• 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.

• Korean Chemical Engineering Research
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• 제56권1호
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• pp.79-84
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• 2018

본 연구에서는 기 개발된 액적 접촉충전 기반의 디지털 전기천공 기술을 이용해 미세조류에 단백질을 전달하는 연구를 수행 하였다. Chlamydomonas reinhardtii 중 세포벽이 존재하는 야생종 cc-125에 적용한 결과, 살아 있는 세포의 핵 내부로 형광 단백질 GFP가 10% 이상의 비교적 높은 효율로 전달될 수 있음을 확인하였다. 또한 인가 전기장의 크기 변화에 따른 단백질 전달 효율을 살펴봄으로써 최적의 단백질 전달 효율을 위한 전기천공 전기장 조건을 도출하였다(960 V/cm). 전달 물질의 크기에 따른 영향 분석을 위해 추가로 수행한 핵산 염색 형광 염료 Yo-Pro-1의 전달 특성 분석 결과, 크기에 따른 차이가 존재함에도 최적의 전달 효율을 나타내는 인가 전기장의 세기 조건은 매우 유사한 경향을 보였다. 마지막으로 본 연구 결과의 의미 및 크리스퍼 유전자 가위 기술의 적용 등 향후 활용방안에 대해서 논의하였다.

• Journal of Microbiology and Biotechnology
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• 제33권12호
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• pp.1681-1691
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• 2023

Flavin mononucleotide-binding proteins or domains emit cyan-green fluorescence under aerobic and anaerobic conditions, but relatively low fluorescence and less thermostability limit their application as reporters. In this work, we incorporated the codon-optimized fluorescent protein from Chlamydomonas reinhardtii with two different linkers independently into the redox-responsive split intein construct, overexpressed the precursors in hyperoxic Escherichia coli SHuffle T7 strain, and cyclized the target proteins in vitro in the presence of the reducing agent. Compared with the purified linear protein, the cyclic protein with the short linker displayed enhanced fluorescence. In contrast, cyclized protein with incorporation of the long linker including the myc-tag and human rhinovirus 3C protease cleavable sequence emitted slightly increased fluorescence compared with the protein linearized with the protease cleavage. The cyclic protein with the short linker also exhibited increased thermal stability and exopeptidase resistance. Moreover, induction of the target proteins in an oxygen-deficient culture rendered fluorescent E. coli BL21 (DE3) cells brighter than those overexpressing the linear construct. Thus, the cyclic reporter can hopefully be used in certain thermophilic anaerobes.

• 한국해양바이오학회지
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• 제11권2호
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• pp.52-61
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• 2019

Over the past few decades, microalgae-based biotechnology conjugated with innovative CRISPR/Cas9-mediated genetic engineering has been attracted much attention for the cost-effective and eco-friendly value-added compounds production. However, the discharge of reproducible living modified organism (LMO) into environmental condition potentially causes serious problem in aquatic environment, and thus it is essential to assess potential environmental risk for human health. Accordingly, in this study, we monitored discharged genetically modified microalgae (GMM) near the research complex which is located in Daejeon, South Korea. After testing samples obtained from 6 points of near streams, several green-colored microalgal colonies were detected under hygromicin-containing agar plate. By identification of selection marker genes, the GMM was not detected from all the samples. For the lab-scale environmental risk assessment of GMM, acute toxicity test using rotifer Brachionus calcyflorus was performed by feeding GMM. After feeding, there was no significant difference in mortality between WT and transformant Chlamydomonas reinhardtii. According to further analysis of horizontal transfer of green fluorescence protein (GFP)-coding gene after 24 h of incubation in synthetic freshwater, we concluded that the GFP-expressed gene not transferred into predator. However, further risk assessments and construction of standard methods including prolonged toxicity test are required for the accurate ecological risk assessment.

• 한국미생물·생명공학회지
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• 제46권3호
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• pp.225-233
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• 2018

In this exploratory study, eight types of microalgae from different phyla (Chlamydomonas reinhardtii, Chlorella species, Haematococcus pluvialis, Porphyridium purpureum, Porphyridium cruentum, Isochrysis species, Isochrysis galbana, and Pavlova lutheri) were tested for their antibacterial activities against eight target pathogenic bacterial strains. The agar well diffusion method and broth micro dilution assay were conducted to estimate the antibacterial activity. Microalgae cell-free supernatants, exopolysaccharides (EPS), water, and organic solvent extracts were used for inhibition analysis. EPS extracted from P. lutheri showed activity against Bacillus subtilis and Pseudomonas aeruginosa. Inhibition zone diameters of 14-20 mm were recorded on agar plates, while the minimum inhibitory concentrations in the broth micro dilution assay were $0.39-25mg\;ml^{-1}$. During this study, haptophyte microalgae, Isochrysis species, and P. lutheri extracts showed the highest activity against most of the tested pathogenic bacterial strains, while most of the extracts were active against the important foodborne pathogen P. aeruginosa. This study showed promising results regarding important microalgae phyla, which will further aid research related to extracts and exploitation of bioactive metabolic compounds in the food and pharmaceutical industries.

• 한국환경과학회지
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• 제23권4호
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• pp.681-695
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• 2014

In a pilot-scale dyeing wastewater treatment using two-type fluidizing media, each thickness of biofilm was 15 and 30 ${\mu}m$, respectively. The numbers of protozoa inhabited in small-size (PEMT A) and big-size (PEMT B) media were $7.5{\times}10^4$ and $1.25{\times}10^5$ cells/ml, respectively, and dominant species were Entosiphon sulcatus var sulcatus in PEMT A and Chlamydomonas reinhardtii in PEMT B, respectively. Flask experiments using the two media revealed that the percentages of color removal were 25.8% in PEMT A and 27.1% in PEMT B after 72-h cultivation, indicating the necessity of bioaugmentation. Experiments for bioaugmentation effect on color removal were carried out in the pilot-scale treatment for 75 d by three-step operation under the control of wastewater loading rate and microbial input rate. Dye degradation occurred mainly in the second reaction tank, and the attachment of augmented dye-degrading microorganisms to media took at least 35 d. Final value of chromaticity in effluent was 227, meeting the required standard. Therefore bioaugmentation onto media was good for color treatment. In summary, thickness of biofilm formed on the media depended upon the size of media, resulting in different ecosystem inside the media. Hence, this affected microbial community and color treatment further. Accordingly, the reduction of operation cost is expected by efficient color-treatment process using bioaugmented media.

• ALGAE
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• 제36권3호
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• pp.207-217
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• 2021

An increase in seawater temperature owing to global warming is expected to substantially limit the growth of marine algae, including Pyropia yezoensis, a commercially valuable red alga. To improve our knowledge of the genes involved in the acquisition of heat tolerance in P. yezoensis, transcriptomes sequences were obtained from both the wild-type SG104 P. yezoensis and heat-tolerant mutant Gy500. We selected 1,251 differentially expressed genes that were up- or downregulated in response to the heat stress condition and in the heat-tolerant mutant Gy500, based on fragment per million reads expression values. Among them, PyHRG1 was downregulated under heat stress in SG104 and expressed at a low level in Gy500. PyHRG1 encodes a secretory protein of 26.5 kDa. PyHRG1 shows no significant sequence homology with any known genes deposited in public databases to date. However, PyHRG1 homologs were found in other red algae, including other Pyropia species. When PyHRG1 was introduced into the single-cell green alga Chlamydomonas reinhardtii, transformed cells overexpressing PyHRG1 showed severely retarded growth. These results demonstrate that PyHRG1 encodes a novel red algae-specific protein and plays a role in heat tolerance in algae. The transcriptome sequences obtained in this study, which include PyHRG1, will facilitate future studies to understand the molecular mechanisms involved in heat tolerance in red algae.

• 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.

• 생태와환경
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• 제41권2호
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• pp.117-127
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• 2008

본 연구에서는 국내 수계에 분포하는 녹조류를 대상으로 수행된 바 있는 국내외 독성 시험법의 시험 세부 조건을 노출 기간, 종말점, 초기접종농도 등을 중심으로 수렴하여 급성 및 만성 독성시험기법의 시험 세부 조건별 범위를 제시하였다. 또한 국내형 생태독성시험기법을 마련하기 위해 현 시점에서의 제한점과 생태독성평가기법 구축을 위한 방향을 제시하였다. 국내 생물종을 대상으로 국내외에서 수행된 바 있는 독성 시험법에 대한 기존 연구 사례를 살펴본 결과, OECD, EC, KS 등의 표준시험법에서 추천한 시험종 Pseudokirchneriella subcapitata, Desmodesmus subspicatus를 대상으로 한 독성자료가 대부분을 차지하였다. 또한 표준시험법의 시험종 이외에도 국내 생물종에 대한 소수의 독성 자료가 있었으나, 대체로 표준시험법을 수정 적용한 것으로 나타났다. 반면 초기접종농도 및 배양액은 생물종마다 상이하게 수행되었으므로 녹조류 성장저해시험에 있어서 가장 주의할 조건인 것으로 나타났다. 이와 같이 국내 생물종을 이용한 생태독성평가 기반이 미약한 현 시점에서 국내생태독성시험법을 보다 효율적으로 개발하기 위해서는 본 연구에서 선정한 녹조류 7종을 대상으로 도출된 생태독성시험기법에 대한 실험 연구를 통한 검증 및 보완, 기존 연구에서 사용된 바 없는 시험종 개발 및 각각의 시험종별 생태독성시험기법 규명이 선행되어야 할 것이다. 따라서 본 연구는 국내 생물종별 독성시험기법 및 방향을 제시함으로써 국내 수계 실정 및 먹이 연쇄에 따른 생물종간 상호 연관성 등을 고려할 수 있는 국내 시험종 및 그 시험법 개발을 위한 중요한 기초가 될 것으로 사료된다.