Method for producing the 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 Art

[0002] For improving the preservation, taste, texture, etc. of foods, various enzymes are used. Enzymes are proteins that originally exist in natural living organisms or extracellularly, such as microorganisms and plants. Enzymes are industrially produced by microorganism culture, cell culture, plant cultivation, etc., and are purified and used. Also, in order to improve the productivity of enzymes, they may be highly expressed by genetic recombination.

[0003] In nature, organisms having plasmid DNA in addition to chromosomal DNA are known. A technique of artificially modifying plasmid DNA as a vector for expressing a target protein and imparting a new function to a host cell is generally known. Expression of a protein by this genetic recombination is performed by introducing a vector containing the gene of the protein into a host cell and culturing the host cell. Here, since the vector is only an unnecessary foreign factor for the host cell, there is a problem that the vector is likely to drop out during continuous culture. To avoid this problem, various methods for stably maintaining the vector in the host cell have been developed.

[0004] The most widely used method for stably maintaining a vector is a method of introducing an antibiotic resistance gene into the vector in addition to the gene of the protein and culturing the host cell in the presence of an antibiotic. Since the host cell cannot survive without the antibiotic resistance gene, the vector containing the antibiotic resistance gene is also stably maintained. However, there is a risk that the antibiotic resistance gene may be mixed into the enzyme product. There is also a concern that the generation of resistant bacteria may result from the outflow of the antibiotic resistance gene and the antibiotic itself into the environment.

[0005] Therefore, a method has been proposed in which antibiotic resistance genes are selectively excised and removed from the vector when the host cell growth reaches a certain level (Patent Documents 1-2). Other known methods include deleting essential genes that are normally present in the host and introducing those genes into the vector (Patent Document 3), introducing recombinase recognition sequences into the vector (Non-Patent Documents 1-2), and maintaining genes that express substances toxic to host cells in the host genome and introducing corresponding detoxification genes into the vector (Non-Patent Document 3). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2008-505620 [Patent Document 2] Japanese Patent Publication No. 2013-533743 [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 [Overview of the project] [Problems that the invention aims to solve]

[0008] While the methods described in Patent Documents 1 and 2 can reduce the amount of antibiotics used, they cannot eliminate the risk of contamination of enzyme products with antibiotic resistance genes. The method in Patent Document 3 requires the deletion of essential genes in the host cell, and the methods in Non-Patent Documents 1 to 3 require the introduction of specific sequences 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 the vector, without performing any special genetic manipulation on the host cell and without using drug resistance genes, etc. [Means for solving the problem]

[0009] As a result of the inventors' research, they discovered that it is possible to stably maintain the vector without performing any special genetic manipulation on host cells and without using drug resistance genes, thus completing the present invention.

[0010] In other words, the present invention relates to a method for producing a target protein, comprising the step of culturing cells transformed with a vector, wherein the vector contains the gene for the target protein but does not contain antibiotic resistance genes, recombinase recognition sequences, or genes essential for cell survival.

[0011] It is preferable that the genes essential for cell survival are genes encoding ribosome recycling factors or genes encoding translation initiation factors.

[0012] It is preferable that the cells are bacteria.

[0013] It is preferable that the bacteria are actinomycetes.

[0014] It is preferable that the target protein is an enzyme.

[0015] The enzyme is preferably 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 or chitinase.

Advantages of the Invention

[0016] In the production method of the present invention, without performing any specific genetic manipulation on the host cell and without using a drug resistance gene, the vector can be stably maintained. The risk of contamination of the enzyme product with the antibiotic resistance gene and the risk of outflow of the antibiotic resistance gene into the environment can be eliminated.

Brief Description of the Drawings

[0017] [Figure 1] [ Shows the detection results of the target protein in Example 1. [Figure 2] Shows the test results of sensitivity to antibiotics in Example 1. [Figure 3] Shows the detection results of the target protein in Example 2. [[ID=SS]] [Figure 4] Shows the test results of sensitivity to antibiotics in Example 2. [Figure 5] Shows an overview of the method for preparing the vector in Example 3. [Figure 6] Shows the detection results of the target protein in Example 3.

Modes for Carrying Out the Invention

[0018] <Method for Producing a Target Protein> The present invention provides a method for producing the target protein, comprising the step of culturing cells transformed with a vector, wherein the vector contains the gene for the target protein but does not contain antibiotic resistance genes, recombinase recognition sequences, or genes essential for cell survival.

[0019] In the cell culture process, cells transformed with the vector, 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 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 or organic acid ammonium salts or 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.

[0020] 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.

[0021] 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 protein. Typically, culture is carried out under aerobic conditions such as shaking culture or aeration stirring culture in 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 is preferably pH 3.0 to 9.0, and more preferably pH 6.0 to 8.0. The culture time is preferably 24 to 96 hours, and more preferably 48 to 72 hours. Alternatively, subculturing may be performed by diluting the culture medium 10 to 250 times. While 2 to 4 subculturing cycles are preferred to maintain high purity of the target protein, the plasmid can be stably maintained even after 10 subculturing cycles.

[0022] The method for producing the target protein preferably includes a step of purifying the target protein after a step of culturing cells. 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 culture supernatant can be used instead of the cell-free extract and purified in the same manner.

[0023] <cell> The cells used are not particularly limited as long as they are capable of expressing the target protein; microbial cells, animal cells, or plant cells can all be used. From the viewpoint of the 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 Streptomyces and Rhodococcus, for which host-vector systems have been developed; and bacteria such as 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 those belonging to the genera Saccharomyces, Kluyveromyces, Schizosaccharomyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidium, 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.

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

[0026] A specific strain of Streptomyces lividan is Streptomyces lividan 1326. This strain is also known as Streptomyces violaceoruber 1326, and its culture and purified products have been confirmed to be safe for use in food.

[0027] An example of the genus Escherichia is Escherichia coli.

[0028] 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.

[0029] The cells described above may be cells that naturally express the target protein, or they may 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 them with a vector.

[0030] <Vector> Any vector capable of expressing the target protein within a cell is acceptable, and specific forms include plasmid vectors, phage vectors, and cosmid vectors. Plasmid vectors are preferred from the viewpoint of ease of transformation. The total length of the vector, including the gene sequence of the target protein described later, is preferably 3,000 to 10,000 bp, and more preferably 4,500 to 6,500 bp. Vectors with a total length exceeding 10,000 bp tend to become unstable within cells, and those with a total length less than 3,000 bp limit the length of the gene sequence of the target protein that can be used.

[0031] <Target Protein> The target protein is not particularly limited as long as it can be expressed within the cell, but examples 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.

[0032] 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. Among these, phospholipase, glucanase, and protease are preferred.

[0033] Examples of DNA encoding phospholipase include the following DNAs: (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 that exhibits 85% or more sequence identity with the sequence shown in Sequence ID No. 1 of the sequence listing and has phospholipase activity; (c) DNA encoding a polypeptide having phospholipase activity, comprising a nucleotide sequence in which one or more nucleotides are deleted, inserted, substituted and / or added in the nucleotide sequence shown in Sequence ID No. 1 of the sequence listing.

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

[0035] 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.

[0036] Examples of DNA encoding glucanase include the following DNAs: (a), (b), or (c). (a) DNA containing the nucleotide sequence shown in Sequence ID No. 2 of the sequence listing; (b) DNA encoding a polypeptide that exhibits 85% or more sequence identity with the sequence shown in Sequence ID No. 2 of the sequence listing and has glucanase activity; (c) DNA encoding a polypeptide having glucanase activity, comprising a nucleotide sequence in which one or more nucleotides are deleted, inserted, substituted and / or added in the nucleotide sequence shown in Sequence ID No. 2 of the sequence listing.

[0037] The sequence identity with the base sequence shown in Sequence ID No. 2 is preferably 90% or higher, more preferably 95% or higher, and even more preferably 98% or higher.

[0038] In the nucleotide sequence shown in Sequence ID No. 2 of the sequence listing, the number of deletions, insertions, substitutions and / or additions 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] <Starting array for duplication> A vector needs to have a replication initiation sequence in order to be maintained and amplified within cells. Examples of replication initiation sequences include those derived from plasmid vector pIJ101, plasmid vector pSG5, and plasmid vector SLP2. Among these, the replication initiation sequence derived from pIJ101 is preferred. The length of the replication initiation sequence is preferably 500 to 2000 bp, and more preferably 1000 to 1500 bp.

[0041] <Promoter Sequence> The vector contains a promoter sequence within the gene encoding the target protein. The promoter sequence may be the promoter inherent to the target protein or a heterologous promoter. When using a heterologous promoter, 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.

[0042] Examples of DNA containing promoter sequences derived from metalloendopeptidase genes include the following DNAs: (a), (b), or (c). (a) DNA containing the nucleotide sequence shown in Sequence ID No. 3 of the sequence listing; (b) DNA that exhibits 85% or more sequence identity with the sequence shown in Sequence ID No. 3 of the sequence listing and induces 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 in the nucleotide sequence shown in Sequence ID No. 3 of the sequence listing, which induces the expression of the target protein in a cell.

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

[0044] 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.

[0045] Examples of DNA containing a promoter sequence derived from the phospholipase D (PLD) gene include the following DNAs: (a), (b), or (c). (a) DNA containing the nucleotide sequence shown in Sequence ID No. 4 of the sequence listing; (b) DNA that exhibits 85% or more sequence identity with the sequence shown in Sequence ID No. 4 of the sequence listing and induces 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 in the nucleotide sequence shown in Sequence ID No. 4 of the sequence listing, which induces the expression of the target protein in a cell.

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

[0047] 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.

[0048] <Terminator Sequence> A vector contains a terminator sequence within the gene encoding the target protein 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, metalloendopeptidase gene, and amylase gene. Among these, the terminator sequence derived from the phospholipase D (PLD) gene is preferred.

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

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

[0051] 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.

[0052] <Antibiotic resistance genes, etc.> The vector used in the present invention is characterized by not containing antibiotic resistance genes, recombinase recognition sequences, and genes essential for cell survival.

[0053] When using antibiotic resistance genes, antibiotics are added to the culture medium as a selective pressure, which can lead to the emergence of resistant bacteria due to the release of the antibiotic itself or the antibiotic resistance gene into the environment. The vector used in this invention does not contain antibiotic resistance genes, thus eliminating these risks. Antibiotic resistance genes refer to genes that maintain the activity to degrade antibiotics or inhibit the action of antibiotics. Examples of antibiotics used here include thiostrepton, penicillin, kanamycin, vancomycin, erythromycin, biomycin, neomycin, streptomycin, tetracycline, and chloramphenicol.

[0054] Recombinase recognition sequences have been used in Patent Documents 1-2 and Non-Patent Documents 1-2 to selectively excise and remove antibiotic resistance genes from vectors. Examples of such recombinases include Cre, Flop, R, XerC, XerD, RipX, and CodV. Other 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 the 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 the toxin-antitoxin system.

[0056] <Vector manufacturing and cell transformation> The vector used in the present invention is obtained by ligating the gene, replication initiation sequence, promoter sequence, etc., of the target protein using a known method. Examples of ligation methods include restriction enzyme cleavage and DNA ligation. The DNA cleaved by the restriction enzyme may be 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 method, and particle gun method 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. Hereinafter, unless otherwise specified, "parts" or "%" means "parts by weight" or "% by weight," respectively.

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

[0060] <Expression plasmid creation and host cell transformation> The extracted pGlu plasmid was digested with restriction enzymes PvuII and EcoRV. The resulting fragments were ligated and then introduced into Streptomyces lividans 1326 strain to produce transformants.

[0061] <Selection of enzyme-producing strains> Each transformed colony was transplanted onto tryptic soy agar (Becton Dinkinson) and cultured at 28°C for 3 days. The bacterial cells on a loop were dispersed in 10 μL of 0.1N NaOH aqueous solution and heat-treated at 95°C for 15 minutes. PCR was performed using this solution as a template. Sense primers (SEQ ID NO: 6) and antisense primers (SEQ ID NO: 7) were designed from the nucleotide sequence of the rep pIJ101 gene, a replication factor of the expression plasmid. The PCR reaction mixture composition was as follows: 3 μL of GO Taq (Bio-Rad), 0.05 μL of 100 μM sense primer, 0.05 μL of 100 μM antisense primer, and distilled water added to a total volume of 10 μL. The PCR reaction conditions were as follows: 98°C, 3 min; step 2; 98°C, 15 sec; step 3; 50°C, 30 sec; step 4; 72°C, 1 min. Steps 2 to 4 were repeated 30 times. This PCR yielded a unique amplification product of approximately 500 bp in 4 out of 48 colonies.

[0062] The transformants from which amplification products were obtained as described above were transferred to 6 mL of triptych soy medium (Becton Dinkinson) in test tubes autoclaved at 121°C for 20 minutes, and incubated at 28°C at 300 rpm with reciprocating shaking for 3 days. After centrifuging 1 mL of the culture medium (10,000 rpm, 10 minutes, room temperature), the supernatant was discarded, and plasmid extraction was performed using the bacterial precipitate in the same manner as described above.

[0063] Furthermore, the culture medium in tryptic soy was transferred to 6 mL of enzyme production medium and incubated at 28°C and 300 rpm for 3 days. The culture medium was centrifuged, and enzyme production was confirmed by subjecting the supernatant to sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE). Electrophoresis was performed at a fixed voltage of 200 V using a Bio-Rad polyacrylamide gel (product name Criterion TGX Stain-Free Any kD precast gel) according to the SDS-PAGE method, and electrophoresis images were captured using a gel imaging system (product name Gel Doc RZ Imager, Bio-Rad Stain-Free system) (Figure 1, Table 1).

[0064] <Confirmation of thiostrepton sensitivity> Selected strains that demonstrated enzyme production were spread on tryptic soy agar containing thiostrepton (50 ppm) and incubated at 28°C for 3 days (Figure 2, Table 1).

[0065] <Confirmation of the nucleotide sequence of the expression plasmid of the enzyme-producing selected strain> For selected strains that confirmed enzyme production and were thiostrepton-sensitive, plasmid DNA was prepared using the same method as described above, analyzed with a DNA sequencer, and the base sequence was determined. The results are shown in Table 1.

[0066] [Table 1] Colonies No. 1, 17, and 26 had partial deletions of the tsr gene via restriction enzymes and did not actually exhibit drug resistance. Colony No. 38 retained the tsr gene and was drug resistant.

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

[0068] Sense primers with an EcoRV moiety (SEQ ID NO: 8) and antisense primers with an EcoRV moiety (SEQ ID NO: 9) were designed. The PCR reaction mixture composition was as follows: 5 μL of 10× PCR Buffer for KOD-plus (Toyobo Co., Ltd.), 300 nM each of primers, 0.2 mM each of dNTP mixture, 1 mM MgSO4, 5% DMSO, and 1.0 unit of KOD-plus-DNA Polymerase, to which distilled water was added to bring the total volume to 50 μL. The PCR reaction conditions were as follows: Step 1; 98°C, 2 min; Step 2; 98°C, 15 sec; Step 3; 60°C, 30 sec; Step 4; 68°C, 5 min. Steps 2 to 4 were repeated 30 times. This PCR yielded a specific amplification product of approximately 5 kbp. This amplified fragment was digested with EcoRV, ligated, and then introduced into Streptomyces lividans 1326 strain to produce transformants.

[0069] Transformants were selected for enzyme production by detecting plasmid DNA using colony PCR, detecting protein production using SDS-PAGE, and confirming thiostrepton sensitivity on agar, similar to Example 1. Plasmid DNA from the selected enzyme-producing strains was analyzed using a DNA sequencer and its base sequence was determined. As a result, the transformants expressed glucanase (Figure 3) and were sensitive to thiostrepton (Figure 4).

[0070] <Evaluation of plasmid retention ability of enzyme-producing strains> Transformants were transferred to 50 mL of tryptic soy medium (Becton Dinkinson) in a 500 mL baffled flask and cultured with shaking at 28 °C and 160 rpm for 3 days. 0.5 mL of this culture was then transferred to a flask containing another 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 onto tryptic soy agar (Becton Dinkinson) and cultured at 28 °C for 3 days. Twenty-four colonies were randomly selected, and the colony PCR method described above was performed. Plasmid retention was calculated from the number of amplified products observed. The results are shown in Table 2.

[0071] [Table 2]

[0072] The transformants maintained an extremely high plasmid retention rate of 96% even after 10 cycles of 3-day incubation at 28°C. Furthermore, the plasmid retention rate remained above 70% throughout the 10 incubation cycles.

[0073] <Example 3> Deletion of drug resistance genes by PCR and restriction enzyme treatment <Construction of a vector that does not contain the thiostrepton resistance gene> A vector that does not contain the 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, using the method described below.

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

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

[0076] <Method for producing actinomycete enzyme-producing strains> Plasmid DNA extracted from the phospholipase D-producing strain Streptomyces violaceoluber pPDN was used as a template for the following PCR reaction.

[0077] Sense primers with a KpnI moiety (SEQ ID NO: 14) and antisense primers with a ClaI moiety (SEQ ID NO: 15) were designed. The PCR reaction mixture composition was as follows: 5 μL of 10× PCR Buffer for KOD-plus (Toyobo Co., Ltd.), 300 nM each of the primers, 0.2 mM each of the dNTP mixture, 1 mM MgSO4, 5% DMSO, and 1.0 unit of KOD-plus-DNA Polymerase, to which distilled water was added to bring the total volume to 50 μL. The PCR reaction conditions were as follows: Step 1; 98°C, 2 min; Step 2; 98°C, 15 sec; Step 3; 60°C, 30 sec; Step 4; 68°C, 2 min. Steps 2 to 4 were repeated 30 times. This PCR yielded a specific amplification product of approximately 5 kbp. This amplified fragment was digested with KpnI and ClaI. Meanwhile, the newly constructed vector pIJ350RM was digested with KpnI and ClaI. After ligation of both fragments, they were introduced into E. coli strain JM109 to produce transformants. The plasmid contained in these transformants was designated pIJ350RM-PDN.

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

[0079] <Selection of enzyme-producing strains> Enzyme-producing strains were selected using the same procedure as in Examples 1 and 2. Plasmid DNA was prepared from the selected strains using the same method as described above, analyzed with a DNA sequencer, and the base sequence was determined. Figure 6 shows the results of confirming protein production by SDS-PAGE. A phospholipase D band (Figure 6) was confirmed at the 50 kDa position in the bred strains.

Claims

1. A method for producing an enzyme, comprising the step of culturing cells transformed with a vector, The aforementioned cells are actinomycetes, The aforementioned enzyme is a protease, A method for producing a vector characterized in that the vector comprises a replication initiation sequence derived from plasmid vector pIJ101 and the gene for the enzyme, but does not contain an antibiotic resistance gene, a recombinase recognition sequence, or a gene essential for cell survival.

2. The manufacturing 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 manufacturing 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 manufacturing 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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