Plasmid vectors, shuttle vectors, and protein manufacturing methods

Abstract

To provide plasmid DNA with a novel replication origin that can stably introduce foreign genes into actinomycetes. [Solution] The plasmid vector according to the present disclosure comprises (a) a nucleotide sequence having a specific sequence, or (b) a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted in the nucleotide sequence having the specific sequence.

Description

[Technical Field]

[0001] This invention relates to plasmid vectors, shuttle vectors, and methods for producing proteins. [Background technology]

[0002] The development of upcycling technology for waste rubber is one of the important challenges toward realizing a circular economy. Research is underway to utilize the function of microorganisms with rubber-degrading capabilities in the biological decomposition of waste rubber and upcycling technologies that convert waste rubber into useful substances.

[0003] In recent years, the Gordonia sp. J1A strain, which possesses the ability to degrade acrylonitrile butadiene rubber (NBR), has been discovered, and its use in the degradation and upcycling of NBR is anticipated (Non-Patent Literature 1).

[0004] In addition to the J1A strain mentioned above, other bacteria from the genus Gordonia have been reported to possess the ability to decompose hydrocarbons, various environmental pollutants, xenobiotics, and naturally occurring compounds that are not readily biodegradable (Non-Patent Literature 2). Furthermore, many bacteria in the order Actinomycetes, to which Gordonia belongs, are used for antibiotic production and as suitable hosts for the secretion of heterologous proteins derived from various Gram-positive bacteria. Therefore, there is a need for the development of genetic engineering technologies that can be used for Gordonia and the order Actinomycetes. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Tsuyoshi Chiba, Takuma Nakaarai, and Daisuke Sugimori, Estimation of the rubber-degrading enzyme gene of the nitrile rubber-degrading bacterium Gordonia sp. J1A strain, Proceedings of the Enzyme Engineering Research Society Conference, November 10, 2023, Vol. 90, p. 77. [Non-Patent Document 2] M. Arenskotter, D. Broker, and A. Steinbuchel. (2004). Biology of the metabolically diverse genus Gordonia. Appl. Environmen. Microbiol. 70(6):3195-3204. [Overview of the project] [Problems that the invention aims to solve]

[0006] The objective of this invention is to provide plasmid DNA that can stably introduce foreign genes into bacteria of the Actinomycetes order, including the genus Goldonia. [Means for solving the problem]

[0007] As a result of diligent research, the inventors identified the origin of replication in the natural plasmid of Gordonia sp.J1A strain, discovered a protein production system containing plasmid DNA that can stably introduce foreign genes into Actinomycetes bacteria, including the genus Gordonia, and thus completed the present invention.

[0008] In other words, the present invention includes the following embodiments. <1> (a) A polynucleotide consisting of bases 3463-4605 and 1-266 of Sequence ID No. 1, or its complementary strand, or (b) A polynucleotide or its complementary chain consisting of a nucleotide sequence in which one or more nucleotides are substituted, deleted, and / or inserted in the nucleotide sequence of (a), and which is replicable in Actinomycetes bacteria. A plasmid vector that can replicate in Actinomycetes bacteria, including the above. <2> (1) (a) A polynucleotide consisting of bases 3463-4605 and 1-266 of Sequence ID No. 1, or its complementary strand, or (b) Polynucleotides or their complementary chains consisting of a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted in the nucleotide sequence of (a), and which are replicable in Actinomycetes bacteria, and (2) Origin of replication polynucleotides that function in E. coli A shuttle vector between Actinomycetes bacteria and Escherichia coli, including the aforementioned. <3> The system further comprises at least one sequence selected from the group consisting of promoter sequences, cloning site sequences, and terminator sequences. <1> Plasmid vector or <2> The shuttle vector described above. <4> Further including drug resistance genes, <1> from <3> A plasmid vector or shuttle vector as described in any one of the following. <5> The aforementioned drug resistance gene is an ampicillin resistance gene, a chloramphenicol resistance gene, or a kanamycin resistance gene. <4> Plasmid vectors or shuttle vectors as described above. <6> The number of copies maintained within the Actinomycetes bacteria is 40 or more. <1> from <6> A plasmid vector or shuttle vector as described in any one of the following. <7> <1> from <6> A method for producing a protein, comprising the step of introducing a plasmid vector or shuttle vector described in any one of the following into a host. <8> The vector is introduced using electroporation, and the electroporation is performed under conditions of 15 kV / cm and 10 μF. <7> A method for producing the protein described above. [Effects of the Invention]

[0009] According to the present invention, a plasmid vector that solves the above problems can be provided, enabling the stable introduction of foreign genes into Actinomycetes bacteria, including the genus Gordonia, and the production of proteins. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 is a photograph of the waste NBR sample. [Figure 2] Figure 2 is a schematic diagram of the natural plasmid of strain J1A whose existence was suggested by genomic analysis. [Figure 3] Figure 3 is an electrophoresis gel photograph of the DNA of the natural plasmid pJ1A of strain J1A. U represents Uncut, C represents Cut by EcoRI, and M represents Size marker respectively. The extraction of J1A natural plasmid DNA requires the cell lysis process by lysozyme. [Figure 4] Figure 4 shows the composition (ori, rep, DNA binding protein) of the pJ1A minimal replicon inferred from bioinformatics analysis. [Figure 5] Figure 5 shows the results of the susceptibility tests of Gordonia sp. strain J1A against ampicillin (Amp), streptomycin (Sm), chloramphenicol (Cm), and kanamycin (Km). The concentration of each is 20 μg / mL. The left shows the state immediately after inoculation, and the right shows the state on the 5th day of culture respectively. [Figure 6] Figure 6 shows the results of the chloramphenicol (Cm) susceptibility test of Gordonia sp. J1A. Concentrations of 0, 1.0, 5.0, 10, and 20 μg / mL Cm were tested respectively. The upper part is liquid culture, and the lower part is solid culture. The left shows the state immediately after inoculation, and the right shows the state on the 5th day of culture respectively. [Figure 7] Figure 7 is a schematic diagram of the experiment to construct a shuttle vector. pBBR1ori, The broad-host-range origin of replication; pBBR1rep, Replication initiation protein gene; Cmr; Chloramphenicol acetyltransferase gene; mob, mobile genetic elements; oriT, origin of transfer; lacZα, β-galactosidase gene lacZα fragment. [Figure 8]Figure 8 shows electrophoretic gel images of the cloning fragments. (A) PCR product before purification, (B) PCR product after purification. M indicates the size marker, I indicates the insert sequence (1,409 bp), and V indicates the vector sequence (4,707 bp). [Figure 9] Figure 9 shows the electrophoresis gel images of plasmid extracts derived from ligation products. M indicates the size marker, C indicates the EcoRV cut, and the numbers indicate the sample numbers. Candidate target plasmids are shown in red. [Figure 10] Figure 10 shows the appearance of microbial colonies obtained from an electroporation experiment (17 days into culture). [Figure 11] Figure 11 shows plasmid extracts from transformants. M indicates the size manufacturer, U indicates uncut, C indicates cut (pJ1A was digested with BamHI, pBBJ1 with EcoRV), and the numbers indicate the sample numbers. [Figure 12] Figure 12 is a schematic diagram illustrating the principle of relative quantification of plasmid copy number. CT stands for Threshold cycle; E stands for Amplification efficiency. [Figure 13] Figure 13 shows the calibration curves for the gyrB set and rep set for validation of ΔΔCT. The black circles and solid lines represent the gyrB set, and the black triangles and dashed lines represent the rep set. The PCR amplification efficiency was calculated from each slope, and the average value was used. [Figure 14] Figure 14 shows the PCR amplification specificity of (A) gyrB set and (B) rep set, with the red line representing the melting curve of the amplification product of the target sample and the gray line representing the melting curve of the calibrator sample. M is the size marker, T is the target sample, and C is the calibrator sample. [Modes for carrying out the invention]

[0011] One embodiment of the present invention will be described in detail below, but the scope of the present invention is not limited to the embodiment described herein, and various modifications can be made without departing from the spirit of the invention. Furthermore, if multiple upper and lower limits are described for a particular parameter, any combination of these upper and lower limits can be used to obtain a suitable numerical range. In this specification, the base position of a base sequence is counted with the first base of the base sequence shown in the specified sequence number as the 1st base.

[0012] The present invention relates to a plasmid vector that can replicate in Actinomycetes bacteria, comprising (a) or (b) below: (a) A polynucleotide consisting of bases 3463-4605 and 1-266 of Sequence ID No. 1, or its complementary strand, or (b) A polynucleotide or its complementary chain consisting of a nucleotide sequence in which one or more nucleotides are substituted, deleted, and / or inserted in the nucleotide sequence of (a), and which is replicable in Actinomycetes bacteria.

[0013] Furthermore, the present invention relates to a shuttle vector between Actinomycetes bacteria and Escherichia coli, comprising (a) or (b) and (2) of (1) below: (1) (a) A polynucleotide consisting of bases 3463-4605 and 1-266 of Sequence ID No. 1, or its complementary strand, or (b) Polynucleotides or their complementary chains consisting of a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted in the nucleotide sequence of (a), and which are replicable in Actinomycetes bacteria, and (2) A replication origin polynucleotide that functions in E. coli.

[0014] The polynucleotides (a) and (1)(a) are common to the plasmid vector, the actinomycetes bacteria, and the shuttle vector, and the polynucleotides (b) and (1)(b) are common to both.

[0015] Sequence ID 1 is the nucleotide sequence of the natural plasmid of Gordonia sp. J1A strain, which is a 4605-base-pair circular plasmid as shown in Figure 2. The 4605th base and the 1st base are consecutive. Gordonia sp. J1A strain is deposited with the National Institute of Technology and Evaluation (NITE) (Accession number: P-03989).

[0016] The vector (hereinafter referred to as plasmid 1) containing the polynucleotide sequence consisting of nucleotides 3463-4605 and 1-266 of Sequence ID No. 1 and its complementary strand contains putative ori, repA, and repB, which were identified in the natural plasmid of Gordonia sp. J1A strain. Plasmid 1 is capable of autonomous replication within Actinomycetes bacteria, including those of the genus Gordonia. The region consisting of nucleotide sequences 3463-4605 and 1-266 of Sequence ID No. 1 is a contiguous region.

[0017] In the polynucleotide of (b), "several" means, for example, in the range of 2 to 50, 2 to 40, 2 to 30, or 2 to 20. From the viewpoint of increasing the likelihood of replication within Actinomycetes bacteria, it is preferably in the range of 2 to 10, more preferably 2 to 8, even more preferably 2 to 6, and even more preferably 2 to 4. Base substitutions, deletions, and / or insertions may be present in sequences other than ori, for example. Substitutions, deletions, and / or insertions of one or more bases in a polynucleotide can be carried out as appropriate by techniques known to those skilled in the art at the time of filing.

[0018] Plasmid 1 was prepared in general as follows. See the examples for details. Gordonia sp. J1A strain was cultured in LB medium containing 2.0 w / v% Tween 80, plasmid isolation was performed, and sequencing was determined. The minimum region required for replication was identified, and a plasmid containing the replication replicon (hereinafter referred to as the replication replicon) was obtained.

[0019] As shown in Figure 4, plasmid 1 contains the putative ori, the RepA gene, and the RepB gene as the replication replicon. The putative Ori is the region from nucleotides 4548-4605 and 1-266 of the sequence of SEQ ID NO: 1, and this region is contiguous. The RepA protein is encoded in the complementary strand of the region from nucleotides 3759-4547 of the sequence of SEQ ID NO: 1. The RepB protein is encoded in the complementary strand of the region from nucleotides 3463-3762 of the sequence of SEQ ID NO: 1. The RepA and RepB genes are transcribed counterclockwise in the plasmid shown in Figure 4. The amino acid sequences of the RepA and RepB proteins are shown in SEQ ID NOs: 2 and 3, respectively.

[0020] The E. coli-functional origin of replication polynucleotides included in the shuttle vector between Actinomycetes and Escherichia coli of the present invention can be appropriately selected from existing E. coli-derived origin of replication polynucleotides or origin of replication polynucleotides that function in E. coli. For example, pBBRoriV from the pBBR1 plasmid isolated from Bordetella bronchiseptica can be cited. pBBRoriV is from positions 1023 to 1792 of Sequence ID No. 4, which shows the entire nucleotide sequence of pBBR1MCS1, and pBBR1rep is from positions 1793 to 2455 of Sequence ID No. 4. Other E. coli-functional origin of replication polynucleotides include pBR322ori and modified pBR322ori (pUC18 / pUC19ori) from the E. coli-derived plasmid pMB1. It can also be ori from the E. coli-derived p15A plasmid or pSC101 plasmid.

[0021] The plasmid vectors and shuttle vectors of the present invention may include at least one sequence selected from the group consisting of a cloning site, a promoter, and a terminator sequence. The cloning site has a specific nucleotide sequence for inserting a protein-coding DNA fragment into the plasmid vector. The specific nucleotide sequence is, for example, a restriction enzyme recognition sequence. The cloning site may also be a multi-cloning site having multiple restriction enzyme sites. The cloning site is located downstream of the promoter. The terminator is ligated downstream of the cloning site.

[0022] The promoter and terminator can be derived from any gene that functions in the host bacterium. For example, the promoter and terminator of the glycolytic enzyme glyceraldehyde 3'-phosphate dehydrogenase (GAPDH) from bacteria of the genus Gordonia or other Actinomycetes, or the promoter and terminator of phosphoglycerate kinase (PGK), may be used.

[0023] The plasmid vectors and shuttle vectors of the present invention may further contain drug resistance genes. The drug resistance genes may be ampicillin resistance genes, chloramphenicol resistance genes, or kanamycin resistance genes.

[0024] The plasmid vectors and shuttle vectors of the present invention are characterized by a high copy number retention within Actinomycetes bacteria. In the examples described below, the copy number retained within Actinomycetes bacteria was 30 to 180 copies when transformed with the shuttle vector of the present invention. This is higher than the approximately 10 to 40 copies retained when transformed with the pJ1A strain possessed by the J1A wild type. This characteristic allows for the stable introduction of foreign genes.

[0025] The present invention further includes a method for producing a protein, comprising the step of introducing the plasmid vector or shuttle vector of the present invention into a host. The host may be any bacterium of the order Actinomycetales, such as species or strains of the genera Goldonia, Nocardia (e.g., N. asteroides), Streptomyces (e.g., S. viridosporus, S. griseus, S. avermitilis, S. venezuelae), Rhodococcus (e.g., R. ruber), Amycolatopsis, or Actinomyces. The shuttle vector can replicate in Escherichia coli as well as in Actinomycetes. The protein produced by the protein production method of the present invention is any protein. For example, when using a bacterium of the genus Goldonia as the host, a plasmid vector or shuttle vector containing an enzyme gene involved in rubber degradation can be introduced into the host to produce a rubber-degrading enzyme for the purpose of promoting the degradation of waste rubber.

[0026] Methods for introducing a vector into a host include transformation methods such as heat shock and electroporation, transgenic conjugation (conjugation transfer), microparticle cannon, and lipofection. In one embodiment, the method for introducing a vector into a host may be electroporation. For example, vector introduction into a Goldonia host can be performed by electroporation under conditions of 15 kV / cm and 10 μF. This allows for efficient introduction of the foreign gene. [Examples]

[0027] Example 1: Culture of Gordonia sp. J1A strain The Gordonia sp. J1A strain (hereinafter referred to as the J1A strain) was provided by Associate Professor Daisuke Sugimori of the Faculty of Symbiotic Systems Science and Engineering, Fukushima University. The J1A strain is a bacterial strain isolated from wastewater from a factory wastewater treatment plant and circulation outlet, and has been reported to degrade acrylonitrile butadiene rubber (Tsuyoshi Chiba, Takuma Nakaarai, and Daisuke Sugimori, Estimation of the rubber-degrading enzyme gene of the nitrile rubber-degrading bacterium Gordonia sp. J1A strain, Proceedings of the Enzyme Engineering Research Conference, November 10, 2023, Vol. 90, p. 77).

[0028] Culture medium composition and culture conditions Culture of the J1A strain The J1A strain formed clumps in standard LB medium. This clumping is undesirable from the perspective of drug resistance and the difficulty of measuring the optical density of the culture medium. Therefore, we attempted to eliminate clumping by adding polyoxyethylene sorbitan monooleate (Tween 80). Ultimately, the addition of 2.0 w / v% Tween 80 successfully eliminated J1A clumping. Accordingly, in the following experiments, the J1A strain was cultured in LB medium containing 2.0 w / v% Tween 80 (TwLB medium).

[0029] Bacterial collection method All of the obtained culture medium was placed in a centrifuge tube and centrifuged (4°C, 5,000×g, 15 min), and the supernatant was discarded. 30 mL of deionized water was added to the resulting precipitate, and it was resuspended using a Pasteur pipette. The entire suspension was transferred to a 50 mL Falcon tube and centrifuged (4°C, 6,600×g, 15 min), and the supernatant was discarded. The process of adding 30 mL of deionized water to the resulting precipitate, resuspending, and centrifuging was repeated a total of three times to wash the bacterial pellet. In the case of culture medium containing NBR, the NBR was collected in a separate Falcon tube and thoroughly washed with deionized water. The Falcon tube samples containing liquid were capped with Parafilm and allowed to solidify overnight in a -30°C freezer. The reason for solidification is that freeze-drying in the liquid state would cause the liquid to boil over due to a decrease in vapor pressure. Dried bacterial cell samples were obtained by freeze-drying frozen bacterial cells overnight using a freeze-dryer, model FDL-2000 (EYELA, Tokyo, Japan). During the freeze-drying process, holes were made in the Parafilm lid to ensure airflow. The weight of the dried bacterial cell samples obtained by freeze-drying was measured using an electronic balance. Furthermore, the NBR mass after culturing was measured and subtracted from the NBR mass measured before culturing to calculate the mass change. This change was then divided by the NBR mass before culturing to obtain the percentage change in NBR mass.

[0030] Example 2: Development of a transformation method for Gordonia sp. J1A strain Confirmation of NBR degradation ability and PHA synthesis ability in the J1A wild-type strain. The J1A strain was cultured in various culture media with different nutritional conditions, and its PHA accumulation capacity was verified. Specifically, the following three media were used: (i) NB medium, (ii) MS medium with glucose as the main source, and (iii) MS medium with NBR as the main carbon source.

[0031] method J1A was cultured overnight in 5.0 mL of LB medium. Next, 2.0 v / v% of the J1A culture solution was seeded onto 100 mL of NB medium (Table 1), 100 mL of MS medium with glucose as the main carbon source (MS / Glc medium) (Table 2), or 200 mL of MS medium with NBR as the main carbon source (MS / NBR medium) (Table 3), and cultured with shaking at 30°C and 120 rpm. Culture in NB medium and MS medium containing glucose was carried out for 5 days, and culture in MS medium containing NBR was carried out for 21 days. NBR samples were cut to approximately 5.0 × 10 mm (Figure 1). After the completion of culture, the cells were collected, and finally, freeze-dried bacterial samples were obtained.

[0032] [Table 1] [Table 2] [Table 3]

[0033] Example 3: Isolation and Characterization of Natural Plasmids of the J1A Strain Whole-genome analysis suggested that strain J1A possesses only one native plasmid (Figure 2). To attempt to isolate this native plasmid, plasmid extraction was performed from strain J1A. Since the standard protocol using commercially available kits yielded little to no extraction of the J1A native plasmid, a method for extracting native plasmids from Rhodococcus bacteria reported by O. Tkachuk-Saad and J. Prescott. ((1991). J. Clin. Microbiol. 29(12):2696-2700) was partially applied. First, strain J1A was cultured in 5 mL LB medium at 30°C for two nights with shaking. The culture medium was centrifuged (4°C, 5,000 × g, 10 min), and the resulting cell pellet was suspended in 5 mL lysozyme buffer (Table 4) and incubated at 37°C, 120 rpm, 60 min. After incubation, the lysate was recovered by centrifugation (4°C, 5,000×g, 10 min), and plasmid DNA was extracted from the resulting pellet according to the prescribed protocol using the kit. The extract was digested with EcoRI and subjected to agarose gel electrophoresis. [Table 4]

[0034] • Purification of DNA by phenol-chloroform extraction and ethanol precipitation. Equal volumes of chloroform and TE-saturated phenol were added to the plasmid DNA solution. The mixtures were vigorously mixed for 20 seconds and then centrifuged (4°C, 15,000×g, 10 min). The upper aqueous layer was carefully collected in a new tube, to which equal volumes of chloroform and TE-saturated phenol were again added. The mixtures were vigorously mixed for 20 seconds and then centrifuged (4°C, 15,000×g, 10 min), and finally the upper aqueous layer was carefully collected. For every 100 μL of the obtained plasmid DNA solution, 10 μL of 3M CH3COONa and 250 μL of cold 100% ethanol were added, and the mixtures were inverted and mixed about 5 times. After standing at room temperature for 10 minutes, the mixtures were centrifuged (4°C, 15,000×g, 20 min), and the supernatant was discarded by decantation. Then, 600 μL of cold 70% ethanol was added, and the mixtures were inverted and mixed about 5 times. The samples were centrifuged (4°C, 15,000×g, 20 min), the supernatant was decanted and discarded, and then air-dried. The dried DNA samples were dissolved in DNase-free water. Plasmid DNA concentration and purity were measured using BioSpec nano (Shimadzu, Kyoto, Japan).

[0035] • Sequencing analysis of extracted plasmids The entire sequence of the extracted plasmid was analyzed using the "Plasmid Reading Service" provided by Eurofins Genomics, Inc. The sequence analysis data was verified by comparing it with existing J1A whole genome analysis data.

[0036] • Bioinformatics analysis We hypothesized that the replicon of the natural J1A plasmid consists of ori and rep, and attempted to identify these on a computer. First, we estimated the ori base sequence using the "Ori-Finder 2022" web service (https: / / tubic.org / Ori-Finder2022 / public / index.php / register), which allows for the prediction and analysis of bacterial DNA replication origins. We then subjected the seven estimated ORFs to the BLAST search web service provided by the National Center for Biotechnology Information (NCBI) to search for ORFs that suggest function as rep genes. To reduce the impact of base substitutions on the search results, we used amino acid sequences as query sequences.

[0037] result • Isolation and sequencing analysis of J1A natural plasmids By applying the modified protocol described above, a single DNA band was extracted from the J1A strain (Figure 3). The extracted DNA contained two specific EcoRI cleavage sites, which are consistent with the results of whole-genome analysis of the J1A strain. In addition, actual sequencing analysis identified a 4605 bp circular DNA molecule whose sequence matched that of previous analyses. Based on these results, we successfully isolated a native plasmid derived from J1A. This plasmid was named pJ1A.

[0038] Identification of ori The 324-base region adjacent to ORF7 was presumed to be the ori. Database analysis revealed that the ori consists of (i) an AT-rich region that undergoes DNA cleavage, and (ii) a DnaA box, which is the recognition sequence for the DnaA initiator protein. [ka]

[0039] • Identification of rep Seven ORFs on pJ1A were subjected to a BLAST search, and when the search results with the highest total score were compared, ORF7 showed high homology to the replication initiation protein gene. Considering that the predicted ori nucleotide sequence was adjacent to ORF7, it was thought that these regions constitute a functional replicon. Furthermore, ORF6, which shares a coding region with ORF7, contains a Helix-turn-helix domain that is generally reported as a DNA-binding domain. Therefore, it was hypothesized that ORF6 may also be involved in the replication cycle of pJ1A.

[0040] The Rhodococcus replicons of the Escherichia coli shuttle vectors pNC9501 and pNC5403, derived from the genus Rhodococcus of the order Actinomycetes, consist of ori, repA, and repB (T. Matsu et al., Curr. Microbiol. 52(6):445-448, T. Matsui et al., Appl. Microbiol. Biotechnol. 74(1):169-175). Amino acid sequence comparisons revealed that ORF6 derived from the natural plasmid of strain J1A showed high homology to repA of pNC9501 and pNC5403 (vs. pNC9501: 57.8%, vs. pNC5403: 33.7%). ORF7 derived from the natural plasmid of the J1A strain also showed high homology to repB of pNC9503 and pNC9501 (vs. pNC9503: 38.4%, vs. pNC9501: 33%). Based on these results, the configuration shown in Figure 4 (estimated ori, rep (repA), DNA-binding protein (repB)) was considered to be the smallest replicon of pJ1A.

[0041] Example 4: Antimicrobial resistance testing and determination of the selection marker to be used. In this experiment, we tested the drug sensitivity of the J1A wild-type strain and determined the selection marker genes to be used for selecting transformants. method J1A glycerol stock was seeded in 5 mL of TwLB medium (LB medium containing 2 w / v% Tween 80) and cultured overnight with shaking at 30°C. 200 μL of the culture solution was then seeded into four 5 mL TwLB medium tubes, each containing ampicillin (Amp), streptomycin (Sm), chloramphenicol (Cm), and kanamycin (Km), respectively. The final concentration of all antibiotics was 20 μg / mL. The culture conditions were 30°C and 120 rpm, and growth was visually checked every other day.

[0042] result In the presence of three antibiotics excluding streptomycin, the growth of J1A was inhibited (Figure 5). It was considered that ampicillin, chloramphenicol, or kanamycin could be used as antibiotics for selecting transformants. Next, the same culture procedure was performed to determine the minimum inhibitory concentration (MIC) required to actually select J1A transformants. The MIC for J1A relative to Cm was suggested to be 5.0–10 μg / mL in liquid medium and 10–20 μg / mL in solid medium (Figure 6). [Table 5]

[0043] Example 5: Construction of the E. coli-Gordonia shuttle vector: pBBJ1 This example aims to construct a shuttle vector that can replicate with the J1A strain and a cloning host (e.g., E. coli) by ligating the minimal replicon of pJ1A from Example 4 into a general-purpose cloning vector (Figure 7).

[0044] method The pBBR1 MCS-1 vector was selected as the cloning target for the minimal pJ1A replicon (ME Kovach et al. (1995). Gene 166(1):176-176). The pBBR1 MCS-1 vector is characterized by its transduction function derived from the RK2 plasmid. This vector was chosen because it was considered possible that the transformation of the J1A strain could be achieved by conjugation rather than the simpler electroporation method described later. The selection marker gene carried in this vector is the chloramphenicol acetyltransferase gene, and its expression confers a Cm-resistant phenotype.

[0045] First, the full-length pJ1A minimal replicon and the linearization vector derived from pBBR1 MCS-1 were amplified by PCR using KOD-Plus-Neo. The amplified products were subjected to DpnI treatment and column purification. Then, the amplified pJ1A minimal replicon fragments were ligated into the linearization vector using the in-fusion method. Chemically competent E. coli JM109 cells were transformed with the ligation product, selected on selective medium, and plasmids were extracted from the resulting single colonies. The extracts were digested with the restriction enzyme EcoRV, and their sizes were confirmed. Candidate target plasmids were sequenced by Sanger sequencing using primers 5 and 6. The obtained vectors were named pBBJ1. The primer sequences used in this experiment are shown in Table 6.

[0046] [Table 6]

[0047] result The pJ1A minimal replicon insert sequence and the pBBR1 MCS-1 linearization vector sequence were amplified and purified by PCR and column purification (Figure 8). Several transformant colonies were obtained from the ligation products, and four samples were subjected to plasmid extraction. As a result, it was considered that the target pBBJ1 sequence was constructed in all four samples (Figure 9). Next, the DNA sequencing of these plasmids was confirmed. Of the four samples, samples 1 and 3 matched the designed target sequence, so it was determined that the target plasmid had been successfully constructed.

[0048] Example 6: Transformation of J1A strain by electroporation Using the shuttle vector pBBJ1 constructed in Example 5, we tried two previously reported electroporation methods for transforming Gordonia bacteria (M. Arenskotter, D. Baumeister, R. Kalscheuer, and A. Steinbuchel. (2003). Appl. Environ. Microbiol. 69(8):4971-4974., P. Singh and P. Srivastava. ((2013). J. Microbiol. Methods. 95(2):114-116). However, under these conditions, we were unable to obtain J1A / pBBJ1 transformants. Therefore, the aim of this experiment was to find a modified electroporation protocol that can transform the J1A strain.

[0049] method • Preparation of electrocompetent cells J1A electrocompetent cells were prepared according to the report by P. Singh and P. Srivastava ((2013). J. Microbiol. Methods. 95(2):114-116). First, J1A cells were cultured in 5 mL of LB medium at 30°C for two nights with shaking. Next, 1 mL of the culture solution was seeded into 100 mL of competent cell LB medium (Table 7) in a 300 mL Erlenmeyer flask, and OD was used. 600The cells were cultured at 30°C with shaking at 120 rpm until the pH reached approximately 0.5. Then, the culture medium was cooled on ice for 30 minutes, followed by centrifugation (4°C, 2760×g, 6 min) to collect the cells. The cells were washed twice with 30 mL of cooled 10 v / v% glycerol, and then resuspended in 10 mL of 10 v / v% glycerol. The suspension was prepared as electrocompetent cells, dispensed into Eppendorf tubes in 100 μL portions, rapidly cooled with liquid nitrogen, and stored in a -80°C freezer. [Table 7]

[0050] • Electroporation and selection of transformants 100 μL of competent cell solution was mixed with less than 10 ng of pBBJ1 and transferred to a cuvette electrode (2.0 mm wide, NEPA gene, Chiba, Japan). The cuvette was thoroughly cooled on ice and then transferred to a MicroPulser. TM An electroporator (Bio-Rad Laboratories, Tokyo, Japan) was used to apply electrical pulses to the cells under the conditions of 15kV / cm, 10μF, 600Ω, and 4ms. Immediately after pulsing, 1 mL of TwLB medium was added, and the cell symposium was transferred to an Eppendorf tube and recovered at 30°C for 4 hours. 200 μL of the recovered culture was seeded onto LB / Cm selective medium and incubated statically at 30°C until colony formation was confirmed.

[0051] • DNA sequencing analysis Plasmid extraction was performed from colonies formed on the selective medium as described in Example 3. The extracted plasmids were sequenced by Sanger sequencing analysis. Furthermore, the fact that the formed colonies were Gordonia sp.J1A strain was confirmed by determining the 16S rRNA gene sequence.

[0052] result • Acquisition of J1A / pBBJ1 transformed organisms When we attempted to transform the J1A strain using the shuttle vector pBBJ1 with a modified protocol, colonies that grew on selective medium were obtained (Figure 10). The formed colonies resembled the colony morphology of wild-type J1A, supporting the possibility that they were J1A transformants. Furthermore, since almost no colony formation was observed at a Cm concentration of 20 μg / mL, the Cm concentration to be used for future transformant selection was determined to be 10 μg / mL. Two colonies were randomly selected, and plasmid extraction was performed, revealing DNA staining bands consistent with pBBJ1 (Figure 11). Importantly, the DNA band of pJ1A was not detected, suggesting that pBBJ1 and pJ1A are incompatible. In addition, sequencing analysis of the 16S rRNA gene and plasmid sequence of the transformants confirmed that the obtained colonies were J1A possessing pBBJ1 (see source data file for sequencing data). Based on these results, we concluded that we have successfully identified new experimental conditions (particularly electric field conditions) that enable the transformation of J1A.

[0053] Example 7: Determination of plasmid copy number The intracellular copy numbers of pJ1A in J1A wild-type cells and pBBJ1 in J1A transformants were investigated. The experimental method followed the guidelines of C. Lee, et al., ((2006). J. Biotechnol. 123(3):273-280), using real-time PCR to measure ΔΔC. T Relative quantification of plasmid copy numbers was performed using a specific method. First, the total DNA for analysis was extracted from the J1A strain using the Bacteria genomicPrep Mini Spin Kit (GE Healthcare Japan, Tokyo, Japan). Three samples each of J1A wild-type and J1A transformant cultures were used for extraction, all in OD (Oral-Dynamic) form. 660A culture medium with a pH of approximately 4.0-5.0 was used. Next, the target region for real-time PCR was determined. The gyrB (DNA gyrase subunit B gene) on the genome and the rep (replication initiation protein gene) on the plasmid were chosen as amplification targets, and primer sets specific to these were designed (gyrB set and rep set, respectively). The rep set was confirmed to have no homology to the genome using Local BLAST. The gyrB set was chosen because the gyrB gene has been reported to be distributed as a single copy on the genome among Gordonia bacteria (FT Shen et al., 2006, Res. Microbiol. 157(4):367-375). Therefore, the quantification of samples using the gyrB and rep sets represents the amount of plasmid corresponding to the amount of genomic DNA. The rep set produces a PCR amplification product of 121 bp, and the gyrB set produces a PCR amplification product of 120 bp.

[0054] Next, calibrator DNA for relative quantification was prepared. The gyrB amplification fragment was cloned into pBBJ1 via PCR and in-fusion reaction. The obtained plasmid was named pBBJ1 rg. Since pBBJ1 rg contains the amplification regions of the gyrB set and the rep set in a 1:1 ratio, ΔΔC T It functions as a calibrator for relative quantification by method (Figure 12).

[0055] Next, real-time PCR was performed using THUNDERBIRD® SYBR® qPCR Mix (Toyobo, Osaka, Japan) (Table 8). [Table 8]

[0056] The PCR system was analyzed in relative quantification mode using a Thermal Cycler Dice (registered trademark) Real Time System (TaKaRa, Shiga, Japan) equipped with Thermal Cycler Dice (registered trademark) Real Time System software. The C T (Threshold cycle) value of each sample was applied to relative quantification by the ΔΔC T method. Next, the amplification efficiency of PCR required for calculating the target relative amount was measured. A 2 x -fold dilution series of pBBJ1 rg was prepared from x = 0 - 8, and a plot was created from the observed C T values, and the amplification efficiency (E, Amplification efficiency) by PCR was calculated from the slope of the approximate straight line using the following formula.

Equation

[0057] To evaluate the influence of non-specific amplification, melting curve analysis was applied in this experiment. Furthermore, the amplification products were subjected to agarose gel electrophoresis, and non-specific amplification was also visually evaluated. Finally, from the change amount of the C T value corresponding to the two detection target regions of the experimental sample, the ΔC T value was calculated. From the change amount of each ΔC T value derived from the target sample and the calibrator, the ΔΔC T value was calculated. Then, the target relative amount was calculated by the formula shown below and used as the plasmid copy number per genome, that is, per cell.

Equation

[0058] The primer sequences used in Example 6 are shown in Table 9.

Table 9

[0059] result The whole DNA samples extracted from cells in the late logarithmic phase were subjected to experiments, and the total DNA was separated at the concentrations and purities shown in Table 10. Subsequently, the obtained whole DNA was used to perform real-time PCR to separate C from each sample. T Values ​​were obtained (Table 11). C obtained from the serial dilution system of pBBJ1 rg. T A plot and a approximation line were created from the values ​​(Figure 12). From the obtained slope, the PCR amplification efficiency within the measurement range was calculated to be 1.16, averaging the results obtained from the gyrB set and the rep set. The final calculated plasmid copy numbers per cell were (i) 43.0, 10.7, and 35.8 copies for pJ1A possessed by J1A wild-type cells, and (ii) 180.8, 43.0, and 58.9 copies for pBBJ1 possessed by J1A transformants. Although some outliers were observed, it was suggested that the pJ1A replicon has the ability to maintain approximately several tens of copies in a single J1A cell. Furthermore, no nonspecific DNA amplification was confirmed in the melting curve analysis (Figure 14), which supports the accuracy of the experimental results. [Table 10] [Table 11]

[0060] The information for sequence numbers 1 through 27 is listed below. [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 12-5] Table 12-6 Table 12-7

Claims

1. (a) A polynucleotide or its complementary strand consisting of the base sequences 3463 to 4605 and 1 to 266 of Sequence ID No. 1, or (b) A polynucleotide or its complementary chain consisting of a nucleotide sequence in which one or more nucleotides are substituted, deleted, and / or inserted in the nucleotide sequence of (a), and which is replicable in Actinomycetes bacteria. A plasmid vector that can replicate in Actinomycetes bacteria, including the above.

2. (1) (a) A polynucleotide consisting of base sequences 3463 to 4605 and 1 to 266 of Sequence ID No. 1, or a complementary strand thereof, (b) Polynucleotides or their complementary chains, which consist of a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted in the nucleotide sequence of (a), and which are replicable in Actinomycetes bacteria, and (2) Origin of replication polynucleotides that function in E. coli A shuttle vector between Actinomycetes bacteria and Escherichia coli, including the aforementioned.

3. A plasmid vector according to claim 1 or a shuttle vector according to claim 2, further comprising at least one sequence selected from the group consisting of a promoter sequence, a cloning site sequence, and a terminator sequence.

4. A plasmid vector according to claim 1 or a shuttle vector according to claim 2, further comprising a drug resistance gene.

5. The plasmid vector or shuttle vector according to claim 4, wherein the drug resistance gene is an ampicillin resistance gene, a chloramphenicol resistance gene, or a kanamycin resistance gene.

6. A plasmid vector according to claim 1 or a shuttle vector according to claim 2, wherein the number of copies maintained in Actinomycetes bacteria is 40 or more.

7. A method for producing a protein, comprising the step of introducing the plasmid vector described in claim 1 or the shuttle vector described in claim 2 into a host.

8. A method for producing a protein according to claim 7, wherein the vector introduction method is electroporation, and the electroporation method is performed under conditions of 15 kV / cm and 10 μF.

Images