• 미생물학회지
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• 제40권4호
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• pp.359-363
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• 2004

자연계에 존재하는 미생물의 대부분이 배양이 불가능하다는 것이 밝혀진 이후, 자연계의 시료로부터 직접 유전자를 클로닝하여 유용유전자를 발굴하는 메타제놈(metagenome) 이용 방법이 주목을 받게 되었다. 그러나 실제로 오염이 심한 환경시료로부터 DNA를 추출하여 유용한 메타제놈 라이브러리(metagenomic library)를 제작하기란 쉽지 않은 일이다. 따라서 본 연구에서는 실제 메타제놈 라이브러리를 제작할 때 만나는 기술적 문제점에 대한 해결 방법을 탐구하였다. 메타제놈 라이브러리 제작에는 fosmid vector가 가장 편리하였으며, 성공적인 라이브러리제작에는 fosmid vector에 클로닝이 가능한 40 kbp 크기의 DNA 조각을 얻는 과정이 중요함을 알았다. 여러 실험 조건을 종합적으로 검사한 후 메타제놈 라이브러리 제작에 대한 최적 방법을 제사하였다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권5호
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• pp.393-399
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• 2004

Useful genes can be Screened from various environments by construction of metagenomic DNA libraries. In this study, water samples were collected from several lakes in mid Korea, and analyzed by T-RFLP to examine diversities of the microbial communities. The crude DNAs r were extracted by the SDS-based freezing-thawing method, and then further purified using an $UltraClean^{TM}$ kit (MoBio, USA). The metagenomic libraries were constructed with the DNAs partially digested with EcoR I, BamH I, and Sac II in Escherichia coli DH 10B using the pBACe3.6 vector. About 44.0 Mb of metagenomic libraries were obtained with average inserts 13-15 kb in size. The bphC genes responsible for degradation of aromatic hydrocarbons via mets-cleavage were identified from the metagenomic libraries by colony hybridization using the bphC specific sequence as a probe. The 2,3-dihydroxybiphenyl (2, 3-DHBP) dioxygenase gene (bphC ), capable of degradation of 2,3-DHBP, was cloned and its nucleotide Sequences analyzed. The genes consisted of 966 and 897 base pairs with an ATG initiation codon and a TGA termination codon. The activity of the 2,3-DHBP dioxygenase was highly expressed to 2,3-DHBP and Showed a broad substrate range to 2,3-DHBP, catechol, 3-methylcatechol and 4-methylcatechol. These results in-dicated that the bphC gene identified from the metagenomes derived from lake water might be useful in the development of a potent strain for degradation of aromatic pollutants.

• The Plant Pathology Journal
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• 제21권2호
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• pp.93-98
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• 2005

Molecular ecological studies of microbial communities revealed that only tiny fraction of total microorganisms in nature have been identified and characterized, because the majority of them have not been cultivated. A concept, metagenome, represents the total microbial genome in natural ecosystem consisting of genomes from both culturable microorganisms and viable but non-culturable bacteria. The construction and screening of metagenomic libraries in culturable bacteria constitute a valuable resource for obtaining novel microbial genes and products. Several novel enzymes and antibiotics have been identified from the metagenomic approaches in many different microbial communities. Phenotypic analysis of the introduced unknown genes in culturable bacteria could be an important way for functional genomics of unculturable bacteria. However, estimation of the number of clones required to uncover the microbial diversity from various environments has been almost impossible due to the enormous microbial diversity and various microbial population structure. Massive construction of metagenomic libraries and development of high throughput screening technology should be necessary to obtain valuable microbial resources. This paper presents the recent progress in metagenomic studies including our results and potential of metagenomics in plant pathology and agriculture.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.108.1-108
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• 2003

Soil metagenome is untapped total microbial genome including that of the majority of unculturable bacteria present in soil. We constructed soil metagenomic library in Escherichia coli using DNA directly extracted from two different soils, pine tree rhizosphere soil and forest topsoil. Metagenomic libraries constructed from pine tree rhizosphere soil and forest topsoil consisted of approximately 33,700 clones and 112,000 clones with average insert DNA size of 35-kb, respectively. Subsequently, we screened the libraries to select clones with antimicrobial activities against Saccharomyces cerevisiae and Agrobacterium tumefaciens using double agar layer method. So far, we have a clone active against S. cerevisiae and a clone active against A. tumefaciens from the forest topsoil library. In vitro mutagenesis and DNA sequence analysis of the antifungal clone revealed the genes involved in the biosynthesis of antimicrobial secondary metabolite. Metagenomic libraries constructed in this study would be subject to search for diverse genetic resources related with useful microbial products.

• 생태와환경
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• 제38권2호통권112호
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• pp.137-145
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• 2005

전통적인 스크린 방법으로는 자연계에 존재하는 99% 이상의 미생물 자원을 확보하지 못했다. 자연생태계의 핵산을 직접 클로닝하는 전략은 배양 가능한 미생물의 유전적인 정보보다 더 광범위한 유전적인 정보를 전체의 미생물 메타지놈에서 확보하기 위한 계획을 세웠다. 그 결과 유용한 유전자를 탐색하는 한 방법으로 다양한 환경에서 메타지놈 DNA 라이브러리를 구축하는 방법이었다. 본 연구는 국내 중부권에 위치한 대청호로부터 시료를 수집하였고, T-RFLP 방법을 사용하여 미생물 군집의 다양성을 분석하였다. 핵산의 추출은 SDS를 사용한 freeing-thawing 방법을 사용하였으며, 추출한 핵산은 $UltraClean^{TM}kit$ (MoBio, USA)을 사용하여 정제하였다. 메타지놈 라이브러리는 제작은 정제한DNA와 pBACe3.6 vector를 EcoRI, BamHI, 그리고 SacII 등의 제한효소로 partial digestion하였고, 이들을 ligation한 다음 Escherichia coli DH10B에 형질전화 시켜 제작하였다. 메타지놈 라이브러는 14 Mb 정도 확보하였는데, 평균 insert size는 약 13 ${\sim}$ 15 kb이었다. Colony hybridization으로 메타지놈 라이브러리로부터 1, 2-dichloroethane (1, 2-DCE) hydrolytic dehalogenation의 분해에 관련된 유전자를 확인하였다. 1, 2-DCE dehalogenas효소는 기질에 대한 높은 활성을 나타내었다. 1, 2-dichloroethane dehalogenase 유전자의 클론을 만들었고, 염기서열을 분석하였다. 이들 결과로 보아 대청호로부터 제작한 메타지놈에서 dhlA 유전자를 확인한 균주는 1, 2-DCE 분해에 탁월한 능력을 나타내었다.

• Journal of Microbiology and Biotechnology
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• 제19권9호
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• pp.888-897
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• 2009

Lipase diversity in glacier soil was assessed by culture-independent metagenomic DNA fragment screening and confirmed by cell culture experiments. A set of degenerate PCR primers specific for lipases of the hormone-sensitive lipase family was designed based on conserved motifs and used to directly PCR amplify metagenomic DNA from glacier soil. These products were used to construct a lipase fragment clone library. Among the 300 clones sequenced for the analysis, 201 clones encoding partiallipases shared 51-82% identity to known lipases in GenBank. Based on a phylogenetic analysis, five divergent clusters were established, one of which may represent a previously unidentified lipase subfamily. In the culture study, 11 lipase-producing bacteria were selectively isolated and characterized by 16S rDNA sequences. Using the above-mentioned degenerate primers, seven lipase gene fragments were cloned, but not all of them could be accounted for by the clones in the library. Two full-length lipase genes obtained by TAIL-PCR were expressed in Pichia pastoris and characterized. Both were authentic lipases with optimum temperatures of ${\le}40^{\circ}C$. Our study indicates the abundant lipase diversity in glacier soil as well as the feasibility of sequence-based screening in discovering new lipase genes from complex environmental samples.

• Environmental Engineering Research
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• 제12권5호
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• pp.231-237
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• 2007

The microbial eco-physiology has been the vital key of microbial ecological research. Unfortunately, available methods for direct identity of microorganisms and for the investigation of their activity in complicated community dynamics are limited. In this study, metagenomics was considered as a promising functional genomics tool for improving our understanding of microbial eco-physiology. Its potential applications and challenges were also reviewed. Because of tremendous diversity in microbial populations in environment, sequence analysis for whole metagenomic libraries from environmental samples seems to be unrealistic to most of environmental engineering researchers. When a target function is of interest, however, sequence analysis for whole metagenomic libraries would not be necessary. For this case, nucleic acids of active populations of interest can be selectively gained using another cutting-edge functional genomic tool, SIP (stable isotope probing) technique. If functional genomes isolated by SIP can be transferred into metagenomic library, sequence analysis for such selected functional genomes would be feasible because the reduced size of clone library may become adequate for sequencing analysis. Herein, integration of metagenomics with SIP was suggested as a novel functional genomics approach to study microbial eco-physiology in environment.

• Journal of Microbiology and Biotechnology
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• 제16권1호
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• pp.153-157
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• 2006

A soil metagenomic library was constructed using an E. coli-fosmid cloning system with environmental DNAs extracted from Kwangreung forest topsoil. We targeted the genes involved in the biosynthesis of bacterial polyketides. Initially, a total of 36 clone pools (10,800 clones) were explored by the PCR-based method using the metagenomic DNAs from each pool and a degenerate primer set, which has been designed based on the highly conserved regions among ketoacyl synthase (KS) domains in actinomycete type I polyketide synthases (PKS Is). Six clone pools were tentatively selected as positive and further examined through a hybridization-based method for selecting a fosmid clone containing PKS I genes. Colony hybridization was performed against fosmid clones from the 6 positive pools, and finally 4 clones were picked out and confirmed to contain the conserved DNA fragment of KS domains. In this study, we present a simple and feasible sorting method for a desired clone from metagenomic libraries.

• Journal of Microbiology and Biotechnology
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• 제24권6호
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• pp.812-822
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• 2014

Metagenomic analysis of the human intestinal microbiota has extended our understanding of the role of these bacteria in improving human intestinal health; however, a number of reports have shown that current total fecal DNA extraction methods and 16S rRNA universal primer sets could affect the species coverage and resolution of these analyses. Here, we improved the extraction method for total DNA from human fecal samples by optimization of the lysis buffer, boiling time (10 min), and bead-beating time (0 min). In addition, we developed a new long-range 16S rRNA universal PCR primer set targeting the V6 to V9 regions with a 580 bp DNA product length. This new 16S rRNA primer set was evaluated by comparison with two previously developed 16S rRNA universal primer sets and showed high species coverage and resolution. The optimized total fecal DNA extraction method and newly designed long-range 16S rRNA universal primer set will be useful for the highly accurate metagenomic analysis of adult and infant intestinal microbiota with minimization of any bias.

• Genomics & Informatics
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• 제11권3호
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• pp.102-113
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• 2013

Metagenomics has become one of the indispensable tools in microbial ecology for the last few decades, and a new revolution in metagenomic studies is now about to begin, with the help of recent advances of sequencing techniques. The massive data production and substantial cost reduction in next-generation sequencing have led to the rapid growth of metagenomic research both quantitatively and qualitatively. It is evident that metagenomics will be a standard tool for studying the diversity and function of microbes in the near future, as fingerprinting methods did previously. As the speed of data accumulation is accelerating, bioinformatic tools and associated databases for handling those datasets have become more urgent and necessary. To facilitate the bioinformatics analysis of metagenomic data, we review some recent tools and databases that are used widely in this field and give insights into the current challenges and future of metagenomics from a bioinformatics perspective.