotide sequence shown in Sequence ID No. 3 of the sequence listing, the number of deletions, insertions, substitutions and / or additions is preferably 44 or less, more preferably 29 or less, even more preferably 14 or less, even more preferably 5 or less, and particularly preferably 4, 3, or 2 or less.

[0048] Examples of DNA containing a promoter sequence derived from the phospholipase D (PLD) gene include the following DNAs: (a) DNA containing the nucleotide sequence shown in Sequence ID No. 4 of the sequence listing; (b) DNA showing 85% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 4 of the sequence listing and inducing the expression of the target protein in cells; (c) DNA consisting of a nucleotide sequence in which one or more nucleotides are deleted, inserted, substituted and / or added to the nucleotide sequence shown in Sequence ID No. 4 of the sequence listing and inducing the expression of the target protein in cells.

[0049] The sequence identity with the base sequence shown in Sequence ID No. 4 is preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more.

[0050] In the nucleotide sequence shown in Sequence ID No. 4 of the sequence listing, the number of deletions, insertions, substitutions and / or additions is preferably 10 or less, more preferably 7 or less, even more preferably 3 or less, and even more preferably 1 or less.

[0051] <Terminator Sequence> The target plasmid (A) contains a terminator sequence within the gene encoding the target substance to stop transcription. The terminator sequence may be the terminator inherent in the target protein, or it may be a heterologous terminator. When using a heterologous terminator, examples of the gene from which the terminator originates include the phospholipase D (PLD) gene, the metalloendopeptidase gene, and the amylase gene. Among these, the terminator sequence derived from the phospholipase D (PLD) gene is preferred.

[0052] Examples of DNA containing a terminator sequence derived from the phospholipase D (PLD) gene include the following DNAs: (a) DNA containing the nucleotide sequence shown in Sequence ID No. 5 of the sequence listing; (b) DNA showing 85% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 5 of the sequence listing and halting the transcription of the target protein gene in a cell; (c) DNA consisting of a nucleotide sequence in which one or more nucleotides are deleted, inserted, substituted and / or added to the nucleotide sequence shown in Sequence ID No. 5 of the sequence listing and halting the transcription of the target protein gene in a cell.

[0053] The sequence identity with the base sequence shown in Sequence ID No. 5 is preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more.

[0054] In the nucleotide sequence shown in Sequence ID No. 5 of the sequence listing, the number of deletions, insertions, substitutions and / or additions is preferably 30 or less, more preferably 20 or less, even more preferably 10 or less, even more preferably 4 or less, and particularly preferably 3 or 2 or less.

[0055] The total length of the target plasmid (A) is preferably 2.5 kbp or more, more preferably 5 kbp or more, and even more preferably 7 kbp or more. If the total length of the target plasmid (A) is less than 2.5 kbp, the retention rate of the target plasmid (A) tends to decrease. Furthermore, the total length of the target plasmid (A) is preferably 10 kbp or less, more preferably 9 kbp or less, and even more preferably 8 kbp or less. When the total length of the target plasmid (A) is greater than 2.5 kbp, the plasmid tends to be more easily and stably retained within the cell.

[0056] <Temperature-sensitive plasmid (B)> Temperature-sensitive plasmid (B) contains essential genes and antibiotic resistance genes.

[0057] The essential genes contained in temperature-sensitive plasmid (B) are essential genes whose function is reduced on the cell's chromosome and genes that have the same function as the essential genes contained in target plasmid (A). Because temperature-sensitive plasmid (B) contains essential genes, cells with reduced function of essential genes on their chromosomes can survive if temperature-sensitive plasmid (B) is present, regardless of the presence or absence of target plasmid (A). Note that the nucleotide sequence of the essential genes contained in temperature-sensitive plasmid (B) does not need to be the same as the nucleotide sequence of the essential genes whose function is reduced on the cell's chromosome and the essential genes contained in target plasmid (A). The sequences of the essential genes contained in temperature-sensitive plasmid (B) may differ from those of the essential genes on the chromosome and the essential genes contained in target plasmid (A), as long as it is within a range that allows for the survival of cells with reduced function of essential genes on their chromosomes.

[0058] The antibiotic resistance genes contained in temperature-sensitive plasmids (B) are genes that maintain the activity to degrade antibiotics or inhibit the action of antibiotics. Antibiotics to which these antibiotic resistance genes show resistance include thiostrepton, penicillin, kanamycin, vancomycin, erythromycin, biomycin, neomycin, streptomycin, tetracycline, chloramphenicol, hygromycin B, tuberactinomycin N, rifampicin, spectinomycin, lincomycin, clarithromycin, apramycin, and ribostamycin. Because temperature-sensitive plasmids (B) contain antibiotic resistance genes, it is easy to confirm that cells contain temperature-sensitive plasmids (B) by using antibiotics.

[0059] A temperature-sensitive plasmid (B) replicates in cells at a certain culture temperature, but loses its ability to replicate and is released from the cells at a different culture temperature. The mechanism by which the temperature-sensitive plasmid (B) becomes temperature-sensitive is not particularly limited, and any mechanism known as common technical knowledge in the relevant field can be used. For example, the temperature-sensitive plasmid (B) may contain a temperature-sensitive plasmid replication factor and may contain a temperature-sensitive origin of replication (ori). A temperature-sensitive origin of replication (ori) is pSG5ori. As a temperature-sensitive plasmid replication factor, for example, the temperature-sensitive replication plasmid factor (pSG5rep) of the Streptomyces ghanaensis plasmid pSG5 can be used.

[0060] Examples of temperature-sensitive replication plasmid factors (pSG5rep) for pSG5 include the following DNAs: (a), (b), or (c): (a) DNA containing the nucleotide sequence shown in Sequence ID No. 6 of the sequence listing; (b) DNA exhibiting 85% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 6 of the sequence listing and functioning as a temperature-sensitive plasmid replication factor; (c) DNA consisting of a nucleotide sequence in which one or more nucleotides are deleted, inserted, substituted, and / or added to the nucleotide sequence shown in Sequence ID No. 6 of the sequence listing and functioning as a temperature-sensitive plasmid replication factor.

[0061] The sequence identity with the base sequence shown in Sequence ID No. 6 is preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more.

[0062] <Step of culturing at a temperature at which temperature-sensitive plasmid (B) is removed> The transformed cells contain the target plasmid (A) and temperature-sensitive plasmid (B), and the cells have reduced function of essential genes on the chromosome. The method includes the step of culturing these cells at a temperature at which temperature-sensitive plasmid (B) is removed.

[0063] This process will be explained with reference to Figure 1. In Figure 1, cell 4 contains the target plasmid (A) 1 and the temperature-sensitive plasmid (B) 2, and has chromosome 3 with reduced function of essential genes. Target plasmid (A) 1 contains the target substance gene 11 and essential gene (A) 12. Temperature-sensitive plasmid (B) 2 contains the temperature-sensitive plasmid replication factor 21, essential gene (B) 22, and antibiotic resistance gene 23. Essential gene (A) 12 and essential gene (B) 22 only need to have the same function; they do not need to have the same base sequence. When cell 4 is cultured at a temperature at which temperature-sensitive plasmid (B) 2 is lost, the function of temperature-sensitive plasmid replication factor 21 decreases, temperature-sensitive plasmid (B) 2 loses its replication ability, and is lost from the cell. As a result, cell 4 containing the target plasmid (A) 1 and having chromosome 3 with reduced function of essential genes can be obtained. In this process, by simply changing the culture temperature to a temperature at which temperature-sensitive plasmid (B) 2 is lost, cell 4 containing the target plasmid (A) 1 can be obtained easily and efficiently.

[0064] In Figure 1, cells 4 obtained in the process of culturing at a temperature at which temperature-sensitive plasmid (B) 2 is lost do not contain temperature-sensitive plasmid (B) 2 but do contain target plasmid (A) 1. Since target plasmid (A) 1 does not contain antibiotic resistance genes, using cells 4 to produce the target substance can prevent contamination of the product with antibiotic resistance genes. Furthermore, it is possible to prevent the emergence of resistant bacteria due to the release of antibiotic resistance genes or the antibiotics themselves into the environment.

[0065] In Figure 1, cells 4 obtained in the process of culturing at a temperature at which temperature-sensitive plasmid (B) 2 is lost have chromosome 3 with reduced function of essential genes. Since the essential gene 12 contained in target plasmid (A) 1 is indispensable for the survival of cells 4, the state in which target plasmid (A) 1 is retained in cells 4 can be maintained.

[0066] Cells containing a target plasmid (A) and a temperature-sensitive plasmid (B), in which the function of essential genes on the chromosome is reduced, require the temperature at which the temperature-sensitive plasmid (B) is maintained within the cell in order to preserve these two plasmids. ＡIt is cultured at T. Ａ T is preferably 20 to 36°C, more preferably 25 to 32°C, and even more preferably 28 to 30°C. For culturing cells containing the target plasmid (A) and the temperature-sensitive plasmid (B) with reduced function of essential genes on the chromosome, either a liquid medium or a solid medium can be used. The composition of the medium is not particularly limited, and the media described above for the cell culturing step can be used.

[0067] The temperature T at which the temperature-sensitive plasmid (B) is lost Ｂ is different from the culturing temperature T at which the temperature-sensitive plasmid (B) is maintained in the cells, Ａ and it may be T Ａ > T Ｂ or it may be T Ａ < T Ｂ but it is preferably T Ａ < T Ｂ The difference between T Ａ and T Ｂ is preferably 1 to 10°C, more preferably 2 to 7°C. T Ｂ is preferably 25 to 40°C, more preferably 28 to 37°C, and even more preferably 30 to 37°C. In FIG. 1, a temperature of 37°C is described as an example of the temperature T at which the temperature-sensitive plasmid (B) is lost. Ｂ

[0068] In the step of culturing at the temperature at which the temperature-sensitive plasmid (B) is lost, the culturing may be performed only once or multiple times. The total culturing time in the step of culturing at the temperature at which the temperature-sensitive plasmid (B) is lost is not particularly limited, but from the viewpoint of sufficiently losing the temperature-sensitive plasmid (B), it is preferably 16 hours or more, more preferably 72 hours or more. On the other hand, from the viewpoint of shortening the cell production time, it is preferably 48 hours or less, more preferably 24 hours or less.

[0069] For the medium used for culturing at the temperature at which the temperature-sensitive plasmid (B) is lost, either a liquid medium or a solid medium can be used, but a solid medium is preferred because it is easy to select the desired strain. The composition of the medium is not particularly limited, and the media described above for the cell culturing step can be used.

[0070] The method for producing the target substance of the present invention may include a step of culturing the cells at a temperature at which the temperature-sensitive plasmid (B) is removed, followed by a step of confirming the antibiotic sensitivity of the obtained cells and selecting antibiotic-sensitive cells. Since the temperature-sensitive plasmid (B) contains an antibiotic resistance gene, cells in which the temperature-sensitive plasmid (B) has not been removed are resistant to antibiotics. On the other hand, cells in which the temperature-sensitive plasmid (B) has been removed cannot survive in a culture medium containing antibiotics and become antibiotic-sensitive cells. Therefore, cells in which the temperature-sensitive plasmid (B) has been removed can be selected, for example, by selecting antibiotic-sensitive cells using a replica method.

[0071] Cells containing the target plasmid (A) and the temperature-sensitive plasmid (B), with reduced function of essential genes on the chromosome, can be obtained through general genetic engineering. For example, when using Streptomyces species as host cells, the method described in the following literature can be used: Practical Streptomyces Genetics (Tobias Kieser, Mervyn J. Bibb, Mark J. Buttner, Keith F. Chater, David A. Hopwood), ISBN 0-7084-0623-8, 2000, The John Innes Foundation

[0072] A specific method for producing cells containing a target plasmid (A) and a temperature-sensitive plasmid (B) in which the function of essential genes on the chromosome is reduced includes, for example, the following steps: - A step of obtaining cells containing the temperature-sensitive plasmid (B) in which the function of essential genes on the chromosome is reduced; and - A step of introducing the target plasmid (A) into the cells.

[0073] In the step of obtaining cells containing a temperature-sensitive plasmid (B) in which the function of essential genes on the chromosome is reduced, the function of essential genes on the chromosome is reduced. The method for reducing the function of essential genes on the chromosome is not particularly limited, and general methods in the art can be used, such as deletion, substitution (e.g., mutation), inhibition, and / or insertion in the nucleic acid gene sequence. It is preferable that the reduction of essential gene function is stable and irreversible.

[0074] If the reduction in the function of an essential gene on a chromosome is due to the deletion of the essential gene, it is preferable to delete the essential gene by homologous recombination. For example, all or part of the essential gene on the chromosome may be deleted by introducing a plasmid containing sequences homologous to the upstream and downstream of the gene to be deleted into cells and inducing homologous recombination within the cells.

[0075] When reducing the function of essential genes on chromosomes, it is preferable that the cells contain a complementary plasmid containing the essential gene. The presence of the complementary plasmid allows the cells with reduced essential gene function to survive. The complementary plasmid may be a temperature-sensitive plasmid (B), or a plasmid other than a temperature-sensitive plasmid (B). If the complementary plasmid is a plasmid other than a temperature-sensitive plasmid (B), cells containing a temperature-sensitive plasmid (B) and with reduced essential gene function on chromosomes can be obtained by removing the complementary plasmid using antibiotic resistance as an indicator and maintaining the temperature-sensitive plasmid (B).

[0076] Since the temperature-sensitive plasmid (B) contains an antibiotic resistance gene, cells containing the temperature-sensitive plasmid (B) can be selected based on their resistance to the antibiotic. Furthermore, if the complementary plasmid is a plasmid other than the temperature-sensitive plasmid (B), it is preferable that the complementary plasmid contains a different antibiotic resistance gene than the temperature-sensitive plasmid (B). This makes it possible to select the desired cells by choosing cells that show antibiotic resistance due to the temperature-sensitive plasmid (B) but not due to the complementary plasmid when exchanging the complementary plasmid for the temperature-sensitive plasmid (B).

[0077] In the step of introducing the target plasmid (A) into cells, the target plasmid (A) is introduced into cells containing the temperature-sensitive plasmid (B) in which the function of essential genes on the chromosome has been reduced. The introduction method is not particularly limited as long as it can introduce the target plasmid (A) into the cells. Well-known methods such as electroporation, protoplast-PEG, calcium chloride method, and particle gun method can be used. It is preferable to culture the cells into which the target plasmid (A) has been introduced in order to increase their size. Either liquid or solid culture media can be used for culture. The composition of the culture medium is not particularly limited, and the media described above for the cell culture step can be used.

[0078] <<Method for Manufacturing Cells>> The present invention relates to a method for manufacturing cells transformed with a target plasmid (A), comprising the steps of culturing cells in which the function of essential genes on the chromosome is reduced, including the target plasmid (A) and a temperature-sensitive plasmid (B), at a temperature at which the temperature-sensitive plasmid (B) is removed, wherein the target plasmid (A) includes the essential genes and the target substance gene, but does not include the antibiotic resistance gene, and the temperature-sensitive plasmid (B) includes the essential genes and the antibiotic resistance gene.

[0079] The method for producing cells according to the present invention corresponds to the method for obtaining cells transformed with the target plasmid (A) in the method for producing the target substance of the present invention. The target plasmid (A), temperature-sensitive plasmid (B), each gene, and each step in the method for producing cells according to the present invention are as described in the method for producing the target substance of the present invention.

[0080] <<Cells according to one aspect of the present invention>> Cells according to one aspect of the present invention contain a target plasmid (A) which includes essential genes and target substance genes but does not contain antibiotic resistance genes, and the function of the essential genes on the chromosome is reduced. Preferably, these cells do not contain a temperature-sensitive plasmid (B).

[0081] These cells can be obtained by the cell production method of the present invention. The target plasmid (A) and each gene in the cells are as described in the method for producing the target substance of the present invention.

[0082] In the cells described above, the essential gene is preferably a non-protein encoding gene, and more preferably a tRNA gene. In this case, the risk of inhibition of the production of the target substance by the expression of the gene in the target plasmid (A) can be reduced, and the risk of excessive expression of the gene being toxic to the cell can be reduced.

[0083] <<Cells according to another aspect of the present invention>> Cells according to another aspect of the present invention include a target plasmid (A) which contains an essential gene and a target substance gene but does not contain an antibiotic resistance gene, and a temperature-sensitive plasmid (B) which contains the essential gene and the antibiotic resistance gene, and the function of the essential gene on the chromosome is reduced.

[0084] The cells described above correspond to the cells before being cultured at the temperature at which the temperature-sensitive plasmid (B) is lost, in the method for producing the target substance of the present invention and the method for producing the cells. The target plasmid (A), temperature-sensitive plasmid (B), and each gene in the above cells are as described in the method for producing the target substance of the present invention.

[0085] In the cells described above, the essential gene is preferably a non-protein encoding gene, and more preferably a tRNA gene. In this case, the risk of inhibition of the production of the target substance by the expression of the gene in the target plasmid (A) can be reduced, and the risk of excessive expression of the gene being toxic to the cell can be reduced.

[0086] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples. Hereinafter, unless otherwise specified, "parts" or "%" means "parts by weight" or "weight percent," respectively.

[0087] [1] Production of phospholipase (PLA2) (1) Preparation of target plasmid (A) The phospholipase (PLA2) gene derived from Streptomyces viola olver was ligated to the NdeI-SphI cleavage site of plasmid pDNX2421 shown in Figure 3. The plasmid was digested with AseI and EcoRV to remove the ampicillin resistance gene (bla) and the E. coli replication origin (pUCori). Subsequently, the target fragment was obtained by gel purification and ligation to obtain the target plasmid (A). This target plasmid (A) contains the tRNA-Trp gene as an essential gene, the phospholipase (PLA2) gene as the target substance gene, and a sequence derived from pIJ101 as the replication origin sequence, and its total length is 6.8 kb. Although EcoRV treatment is not essential in this process, it was performed to prevent digestion fragments generated by AseI treatment alone from being introduced into the ligation site.

[0088] (2) Cells containing the target plasmid (A) and the temperature-sensitive plasmid (B) were prepared by conventional methods. Host cells were transformed using the target plasmid (A) and the temperature-sensitive plasmid (B). As host cells, Streptomyces viola seolber 1326 cells were used in which the tRNA-Trp gene on the chromosome was removed and replaced with the aac(3)IV gene. As the temperature-sensitive plasmid (B), pDNKO3::tRNA-Trp, which contains tRNA-Trp as the essential gene, a thiostrepton resistance gene as the antibiotic resistance gene, and pSG5ori as the replication initiation sequence, was used (see Figure 2). The transformed cells were used as cells in which the function of the tRNA gene on the chromosome was reduced.

[0089] As a transformation method, the protoplast transformation method, which is common in actinomycetes, was used. The following three types of cells were expected to be present in the transformed cell population: (i) cells containing only the temperature-sensitive plasmid (B) (ii) cells containing both the temperature-sensitive plasmid (B) and the target plasmid (A) (iii) cells containing only the target plasmid (A) Subsequently, the transformed cells were spread onto regeneration medium (R2YE) to regenerate the cell wall. R2YE is a solid agar medium with adjusted osmotic pressure to prevent protoplast rupture.

[0090] (3) Culturing at a temperature at which the temperature-sensitive plasmid (B) is lost The cells coated on the regenerated medium in (2) were cultured at 37°C for 16 hours to select cells (ii) and cells (iii). At 37°C, the temperature-sensitive pDNKO3::tRNA-Trp was not easily replicated, and it was expected that cells (i) would not grow or would grow very poorly. The colonies that grew on the regenerated medium were washed twice with sterile water and suspended in 400 μl of sterile water.

[0091] Furthermore, to reliably select cells (ii) and (iii), the cell suspension was spread at a rate of 200 μl / plate onto thiostrepton-free SFM agar medium and cultured at 37°C to induce spore formation. The spores were collected, diluted, and spread onto thiostrepton-free SFM agar medium, and cultured at 30°C for several days to form single colonies.

[0092] The thiostrepton sensitivity of the colonies was confirmed by the replica method, and transformants with pDNKO3::tRNA-Trp removed (cells (iii)) were selected. Figure 4 shows the results of the replica method. Cells were grown from colonies that were grown in a thiostrepton-free medium and not in a thiostrepton-containing medium, and it was confirmed that they contained the target plasmid (A). Approximately 90% of the total number of colonies grown on the plate before replicating were cells (iii) containing only the target plasmid (A).

[0093] (4) Expression of the target protein The cells (iii) were cultured in antibiotic-free tryptosawyer broth medium at 30°C for 96 hours with shaking. The cells were then removed from the culture medium. Phospholipase (PLA2) was confirmed to be expressed in the recovered culture supernatant by SDS-PAGE.

[0094] The expression of phospholipase (PLA2) was confirmed by performing a specific quantitative enzyme activity measurement method on the recovered culture supernatant. The results are shown in Figure 5. Figure 5 shows the relative activity of PLA2 by the method of the present invention, with the activity of PLA2 in the control test set to 100%. In the control test, wild-type Streptomyces viola seolver 1326, in which the tRNA-Trp gene on the chromosome was not disrupted, was transformed with the target plasmid (A) that carries the thiostrepton resistance gene. These transformed cells were cultured in antibiotic-free tryptosawyer broth medium with shaking at 30°C for 96 hours, and the activity of PLA2 in the culture supernatant was measured. The method of the present invention confirmed PLA2 activity that was almost equivalent to that of the control test. The specific quantitative enzyme activity measurement method for PLA2 was performed using NEFA C-Test Wako manufactured by FUJIFILM, according to the attached manual.

[0095] Furthermore, whole-nucleotide sequence analysis of plasmids containing the target gene confirmed that the target plasmid (A) was retained in 100% of the cultured cells.

[0096] [2] Examination of essential genes As host cells, Streptomyces viola seolber 1326 cells in which the tRNA-Phe gene on the chromosome was removed and replaced with the aac(3)IV gene were used. When pDNKO3::tRNA-Phe, which contains tRNA-Phe as the essential gene, a thiostrepton resistance gene as the antibiotic resistance gene, and pSG5ori as the replication initiation sequence, was used as the temperature-sensitive plasmid (B), the host cells could not grow without the temperature-sensitive plasmid (B). Therefore, it was confirmed that tRNA-Phe can be used as an essential gene in the same way as tRNA-Trp.

[0097] [3] Production of glucanase and protease Cells containing the target plasmid (A) and temperature-sensitive plasmid (B) were prepared using the same method as for producing phospholipase (PLA2), except that the glucanase gene (BAC70420) from Streptomyces evamethylis or the protease gene (WP_075032007) from Streptomyces mirabilis were used instead of the phospholipase (PLA2) gene from Streptomyces viola. The cells were cultured at a temperature at which the temperature-sensitive plasmid (B) was removed. The resulting cells containing only the target plasmid (A) were cultured with shaking at 30°C for 96 hours in antibiotic-free tryptosawyer broth medium. The cells were then removed from the culture medium. SDS-PAGE was performed on the recovered culture supernatant to confirm the expression of glucanase or protease.

[0098] The glucanase activity of the recovered culture supernatant was measured using the D-Fructose / D-Glucose Kit from NZYTECH, according to the attached manual. Protease activity was measured according to Method 4 of the Protease Activity Test Method described in the 10th edition of the Japanese Food Additives Standards. The results are shown in Figures 6 and 7.

[0099] Figure 6 shows the relative activity of glucanase obtained by the method of the present invention, with the glucanase activity in the control test set to 100%. The method of the present invention confirmed glucanase activity that was almost equivalent to that of the control test.

[0100] Figure 7 shows the relative activity of the protease obtained by the method of the present invention, with the protease activity in the control test set to 100%. The method of the present invention demonstrated protease activity more than 10 times higher than that in the control test.

[0101] 1. Target plasmid (A) 11. Target substance gene 12. Essential gene (A) 2. Temperature-sensitive plasmid (B) 21. Temperature-sensitive plasmid replication factor 22. Essential gene (B) 23. Antibiotic resistance gene 3. Chromosome with reduced function of essential gene 4. Cell

Claims

1. A method for producing a target substance, comprising the step of culturing cells transformed with a target plasmid (A), wherein the transformed cells comprise the target plasmid (A) and a temperature-sensitive plasmid (B), and the transformed cells are obtained by a method comprising the step of culturing the cells, in which the function of essential genes on the chromosome is reduced, at a temperature at which the temperature-sensitive plasmid (B) is removed, wherein the target plasmid (A) comprises the essential genes and the target substance gene, but does not contain antibiotic resistance genes, and the temperature-sensitive plasmid (B) comprises the essential genes and the antibiotic resistance genes.

2. A method for producing the target substance according to claim 1, further comprising: a step of obtaining cells containing the temperature-sensitive plasmid (B) and in which the function of essential genes on the chromosome is reduced, before the step of culturing at a temperature at which the temperature-sensitive plasmid (B) is lost; and a step of introducing the target plasmid (A) into the cells.

3. The method for producing the target substance according to claim 1 or 2, wherein the essential gene is a gene that does not code for a protein.

4. The method for producing the target substance according to claim 3, wherein the essential gene is a tRNA gene.

5. A method for producing the target substance according to any one of claims 1 to 4, wherein the total length of the target plasmid (A) is 2.5 kbp or more.

6. A method for producing the target substance according to any one of claims 1 to 5, wherein the cells are actinomycetes.

7. A method for producing the target substance according to any one of claims 1 to 6, wherein the target substance is a protein.

8. A method for producing cells transformed with a target plasmid (A), comprising the step of culturing cells comprising the target plasmid (A) and a temperature-sensitive plasmid (B), wherein the function of essential genes on the chromosome is reduced, at a temperature at which the temperature-sensitive plasmid (B) is shed, wherein the target plasmid (A) comprises the essential genes and the target substance gene, but does not contain the antibiotic resistance gene, and the temperature-sensitive plasmid (B) comprises the essential genes and the antibiotic resistance gene.

9. A cell containing a target plasmid (A) that includes essential genes and target substance genes but does not contain antibiotic resistance genes, and in which the function of the said essential genes on the chromosome is reduced.

10. The cell according to claim 9, wherein the essential gene is a tRNA gene.

11. A cell comprising a target plasmid (A) containing essential genes and target substance genes but not antibiotic resistance genes, and a temperature-sensitive plasmid (B) containing the essential genes and antibiotic resistance genes, wherein the function of the essential genes on the chromosome is reduced.

12. The cell according to claim 11, wherein the essential gene is a tRNA gene.

Images