Patent Application
20250122511
This patent application claims priority to and the benefit of Korean Patent Application No. 10-2021-0097952 filed with the Korean Intellectual Property Office on Jul. 26, 2021, the disclosure of which is incorporated herein by reference.
This patent application claims priority to and the benefit of Korean Patent Application No. 10-2021-0097953 filed with the Korean Intellectual Property Office on Jul. 26, 2021, the disclosure of which is incorporated herein by reference.
The present disclosure relates to a vector system for the transformation of Chlorella vulgaris, a method for transforming Chlorella vulgaris using same, and a Chlorella vulgaris transformant.
Chlorella vulgaris is a unicellular microalga in the Chlorophyta phylum. Chlorella vulgaris, possessing a photosynthetic apparatus, can grow rapidly using only light, carbon dioxide, water, and a small amount of minerals through photosynthesis and thus is widely used in research for mass production of useful substances (photobioreactors).
Additionally, with a lipid content of about 42% of its biomass, Chlorella vulgaris is also extensively used in research for biofuel development as a potential alternative to biodiesel. Recent studies have shown that Chlorella vulgaris can be used to remove the radioactive isotope 90Sr.
Selectable markers such as the hygromycin B resistance gene, zeocin resistance gene, and chloramphenicol acetyltransferase gene (CAT gene) have been used in electroporation, resulting in integration into chromosomal DNA. However, the transformants were only temporary, losing their resistance after a short period.
Transformation methods include the use of glass beads, electroporation, and Agrobacterium-mediated transformation. However, due to the lack of efficient selectable markers and transformation systems, the use of Chlorella vulgaris remains limited.
In pursuit of developing a transformation method for Chlorella vulgaris, the present inventors, after thorough and intensive research, predicted and obtained the sequences of the promoter and terminator regions of Coccomyxa C-169 (hereinafter referred to as C-169; previously known as Chlorella vulgaris but renamed) and fused the sequences with the Sh ble gene from Streptomyces verticillus bleomycin (Sh ble). No introns were inserted in the middle of the Sh ble gene, and the synthesized DNA fragment was treated with SwaI/KpnI restriction enzymes and cloned into the pSP124S vector, which was then named pKA650.
Subsequently, after transformation via gold particle bombardment, the cells were cultured in a medium containing zeocin for selective selection. The transformation was completed by obtaining colonies through selection, thus producing transformants.
Therefore, an aspect of the present disclosure is to provide a vector system for the transformation of Chlorella vulgaris.
Also, another aspect of the present disclosure is to provide a method for the transformation of Chlorella vulgaris.
Furthermore, a further aspect of the present disclosure is to provide a Chlorella vulgaris transformant.
The present disclosure pertains to a vector system for the transformation of Chlorella vulgaris, a method for transforming Chlorella vulgaris, using same, and a microalga transformant.
Below, a detailed description will be given of the present disclosure.
An aspect of the present disclosure contemplates a vector system for the transformation of Chlorella vulgaris, comprising:
In the present disclosure, the promoter may include the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence substantially identical to that of SEQ ID NO: 2, for example, consisting of the nucleotide sequence of SEQ ID NO: 2, but with no limitations thereto.
In an embodiment of the present disclosure, the promoter including the nucleotide sequence of SEQ ID NO: 2, which corresponds to a part of the nucleotide sequence of SEQ ID NO: 1, may additionally contain 1 to 50 base pairs (bp) at the 3′ end of the sequence of SEQ ID NO: 2, but with no limitations thereto.
The terminator sequence in the present disclosure may include the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence substantially identical to that of SEQ ID NO: 3, for example, consisting of the nucleotide sequence of SEQ ID NO: 3, but with no limitations thereto.
The target protein-encoding nucleotide sequence in the present disclosure may be operatively linked to the promoter.
As used herein, the term “operatively linked” refers to a functional connection between a nucleic acid expression regulatory sequence (e.g., promoter sequence, signal sequence, or array of transcription factor binding sites) and another nucleic acid sequence, whereby the regulatory sequence regulates transcription and/or translation of the other nucleic acid sequence.
In the present disclosure, the target protein-encoding nucleotide sequence may be linked to the 3′ end of the terminator sequence, but with no limitations thereto.
The vector system in the present disclosure may additionally include a selectable marker.
The selectable marker in the present disclosure may be an antibiotic resistance gene, but is not limited thereto.
In the present disclosure, 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 disclosure, selectable markers can include various selectable marker genes for the desired antibiotic. Examples include aminoglycoside phosphotransferase, which confers kanamycin resistance, and chloramphenicol acetyltransferase, which is involved in chloramphenicol resistance.
As for a selectable marker for bleomycin, its nucleotide sequence may include the nucleotide sequence of SEQ ID NO: 4, or a nucleotide sequence substantially identical to SEQ ID NO: 4, for example, consisting of the nucleotide sequence of SEQ ID NO: 4, but is not limited thereto.
In the present disclosure, the method for selecting selectable marker-introduced, transformed Chlorella vulgaris may be easily carried out using the phenotype expressed by the selectable marker in a manner well known in the art. For example, if the selectable marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing in a medium containing the antibiotic.
The vector system in the present disclosure may additionally include a gene coding for a reporter molecule.
The reporter molecule in the present disclosure may be one or more selected from the group consisting of growth-promoting proteins, fluorescent proteins, and hydrolases, but is not limited thereto.
The fluorescent protein in the present disclosure may be luciferase, but is not limited thereto.
The hydrolase in the present disclosure may be β-glucuronidase, but is not limited thereto.
The vector system in the present disclosure may include the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence substantially identical to SEQ ID NO: 1, for example, consisting of the nucleotide sequence of SEQ ID NO: 1, but with no limitations thereto.
The vector system in the present disclosure may be for transformation of Chlorella vulgaris using gold particle bombardment.
As used herein, the term “vector” refers to a means for expressing a target gene in a host cell. Examples include plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors such as adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors.
Vectors available as recombinant vectors in the present disclosure may be constructed by manipulating plasmids (e.g., pSC101, pGV1106, pACYC177, CoIE1, 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). For example, it may be based on the pSP124S vector backbone, but with no limitations thereto.
In the present disclosure, the vector system can typically be constructed as a cloning vector or as an expression vector.
For expression vectors in the present disclosure, conventional vectors used in the industry for expressing target proteins in plants, animals, or microorganisms can be used.
The vector system in the present disclosure may be constructed through various methods well known in the art.
As used herein, the term “substantial identity” means that two sequences are aligned to correspond to each other as much as possible and show a homology of 70% or greater, 90% or greater, or 98% or greater therebetween as analyzed.
Another aspect of the present disclosure pertains to a method for producing a Chlorella vulgaris transformant, the method comprising a transforming step of:
In the present disclosure, the transformation step may be performed using gold particle bombardment.
In the present disclosure, the cells used for the transformation step may be in a log phase. The most efficient transformation can be obtained in the log phase.
In the present disclosure, the log phase may be when the OD686 value is between 0.4 and 0.6, between 0.45 and 0.6, between 0.5 and 0.6, or between 0.55 and 0.6, for example, 0.6.
In the present disclosure, the cells in the transformation step may have a density of 5.0*106 to 8.0*107 per 60 mm diameter, 1.0*107 to 8.0*107 per 60 mm diameter, 2.0*107 to 8.0*107 per 60 mm diameter, 3.0*107 to 8.0*107 per 60 mm diameter, 4.0*107 to 8.0*107 per 60 mm diameter, 1.0*107 to 7.0*107 per 60 mm diameter, 2.0*107 to 7.0*107 per 60 mm diameter, 3.0*107 to 7.0*107 per 60 mm diameter, 4.0*107 to 7.0*107 per 60 mm diameter, 1.0*107 to 6.0*107 per 60 mm diameter, 2.0*107 to 6.0*107 per 60 mm diameter, 3.0*107 to 6.0*107 per 60 mm diameter, 4.0*107 to 6.0*107 per 60 mm diameter, 1.0*107 to 5.0*107 per 60 mm diameter, 2.0*107 to 5.0*107 per 60 mm diameter, 3.0*107 to 5.0*107 per 60 mm diameter, or 4.0*107 to 5.0*107 per 60 mm diameter, for example, 4.8*107 per 60 mm diameter.
In the present disclosure, the transformation step may be carried out under a vacuum of 27.0 to 29.0 inches Hg, 27.5 to 29.0 inches Hg, 28.0 to 29.0 inches Hg, 28.5 to 29.0 inches Hg, for example, 29.0 inches Hg.
In the present disclosure, the target distance in the transformation step may be 3 to 9, for example, 3, 6, or 9.
In the present disclosure, the transformation step may be carried out at a pressure of 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.
In the present disclosure, the promoter may include the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence substantially identical to SEQ ID NO: 2, for example, consisting of the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.
In an embodiment of the present disclosure, the promoter including the nucleotide sequence of SEQ ID NO: 2, which corresponds to a part of the nucleotide sequence of SEQ ID NO: 1, may additionally contain 1 to 50 base pairs (bp) at the 3′ end of the sequence of SEQ ID NO: 2, but with no limitations thereto.
The terminator sequence in the present disclosure may include the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence substantially identical to SEQ ID NO: 3, for example, consisting of the nucleotide sequence of SEQ ID NO: 3, but is not limited thereto.
In the present disclosure, the target protein-encoding nucleotide sequence may be operatively linked to the promoter.
The target protein-encoding nucleotide sequence in the present disclosure may be linked at the 3′ end of the terminator sequence, but with no limitations thereto.
The vector in the present disclosure may additionally include a selectable marker.
In the present disclosure, the selectable marker may be an antibiotic resistance gene, but is not limited thereto.
The antibiotic may be one or more selected from a group consisting of spectinomycin, paromomycin, ampicillin, zeocin, and bleomycin, but is not limited thereto.
The selectable marker may be selected from various selectable marker genes for conventional desired antibiotics. Examples include aminoglycoside phosphotransferase, which confers kanamycin resistance, and chloramphenicol acetyltransferase, which is involved in chloramphenicol resistance.
In the present disclosure, the method for selecting selectable marker-introduced, transformed Chlorella vulgaris may be easily performed using the phenotype expressed by the selectable marker in a manner known in the art. For example, if the selectable marker is a specific antibiotic resistance gene, the transformant may be easily selected by culturing in a medium containing the antibiotic.
The vector system in the present disclosure may additionally include a gene coding for a reporter molecule.
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.
The fluorescent protein in the present disclosure may be luciferase, but is not limited thereto.
The hydrolase in the present disclosure may be β-glucuronidase, but is not limited thereto.
The vector in the present disclosure may include the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence substantially identical to SEQ ID NO: 1, for example, consisting of the nucleotide sequence of SEQ ID NO: 1, but with no limitations thereto.
The method for constructing a transformant in the present disclosure provides a Chlorella vulgaris transformant.
Another aspect of the present disclosure pertains to a Chlorella vulgaris transformant transformed with the vector system.
In an embodiment of the present disclosure, the transformant may be constructed by inserting a vector system carrying a protein-encoding nucleotide sequence into Chlorella vulgaris. The transformant may be obtained by introducing the vector system to Chlorella vulgaris.
Another aspect of the present disclosure relates to a method for producing a target protein, comprising the steps of:
The target protein-coding nucleotide sequence in the present disclosure may be operatively linked to the promoter, but with no limitations thereto.
The transformation step in the present disclosure may be performed using gold particle bombardment.
The present disclosure relates to a vector system for the transformation of Chlorella vulgaris, a method for transforming Chlorella vulgaris using same, and a Chlorella vulgaris transformant.
A vector system for transformation of Chlorella vulgaris, comprising:
A better understanding of the present disclosure may be obtained through the following examples that are set forth to illustrate, but are not to be construed to limit the present disclosure.
The sequence of the bleomycin resistance gene (Sh ble) was obtained from the genomic DNA of Streptomyces verticillus. A promoter sequence for Coccomyxa C-169 rbcS2 gene and a terminator sequence of for Coccomyxa C-169 rbcS2 gene were joined to the 5′ and 3′ ends of Coccomyxa C-169 rbcS2, respectively, to synthesize the entire sequence Sh-ble (GenScript, USA). The synthesized gene was treated with restriction enzymes Swa I and Kpn I and then inserted into the pSP124S vector. The obtained plasmid was named pKA650, and the vector map thereof is represented in
As can be seen in
A Simple and Rapid Glass Bead Transformation Method was used through glass beads. In this method, glass beads are added to cells and physical force is applied to create holes in the cell membrane through physical friction with the cell, thereby introducing a desired plasmid into the cells.
The experiment was conducted according to the conditions described in Table 1. Specifically, cells were lysed at room temperature (25° C.) by vortexing with glass beads. Then, beads and supernatant were separated by gravity, and DNA was added to the supernatant. Next, recovery was carried out by slowly rotating at 37° C. After applying onto an antibiotic-containing solid medium, transformants were confirmed. The results are shown in
As shown in
Transformation was carried out by the plant transformation technique gold particle bombardment using a gene gun. This technique, also referred to as microprojectile acceleration or biolistics, and the gene gun is officially called microparticle bombardment. In the method, transformation is performed by coating a plasmid with micro particles. The micro particles, being very heavy relative to their size, penetrate the cells effectively. By shooting the micro particles towards the cells at high speed and surrounding them with a steel net, more particles are directed towards the cells. The DNA coated with the micro particles that enter the cells are released and can integrate into the plant's genome. For use in transformation, gold particles can be used as the micro particles.
Specifically, experiments were carried out using gold particle bombardment for transformation under various conditions shown in Table 2, below. The experiments were performed in consideration of cell stages and concentrations, shooting ranges of gold particles, target distances, and experimental environments of cells, while the vacuum and helium pressure conditions were fixed.
During the condition-establishment process, the experiments were performed on dishes with diameters of 47 mm, 50 mm, and 60 mm in consideration with the gold particle range and pressure of the gene gun, and the appropriate Target Distance was established as Target Distance 3. Also, as the cell stage advances, the algal membrane thickens, making experiments difficult. Thus, experiments were conducted considering changes in OD values from the initial stage. Additionally, cell density was adjusted according to the diameter.
As shown in Table 2 and
To confirm the genetic information, genomic DNA was secured and the introduced gene was confirmed through PCR and DNA sequencing. Specifically, for use in PCR, the primer set of Table 3 was constructed. In the presence of the primer set, PCR started by pre-denaturation at 98° C. for 8 minutes and was continued with 30 cycles of 98° C. for 1 minute, 53.5° C. for 30 seconds, and 72° C. for 1 minute, followed by 72° C. for 7 minutes. The PCR product was analyzed by sequencing, and the results are summarized in Table 3.
Additionally, to confirm the entire sequence Sh-ble, obtained by joining the promoter sequence for the Coccomyxa C-169 rbcS2 gene at the 5′ end and the terminator sequence of the Coccomyxa C-169 rbcS2 gene at the 3′ end to the bleomycin resistance gene, PCR was performed using the M13 primer set. Starting at 98° C. for 8 minutes for pre-denaturation, PCR was carried out with 30 cycles of 98° C. for 1 minute, 54° C. for 40 seconds, 72° C. for 2 minutes 30 seconds, followed by extension at 72° C. for 7 minutes, and the target DNA band was confirmed. The results are shown in
As can be seen in
As can be seen in Table 4, a complete match was confirmed when comparing the DNA nucleotide sequence of the target gene, Sh-ble, with the acquired genetic information, as analyzed by CLUSTAL 2.1 multiple sequence alignment.
To confirm the maintenance of mutants, passages were performed, and the results are shown in
pKA650 vector was constructed for expressing and cloning a gene for a target protein, with resistance to zeocin given thereto. In this regard, the bleomycin resistance gene sequence (Sh ble) was obtained from the genomic DNA of Streptomyces verticillus (Sh ble). An overall sequence was synthesized by linking the promoter and terminator sequences of the Coccomyxa C-169 rbcS2 (ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit) gene to the 5′ and 3′ ends of Sh ble, respectively. The synthesized sequence was named pKA650. In the synthesized pKA650, the intermediate gene BleoR of the entire sequence Sh-ble was substituted for C-169 beta-type carbonic anhydrase, after which repeated promoter region was inserted with the aid of KpnI and ApaI. The cloned plasmid is depicted in
The method of transformation using the pJG002 vector is same as that using the pKA650 vector.
Specifically, competent cells were created. In this regard, 300 ml of sterile MBM medium (KNO3 2.5 mM, MgSO47H2O 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 mix ml/L) was prepared in a 500 mL flask and inoculated with Chlorella vulgaris, followed by incubation at 23° C. and 100 rpm. When OD686 reached 0.5-0.6, Chlorella vulgaris was collected by centrifugation and counted.
After being counted, Chlorella vulgaris was prepared at a density of 4.8*107 cells. For gold particle bombardment, the introduced pKA650 was linearized using the restriction enzyme KpnI. A retaining cap, a brass adjustable nest, a microcarrier holder, a stopping screen, and a macrocarrier were prepared as being sterilized, and a rupture disk was washed with 70% isopropanol. While drying the rupture disk, a plasmid and gold particle mixture was prepared. For 20 bombardments, 12 mg of gold particles was put in a microfuge tube, and 1 ml of 70% ethanol was added thereto. The tube was vortexed at maximum speed for 5 minutes and briefly spun down for 5 seconds, followed by decanting the supernatant.
The cycle of washing gold particles with 1 ml of distilled water, vortexing for 1 minutes, leaving the gold particles for 1 minutes, spinning down for 5 seconds, and decanting the supernatant was repeated three times. Then, the addition of 205 μL of 50% glycerol was followed by vortexing for 5 minutes to suspend the pellet. Vortexing was continued at speed 2-3. One hundred μL of gold particles was transferred to a new microfuge tube, and 10 μL of DNA (0.5-20 μg/μL), 100 μL of 2.5M CaCl2, and 40 μL of 0.1 M spermidine were added sequentially while being mixed by pipetting. After mixing, the mixture was vortexed continued for 2 minutes, left for 1 minute, and spun down for 2 seconds. After removal of the supernatant, the pellet was added with 300 μL of 70% ethanol and left for 1 minute. The supernatant was decanted after which 300 μL of 100% ethanol was added and left for 1 minute. Again, the supernatant was removed, and 110 μL of 100% ethanol was added and continuously vortex to suspend the pellet.
Once prepared, the Gold Particle Mix was placed in an amount of 11 μL in the center of the microcarrier and dried for 5-10 minutes. Meanwhile, the prepared cells were evenly spread on the plate with a diameter of 60 mm. The stopping screen was mounted on the brass adjustable nest, followed by loading the dried microcarrier thereon. After fixation with a holder, the gene gun instrument was turned on and preheated for 5 minutes. When preheating was completed, the rupture disk was loaded into a retaining cap and inserted into the gun part by screwing. Then, the microcarrier-assembled brass adjustable nest was inserted and the plate having the cells spread thereon was mounted at target distance 3 before closing the door. Helium gas was introduced and the vacuum button was pressed. When the vacuum and the helium pressure reached 29 (inches Hg) and 1300 psi, respectively, the fire button was pressed to increase the pressure to 1100 psi at which gold particles are shot. After bombardment, the plate was incubated at 23° C. for recovery.
The transformation was performed biolistically as in Example 2, and after passages, the transformants were tested for maintaining antibiotic resistance at various concentrations. The results are shown in
The genetic and protein expression of the transformants was examined. To this end, the target protein, beta-type carbonic anhydrase was detected at a band size of approximately 43 kDa as analyzed by whole protein SDS-PAGE. The results are shown in
The band for the target protein was determined by MALDI-TOF, and the results are depicted in
In the present disclosure, the transformants were found to express the target protein and maintain antibiotic resistance over passages. For the genetic aspect, the presence or absence of the target gene was determined by southern blotting, and the results are depicted in
As seen in
The function of the transformant was to provide an increase in Sr biomineralization capacity. This was confirmed by visualizing crystal formation under a microscope with equal amounts. Initially, the Sr biomineralization capability of the wild-type (WT) Chlorella vulgaris was confirmed.
In brief, Chlorella vulgaris was cultured under light conditions until OD0.6. After cultivation, the microalgae were collected at 25° C. and 4000 rpm for 15 minutes in a sterile condition. They were then washed three times with 3 mM NaHCO3. Subsequently, 3 mM NaHCO3 was added with 200 ppm Sr, mixed with 1*107 Chlorella vulgaris, and incubated at 4° C. for 4 hours or longer. The cells were then stained with 0.004% sodium rhodizonate and observed under a microscope, with the results shown in
As can be seen in
As seen in
The present disclosure relates to a vector system for the transformation of Chlorella vulgaris, a method for transforming Chlorella vulgaris using same, and a Chlorella vulgaris transformant.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2021-0097952 | Jul 2021 | KR | national |
| 10-2021-0097953 | Jul 2021 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2021/018445 | 12/7/2021 | WO |
