• Korean Journal of Microbiology
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• v.45 no.2
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• pp.228-231
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• 2009

We have purified Phytophthora capsici alpha and beta tubulin from Escherchia coli BL21(DE3). The recombinant alpha and beta tubulins were assembled into microtubule in vitro with specific conditions. The metagenome library was isolated from soil in the Mt. Yeo-Ki, Suwon, Korea and manufactured with the method mentioned in experiment contents for in vitro screening of microtubule assembly screening. FRET effect was used for microtubule assembly inhibitor screening with metagenome library. We got 2 metagenome clones from in vitro screening, and these 2 hit clones showed P. capsici growth inhibition activity on the growing pepper plants. These results suggest that new development of potent inhibitor for pepper blight disease and new approach to prevention of pepper blight disease.

• Journal of Microbiology and Biotechnology
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• v.26 no.3
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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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• v.11 no.3
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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.

• Journal of Life Science
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• v.16 no.2 s.75
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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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• v.18 no.10
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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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• v.25 no.5
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• pp.490-496
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• 2010

As sequencing technologies are steadily improving, massive sequence data have been accumulated in public databases. Thereby, programs based on various algorithms are developed to mine useful information, such as genes, operons and regulatory factors,from these sequences. However, despite its usefulness in a wide range of applications, comprehensive analyses of metagenome using these programs have some drawbacks, thereby yielding inaccurate or complex results. We here provide a possibility of signature sequences (cis-acting transcriptional and translational factors of metagenome) as a hallmark of ORFs finding from metagenome.

• Korean Journal of Agricultural Science
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• v.48 no.3
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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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• v.24 no.5
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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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• v.31 no.2
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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.

• Microbiology and Biotechnology Letters
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• v.36 no.4
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• pp.260-267
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• 2008

The choice of expression vector is very important for industrial production of proteins. Therefore, the systematic mining of promoters over a wider range of genetic resource and/or host is required. We previously reported a novel bidirectional reporting system (pBGR) for the isolation of promoters from metagenome and screened useful promoters that functioned constitutively in E. coli under general culture conditions. Among them, three promoter sequences including each upstream region were amplified by PCR and used to construct new expression vectors. To facilitate subcloning, a multi-cloning site was incorporated into the downstream region of the revere primer sequence. At these sites, GFP, esterase and $\beta$-glucosidase were subcloned and analyzed the constitutive expression ability of new promoter in terms of protein solubility and expression level. As a result, these vectors expressed the proteins constitutively to a level of $2{\sim}3%$ of the total cell protein in soluble fraction (>80 %). This study suggested that excavation of metagenomic promoters for construction of expression vector in a certain strain could provide a way for the development of the expression systems.