토양 Metagenome Library로부터 고추역병 저해 클론 탐색

박해철,성소라,김동관,구본성,정병문,김진흥,윤문영,Park, Hae-Chul,Sung, So-Ra,Kim, Dong-Gwan,Koo, Bon-Sung,Jeong, Byeong-Moon,Kim, Jin-Heung,Yoon, Moon-Young 한국미생물학회 2009 미생물학회지 Vol.45 No.2

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. 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.

Functional gene networks based on the gene neighborhood in metagenomes

김찬영,이인석 한국통합생물학회 2017 Animal cells and systems Vol.21 No.5

The gene neighborhood in prokaryotic genomes has been effectively utilized in inferring cofunctional networks in various organisms. Previously, such genomic context information has been sought among completely assembled prokaryotic genomes. Here, we present a method to infer functional gene networks according to the gene neighborhood in metagenome contigs, which are incompletely assembled genomic fragments. Given that the amount of metagenome sequence data has now surpassed that of completely assembled prokaryotic genomes in the public domain, we expect benefits of inferring networks by the metagenome-based gene neighborhood. We generated co-functional networks for diverse taxonomical species using metagenomics contigs derived from the human microbiome and the ocean microbiome. We found that the networks based on the metagenome gene neighborhood outperformed those based on 1748 completely assembled prokaryotic genomes. We also demonstrated that the metagenome-based gene neighborhood could predict genes related to virulence-associated phenotypes in a bacterial pathogen, indicating that metagenome-based functional links could be sufficiently predictive for some phenotypes of medical importance. Owing to the exponential growth of metagenome sequence data in public repositories, metagenome-based inference of cofunctional networks will facilitate understanding of gene functions and pathways in diverse species.

Metagenome Resource for D-Serine Utilization in a DsdA-Disrupted Escherichia coli

( Mi Young Lim ),( Hyo Jeong Lee ),( Pil Kim ) 한국미생물 · 생명공학회 2011 Journal of microbiology and biotechnology Vol.21 No.4

To find alternative genetic resources for D-serine dehydratase (E.C. 4.3.1.18, dsdA) mediating the deamination of D-serine into pyruvate, metagenomic libraries were screened. The chromosomal dsdA gene of a wild-type Escherichia coli W3110 strain was disrupted by inserting the tetracycline resistance gene (tet), using double-crossover, for use as a screening host. The W3110 dsdA::tet strain was not able to grow in a medium containing D-serine as a sole carbon source, whereas wild-type W3110 and the complement W3110 dsdA::tet strain containing a dsdA-expression plasmid were able to grow. After introducing metagenome libraries into the screening host, a strain containing a 40-kb DNA fragment obtained from the metagenomic souce derived from a compost was selected based on its capability to grow on the agar plate containing D-serine as a sole carbon source. For identification of the genetic resource responsible for the D-serine degrading capability, transposon- μ was randomly inserted into the 40-kb metagenome. Two strains that had lost their D-serine degrading ability were negatively selected, and the two 6-kb contigs responsible for the D-serine degrading capability were sequenced and deposited (GenBank code: HQ829474.1 and HQ829475.1). Therefore, new alternative genetic resources for D-serine dehydratase was found from the metagenomic resource, and the corresponding ORFs are discussed.

A new metagenome binning method based on gene uniqueness

Yulin Kang,Cheng Wan,Sifen Lu,Zhehan Fu,Antony K. Chen,Zuhong Lu 한국유전학회 2020 Genes & Genomics Vol.42 No.8

Background The human gut microbiome contains millions of genes and many undetected bacteria species. Recovering bacterial genomes from large complex metagenomes remains highly challenging, and current binning methods show insufficient recall rates. Objective This study was performed to put forward a new metagenome binning method with promising recall rate and accuracy. Methods We found that more than 85% of the genes could be aligned to only one bacteria species by using strict BLAST parameters (identity > 90% and aligning length > 100 bp). This phenomenon was called “the gene uniqueness”, which indicated that the most bacterial genes could be exclusive to the species’ taxonomy. In our new metagenome binning method, we could cluster contigs based on gene similarity via a graph model. Any contig shared with same gene under Strict Blast parameters would be clustered into one bin. Results we obtained 1,131 bins and reconstructed the genomes of 12 unknown species for MetaHIT data Our method exhibited a more promising recall rate, faster running speed and lower time complexity than the current methods. Conclusions The present new metagenome binning method based on gene uniqueness had high recall rate and low error, which could be applied to assemble the bacterial genomes efficiently in complex metagenome.

Comparison of methods for library construction and short read annotation of shellfish viral metagenomes

Hong‑Ying Wei,Sheng Huang,Jiang‑Yong Wang,Fang Gao,Jing‑Zhe Jiang 한국유전학회 2018 Genes & Genomics Vol.40 No.3

The emergence and widespread use of high-throughput sequencing technologies have promoted metagenomic studies on environmental or animal samples. Library construction for metagenome sequencing and annotation of the produced sequence reads are important steps in such studies and influence the quality of metagenomic data. In this study, we collected some marine mollusk samples, such as Crassostrea hongkongensis, Chlamys farreri, and Ruditapes philippinarum, from coastal areas in South China. These samples were divided into two batches to compare two library construction methods for shellfish viral metagenome. Our analysis showed that reverse-transcribing RNA into cDNA and then amplifying it simultaneously with DNA by whole genome amplification (WGA) yielded a larger amount of DNA compared to using only WGA or WTA (whole transcriptome amplification). Moreover, higher quality libraries were obtained by agarose gel extraction rather than with AMPure bead size selection. However, the latter can also provide good results if combined with the adjustment of the filter parameters. This, together with its simplicity, makes it a viable alternative. Finally, we compared three annotation tools (BLAST, DIAMOND, and Taxonomer) and two reference databases (NCBI’s NR and Uniprot’s Uniref). Considering the limitations of computing resources and data transfer speed, we propose the use of DIAMOND with Uniref for annotating metagenomic short reads as its running speed can guarantee a good annotation rate. This study may serve as a useful reference for selecting methods for Shellfish viral metagenome library construction and read annotation.

Estimating the composition of species in metagenomes by clustering of next-generation read sequences

Seok, H.S.,Hong, W.,Kim, J. Academic Press 2014 Methods Vol.69 No.3

Faster and cheaper sequencing technologies together with the ability to sequence uncultured microbes collected from any environment present us an opportunity to distill meaningful information from the millions of new genomic sequences from environmental samples, called metagenome. Contrary to conventional cultured microbes, however, the metagenomic data is extremely heterogeneous and noisy. Therefore the separation of the sets of sequenced genomic fragments that belong to different microbes is essential for successful assembly of microbial genomes. In this paper, we present a novel clustering method for a given metagenomic dataset. The metagenomic dataset has some distinguished features because (i) it is possible that similar sequence patterns may exist in different species and (ii) each species has different number of individuals in the given metagenomic dataset. Our method overcomes these obstacles by using the Gaussian mixture model and analysis of mixture profiles, and taking advantage of genomic signatures extracted from the metagenomic dataset. Unlike conventional clustering methods where clusters are discovered through global similarities of data instances, our method builds clusters by combining the data instances sharing local similarities captured by mixture analysis. By considering shared mixture components, our method is able to create clusters of genomic sequences although they are globally distinct each other. We applied our method to an artificial metagenomic dataset comprised of simulated 47 million reads from 25 real microbial genomes, and analyzed the resulting clusters in terms of the number of clusters, the number of participating species and dominant species in each cluster. Even though our approach cannot address all challenges in the field of metagenome sequence clustering, we believe that out method can contribute to take a step forward to achieve the goals.

BARM : 유전자 조립과 레퍼런스 얼라인먼트를 접목한 메타유전체 비닝 방법

여윤구(Yunku Yeo),문명진(Myungjin Moon),김우철(Woocheol Kim),박상현(Sanghyun Park) 한국정보과학회 2011 정보과학회논문지 : 데이타베이스 Vol.38 No.2

메타유전체는 환경에서 직접 채취한 유전체 정보의 집합으로서, 이를 통해 연구실 환경에서 얻을 수 없는 다양한 유전체 정보를 얻을 수 있다. 메타유전체에는 수많은 생물체의 유전체가 뒤섞여 있기 때문에, 그 안에 존재하는 생물의 구성과 비율을 알아내는 것이 중요한 문제가 된다. 이러한 문제를 비닝이라고 하는데, 16S rRNA를 이용하는 것이 대표적인 비닝 방법이다. 16S rRNA를 이용하면 매우 정확한 결과를 얻을 수 있지만, 별도의 라이브러리를 구축해야 하기 때문에 시간과 비용이 많이 필요하다. 이 때문에, 컴퓨터 계산을 기반으로 하는 여러 가지 비닝 방법이 개발되고 있다. 본 논문은 레퍼런스 얼라인먼트 방식의 비닝 방법에 유전체 조립(genome assembly) 알고리즘을 융합하여 새로운 비닝 방법인 BARM을 개발하였다. 가상 변이 생성기를 이용하여 메타유전체 환경을 시뮬레이션하여 실험한 결과, 레퍼런스 데이터가 부족한 종의 비닝에 있어서 기존 비닝 방법보다 더 우수한 결과를 나타내었다. Metagenome is a large set of genomic information, collected directly from the environmental sample. We can acquire much information which cannot be obtained under laboratory conditions. Since the metagenome is a complicate mixture of numerous species, it is an important problem to infer the composition of species in metagenome - the binning problem. Binning with 16S rRNA is the most widely-used and accurate binning method. But it requires much time and high cost for the construction of additional biological library. To solve this problem, many computational binning methods such as Random Sequence Read(RSR) are developed. In this paper, we suggest a new binning approach BARM - a conjunction of reference alignment and genome assembly. We compare BARM with RSR using the synthetic genomic-variants generator, and BARM produced more superior result in genomic diversity of metagenome.

Sequence-based screening for self-sufficient P450 monooxygenase from a metagenome library

Kim, B.S.,Kim, S.Y.,Park, J.,Park, W.,Hwang, K.Y.,Yoon, Y.J.,Oh, W.K.,Kim, B.Y.,Ahn, J.S. Published for the Society for Applied Bacteriology 2007 Journal of applied microbiology Vol.102 No.5

<P>Abstract</P><P>Aims: </P><P>Cytochrome P450 monooxygenases (CYPs) are useful catalysts for oxidation reactions. Self-sufficient CYPs harbour a reductive domain covalently connected to a P450 domain and are known for their robust catalytic activity with great potential as biocatalysts. In an effort to expand genetic sources of self-sufficient CYPs, we devised a sequence-based screening system to identify them in a soil metagenome.</P><P>Methods and Results: </P><P>We constructed a soil metagenome library and performed sequence-based screening for self-sufficient CYP genes. A new CYP gene, <I>syk181</I>, was identified from the metagenome library. Phylogenetic analysis revealed that SYK181 formed a distinct phylogenic line with 46% amino-acid-sequence identity to CYP102A1 which has been extensively studied as a fatty acid hydroxylase. The heterologously expressed SYK181 showed significant hydroxylase activity towards naphthalene and phenanthrene as well as towards fatty acids.</P><P>Conclusions: </P><P>Sequence-based screening of metagenome libraries is expected to be a useful approach for searching self-sufficient CYP genes. The translated product of <I>syk181</I> shows self-sufficient hydroxylase activity towards fatty acids and aromatic compounds.</P><P>Significance and Impact of the Study: </P><P>SYK181 is the first self-sufficient CYP obtained directly from a metagenome library. The genetic and biochemical information on SYK181 are expected to be helpful for engineering self-sufficient CYPs with broader catalytic activities towards various substrates, which would be useful for bioconversion of natural products and biodegradation of organic chemicals.</P>

메타유전체 서열 조립의 문제점에 대한 연구

김우철,박상현,여윤구,김종현 한국정보과학회 2009 데이타베이스 연구 Vol.25 No.1

미생물의 경우 전체 생물종 중에서 많은 비율을 차지함에도 불구하고, 대부분의 미생물은 자연상태에서 분리시켜 배양하여 연구하기 힘들다. 최근의 미생물학은 자연상태에 있는 미생물을 직접 연구하려는 시도를 하고 있는데, 이런 연구를 가능하게 하기 위해서는 메타유전체의 염기서열을 밝혀내야 한다. 메타유전체의 염기서열을 밝혀내면, 현재 유전체학(Genomics)에 이용되는 분석방법을 적용할 수 있게 된다. 그러나, 단일 유전체가 아닌 특정 환경에서 살고 있는 다양한 미생물 유전체들의 집합인 메타유전체(Metagenome)의 경우에는 현재 확립된 서열조립 방법론이 없는 실정이다. 유전체의 염기 서열을 밝히는데 일반적으로 널리 쓰이고 있는 방법은 Whole Genome Shotgun Sequencing(WGSS)이다. 기존의 WGSS방식은 염기변이가 낮은 단일 유전체를 서열화(sequencing)하는데 초점을 맞춰서 개발되었기 때문에 염기변이율이 높은 유전체(polymorphic genome)의 서열화에 적용시킬 경우에는 염기서열의 연속성(continuity)이 심각하게 손상된다. 염기변이로 인한 염기서열 연속성의 손상은 여러 미생물들의 유전체서열이 동시에 조립되는 메타유전체의 경우에는 더욱 심각해 진다. 이와 같이 메타유전체를 대상으로 하는 서열 조립에 대한 후속 연구들이 필요하다. 따라서 본 논문은 이러한 연구들의 기초 연구가 될 수 있는 메타유전체 조립 알고리즘에 대한 연구의 중요성과 메타유전체 서열화 과정에서 발생하는 문제점들의 원인을 분석한다. 이를 바탕으로 후속 연구들이 파생될 수 있는 기초 연구를 제공한다. Although microbial organisms take a significant portion of taxa, most of them are not trivially cultured under the laboratory condition. Recent advances in microbiology and availability of Metagenome sequences enable the study of microbial organisms under the natural condition. Although Metagenome sequences are essential to apply genomics technologies to the study of microbial organisms, the strategy to assemble Metagenome sequences is yet to be established. Whole-genome Shotgun Sequencing (WGSS) has been widely used to sequence genomes. Because the assembly strategy of WGSS has focused on assembling less polymorphic genomes, it undermines the continuity of assembled genome sequences, applied to highly polymorphic genomes. The impairment of the sequence continuity becomes more serious in assembling metagenomes. In this paper, we note the problem of the impaired continuity, and emphasize the urgency of optimizing assembly strategy for metagenomes.

메타게놈 서열에 존재하는 보존적인 전사와 번역 인자를 이용한 ORF 예측

정대은(Dea-Eun Cheong),김근중(Geun-Joong Kim) 한국생물공학회 2010 KSBB Journal Vol.25 No.5

미생물은 지구상에 약 5 × 10<SUP>30</SUP> 정도의 개체가 존재하며, 350~550 Pg (1Pg = 10<SUP>15</SUP>g)의 탄소, 85~130 Pg의 질소, 9~14 Pg의 인 등, 지구상의 어떠한 생물 종보다 거대한 양의 원소를 포함하고 있다 [28]. 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 (training 이나 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 개량여하에 따라 광범위한 적용 범위를 지닐 것이다. 현 수준에 서도 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. 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.