• 청정기술
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• 제21권2호
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• pp.90-95
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• 2015

디지털 미세유체 전기천공 시스템을 활용하여 미세녹조류에 대한 형질전환 실험을 통해 기존 상용화 장치 대비 높은 유전자 전달 효율과 세포 생존도를 확인하였다. 전기천공의 주요 파라미터인 인가전압 및 인가시간 변화를 주며 세포벽이 존재하는 세포종과 세포벽이 없는 세포종에 대한 비교 실험을 수행하였다. 이를 통해 식물 세포의 전기천공에서 세포벽의 역할은 단순히 유전체의 전달을 방해하는 부정적 요소로만 작용하지 않는 다는 사실을 확인하였다. 병렬화 및 온 칩 세포 배양 등을 통해 제안된 디지털 전기천공 기술이 향후 새로운 청정 형질전환 방법으로써의 가능성도 확인하였다.

• 한국해양바이오학회지
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• 제1권1호
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• pp.34-39
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• 2006

Molecular biology and microalgal biotechnology have the potential to play a major role in improving the production efficiency of a vast variety of products including functional foods, industrial chemicals, compounds with therapeutic applications and bioremediation solutions from a virtually untapped source. Microalgae are a source of natural products and have been recently studied for biotechnological applications. Efficient genetic transformation systems in microalgae are necessary to enhance their potential to be used for human health. A microalga such as Chlarella is a eukaryotic organism sharing its metabolic pathways with higher plants. This microalga is capable of expressing, glycosylating, and correctly processing proteins which normally undergo post-translational modification. Moreover, it can be cultured inexpensively because it requires only limited amount of sunlight and carbon dioxide as energy sources. Because of these advantages, Chlarella may be of great potential interest in biotechnology as a good candidate for bioreactor in the production of pharmaceutical and industrial compounds for human functional foods. Here, we briefly discuss recent progress in microalgal transgenesis that has utilized molecular biology to produce functional proteins and bioactive compounds.

• Journal of Microbiology and Biotechnology
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• 제25권12호
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• pp.2116-2124
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• 2015

To understand the effects of physicochemical factors on nitrite transformation by microalgae, a lipid-rich Chlorella with high nitrite tolerance was cultured with 8 mmol/l sodium nitrite as sole nitrogen source under different conditions. The results showed that nitrite transformation was mainly dependent on the metabolic activities of algal cells rather than oxidation of nitrite by dissolved oxygen. Light intensity, temperature, pH, NaHCO₃ concentrations, and initial cell densities had significant effects on the rate of nitrite transformation. Single-factor experiments revealed that the optimum conditions for nitrite transformation were light intensity: 300 μmol/m²/s; temperature: 30℃ pH: 7-8; NaHCO₃ concentration: 2.0 g/l; and initial cell density: 0.15 g/l; and the highest nitrite transformation rate of 1.36 mmol/l/d was achieved. There was a positive correlation between nitrite transformation rate and the growth of Chlorella. The relationship between nitrite transformation rate (mg/l/d) and biomass productivity (g/l/d) could be described by the regression equation y = 61.3x (R² = 0.9665), meaning that 61.3 mg N element was assimilated by 1.0 g dry biomass on average, which indicated that the nitrite transformation is a process of consuming nitrite as nitrogen source by Chlorella. The results demonstrated that the Chlorella suspension was able to assimilate nitrite efficiently, which implied the feasibility of using flue gas for mass production of Chlorella without preliminary removal of NO_X.

• 한국산학기술학회논문지
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• 제15권6호
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• pp.3799-3804
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• 2014

Mycosporine-like 아미노산(MAAs)은 UV 흡수물질이며, 다양한 해양생물들은 MAAs의 합성과 축적을 통하여 환경자외선의 직 간접적인 영향을 감소시키는 기능을 진화시켜 왔다. 이 연구에서는 미세조류, Chlamydomonas hedleyi에 포도당 전달 단백질인 Glucose transporter 1(Glut-1) 유전자를 pCAM1303 벡터에 도입한 형질전환체를 제작하여, 형질전환체의 바이오매스를 최대로 증가시킬 수 있는 최적의 Glucose 농도와 NH4Cl농도를 결정하고, 고주파(Radiofrequency) 발생장치를 활용한 바이오매스 증가와 함께 MAA를 대량 생산할 수 있는 배양 조건을 확립하였다. 연구결과 고주파 처리를 통한 형질전환 미세조류는 4.13 mg/L(MAAs/DCW)으로 3.23 mg/L(MAAs/DCW)의 고주파 처리 없이 배양한 형질전환체보다 효율이 증가하였다. 이러한 결과는 자외선 A 흡수물질을 인위적으로 증폭시킬 수 있어서, 대량배양한 후 MAAs물질을 분리 및 정제하여 피부자극성이 없는 친환경적인 자외선 차단 화장품 산업화에 크게 기여할 수 있음을 의미한다.

• Journal of Microbiology and Biotechnology
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• 제33권7호
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• pp.955-963
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• 2023

Chlorella is a eukaryotic organism that can be used as an industrial host to produce recombinant proteins. In this study, a salt-inducible promoter (SIP) was isolated from the freshwater species Chlorella vulgaris PKVL7422 from the screening of genes that were upregulated after salt treatment. Several cis-acting elements, including stress response elements, were identified in the isolated SIP. Moreover, the Gaussia luciferase gene was cloned after the SIP and transformed into C. vulgaris to test the inducibility of this promoter. Reexamination of transcriptome of C. vulgaris revealed that genes involved in the synthesis of methyl jasmonic acid (MeJA), gibberellin (GA), and abscisic acid (ABA) were upregulated when C. vulgaris was treated with salt. Furthermore, the expression level of recombinant luciferase increased when the transformed C. vulgaris was treated with salt and MeJA, GA, and ABA. This study represents the first report of the C. vulgaris SIP and highlights how transformed microalgae could be used for robust expression of recombinant proteins.

• The Plant Pathology Journal
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• 제21권1호
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• pp.13-20
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• 2005

Chlorella viruses are large icosahedral, plaque-forming, dsDNA viruses that infect certain unicellular, chlorellalike green algae. The genomic DNA of over 300 kb contains many useful genes and promoters. Over 40 chlorella viruses have been isolated from fresh water in Korea since 1998. The viruses were amplified initially in chlorella strain NC64A, and pure isolates were obtained by repeated plaque isolation. SDS-PAGE analysis revealed similar but distinct protein patterns, both among the group of purified viruses and in comparison with the prototype chlorella virus PBCV-1. Digestions of the 330- to 350-kb genomic DNAs with 10 restriction enzymes revealed different restriction fragment patterns among the isolates. The tRNA-coding regions of 8 chlorella viruses were cloned and sequenced. These viruses contain 14-16 tRNA genes within a 1.2- to 2-kb region, except for the SS-1 isolate, which has a 1039-bp spacer in a cluster of 11 tRNA genes. Promoter regions of several early genes were isolated and their activities were analyzed in transformed chlorella. Some promoters showed stronger activity than commonly used CaMV 35S promoter and chlorella transformation vectors for heterologous protein are beings constructed using these promoters.

• The Plant Pathology Journal
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• 제20권2호
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• pp.158-163
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• 2004

Chlorella is a eukaryotic microalgae which shares metabolic pathways with higher plants. These charac-teristics make chlorella a potential candidate for eukaryotic overexpression systems. Recently, a foreign flounder growth hormone gene was stably introduced and expressed in transformed Chlorella ellipsoidea by using a modified plant transformation vector that contains cauliflower mosaic virus (CaMV) 35S pro-moter and the phleomycin resistant Sh ble gene as a selection marker. In this study, this same vector was modified by incorporating a promoter and a 3' UTR region of the 33kDa peptide gene from a chlorella virus that was isolated in our laboratory. The 33kDa gene promoter was used to replace the 35S promoter and the 3' UTR was introduced to separate the target gene and downstream Sh ble gene. Three different chlorella transformation vectors containing human erythropoietin (EPO) gene were constructed. The mp335EPO vector consists of a promoter from the 33kDa peptide gene, whereas the mp3353EPO vector contains the same promoter from the 33kDa peptide gene and its 3' UTR. The mp35S33pEPO vector contains the 35S promoter and the 3' UTR from the 33 kDa peptide gene. There was no significant difference in the expression levels of EPO protein in chlorella cells transformed with either of three of the transformation vectors. These data indicate that the promoters from the chlorella virus are comparable to the most common CaMV 35S promoter. Furthermore, these data suggest that other promoters from this virus can be used in future construction of chlorella transformation system for higher expression of target proteins.

• ALGAE
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• 제26권1호
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• pp.3-20
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• 2011

The physiology of the unicellular green alga Dunaliella salina in response to abiotic stress has been studied for several decades. Early D. salina research focused on its remarkable salinity tolerance and ability, upon exposure to various abiotic stresses, to accumulate high concentrations of $\beta$-carotene and other carotenoid pigments valued highly as nutraceuticals. The simple life cycle and growth requirements of D. salina make this organism one of the large-scale commercially exploited microalgae for natural carotenoids. Recent advances in genomics and proteomics now allow investigation of abiotic stress responses at the molecular level. Detailed knowledge of isoprenoid biosynthesis mechanisms and the development of molecular tools and techniques for D. salina will allow the improvement of physiological characteristics of algal strains and the use of transgenic algae in bioreactors. Here we review D. salina isoprenoid and carotenoid biosynthesis regulation, and also the biotechnological and genetic transformation procedures developed for this alga that set the stage for its future use as a production system.

• 한국해양바이오학회지
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• 제16권1호
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• pp.55-62
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• 2024

Various antimicrobial peptides (AMPs) from microalgae have shown antibacterial, antiviral, antifungal, anticancer, and antioxidant effects, and play crucial roles in medical applications, aquaculture-related disease management, and the food industry. Magainin 2 (MAG2), an AMP, exhibits high antibacterial and antitumor activity, necessitating an efficient recombinant expression system for low-cost, large-scale production. To enhance MAG2 secretion efficiency in Chlorella, we constructed the SS:MAG2:His vector using the known Chlamydomonas reinhardtii CA1 signal sequence (SS) and obtained a stable transformant via an Agrobacterium-mediated transformation method and RT-qPCR. ELISA results revealed that the MAG2 content secreted into the medium by the SS:MAG2:His transformants increased proportionally with mRNA expression. These findings offer a strategy for high MAG2 secretion in the Chlorella vulgaris platform, potentially minimizing downstream processing costs.

• 한국해양바이오학회지
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• 제16권1호
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• pp.45-54
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• 2024

Microalgae have great potential in the biomedical and pharmaceutical industries as a new type of bioreactor that can produce proteins for specific purposes, including recombinant proteins, pharmaceuticals, and industrial enzymes. Despite the production advantages and importance of microalgae-based expression systems, studies on secretion efficiency are limited. In this study, for stable expression and efficient secretion of the heterologous protein (human SCF and human INFγ) in Chlorella vulgaris, we constructed SP:hSCF:His and SP:hINFγ:His plant binary vectors using the signal peptide (SP) of Chlamydomonas reinhardtii, and we obtained stable transformants through the effective agrobacterium-mediated transformation of these vectors. Transformants with accurately inserted hSCF and hINFγ demonstrated stably increased mRNA and protein expression using RT-PCR and western blotting under the same culture conditions. Following the analysis of the proteins secreted into the culture medium using ELISA, it was confirmed that hINFγ was effectively produced in the transformed C. vulgaris culture medium. The overall findings indicate that the combination of heterologous protein and SP may be crucial for ensuring the expression and secretion of recombinant proteins in Chlorella culture systems.