• 미생물학회지
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• 제45권2호
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• pp.228-231
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• 2009

고추 역병을 야기하는 Phytophthora capsici 는 짧은 시간 내에 많은 면적에 피해를 주는 병으로 한번 발생하면 방제가 어려운 병으로 알려져 있다. 이러한 역병 곰팡이의 방제를 위하여 본 연구에서는 P. capsici의 염색체 복제 및 세포 골격 유지 등에 관여하는 단백질인 microtubule의 형성 저해를 유도하는 물질을 탐색하여 궁극적으로 고추역병 방제를 위한 연구를 진행하였다. 먼저 P. capsici alpha 및 beta tubulin을 E. coli BL21(DE3)에서 발현시켜 분리 정제하여 in vitro microtubule 형성을 확인하였다. P. capsici microtubule 형성 저해 metagenome clone 스크리닝을 위하여 경기도 수원의 여기산 토양에서 metagenome을 분리하여 library를 제작하여 Fluorescence Resonance Energy Transfer (FRET) 방법을 이용하여 P. capsici microtubule 형성을 저해하는 화합물을 탐색하였다. In vitro 스크리닝에서 약 384개의 metagenome library에서 2종의 clone을 선택하여 고추작물에 직접 방제하여 역병균의 생장 억제를 확인하였다. 이는 차후 고추역병 방제제 개발에 있어 중요한 후보물질뿐만 아니라 metagenome library를 이용한 새로운 방법의 개발이라 사료 된다. 또한 in vitro 스크리닝에서 얻어진 2종의 metagenome clone의 염기서열을 분석하여 항역병 활성에 관련하는 DNA 서열을 확보하고 이를 응용하여 물질을 생산 할 경우, 현장에서 활용 할 수 있는 효과 큰 친환경 천연고추역병 방제제로서의 개발 가능성을 가진다는 점에서 본 연구결과는 매우 의미 있는 결과라 생각된다.

• Journal of Microbiology and Biotechnology
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• 제26권3호
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• pp.521-529
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• 2016

Extensive use of antibiotics over recent decades has led to bacterial resistance against antibiotics, including gentamicin, one of the most effective aminoglycosides. The emergence of resistance is problematic for hospitals, since gentamicin is an important broad-spectrum antibiotic for the control of bacterial pathogens in the clinic. Previous study to identify gentamicin resistance genes from environmental samples have been conducted using culture-dependent screening methods. To overcome these limitations, we employed a metagenome-based culture-independent protocol to identify gentamicin resistance genes. Through functional screening of metagenome libraries derived from soil samples, a fosmid clone was selected as it conferred strong gentamicin resistance. To identify a specific functioning gene conferring gentamicin resistance from a selected fosmid clone (35-40 kb), a shot-gun library was constructed and four shot-gun clones (2-3 kb) were selected. Further characterization of these clones revealed that they contained sequences similar to that of the RNA ligase, T4 rnlA that is known as a toxin gene. The overexpression of the rnlA-like gene in Escherichia coli increased gentamicin resistance, indicating that this toxin gene modulates this trait. The results of our metagenome library analysis suggest that the rnlA-like gene may represent a new class of gentamicin resistance genes in pathogenic bacteria. In addition, we demonstrate that the soil metagenome can provide an important resource for the identification of antibiotic resistance genes, which are valuable molecular targets in efforts to overcome antibiotic resistance.

• Genomics & Informatics
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• 제11권3호
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• pp.114-120
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• 2013

The microbial diversity in soil ecosystems is higher than in any other microbial ecosystem. The majority of soil microorganisms has not been characterized, because the dominant members have not been readily culturable on standard cultivation media; therefore, the soil ecosystem is a great reservoir for the discovery of novel microbial enzymes and bioactivities. The soil metagenome, the collective microbial genome, could be cloned and sequenced directly from soils to search for novel microbial resources. This review summarizes the microbial diversity in soils and the efforts to search for microbial resources from the soil metagenome, with more emphasis on the potential of bioprospecting metagenomics and recent discoveries.

• 생명과학회지
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• 제16권2호
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• pp.360-364
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• 2006

Fibrin clots of blood vessels are one of the serious factor caused cardiovascular disease. The development of a antithrombotic and thrombolysis solvent is necessary to prevent and treat these diseases. It has been reported that a strong fibrin-specific fibrinolytic enzyme was produced from a Korean fermented soybean paste similar to Japanese miso. We have been screened the known or novel fibrinolytic enzymes by activity-based and sequence-based screening from soil DNA metagenome library containing all kinds of environmental genomic DNA. The activity-based screening was determined the protease activity on 0.5% skim milk. For sequence-based screening, we designed a set of primer expanding gene sequence of fibrinolytic enzyme, performed PCR and selected clones showing the expected size of amplicons from metagenome library. Transformation of the gene encoding fibrinolytic enzyme was carried out with commercial vectors and their transformants were selected. Finally, we found 15 positive clones from metagenome library. Then each of sequences were analyzed and identified as similar or known the clones of nattokinase. We are going to perform full sequence of each clones, ligate with expression vector, transform into competent cells and then determine activity of expressed enzymes.

• Journal of Microbiology and Biotechnology
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• 제18권10호
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• pp.1634-1640
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• 2008

This study was focused on the screening of valuable genetic resources, such as promoters from metagenome, and describes a promoter trapping system with a bidirectional probe concept, which can select promoters or operons from various biological resources including metagenomic DNA. A pair of reporters, GFP and DsRed, facing the opposite direction without promoters, is an effective system that can function regardless of the direction of inserted promoters. The feasibility of this system was tested for the isolation of constitutively expressed promoters in E. coli from a soil metagenome, resulting in a potential pool of various promoters for practical application. The analyses of structural organization of the trapped genes demonstrated that constitutively expressible promoters in E. coli were broadly distributed within the metagenome, and suggested that some promoters were useful for the construction of expression vectors. Based on these observations, three constitutive promoters were employed in the expression vector system and their potentials for practical application were evaluated in terms of expression level, protein solubility, and effects on host growth.

• KSBB Journal
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• 제25권5호
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• pp.490-496
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• 2010

미생물은 지구상에 약 $5\;{\times}\;10^{30}$ 정도의 개체가 존재하며, 350~550 Pg (1Pg = 1015g)의 탄소, 85~130 Pg의 질소, 9~14 Pg의 인 등, 지구상의 어떠한 생물 종보다 거대한 양의 원소를 포함하고 있다. 또한 이러한 미생물과 생태계를 구성하는 다른 유기체나 무기물과의 관계가 지속적으로 밝혀지고 있다. 이러한 연구들의 기본적인 목표는 상호작용에 중요한 인자들의 규명 (대표적으로 유전자)하는 것이기 때문에, 염색체에 존재하는 true ORF의 검색과 확인은 가장 중요한 기본 수단이 된다. 그러나 다양한 미생물로 구성된 환경 유전체는 기존 정보로 검색 가능한 비율을 정확하게 유추할 수 없기에 많은 어려움이 있다. 이렇게 경계가 불분명한 자료의 검색을 위해서는 보다 많은 정보를 필요 (training이나 space를 규정하기 위한 보다 많은 유전자 서열)로 하며, 다른 검색 방법이나 기법들이 추가적으로 개발되어야 할 것이다. 이러한 방법의 대안으로써, 미생물의 유전자간 서열에 존재하는 전사/번역인자의 보존성에 근거한 검색방법은 개량 여하에 따라 광범위한 적용 범위를 지닐 것이다. 현 수준에서도 조합 탐색, 즉 기존의 방법과 혼용하거나 기존의 방법을 보완하는 과정으로 충분한 가치를 지니고 있다. 이러한 추정은, 기존의 ORF 중심의 발굴 결과와 전혀 일치되지 않는 경우에서부터 90% 이상 일치하는 등의 결과로서 확인하였다. 일치 되지 않는 많은 경우가 BLASTing으로 검색되지 않는 새로운 ORF를 포함하기 때문이다.

• 농업과학연구
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• 제48권3호
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• pp.493-503
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• 2021

Tricholoma matsutake (Pinus mushroom, PM) is one of the most valued ectomycorrhizal fungi in Asia because it is an expensive forest product with a unique flavor and taste. Therefore, many studies have tried to successfully cultivate Tricholoma matsutake artificially in Korea and other countries. However, its physiological and ecological characteristics are still unknown. Thus, we need to understand the diversity and clusters of microorganisms related to Tricholoma matsutake and to identify their core microorganisms related to their growth and production. In this study, we obtained an average of 11,661 fragments from three pine mushrooms with metagenome (an assemblage of genes of all microorganisms in the natural world) analysis from a pine forest located in Pohang, Gyeongsang-Bukdo. Of these, the valid reads were on average 5,073 per sample available for analysis, and the average length of a read was 456 bp. There were an average of 33.3 phyla in the metagenome analysis. Firmicutes phylum made up on an average 46% of the phyla and was dominant among the phyla. The next dominant phylum was Proteobacteria at 27% followed by Bacteroidetes at 17%, Actinobacteria at 5% and Verrucomicrobia at 2%. The Proteobacteria phylum consisted of the γ-proteobacteria class at 54% followed by β-proteobacteria at 37%, α-proteobacteria at 6%, δ-proteobacteria at 2% and ζ-proteobacteria at 0%. The metagenome consisted of the Ruminococcaceae family at 17% followed by Pseudomonadaceae at 13%, Burkholderiaceae at 7%, Bacteroidaceae at 7%, Lachnospiraceae at 7% and Clostridiaceae at 6%.

• Journal of Microbiology and Biotechnology
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• 제24권5호
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• pp.592-596
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• 2014

HMO-2011, a recently isolated lytic phage that infects the SAR116 bacterial clade, represents one of the most abundant phage types in the oceans. In this study, the HMO-2011 genome sequence was compared with virome sequences obtained from various depths of the Pacific Ocean regions using metagenome binning. HMO-2011 was confirmed to be one of the most highly assigned viruses, with a maximum of 7.6% of total reads assigned. The HMO-2011-type phages demonstrated a depth-specific distribution, showing more abundance in the euphotic zone of coastal, transition, and open ocean regions as compared with the dark ocean.

• ALGAE
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• 제31권2호
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• pp.137-154
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• 2016

Next generation sequencing (NGS) technologies have revolutionized many areas of biological research due to the sharp reduction in costs that has led to the generation of massive amounts of sequence information. Analysis of large genome data sets is however still a challenging task because it often requires significant computer resources and knowledge of bioinformatics. Here, we provide a guide for an uninitiated who wish to analyze high-throughput NGS data. We focus specifically on the analysis of organelle genome and metagenome data and describe the current bioinformatic pipelines suited for this purpose.

• 한국미생물·생명공학회지
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• 제36권4호
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• pp.260-267
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• 2008

단백질의 산업적 생산을 위해 발현벡터의 선정이 중요하지만 이용 가능한 프로모터가 극히 제한적이며 많은 경우 과발현되는 특성과 함께 불용성 응집체가 형성되는 단점을 지닌다. 따라서 다양한 생물로부터 유래된 잠재성이 큰 유전자원(metagenome)에서의 프로모터 발굴과 한정된 숙주를 해결하려는 노력이 요구된다. 선행연구에서 발굴한 metagenome 유래의 항시발현 프로모터를 이용해 대장균의 일반적인 배양조건에서 세포생리에 영향이 적은 신규 항시발현 벡터를 제작하였다. 이를 위해 예측된 프로모터 서열과 MCS를 포함하는 합성 primer를 제작한 후 PCR로 증폭해 발현벡터를 구성한 후, 프로모터 구동여부와 단백질 발현양상 등을 관찰하였다. 인위적으로 도입된 MCS에 GFP, esterase, $\beta$-glucosidase를 클로닝해 단백질 발현양과 가용성을 분석한 결과, 안정적으로 전체단백질의 $2{\sim}3%$ 정도로 발현되며 80% 이상의 높은 가용성을 지닌 단백질의 발현이 유도되는 것으로 확인되었다. 이와 같은 결과는 잠재적인 생물자원의 보고로서 metagenome의 활용가능성을 제시하고 있다. 따라서 다양한 숙주에서 작동하는 프로모터의 발굴 및 발현벡터의 제작을 시도할 경우 단백질의 생산이나 대사공학에 의한 균주개량에 유용하게 활용할 수 있을 것이다.