• Genomics & Informatics
• /
• v.11 no.3
• /
• pp.114-120
• /
• 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 Microbiology and Biotechnology
• /
• v.26 no.3
• /
• pp.521-529
• /
• 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.

• Journal of Life Science
• /
• v.16 no.2 s.75
• /
• pp.360-364
• /
• 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
• /
• v.25 no.6
• /
• pp.930-935
• /
• 2015

A phytase gene was identified in a publicly available metagenome derived from subsurface groundwater, which was deduced to encode for a protein of the histidine acid phosphatase (HAP) family. The nucleotide sequence of the phytase gene was chemically synthesized and cloned, in order to further overexpress the phytase in Escherichia coli. Purified protein of the recombinant phytase demonstrated an activity for phytic acid of 298 ± 17 µmol P/min/mg, at the pH optimum of 2.0 with the temperature of 37℃. Interestingly, the pH optimum of this phytase is much lower in comparison with most HAP phytases known to date. It suggests that the phytase could possess improved adaptability to the low pH condition caused by the gastric acid in livestock and poultry stomachs.

• Korean Journal of Microbiology
• /
• v.45 no.2
• /
• pp.228-231
• /
• 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
• /
• v.17 no.10
• /
• pp.1655-1660
• /
• 2007

A metagenome is a unique resource to search for novel microbial enzymes from the unculturable microorganisms in soil. A forest soil metagenomic library using a fosmid and soil microbial DNA from Gwangneung forest, Korea, was constructed in Escherichia coli and screened to select lipolytic genes. A total of seven unique lipolytic clones were selected by screening of the 31,000-member forest soil metagenome library based on tributyrin hydrolysis. The ORFs for lipolytic activity were subcloned in a high copy number plasmid by screening the secondary shortgun libraries from the seven clones. Since the lipolytic enzymes were well secreted in E. coli into the culture broth, the lipolytic activity of the subclones was confirmed by the hydrolysis of p-nitrophenyl butyrate using culture supernatant. Deduced amino acid sequence analysis of the identified ORFs for lipolytic activity revealed that 4 genes encode hormone-sensitive lipase (HSL) in lipase family IV. Phylogenetic analysis indicated that 4 proteins were clustered with HSL in the database and other metagenomic HSLs. The other 2 genes and 1 gene encode non-heme peroxidase-like enzymes of lipase family V and a GDSL family esterase/lipase in family II, respectively. The gene for the GDSL enzyme is the first description of the enzyme from metagenomic screening.

• Journal of Microbiology and Biotechnology
• /
• v.26 no.2
• /
• pp.248-254
• /
• 2016

A soil metagenome contains the genomes of all microbes included in a soil sample, including those that cannot be cultured. In this study, soil metagenome libraries were searched for microbial genes exhibiting lipolytic activity and those involved in potential lipid metabolism that could yield valuable products in microorganisms. One of the subclones derived from the original fosmid clone, pELP120, was selected for further analysis. A subclone spanning a 3.3 kb DNA fragment was found to encode for lipase/esterase and contained an additional partial open reading frame encoding a wax ester synthase (WES) motif. Consequently, both pELP120 and the full length of the gene potentially encoding WES were sequenced. To determine if the wes gene encoded a functioning WES protein that produced wax esters, gas chromatography-mass spectroscopy was conducted using ethyl acetate extract from an Escherichia coli strain that expressed the wes gene and was grown with hexadecanol. The ethyl acetate extract from this E. coli strain did indeed produce wax ester compounds of various carbon-chain lengths. DNA sequence analysis of the full-length gene revealed that the gene cluster may be derived from a member of Proteobacteria, whereas the clone does not contain any clear phylogenetic markers. These results suggest that the wes gene discovered in this study encodes a functional protein in E. coli and produces wax esters through a heterologous expression system.

• Proceedings of the Microbiological Society of Korea Conference
• /
• 2009.05a
• /
• pp.114-115
• /
• 2009

• Journal of Microbiology and Biotechnology
• /
• v.18 no.10
• /
• pp.1634-1640
• /
• 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.

• Journal of Microbiology and Biotechnology
• /
• v.16 no.1
• /
• pp.153-157
• /
• 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.