Method for producing target protein

Abstract

To provide a method for producing target protein while stably maintaining a vector without performing special genetic engineering to a host cell and without using a drug-resistant gene or the like.SOLUTION: A method for producing target protein including a process of culturing a cell transformed by a vector, in which the vector includes a gene of target protein, and does not include an antibiotic-resistant gene, a recognition sequence of recombinase, and a gene necessary for survival of a cell.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing a target protein. [Background technology]

[0002] Various enzymes are used to improve the shelf life, taste, texture, etc. of foods. Enzymes are proteins that are naturally present inside or outside living organisms such as microorganisms and plants. Enzymes are industrially produced by microbial culture, cell culture, plant cultivation, etc., and are purified before use. In addition, to improve the productivity of enzymes, they may be mass-expressed by genetic recombination.

[0003] In nature, organisms that have plasmid DNA in addition to chromosomal DNA are known. A commonly known technology is to improve plasmid DNA as a vector for artificially expressing a target protein and impart new functions to host cells. This expression of a protein by genetic recombination is achieved by introducing a vector containing the gene for the protein into a host cell and culturing the host cell. Here, since the vector is merely an unnecessary foreign factor for the host cell, there is a problem that the vector is easily dropped off during continued culturing. To avoid this problem, various methods have been developed to stably maintain the vector in the host cell.

[0004] The most widely used method for stably maintaining vectors is to introduce an antibiotic resistance gene into the vector in addition to the gene for the protein, and then culture the host cells in the presence of antibiotics. Since host cells cannot live without the antibiotic resistance gene, vectors containing the antibiotic resistance gene are also stably maintained. However, there is a risk that the antibiotic resistance gene will be contaminated into enzyme products. There is also a concern that the antibiotic resistance gene or the antibiotic itself may leak into the environment, leading to the emergence of resistant bacteria.

[0005] Therefore, a method has been proposed in which an antibiotic resistance gene is selectively excised and removed from a vector when the growth of the host cell reaches a certain level (Patent Documents 1 and 2).Other methods known include a method in which an essential gene that the host originally possesses is deleted from the host and the gene is introduced into a vector (Patent Document 3), a method in which a recognition sequence for a recombinase is introduced into a vector (Non-Patent Documents 1 and 2), and a method in which a gene that expresses a substance that is toxic to the host cell is retained in the genome of the host and a corresponding antidote gene is introduced into a vector (Non-Patent Document 3). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2008-505620 A [Patent Document 2] JP 2013-533743 A [Patent Document 3] Special Publication No. 2017-500042 [Non-patent literature]

[0007] [Non-Patent Document 1] Bo Zhang et al.PLoS ONE 8(2):e55906 (2013) [Non-Patent Document 2] Yuan Yu et aL.PLoS ONE 8(5):e62457(2013) [Non-Patent Document 3] Sevillano et al.Microb Cell Fact(2017)16:164 Summary of the Invention [Problem to be solved by the invention]

[0008] The methods of Patent Documents 1 and 2 can reduce the amount of antibiotics used, but cannot eliminate the risk of antibiotic resistance genes being mixed into enzyme products. The method of Patent Document 3 requires the deletion of essential genes in the host cells, while the methods of Non-Patent Documents 1 to 3 require the introduction of a specific sequence other than the target protein into the vector. The object of the present invention is to provide a method for producing a target protein while stably maintaining a vector, without performing any particular genetic manipulation on the host cells or using drug resistance genes or the like. [Means for solving the problem]

[0009] As a result of studies conducted by the present inventors, it was found that a vector can be stably maintained without any particular genetic manipulation of the host cell or using a drug resistance gene, and thus the present invention was completed.

[0010] That is, the present invention relates to a method for producing a target protein, which comprises a step of culturing a cell transformed with a vector, characterized in that the vector contains a gene for the target protein and does not contain an antibiotic resistance gene, a recognition sequence for a recombinase, or a gene essential for cell survival.

[0011] Preferably, the gene essential for cell viability is a gene encoding a ribosome recycling factor or a gene encoding a translation initiation factor.

[0012] Preferably, the cell is a bacterium.

[0013] The bacterium is preferably an actinomycete.

[0014] The target protein is preferably an enzyme.

[0015] Preferably, the enzyme is a phospholipase, glucanase, protease, α-amylase, β-amylase, maltogenic amylase, glucan 1,4-α-maltotriohydrolase, glucan 1,4-α-maltohexaohydrolase, cellulase, hemicellulase, galactolipase, glucose oxidase, ascorbate oxidase, peroxidase, lipoxygenase, catalase, glutathione dehydrogenase, peptidase, transglutaminase, cyclodextrin glucanotransferase, triacylglycerol lipase, phosphodiesterase, esterase, muramidase, phosphatase, glutaminase, chitosanase or chitinase. Effect of the Invention

[0016] In the production method of the present invention, the vector can be stably maintained without any particular genetic manipulation of the host cell or the use of a drug resistance gene, and the risk of the antibiotic resistance gene being contaminated into the enzyme product or leaking into the environment can be eliminated. [Brief description of the drawings]

[0017] [Figure 1] 1 shows the detection results of the target protein in Example 1. [Diagram 2] 1 shows the results of antibiotic susceptibility tests in Example 1. [Diagram 3] 1 shows the detection results of the target protein in Example 2. [Figure 4] 1 shows the results of antibiotic susceptibility tests in Example 2. [Diagram 5] 1 shows an outline of a method for producing a vector in Example 3. [Figure 6] 1 shows the detection results of the target protein in Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] <Method of producing target protein> The method for producing a target protein of the present invention includes a step of culturing a cell transformed with a vector, and is characterized in that the vector contains a gene for the target protein but does not contain an antibiotic resistance gene, a recognition sequence for a recombinase, or a gene essential for cell survival.

[0019] In the step of culturing cells, cells transformed with a vector, which will be described later, are cultured. The culture medium may be either a natural medium or a synthetic medium, as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the cells and can efficiently culture the cells. Examples of carbon sources include carbohydrates such as 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 inorganic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, or ammonium salts of organic acids, or other nitrogen-containing compounds. Other examples include peptone, meat extract, fish extract, corn steep liquor, yeast extract, and various amino acids. Examples of inorganic substances include potassium dibasic phosphate, potassium dibasic phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate. In addition, defoamers such as vegetable oil, surfactants, and silicon may be added as necessary.

[0020] Among the above, a medium containing peptone, fish extract, corn steep liquor or yeast extract as a main component is preferred, and a medium containing yeast extract is more preferred. The concentration of yeast extract in the medium is preferably 0.5 to 10% by weight, more preferably 1 to 5% by weight.

[0021] The culture conditions may be appropriately selected depending on the type of medium, the culture method, etc., and are not particularly limited as long as the cells can grow and produce the target protein. Usually, the culture is performed under aerobic conditions such as shaking culture or aeration and agitation culture in a liquid medium. The shaking speed during shaking culture is preferably 50 to 300 rpm, more preferably 100 to 200 rpm. The culture temperature is preferably 25 to 35°C, more preferably 27 to 30°C. The pH is preferably 3.0 to 9.0, more preferably 6.0 to 8.0. The culture time is preferably 24 to 96 hours, more preferably 48 to 72 hours. Subculture may be performed by diluting the culture solution 10 to 250 times. The number of subcultures is preferably 2 to 4 times in order to maintain high purity of the target protein, but the plasmid can be stably maintained even after 10 subcultures.

[0022] The method for producing a target protein preferably includes a step of purifying the target protein after the step of culturing the cells. In the step of purifying the target protein, when the target protein accumulates within the cells, the cells are collected by centrifugation or filter filtration, and the collected cells are disrupted by ultrasonic treatment 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 a general protein purification method such as salting out or various chromatographies such as ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, and affinity chromatography. When the target protein is secreted outside the cells, it can be purified in the same manner using the culture supernatant instead of the cell-free extract.

[0023] <cell> The cells are not particularly limited as long as they can express the target protein, and any of microbial cells, animal cells, and plant cells can be used. From the viewpoints of productivity of the target protein and ease of culture, microbial cells are preferred, and bacteria are more preferred.

[0024] Examples of bacteria include actinomycetes such as the genus Streptomyces and Rhodococcus, for which host vector systems have been developed; and bacteria such as the genus Escherichia, Bacillus, Pseudomonas, Serratia, Brevibacterium, Corynebacterium, Streptococcus, and Lactobacillus, for which host vector systems have been developed. Other examples include yeasts for which host vector systems have been developed, such as the genus Saccharomyces, Kluyveromyces, Schizosaccharomyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidium, Pichia, and Candida; and molds for which host vector systems have been developed, such as the genus Neurospora, Aspergillus, Cephalosporium, and Trichoderma. Among these, actinomycetes are preferred, and the genus Streptomyces is more preferred.

[0025] Examples of the genus Streptomyces include Streptomyces lividans, Streptomyces violaceoruber, Streptomyces cinnamoneus, Streptomyces avermetilis, Streptomyces thermoviolaceus, Streptomyces halstedii, and the like.

[0026] A specific example of a 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.

[0027] Examples of the genus Escherichia include Escherichia coli.

[0028] Examples of animal cells include cells derived from humans, mice, rats, dogs, monkeys, Chinese hamsters, Drosophila melanogaster, armyworm moths, and nettle loopers. Examples of plant cells include cells derived from tobacco, corn, rice, and the like.

[0029] The above cells may be cells that naturally express the target protein, or may be cells that do not naturally express the target protein. Even in the case of cells that naturally express the target protein, the productivity of the target protein can be improved by transforming the cells with a vector.

[0030] <vector> The vector may be any vector capable of expressing a target protein in cells, and specific examples thereof include a plasmid vector, a phage vector, and a cosmid vector. From the viewpoint of ease of transformation, a plasmid vector is preferred. The total length of the vector, including the gene sequence of the target protein described below, is preferably 3,000 to 10,000 bp, and more preferably 4,500 to 6,500 bp. If the total length exceeds 10,000 bp, the vector tends to be unstable in cells, and if it is less than 3,000 bp, the length of the gene sequence of the target protein that can be used is limited.

[0031] <Target protein> The target protein is not particularly limited as long as it can be expressed in cells, and examples of the target protein include enzymes, hormones, receptors, structural proteins such as collagen, transport proteins such as hemoglobin, contractile proteins such as myosin, etc. Among these, enzymes are preferred because they are easily applied to foods.

[0032] Examples of the enzyme 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, chitinase, etc. Among these, phospholipase, glucanase, and protease are preferred.

[0033] Examples of DNA encoding phospholipase include the following DNA (a), (b), or (c): (a) a DNA containing the nucleotide sequence shown in SEQ ID NO:1 in the sequence listing; (b) a DNA which shows 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:1 in the sequence listing and encodes a polypeptide having phospholipase activity; (c) A DNA having a base sequence in which one or more bases have been deleted, inserted, substituted and / or added in the base sequence shown in SEQ ID NO:1 in the sequence listing, and which encodes a polypeptide having phospholipase activity.

[0034] The sequence identity with the base sequence shown in SEQ ID NO:1 is preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more.

[0035] In the base sequence shown in SEQ ID NO:1 in the sequence listing, the number of deleted, inserted, substituted and / or added bases 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.

[0036] Examples of DNA encoding glucanase include the following DNA (a), (b), or (c): (a) a DNA containing the nucleotide sequence shown in SEQ ID NO:2 in the sequence listing; (b) a DNA which shows 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:2 in the sequence listing and encodes a polypeptide having glucanase activity; (c) A DNA having a base sequence in which one or more bases have been deleted, inserted, substituted and / or added in the base sequence shown in SEQ ID NO:2 in the sequence listing, and which encodes a polypeptide having glucanase activity.

[0037] 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.

[0038] In the base sequence shown in SEQ ID NO:2 in the sequence listing, the number of deleted, inserted, substituted and / or added bases 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.

[0039] Examples of hormones include growth hormone, follicle-stimulating hormone, insulin, and calcitonin.

[0040] <Replication origin sequence> A vector needs to have a replication origin in order to be maintained and amplified in a cell. Examples of the replication origin include the replication origin derived from the plasmid vector pIJ101, the replication origin derived from the plasmid vector pSG5, and the replication origin derived from the plasmid vector SLP2. Among these, the replication origin derived from pIJ101 is preferred. The length of the replication origin is preferably 500 to 2000 bp, more preferably 1000 to 1500 bp.

[0041] <Promoter sequence> The vector has a promoter sequence in a gene encoding a target protein. The promoter sequence may be a promoter inherent to the target protein, or a heterologous promoter. When a heterologous promoter is used, examples of genes from which the promoter is derived include a metalloendopeptidase gene, a phospholipase D (PLD) gene, a xylose isomerase gene, a xylanase gene, an amylase gene, and a protease gene. Among these, promoter sequences derived from a metalloendopeptidase gene and a phospholipase D (PLD) gene are preferred. The length of the promoter sequence is preferably 50 to 2000 bp, more preferably 60 to 400 bp.

[0042] Examples of DNA having a promoter sequence derived from a metalloendopeptidase gene include the following DNA (a), (b), or (c): (a) a DNA containing the nucleotide sequence shown in SEQ ID NO:3 in the sequence listing; (b) a DNA that shows 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:3 in the sequence listing and induces expression of a target protein in a cell; (c) A DNA consisting of a base sequence in which one or more bases have been deleted, inserted, substituted and / or added in the base sequence shown in SEQ ID NO:3 in the sequence listing, and which induces the expression of a target protein in cells.

[0043] The sequence identity with the base sequence shown in SEQ ID NO:3 is preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more.

[0044] In the base sequence shown in SEQ ID NO:3 in the sequence listing, the number of deleted, inserted, substituted and / or added bases 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.

[0045] Examples of DNA having a promoter sequence derived from the phospholipase D (PLD) gene include the following DNA (a), (b), or (c): (a) a DNA containing the nucleotide sequence shown in SEQ ID NO:4 in the sequence listing; (b) a DNA that shows 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing and induces expression of a target protein in a cell; (c) A DNA consisting of a base sequence in which one or more bases have been deleted, inserted, substituted and / or added in the base sequence shown in SEQ ID NO:4 in the sequence listing, and which induces the expression of a target protein in cells.

[0046] The sequence identity with the base sequence shown in SEQ ID NO:4 is preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more.

[0047] In the base sequence shown in SEQ ID NO:4 in the sequence listing, the number of deleted, inserted, substituted and / or added bases is preferably 10 or less, more preferably 7 or less, even more preferably 3 or less, and even more preferably 1 or less.

[0048] <Terminator sequence> The vector has a terminator sequence for terminating transcription in a gene encoding a target protein. The terminator sequence may be a terminator that the target protein originally has, or may be a heterologous terminator. When a heterologous terminator is used, the gene from which the terminator is derived may include a phospholipase D (PLD) gene, a metalloendopeptidase gene, an amylase gene, etc. Among these, a terminator sequence derived from a phospholipase D (PLD) gene is preferred.

[0049] Examples of DNA having a terminator sequence derived from the phospholipase D (PLD) gene include the following DNA (a), (b), or (c): (a) a DNA containing the nucleotide sequence shown in SEQ ID NO:5 in the sequence listing; (b) a DNA that has a sequence identity of 85% or more with the nucleotide sequence shown in SEQ ID NO:5 in the sequence listing and that stops the transcription of a gene for a target protein in a cell; (c) A DNA consisting of a base sequence in which one or more bases are deleted, inserted, substituted and / or added in the base sequence shown in SEQ ID NO:5 in the sequence listing, and which stops the transcription of a gene for a target protein in a cell.

[0050] The sequence identity with the base sequence shown in SEQ ID NO:5 is preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more.

[0051] In the base sequence shown in SEQ ID NO:5 in the sequence listing, the number of deleted, inserted, substituted and / or added bases 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.

[0052] <Antibiotic resistance genes, etc.> The vector used in the present invention is characterized in that it does not contain antibiotic resistance genes, recombinase recognition sequences, or genes essential for cell survival.

[0053] When an antibiotic resistance gene is used, an antibiotic is added to the culture medium as a selective pressure, which may lead to the emergence of resistant bacteria due to the leakage of the antibiotic itself or the antibiotic resistance gene into the environment. The vector used in the present invention does not contain an antibiotic resistance gene, so these risks can be eliminated. An antibiotic resistance gene refers to a gene that maintains the activity of decomposing an antibiotic or the activity of inhibiting the action of an antibiotic. Examples of antibiotics referred to here include thiostrepton, penicillin, kanamycin, vancomycin, erythromycin, viomycin, neomycin, streptomycin, tetracycline, and chloramphenicol.

[0054] Recombinase recognition sequences are used to selectively cut out and remove antibiotic resistance genes from vectors in Patent Documents 1 and 2 and Non-Patent Documents 1 and 2. Examples of such recombinases include Cre, Flp, R, XerC, XerD, RipX, and CodV. Examples of recombinase recognition sequences include transposase target sites, Ecdif, cer, psi, pif, mwr, Bsdif, loxP, FRT, and RS.

[0055] In Patent Document 3, genes essential for cell survival are introduced into a vector instead of being deleted from the host cell, thereby stably maintaining the vector. Examples of such genes include genes encoding ribosome recycling factors, genes encoding translation initiation factors, and genes encoding toxin-antitoxin systems.

[0056] <Vector construction and cell transformation> The vector used in the present invention can be obtained by linking the gene of the target protein, a replication origin sequence, a promoter sequence, etc., by a known method. Examples of the linking method include a linking method using restriction enzyme digestion and ligation using DNA ligase. The DNA to be digested with the restriction enzyme may be a plasmid DNA or a PCR product.

[0057] The transformation method is not particularly limited as long as the vector can be introduced into the cells. Well-known methods such as electroporation, protoplast-PEG, calcium chloride, and particle gun methods can be used. After transformation, the presence of the vector in the cells can be confirmed by well-known methods such as colony PCR. Furthermore, the expression of the target protein from the cells can be confirmed by SDS-PAGE. EXAMPLES

[0058] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples. In the following, "parts" and "%" mean "parts by weight" and "% by weight", respectively, unless otherwise specified.

[0059] <Example 1> Deletion of drug resistance genes by restriction enzyme treatment <Isolation of plasmid DNA> The glucanase-producing strain Streptomyces violaceum pGlu was cultured in 5 mL of tryptic soy medium (Becton-Dinkinson) at 28°C for 3 days and then harvested. The expression plasmid pGlu was extracted using a QIAprep Miniprep kit (QIAGEN). However, lysozyme (Sigma) was added to Buffer P1 to a final concentration of 2.5 mg / mL, and the cells of the pGlu strain were suspended in Buffer P1 containing lysozyme and allowed to stand at 37°C for 30 minutes, after which the expression plasmid pGlu was extracted according to the attached instructions.

[0060] <Construction of expression plasmid and transformation of host cells> The extracted pGlu plasmid was digested with restriction enzymes PvuII and EcoRV, and the resulting fragment was ligated and then introduced into Streptomyces lividans 1326 to generate transformants.

[0061] <Selection of enzyme-producing strains> After transformation, each colony was transferred to a tryptic soy agar medium (Becton-Dinkinson) and cultured at 28°C for 3 days. A loopful of the cells was dispersed in 10μL of 0.1N NaOH aqueous solution and heated at 95°C for 15 minutes. PCR was performed using this solution as a template. A sense primer (sequence number 6) and an antisense primer (sequence number 7) were designed based on the base sequence of the rep pIJ101 gene, which is a replication factor of the expression plasmid. The composition of the PCR reaction solution was as follows: GO Taq (Bio-Rad), 3μL, 100μM sense primer, 0.05μL, 100μM antisense primer, and distilled water were added to a total volume of 10μL. The PCR reaction conditions were as follows: 98°C, 3 minutes; step 2; 98°C, 15 seconds; step 3; 50°C, 30 seconds; step 4; 72°C, 1 minute. Steps 2 to 4 were repeated 30 times. This PCR yielded a unique amplification product of approximately 500 bp in 4 of 48 colonies.

[0062] The transformants from which the amplification products were obtained were transferred to 6 mL of tryptic soy medium (Becton-Dinkinson) in a test tube that had been autoclaved at 121°C for 20 minutes, and cultured at 28°C for 3 days with reciprocal shaking at 300 rpm. After centrifuging 1 mL of the culture (10,000 rpm, 10 minutes, room temperature), the supernatant was discarded, and the plasmid was extracted from the bacterial sediment in the same manner as described above.

[0063] The culture in tryptic soy medium was transferred to 6 mL of enzyme production medium and cultured at 28°C, 300 rpm for 3 days. The culture was centrifuged, and the supernatant was subjected to sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) to confirm enzyme production. According to the SDS-PAGE method, electrophoresis was performed at a fixed voltage of 200 V using Bio-Rad polyacrylamide gel (product name: Criterion TGX Stain-Free Any kD precast gel), and the electrophoretic image was photographed with a gel imaging device (product name: Gel Doc RZ Imager, Bio-Rad Stain-Free system) (Figure 1, Table 1).

[0064] <Confirmation of thiostrepton sensitivity> The selected strains that were confirmed to produce the enzyme were spread on tryptic soy agar medium containing thiostrepton (50 ppm) and cultured at 28°C for 3 days (Figure 2, Table 1).

[0065] <Confirmation of the base sequence of the expression plasmid of the enzyme-producing selected strain> After confirming enzyme production, the selected strains that were sensitive to thiostrepton were subjected to plasmid DNA preparation in the same manner as above, and analyzed with a DNA sequencer to determine the base sequence. The results are shown in Table 1.

[0066] [Table 1] Colonies No. 1, 17, and 26 had a portion of the tsr gene deleted by restriction enzyme digestion and did not actually show drug resistance, whereas colony No. 38 maintained the tsr gene and was drug resistant.

[0067] <Example 2> Deletion of drug resistance genes by PCR The following PCR reaction was carried out using plasmid DNA extracted from the glucanase-producing strain Streptomyces violaceum pGlu as a template.

[0068] A sense primer (SEQ ID NO: 8) with an EcoRV site added and an antisense primer (SEQ ID NO: 9) with an EcoRV site added were designed. The PCR reaction solution consisted of 5 μL of 10×PCR Buffer for KOD-plus (Toyobo Co., Ltd.), 300 nM of each primer, 0.2 mM of each dNTP mixture, 1 mM MgSO4, 5% DMSO, and 1.0 unit of KOD-plus-DNA Polymerase, to which distilled water was added to make a total volume of 50 μL. The PCR reaction conditions were as follows: Step 1; 98°C, 2 minutes; Step 2; 98°C, 15 seconds; Step 3; 60°C, 30 seconds; Step 4; 68°C, 5 minutes. Steps 2 to 4 were repeated 30 times. A unique amplification product of approximately 5 Kbp was obtained by this PCR. This amplified fragment was digested with EcoRV, ligated, and then introduced into Streptomyces lividans 1326 strain to produce a transformant.

[0069] The transformants were subjected to detection of plasmid DNA by colony PCR, detection of protein production by SDS-PAGE, and confirmation of thiostrepton sensitivity on agar medium in the same manner as in Example 1, and enzyme-producing strains were selected. The plasmid DNA of the selected enzyme-producing strains was analyzed with a DNA sequencer to determine the base sequence. As a result, the transformants expressed glucanase (Figure 3) and were thiostrepton sensitive (Figure 4).

[0070] <Evaluation of plasmid retention ability of enzyme-producing strains> The transformants were transferred to 50 mL of tryptic soy medium (Becton-Dinkinson) in a 500 mL baffled flask, and cultured at 28°C and 160 rpm for 3 days with shaking. 0.5 mL of this culture was transferred again to a flask containing 50 mL of tryptic soy medium and cultured in the same manner. This procedure was repeated 10 times. 0.1 mL of each culture was spread on tryptic soy agar medium (Becton-Dinkinson) and cultured at 28°C for 3 days. 24 colonies that appeared were randomly selected and subjected to the colony PCR method described above. The plasmid retention rate was calculated from the number of amplified products. The results are shown in Table 2.

[0071] [Table 2]

[0072] The transformants showed a very high plasmid retention rate of 96% even after 10 cycles of 3-day culture at 28°C, and the plasmid retention rate was maintained at 70% or higher throughout the 10 cycles.

[0073] <Example 3> Deletion of drug resistance genes by PCR and restriction enzyme treatment <Creation of a vector that does not contain a thiostrepton resistance gene> A vector not containing a thiostrepton resistance gene was prepared by ligating a PCR fragment obtained using the actinomycete plasmid pIJ702 as a template with a PCR fragment obtained using the E. coli plasmid pBluescript II KS+ as a template, as described below.

[0074] Using actinomycete plasmid pIJ702 as a template, a sense primer (SEQ ID NO: 10) with an EcoRV site added and an antisense primer (SEQ ID NO: 11) with an EcoRV site added were designed. The PCR reaction solution consisted of 5 μL of 10×PCR Buffer for KOD-plus (Toyobo Co., Ltd.), 300 nM of each primer, 0.2 mM of each dNTP mixture, 1 mM MgSO4, 5% DMSO, and 1.0 unit of KOD-plus-DNA Polymerase, to which distilled water was added to make a total volume of 50 μL. The PCR reaction conditions were as follows: Step 1; 98°C, 2 minutes; Step 2; 98°C, 15 seconds; Step 3; 60°C, 30 seconds; Step 4; 68°C, 3 minutes. Steps 2 to 4 were repeated 30 times. A specific amplification product of approximately 3 kbp was obtained by this PCR. This amplified fragment was digested with EcoRV.

[0075] Using the E. coli plasmid pBluescript II KS+ as a template, a sense primer (SEQ ID NO: 12) with an EcoRV site added and an antisense primer (SEQ ID NO: 13) with an EcoRV site added were designed. The PCR reaction solution consisted of 5 μL of 10×PCR Buffer for KOD-plus (Toyobo Co., Ltd.), 300 nM of each primer, 0.2 mM of each dNTP mixture, 1 mM MgSO4, 5% DMSO, and 1.0 unit of KOD-plus-DNA Polymerase, to which distilled water was added to make a total volume of 50 μL. The PCR reaction conditions were as follows: Step 1; 98°C, 2 minutes; Step 2; 98°C, 15 seconds; Step 3; 60°C, 30 seconds; Step 4; 68°C, 3 minutes and 30 seconds. Steps 2 to 4 were repeated 30 times. A specific amplification product of about 3.3 kbp was obtained by this PCR. This amplified fragment was digested with EcoRV. The two fragments were ligated and introduced into E. coli JM109 strain to produce a transformant. The plasmid contained in this transformant was named pIJ350RM. pIJ350RM is a shuttle vector between actinomycetes and E. coli.

[0076] <How to create actinomycete enzyme-producing strains> The following PCR reaction was carried out using plasmid DNA extracted from the phospholipase D-producing strain Streptomyces violaceum pPDN as a template.

[0077] A sense primer (SEQ ID NO: 14) with a KpnI site and an antisense primer (SEQ ID NO: 15) with a ClaI site were designed. The PCR reaction solution consisted of 5 μL of 10×PCR Buffer for KOD-plus (Toyobo Co., Ltd.), 300 nM of each primer, 0.2 mM of each dNTP mixture, 1 mM MgSO4, 5% DMSO, and 1.0 unit of KOD-plus-DNA Polymerase, to which distilled water was added to make a total volume of 50 μL. The PCR reaction conditions were as follows: Step 1; 98°C, 2 minutes; Step 2; 98°C, 15 seconds; Step 3; 60°C, 30 seconds; Step 4; 68°C, 2 minutes. Steps 2 to 4 were repeated 30 times. A specific amplification product of approximately 5 kbp was obtained by this PCR. This amplified fragment was digested with KpnI and ClaI. On the other hand, the newly constructed vector pIJ350RM was digested with KpnI and ClaI. After ligating both fragments, the resulting vector was introduced into E. coli JM109 strain to produce a transformant. The plasmid contained in this transformant was designated pIJ350RM-PDN.

[0078] The plasmid DNA (pIJ350RM-PDN) extracted from the transformant of E. coli JM109 was digested with EcoRV, ligated, and then introduced into Streptomyces lividans 1326 to produce a transformant. The plasmid contained in this transformant was designated pPDNΔtsr (Figure 5).

[0079] <Selection of enzyme-producing strains> Enzyme-producing strains were selected using the same procedures as in Examples 1 and 2. Plasmid DNA was prepared from the selected strains using the same method as above, and the selected strains were analyzed using a DNA sequencer to determine their base sequences. The results of confirming protein production using SDS-PAGE are shown in Figure 6. In the breeding strain, a band for phospholipase D (Figure 6) was confirmed at the 50 kDa position.

Claims

1. A method for producing an enzyme, comprising a step of culturing a cell transformed with a vector, the cell is an actinomycete; the enzyme is a protease, The method for producing the vector is characterized in that it contains a replication origin sequence derived from the plasmid vector pIJ101 and a gene for the enzyme, but does not contain an antibiotic resistance gene, a recombinase recognition sequence, a gene essential for cell survival, a sti sequence, or a lysis gene.

2. The method according to claim 1, wherein the gene essential for cell survival is a gene encoding a ribosome recycling factor or a gene encoding a translation initiation factor.

3. The method according to claim 1 or 2, wherein the gene for the enzyme is under the control of a promoter derived from a metalloendopeptidase gene or a phospholipase D (PLD) gene.

4. The method according to claim 1 or 2, wherein the gene for the enzyme is under the control of a terminator derived from the phospholipase D (PLD) gene.

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