Biomineralization vector for transformation of Chlorella vulgaris and transformant

Abstract

The present invention relates to Chlorella vulgaris ( Chlorella vulgaris The present invention relates to a vector system for transformation, a method for transforming Chlorella vulgaris using the same, and a Chlorella vulgaris transformant.

Description

[Technical Field]

[0001] This patent application claims priority to Korean Patent Application No. 10-2021-0097954, filed with the Korean Intellectual Property Office on July 26, 2021, the disclosure of which is incorporated herein by reference. The present invention relates to chlorella vulgaris ( Chlorella vulgaris The present invention relates to a vector system for transformation, a method for transforming Chlorella vulgaris using the same, and Chlorella vulgaris transformants. [Background technology]

[0002] Chlorella vulgaris ( Chlorella vulgaris Chlorella vulgaris is a unicellular microalgae belonging to the Chlorophyta phylum. Chlorella vulgaris has a photosynthetic mechanism, and can grow rapidly using only light, carbon dioxide, water, and a small amount of minerals. This is why it is often used in photobioreactor research.

[0003] In addition, the lipid content per biomass is relatively high at about 42%, so it is often used in research on developing biofuels that can be used as a substitute for biodiesel. Recently, Chlorella vulgaris has been used to detect radioactive isotopes. 90 Research has been published showing that Sr can be removed.

[0004] Electroporation using the hygromycin B resistance gene, zeocin resistance gene, and chloramphenicol acetyltransferase gene (CAT gene) as selection markers resulted in integration into chromosomal DNA, but transient transformants were produced and resistance was soon lost.

[0005] Transformation methods include glass beads, electroporation, and Agrobacterium-mediated transformation. However, the lack of an efficient selectable marker and transformation system currently limits the use of Chlorella vulgaris.

[0006] With the development of technology, the amount of electricity used has increased dramatically, and the efficiency of energy consumption and production has increased. Due to the rapid increase in electricity use and the resulting efficiency of production, nuclear power generation has attracted global attention. Along with this efficiency of production, there are also constantly emerging issues, such as nuclear power plant accidents, radioactive contamination from contaminated water, and contamination by metal ions. Furthermore, the country faces a national problem of being dependent on imports of rare metals used in industry, such as Sr, Cs, and Li. This problem is becoming more serious as the market for secondary batteries expands, and the issue of recycling them is also emerging as a major industrial and national problem. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, the present inventors have investigated the effects of Chlorella vulgaris ( Chlorella vulgaris ) as a result of our efforts to develop a transformation method for Coccomyxa The promoter and terminator regions of C-169 (hereinafter referred to as C-169; previously known as Chlorella vulgaris but renamed) were predicted and sequences were obtained. Sh ble It was synthesized by fusion with the gene. Streptomyces verticillus Bleomycin ( Sh ble No intron was inserted into the middle of the . ) gene, and the synthesized DNA fragment was digested with SwaI / KpnI restriction enzymes and cloned into pSP124S vector, which was named pKA650. In addition, in the pKA650 vector Streptomyces verticillus The target gene, beta-type carbonic anhydrase, was cloned into the middle of the bleomycin (Sh ble) gene, and the resulting vector was named pJG002.

[0008] The transformants were then transformed by gold particle bombardment, and selectively cultured in a medium containing Zeocin®. The transformants were then produced by culturing the transformants selectively to obtain colonies, thereby completing the present invention. It is therefore an object of the present invention to provide a vector system for transforming Chlorella vulgaris. Another object of the present invention is to provide a method for transforming Chlorella vulgaris. A further object of the present invention is to provide a Chlorella vulgaris transformant. [Means for solving the problem]

[0009] The present invention relates to Chlorella vulgaris ( Chlorella vulgaris The present invention relates to a transformation vector system, a method for transforming Chlorella vulgaris using the same, and a microalgae transformant. The present invention will now be described in more detail.

[0010] One aspect of the present invention relates to a vector system for transforming Chlorella vulgaris comprising: Coccomyxa C-169 rbcS2( Coccomyxa C-169 ribulose-1,5-bisphosphate carboxylase / oxygenase(Rubisco)small subunit 2; Coccomyxa C-169 Ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco) small subunit 2 gene promoter; a nucleotide sequence encoding a protein of interest; and CoccomyxaC-169 Terminator sequence of the rbcS2 gene.

[0011] In the present invention, the promoter may comprise the base sequence of SEQ ID NO: 2, or may comprise a base sequence having substantial identity to the base sequence of SEQ ID NO: 2, for example, consisting of the base sequence of SEQ ID NO: 2, but is not limited thereto. In one example of the present invention, a promoter containing the base sequence of SEQ ID NO: 2 may be a sequence that further contains 1 to 50 bp of bases at the 3' end of the sequence of SEQ ID NO: 2 contained in SEQ ID NO: 1, but is not limited to this.

[0012] In the present invention, the terminator sequence may include the base sequence of SEQ ID NO: 3, or may include a base sequence that is substantially identical to the base sequence of SEQ ID NO: 3, for example, consisting of the base sequence of SEQ ID NO: 3, but is not limited thereto.

[0013] In the present invention, the nucleotide sequence encoding the protein of interest may be operatively linked to a promoter. In the present invention, the term "operatively linked" refers to the functional association of a nucleic acid expression control sequence (e.g., a promoter sequence, a signal sequence, or an array of transcriptional regulator binding sites) with another nucleic acid sequence, whereby the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0014] In the present invention, the nucleotide sequence encoding the target protein may be linked to the 3' end of the terminator sequence, but is not limited thereto.

[0015] In the present invention, the vector system may further comprise a selection marker. In the present invention, the selection marker may be, but is not limited to, an antibiotic resistance gene. In the present invention, the antibiotic may be one or more selected from the group consisting of spectinomycin, paromomycin, ampicillin, zeocin, and bleomycin, but is not limited thereto. In the present invention, various selectable marker genes can be selected for resistance to the antibiotic to be used, such as aminoglycoside phosphotransferase, which confers resistance to the kanamycin antibiotic, and chloramphenicol acetyltransferase, which is involved in resistance to the chloramphenicol antibiotic. In the present invention, when the selection marker is bleomycin, the selection marker may contain the base sequence of SEQ ID NO: 4, or may contain a base sequence that is substantially identical to the base sequence of SEQ ID NO: 4, for example, it may consist of the base sequence of SEQ ID NO: 4, but is not limited to this. In the present invention, a method for selecting transformed Chlorella vulgaris into which a selection marker has been introduced can be easily carried out by a method well known in the art using the phenotype expressed by the selection marker. For example, if the selection marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

[0016] In the present invention, the vector system may further comprise a gene encoding a reporter molecule. In the present invention, the reporter molecule may be one or more selected from the group consisting of growth-promoting proteins, fluorescent proteins, and hydrolases, but is not limited thereto. In the present invention, the fluorescent protein may be, but is not limited to, luciferase. In the present invention, the hydrolase may be, but is not limited to, β-glucuronidase.

[0017] In the present invention, the vector system may contain the base sequence of SEQ ID NO: 1, or may contain a base sequence that is substantially identical to the base sequence of SEQ ID NO: 1, for example, may consist of the base sequence of SEQ ID NO: 1, but is not limited to this.

[0018] In the present invention, the vector system may be for transformation of Chlorella vulgaris using gold particles bombardment. In the present invention, the term "vector" refers to a means for expressing a gene of interest in a host cell, including, for example, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector such as an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. In the present invention, vectors that can be used as recombinant vectors may be prepared by manipulating plasmids commonly used in the art (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19), phages (e.g., λgt4λB, λ-Charon, λΔz1, and M13), or viruses (e.g., SV40), and may be, for example, but not limited to, the pSP124S vector backbone. In the present invention, the vector system may typically be constructed as a vector for cloning or a vector for expression.

[0019] In the present invention, vectors for expression can be any vectors commonly used in the art for expressing target proteins in plants, animals or microorganisms.

[0020] In the present invention, the vector system may be constructed by various methods known in the art. In the present invention, the term "substantial identity" means that when each base sequence and any other base sequence are aligned to correspond as closely as possible and the sequences are analyzed, the any other base sequence has a sequence homology of 70% or more, 90% or more, or 98% or more with the respective base sequence. Another aspect of the present invention relates to a method for producing a Chlorella vulgaris transformant, comprising the steps of: Coccomyxa C-169 rbcS2 gene promoter, target protein-coding nucleotide sequence, and Coccomyxa C-169 A transformation step of introducing a vector containing the terminator sequence of the rbcS2 gene into Chlorella vulgaris.

[0021] In the present invention, the transformation step can be carried out using gold particle bombardment. In the present invention, the cell stage in the transformation step may be the log phase, as the highest efficiency is achieved in the log phase. In the present invention, the logarithmic phase may have an OD686 value of 0.4 to 0.6, 0.45 to 0.6, 0.5 to 0.6, or 0.55 to 0.6, for example, 0.6. In the present invention, the cell density during the transformation step is 5.0*10 per 60 mm diameter. 6 ~8.0*10 7 , 1.0*10 per 60mm diameter 7 ~8.0*10 7 , 2.0*10 per 60mm diameter 7 ~8.0*10 7 , 3.0*10 per 60mm diameter 7 ~8.0*10 7 , 4.0*10 per 60mm diameter 7 ~8.0*10 7 , 1.0*10 per 60mm diameter 7 ~7.0*10 7 , 2.0*10 per 60mm diameter 7 ~7.0*10 7 , 3.0*10 per 60mm diameter 7~7.0*10 7 , 4.0*10 per 60mm diameter 7 ~7.0*10 7 , 1.0*10 per 60mm diameter 7 ~6.0*10 7 , 2.0*10 per 60mm diameter 7 ~6.0*10 7 , 3.0*10 per 60mm diameter 7 ~6.0*10 7 , 4.0*10 per 60mm diameter 7 ~6.0*10 7 , 1.0*10 per 60mm diameter 7 ~5.0*10 7 , 2.0*10 per 60mm diameter 7 ~5.0*10 7 , 3.0*10 per 60mm diameter 7 ~5.0*10 7 , 4.0*10 per 60mm diameter 7 ~5.0*10 7 For example, 4.8*10 per 60mm diameter 7 It may be.

[0022] In the present invention, the vacuum in the transformation step may be 27.0 to 29.0 inches Hg, 27.5 to 29.0 inches Hg, 28.0 to 29.0 inches Hg, or 28.5 to 29.0 inches Hg, for example, 29.0 inches Hg. In the present invention, the target distance in the transformation step may be 3 to 9, for example, 3, 6 or 9. In the present invention, the pressure in the transformation step may be 1200 to 1300 psi, 1210 to 1300 psi, 1220 to 1300 psi, 1230 to 1300 psi, 1240 to 1300 psi, 1250 to 1300 psi, 1260 to 1300 psi, 1270 to 1300 psi, 1280 to 1300 psi, 1290 to 1300 psi, for example, 1300 psi.

[0023] In the present invention, the promoter may comprise the base sequence of SEQ ID NO: 2, or may comprise a base sequence having substantial identity to the base sequence of SEQ ID NO: 2, for example, consisting of the base sequence of SEQ ID NO: 2, but is not limited thereto. In one example of the present invention, a promoter containing the base sequence of SEQ ID NO: 2 may be a sequence that further contains 1 to 50 bp of bases at the 3' end of the sequence of SEQ ID NO: 2 contained in SEQ ID NO: 1, but is not limited to this.

[0024] In the present invention, the terminator sequence may include the base sequence of SEQ ID NO: 3, or may include a base sequence that is substantially identical to the base sequence of SEQ ID NO: 3, for example, consisting of the base sequence of SEQ ID NO: 3, but is not limited thereto. In the present invention, the nucleotide sequence encoding the protein of interest may be operatively linked to a promoter. In the present invention, the nucleotide sequence encoding the target protein may be linked to the 3' end of the terminator sequence, but is not limited thereto.

[0025] In the present invention, the vector may further comprise a selection marker. In the present invention, the selection marker may be, but is not limited to, an antibiotic resistance gene. In the present invention, the antibiotic may be one or more selected from the group consisting of spectinomycin, paromomycin, ampicillin, zeocin, and bleomycin, but is not limited thereto. In the present invention, various selectable marker genes can be selected for resistance to the antibiotic to be used, such as aminoglycoside phosphotransferase, which confers resistance to the kanamycin antibiotic, and chloramphenicol acetyltransferase, which is involved in resistance to the chloramphenicol antibiotic. In the present invention, a method for selecting transformed Chlorella vulgaris into which a selection marker has been introduced can be easily carried out by a method well known in the art using the phenotype expressed by the selection marker. For example, if the selection marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

[0026] In the present invention, the vector system may further comprise a gene encoding a reporter molecule. In the present invention, the reporter molecule may be one or more selected from the group consisting of growth-promoting proteins, fluorescent proteins, and hydrolases, but is not limited thereto. In the present invention, the fluorescent protein may be, but is not limited to, luciferase. In the present invention, the hydrolase may be, but is not limited to, β-glucuronidase.

[0027] In the present invention, the vector may contain the base sequence of SEQ ID NO: 1, or may contain a base sequence that is substantially identical to the base sequence of SEQ ID NO: 1, for example, the vector may consist of the base sequence of SEQ ID NO: 1, but is not limited to this.

[0028] The method for producing a transformant of the present invention provides a transformant that maintains a Chlorella vulgaris transformant. Yet another aspect of the present invention relates to a biomineralization vector for transforming Chlorella vulgaris using the vector system, and a transformant into which the vector has been introduced. One aspect of the present invention relates to a biomineralization vector for transforming Chlorella vulgaris, comprising: Coccomyxa C-169 rbcS2( Coccomyxa C-169 ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCo) small subunit 2) gene promoter; A nucleotide sequence encoding the beta-carbonic anhydrase of Coccomyxa subellipsoidea C-169; and Coccomyxa C-169 Terminator sequence of the rbcS2 gene. In the present invention, the promoter may comprise the base sequence of SEQ ID NO: 2, or may comprise a base sequence having substantial identity to the base sequence of SEQ ID NO: 2, for example, consisting of the base sequence of SEQ ID NO: 2, but is not limited thereto.

[0029] In one example of the present invention, a promoter containing the base sequence of SEQ ID NO: 2 may be a sequence that further contains 1 to 50 bp of bases at the 3' end of the sequence of SEQ ID NO: 2 contained in SEQ ID NO: 7, but is not limited to this. In the present invention, the terminator sequence may include the base sequence of SEQ ID NO: 3, or may include a base sequence that is substantially identical to the base sequence of SEQ ID NO: 3, for example, consisting of the base sequence of SEQ ID NO: 3, but is not limited thereto.

[0030] In the present invention, the nucleotide sequence encoding the beta carbonic anhydrase of Coccomyxa subellipsoidea C-169 may be operatively linked to a promoter. As used herein, the term "operatively linked" refers to the functional association of a nucleic acid expression control sequence (e.g., a promoter sequence, a signal sequence, or an array of transcriptional regulator binding sites) with another nucleic acid sequence, whereby the control sequence controls the transcription and / or translation of the other nucleic acid sequence. In the present invention, the nucleotide sequence encoding the beta-carbonic anhydrase of Coccomyxa subellipsoidea C-169 may be linked to the 3' end of a terminator sequence, but is not limited thereto. In the present invention, the vector may further comprise a selection marker. In the present invention, the selection marker may be, but is not limited to, an antibiotic resistance gene.

[0031] In the present invention, the antibiotic may be one or more selected from the group consisting of spectinomycin, paromomycin, ampicillin, zeocin, and bleomycin, but is not limited thereto. In the present invention, various selectable marker genes for the antibiotics to be used may be selected as the selectable marker, such as aminoglycoside phosphotransferase, which confers resistance to the kanamycin antibiotic, and chloramphenicol acetyltransferase, which is involved in resistance to the chloramphenicol antibiotic. In the present invention, when the selection marker is bleomycin, the selection marker may contain the base sequence of SEQ ID NO: 4, or may contain a base sequence that is substantially identical to the base sequence of SEQ ID NO: 4, for example, it may consist of the base sequence of SEQ ID NO: 4, but is not limited to this. In the present invention, a method for selecting transformed Chlorella vulgaris into which a selection marker has been introduced can be easily carried out by a method well known in the art using the phenotype expressed by the selection marker. For example, if the selection marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic. In the present invention, the vector may further comprise a gene encoding a reporter molecule.

[0032] In the present invention, the reporter molecule may be one or more selected from the group consisting of growth-promoting proteins, fluorescent proteins, and hydrolases, but is not limited thereto. In the present invention, the fluorescent protein may be, but is not limited to, luciferase. In the present invention, the hydrolase may be, but is not limited to, β-glucuronidase. In the present invention, the vector may contain the base sequence of SEQ ID NO: 7, or may contain a base sequence that is substantially identical to the base sequence of SEQ ID NO: 7, for example, the vector may consist of the base sequence of SEQ ID NO: 7, but is not limited to this. One aspect of the present invention relates to a transformant transformed with a biomineralization vector for transforming Chlorella vulgaris, comprising: Coccomyxa C-169 rbcS2( Coccomyxa C-169 ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCo) small subunit 2) gene promoter; beta-carbonic anhydrase-encoding nucleotide sequence of Coccomyxa subellipsoidea C-169; and Coccomyxa C-169 Terminator sequence of the rbcS2 gene.

[0033] To avoid complicating the description, the details of the biomineralization vector for transforming C. chlorella vulgaris are omitted, as described above. Yet another aspect of the present invention relates to a method for producing a protein of interest, comprising the steps of: Coccomyxa C-169 rbcS2 gene promoter and target protein-encoding nucleotide sequence; and Coccomyxa C-169 A transformation step of introducing a vector containing the terminator sequence of the rbcS2 gene into Chlorella vulgaris; and The culturing step involves culturing the transformed Chlorella vulgaris to obtain the target protein. In the present invention, the nucleotide sequence encoding the protein of interest may be, but is not limited to, operatively linked to a promoter. In the present invention, the transformation step can be performed using gold particle bombardment. [Effects of the Invention]

[0034] The present invention relates to Chlorella vulgaris ( Chlorella vulgaris The present invention relates to a transformation vector system, a method for transforming Chlorella vulgaris using the same, and a Chlorella vulgaris transformant. [Brief explanation of the drawings]

[0035]

Figure 1

Figure 2

[0036] Chlorella vulgaris (including ​ ) Biomineralization vector for transformation: ​ C-169 rbcS2( ​ C-169 ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCo) small subunit 2) gene promoter; beta-carbonic anhydrase-encoding nucleotide sequence of Coccomyxa subellipsoidea C-169; and ​ C-169 Terminator sequence of the rbcS2 gene.

[0037] [Example] The present invention will be described in more detail below with reference to the following examples, but these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0038] Preparation Example 1. Plasmid construction and cloning actinomycetes ( ​ ) from the genomic DNA of the bleomycin resistance gene (​ The 5' part of the bleomycin resistance gene contains ​ C-169 ​ The promoter sequence of the gene is ​ C-169 ​ This is the entire sequence obtained by joining the gene's terminator sequence. ​ was synthesized (GenScript, USA). The synthesized gene was digested with restriction enzymes Swa I and Kpn I and inserted into the pSP124S vector. The resulting plasmid was named pKA650, and the vector map is shown in Figure 1. As can be seen in Figure 1, OriV in the vector map stands for the replication origin, ​ teeth, ​ The bleomycin promoter and terminator are linked together ​ gene, AmpR is the ampicillin resistance gene, and C-169-ble is ​ C-169 ​ The promoter sequence of the gene, and the 3' part is ​ C-169 ​ It is the terminator part of the gene.

[0039] Preparation Example 2: Transformation into Chlorella vulgaris 2-1. Transformation using the glass bead method We used a simple and rapid glass bead transformation method, which involves inserting glass beads into cells and applying physical force to create holes in the cell membrane through physical friction with the cells, allowing the desired plasmid to be introduced. The experiment was performed under the conditions listed in Table 1. Specifically, the cells were vortexed using glass beads at room temperature (25°C) to partially disrupt the cells. The beads and supernatant were then separated by gravity, and DNA was added to the supernatant. Recovery was then allowed to proceed with slow rotation at 37°C. After dispensing onto solid medium containing antibiotics, transformants were confirmed. The results are shown in Figure 2.

[0040] [Table 1] As can be seen from FIG. 2, the efficiency was reduced and transformation was not successful.

[0041] 2-2. Transformation using the gene gun method Transformation was performed using gold particle bombardment with a gene gun, a method of plant transformation. This technology is also called microparticle acceleration or bioballistics, but the official name of the machine called a gene gun is microparticle bombardment. This method involves coating a plasmid with microparticles for transformation. Microparticles are very heavy compared to their size, so they penetrate cells easily. By projecting the microparticles toward the cells at high speed and surrounding them with a steel mesh, more particles can be projected toward the cells. Once inside the cells, the DNA coated on the microparticles is released and can enter the plant's genetic code. Transformation was performed using gold as the microparticles.

[0042] Specifically, experiments were conducted while changing various conditions to perform transformation using the gold particle bombardment method, as shown in Table 2 below. First, the vacuum and helium pressure were fixed, and experiments were conducted while taking into consideration the cell stage, concentration, gold particle injection range, target distance, and experimental environment of the cells. In the process of establishing the conditions, experiments were conducted at 47mm, 50mm, and 60mm, taking into consideration the injection range and pressure of the gold particles from the gene gun, and the experiment was conducted at a target distance of 3 as the optimum position. Also, as the membrane of the microalgae becomes thicker as the cell division stage progresses, making the experiment difficult, experiments were conducted from the early division stage according to the change in OD value. The cell density was also adjusted depending on the diameter.

[0043] [Table 2] As can be seen from Table 2 and Figure 3, in the case of failure, the strain was unable to grow on solid medium containing antibiotics, whereas in the case of success, colonies were formed, but promoter operation (mutants) was confirmed only under the condition of 160704. Subsequently, the target protein (carbonic anhydrase) was introduced under the same conditions to form mutants.

[0044] Experimental example 1. Confirmation of genetic information To confirm the genetic information, genomic DNA was isolated and the transgene was confirmed by PCR and sequencing. Specifically, to perform PCR to confirm the gene, the primer set shown in Table 3 below was prepared. Using the primer set, pre-denaturation was performed at 98°C for 8 minutes, followed by 30 cycles of 98°C for 1 minute, 53.5°C for 30 seconds, and 72°C for 1 minute, followed by PCR for 7 minutes at 72°C. This was confirmed by sequencing analysis. The results are shown in Table 3. The 5' region of the bleomycin resistance gene that the inventors are interested in is ​ C-169 ​ The promoter sequence of the gene is ​ C-169 ​ This is the entire sequence obtained by joining the gene's terminator sequence. ​ To confirm this, PCR was performed using the M13 primer set. Pre-denaturation was performed at 98°C for 8 minutes, followed by 30 cycles of 98°C for 1 minute, 54°C for 40 seconds, and 72°C for 2 minutes 30 seconds, followed by PCR for 7 minutes at 72°C. The target DNA band was confirmed by PCR. The results are shown in Figure 4.

[0045] [Table 3] As can be seen from Figure 4, a band was observed at approximately 2 kb, which is the total size of the target gene. This was confirmed to be identical to the target gene using DNA gene analysis, and the results are shown in Table 4.

[0046] [Table 4] As can be seen from Table 4, the results of CLUSTAL 2.1 multiple sequence alignment confirmed that the DNA base sequence of the target gene, Sh-ble, was completely consistent with the acquired gene information.

[0047] Experimental Example 2: Confirmation of mutant maintenance To maintain the mutants, subculture was used to confirm the maintenance of the mutants, and the results are shown in Figure 5. As can be seen from Figure 5, to maintain the mutants, subculture was used to confirm the maintenance of the mutants.

[0048] Experimental Example 3: Biomineralization Example 1: Plasmid construction and cloning This involves constructing and cloning a plasmid based on the pKA650 vector, inserting a gene for a target protein to confer resistance to Zeocin and expressing the target protein. ​ ( ​ ) from the genomic DNA of the bleomycin resistance gene ( ​ ) sequence was obtained. ​ C-169 The promoter and terminator sequences of the rbcS2 (ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit) gene were ​ The entire sequence was synthesized by adding the 5' and 3' portions of the above. This sequence was named pKA650, and the entire sequence ​ It is an intermediate gene of ​ After synthesizing the C-169 beta-carbonic anhydrase gene, restriction enzyme ​ , ​ The duplicated promoter portion was inserted using the following sequence: The cloned plasmid is shown in Figure 6 and was designated pJG002.

[0049] Example 2: Chlorella vulgaris transformation The transformation method using the pJG002 vector was the same as the transformation method using the pKA650 vector. Specifically, to prepare the responsive cells, 300 ml of sterilized MBM medium (KNO3 2.5 mM, MgSO4 7H2O 0.3 mM, K2HPO4 0.43 mM, KH2PO4 1.29 mM, NaCl 0.43 mM, CaCl2 * 2H2O 0.068 mM, FeSO4 * 7H2O 0.1 g, A5 metal mixture ml / L) was prepared in a 500 ml flask, and Chlorella vulgaris was inoculated and cultured at 23°C and 100 rpm. OD 686 When the value reached 0.5 to 0.6, the Chlorella vulgaris was centrifuged and the cell count was confirmed.

[0050] After checking the cell count, the cell density was set to 4.8*10 7 Next, pKA650 was prepared for gold particle bombardment by linearizing it with the restriction enzyme KpnI. A retaining cap, brass adjustable nest, microcarrier holder, stopping screen, and microcarrier were prepared in a sterile condition, and the rupture disk was washed with 70% isopropanol. While the rupture disk was drying, a mixture of plasmid and gold particles was prepared. First, for 20 bombardments, 12 mg of gold was placed in a microfuge tube and 1 ml of 70% ethanol was added. The mixture was vortexed at maximum speed for 5 minutes, centrifuged for 5 seconds, and the supernatant was removed.

[0051] Wash the gold particles three times with 1 ml of distilled water, vortex for 1 minute, stop for 1 minute, centrifuge for 5 seconds, and then remove the supernatant. Then, add 205 μl of 50% glycerol and dissolve by vortexing for 5 minutes. Vortex at speeds 2-3 for 10 swirls. Transfer 100 μl of gold particles to a new microfuge tube, then add 10 μl of DNA (0.5-20 μg / μl), 100 μl of 2.5 M CaCl2, and 40 μl of 0.1 M spermidine in that order while vortexing. Mix by pipetting while vortexing. Vortex for 2 minutes, stop for 1 minute, and then centrifuge for 2 seconds to remove the supernatant. After removing the supernatant, add 300 μl of 70% ethanol and wait 1 minute. After removing the supernatant, add 300 μl of 100% ethanol and wait 1 minute. Remove again and add 110 ul of 100% ethanol and dissolve the pellet with sustained vortexing.

[0052] Once the gold particle mix is ​​complete, place 11 μl of it in the center of the microcarrier and let it dry for 5-10 minutes. Meanwhile, spread the prepared cells evenly over the medium with a 60 mm diameter. Then, place a stopping screen on the brass adjustable nest and place the dried microcarriers on it. Then, secure it in the holder, turn on the gene gun instrument, and preheat it for 5 minutes. Once preheating is complete, insert the rupture disk into the retaining cap and rotate it into the gun. Then, insert the brass adjustable nest with the microcarriers, place the medium containing the cells at target distance 3, and close the door. Turn on the helium gas and press the vacuum. When the vacuum reaches 29 inches Hg and the helium pressure reaches 1300 psi, hold down the fire button until the pressure increases to 1100 psi, at which point the gold particles are fired. After bombardment, the medium has a recovery period using a 23°C incubation.

[0053] Example 3: Confirmation of Chlorella vulgaris transformants Bioballistic transformation was performed as in Example 2, and the maintenance of antibiotic resistance after subculture was confirmed by concentration. The results are shown in Figure 7. A total of 42 transformants were obtained. These were subcultured using the medium in Example 2 under light irradiation. To confirm the gene and protein expression of such transformants, total protein SDS-PAGE was used to confirm the band size of approximately 43 kDa of the target protein, beta-carbonic anhydrase, and the results are shown in Figure 8. To confirm the band of such a target protein, MALDI-TOF was performed, and the results are shown in FIG. The present invention was advanced by confirming the protein expression of the transformants and the maintenance of resistance through subcultures. For genetic confirmation, the presence or absence of the target gene was confirmed by Southern blotting, and the results are shown in Figure 10. As can be seen from FIG. 10, a DNA band was observed at the same position as the size of the target gene, and this was also confirmed by DNA sequencing using PCR.

[0054] Example 4: Sr biomineralization The functionality of the transformants was confirmed by visualizing the biomineralization crystals in the same amount using a microscope to confirm the Sr biomineralization ability of wild-type (WT) Chlorella vulgaris. Specifically, Chlorella vulgaris was cultured under light conditions until the OD reached 0.6. After cultivation, the microalgae were collected under sterilized conditions at 25°C, 4000 rpm, and 15 minutes. The microalgae were then washed three times with 3 mM NaHCO3. After that, 200 ppm of Sr was added to 3 mM NaHCO3 prepared to adjust the cell concentration to 1*10^7, and the prepared Chlorella vulgaris was mixed and cultured at 4°C for at least four hours. The cells were then stained with 0.004% sodium rhodizonate and observed under a microscope. The results are shown in Figure 11. As can be seen in Figure 11, the WT crystallized less biomineralized crystals than the transformant. For quantitative comparison, the WT was analyzed by ICP-MS, and the results are shown in Figure 12 and Table 5.

[0055] [Table 5] As can be seen from FIG. 12 and Table 5, the transformants were found to have biomineralized crystals that were increased by up to about 160% compared to the wild type. [Industrial Applicability]

[0056] The present invention relates to Chlorella vulgaris ( ​ The present invention relates to a transformation vector system, a method for transforming Chlorella vulgaris using the same, and a Chlorella vulgaris transformant.

Claims

1. Contains chlorella with carbonic anhydrase introduced, A biomineralization composition characterized in that the chlorella is transformed with a vector containing a promoter of the Coccomyxa C-169 ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco) small subunit 2 (rbcS2) gene, and the promoter contains the base sequence of SEQ ID NO:

2.

2. A biomineralization composition as described in claim 1, wherein the transformed chlorella increases biomineralization crystals.

3. A biomineralization composition as described in claim 1, wherein the carbonic anhydrase is beta-carbonic anhydrase.

4. A biomineralization composition as described in claim 1, wherein the chlorella into which the carbonic anhydrase has been introduced has been transformed with a vector containing a base sequence encoding the carbonic anhydrase.

5. The biomineralization composition of claim 4, wherein the nucleotide sequence encoding the carbonic anhydrase is operably linked to the promoter.

6. The biomineralization composition of claim 1, wherein the chlorella into which the carbonic anhydrase has been introduced has been transformed using gold particle bombardment.

7. A biomineralization method comprising culturing a biomineralization composition according to any one of claims 1 to 6.

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