Construction Of Metagenomic Libraries Research Articles

Functional Metagenomics to Study Antibiotic Resistance.

The construction and screening of metagenomic expression libraries have a great potential to identify novel genes with desired functions. Here, we describe metagenomic library preparation from fecal DNA, screening of libraries for antibiotic resistance genes (ARGs), massively parallel DNA sequencing of the enriched DNA fragments, and a computational pipeline for high-throughput assembly and annotation of functionally selected DNA.

Construction of Small-Insert and Large-Insert Metagenomic Libraries.

The vast majority of the Earth's biological diversity are hidden in uncultured and yet uncharacterized microbial genomes. The construction of metagenomic libraries is one cultivation-independent molecular approach to assess this unexplored genetic reservoir. High numbers of novel biocatalysts have been identified by function-based or sequence-based screening of metagenomic libraries derived from various environments. Here, we describe detailed protocols for the construction of metagenomic small-insert and large-insert libraries in plasmids and fosmids, respectively, from environmental DNA.

Identification of PKS Gene Clusters from Metagenomic Libraries Using a Next-Generation Sequencing Approach.

Microbial secondary metabolites have been an important source of bioactive compounds with diverse applications from medicine to agriculture, noticeably those encoded by polyketide synthase (PKS) clusters due to their astounding chemical diversity. While most discovered compounds originate from culturable microorganisms, yet-to-be cultured microbes represent a reservoir of previously inaccessible compounds. The advent and development of metagenomics have allowed not only the characterization of these microorganisms but also their metabolic potential, making viable the prospection of environmental PKS for natural product discovery.Study of environmental PKSs often relies on the construction of metagenomic libraries and their mining, with clones containing PKS clusters identified via amplification of conserved domains and then screened for an activity of interest. Compounds produced by clones exhibiting the desired bioactivity can be isolated and characterized. However, these approaches can be less sensitive and biased against more divergent clusters, in addition to precluding the use of bioinformatics for cluster characterization prior to expression. While direct shotgun sequencing of metagenomes has identified and profiled a great number of PKSs from different environments and yet-to-be cultured microorganisms, it does not lend itself well to heterologous expression, the cruxes of natural product discovery.Here, we describe a strategy for sequencing entire metagenomic libraries while maintaining correspondence between sequence and clone, allowing the full characterization and annotation of all clusters present in a library using bioinformatic tools and then seamlessly passing clones of interest for activity screening through heterologous expression. Once a library is sequenced, the methods herein can be adapted for the mining of any biosynthetic gene cluster of interest within a metagenomic library.

Mosaic Ends Tagmentation (METa) Assembly for Highly Efficient Construction of Functional Metagenomic Libraries.

ABSTRACTFunctional metagenomic libraries, physical bacterial libraries which allow the high-throughput capture and expression of microbiome genes, have been instrumental in the sequence-naive and cultivation-independent exploration of metagenomes. However, preparation of these libraries is often limited by their high DNA input requirement and their low cloning efficiency. Here, we describe a new method, mosaic ends tagmentation (METa) assembly, for highly efficient functional metagenomic library preparation. We applied tagmentation to metagenomic DNA from soil and gut microbiomes to prepare DNA inserts for high-throughput cloning into functional metagenomic libraries. The presence of mosaic end sequences in the resulting DNA fragments synergized with homology-based assembly cloning to result in a 300-fold increase in cloning efficiency compared to traditional blunt-cloning-based protocols. We show that compared to published libraries prepared by state-of-the-art protocols, METa assembly is on average ca. 20- to 200-fold more efficient and can prepare gigabase-sized libraries with as little as 200 ng of input DNA. We show the usefulness of METa assembly first by using a normative 5-μg mass of soil metagenomic DNA to prepare a 700-Gb library that allowed us to discover novel nourseothricin resistance genes and a potentially new mode of resistance to this antibiotic and second by using only 300 ng of goose fecal metagenomic DNA to prepare a 27-Gb library that captured numerous tetracycline and colistin resistance genes. METa assembly provides a streamlined, flexible, and efficient method for preparing functional metagenomic libraries, enabling new avenues of genetic and biochemical research into low-biomass or scarce microbiomes.IMPORTANCE Medically and industrially important genes can be recovered from microbial communities by high-throughput sequencing, but precise annotation is often limited to characterized genes and their relatives. Cloning a metagenome en masse into an expression host to produce a functional metagenomic library, directly connecting genes to functions, is a sequence-naive and cultivation-independent method to discover novel genes. The process of preparing these libraries is DNA greedy and inefficient, however. Here, we describe a library preparation method that is an order of magnitude more efficient and less DNA greedy. This method is consistently efficient across libraries prepared from cultures, a soil microbiome, and a goose fecal microbiome and allowed us to discover new antibiotic resistance genes and mechanisms. This library preparation method will potentially allow the functional metagenomic exploration of microbiomes that were previously off limits due to their rarity or low microbial biomass, such as biomedical swabs or exotic samples.

Strategies for Natural Products Discovery from Uncultured Microorganisms

Microorganisms are highly regarded as a prominent source of natural products that have significant importance in many fields such as medicine, farming, environmental safety, and material production. Due to this, only tiny amounts of microorganisms can be cultivated under standard laboratory conditions, and the bulk of microorganisms in the ecosystems are still unidentified, which restricts our knowledge of uncultured microbial metabolism. However, they could hypothetically provide a large collection of innovative natural products. Culture-independent metagenomics study has the ability to address core questions in the potential of NP production by cloning and analysis of microbial DNA derived directly from environmental samples. Latest advancements in next generation sequencing and genetic engineering tools for genome assembly have broadened the scope of metagenomics to offer perspectives into the life of uncultured microorganisms. In this review, we cover the methods of metagenomic library construction, and heterologous expression for the exploration and development of the environmental metabolome and focus on the function-based metagenomics, sequencing-based metagenomics, and single-cell metagenomics of uncultured microorganisms.

Discovery of a Novel Gene Conferring Tellurite Tolerance Through Tellurite Reduction to Escherichia coli Transformant in Marine Sediment Metagenomic Library.

Metagenomic library construction using a marine sediment-enrichment was employed in order to recover tellurium from tellurite, a tellurium oxyanion, dissolved in water and then functional screening was performed to discover a novel gene related to tellurite reduction. Transmission electron microscopy (TEM) revealed the formation of intracellular Te crystals in Escherichia coli cells transformed with a specific DNA fragment from the marine sediment metagenome. The metagenome fragment was composed of 691bp and showed low homology to known proteins. Phylogenetic analysis suggested that the metagenome fragment was related to Pseudomonas stutzeri. Cloning and expression of an open reading frame (ORF) on the metagenome fragment validated the role of the fragment in conferring tellurite resistance and tellurite-reducing activity to E. coli host cells. E. coli transformant containing the ORF1 showed resistance to 1mM Na2TeO3. The optimal tellurite-reducing activity of cells containing the ORF1 was recorded at 37°C and pH7.0.

Construction of Soil Metagenomic Library to Obtain Recombinant Clones with an Indigenous Lipase Activity

Penelitian ini bertujuan untuk mengonstruksi perpustakaan metagenom tanah dari sekitar lokasi industri pengolahan minyak kelapa di Kabupaten Banyumas, Jawa Tengah, dalam upaya mendapatkan klon rekombinan dengan aktivitas lipase indigenuos. Penelitian dilakukan di Laboratorium Genetika, Fakultas Biologi, Universitas Jenderal Soedirman mulai bulan Mei hingga Desember 2006. Secara umum tahapan penelitian terdiri atas ekstraksi dan pemurnian DNA tanah, isolasi pUC19, pemotongan DNA tanah, pemotongan dan defosforilasi pUC19, ligasi fragmen-fragmen DNA tanah ke dalam pUC19 untuk menghasilkan molekul DNA rekombinan, dan transformasi sel inang E. coli JM109 menggunakan molekul DNA rekombinan yang diperoleh untuk mengonstruksi perpustakaan metagenom tanah. Pemotongan DNA tanah menghasilkan fragmen-fragmen yang sebagian besar berukuran lebih kurang 5 kb sehingga gen lipase dimungkinkan untuk dibawa di dalamnya. Transformasi sel inang E. coli JM109 memperlihatkan efisiensi 1.7 x 105 cfu/μg plasmid. Di antara koloni-koloni yang diperoleh, terdapat 102 koloni berwarna putih, yang menunjukkan keberadaan molekul DNA rekombinan. Koloni putih ini dapat digunakan sebagai materi untuk skrining klon rekombinan dengan aktivitas lipase.

Rapid and economical protocols for genomic and metagenomic DNA extraction from oak (Quercus brantii Lindl.)

Two new efficient, fast and low-cost metagenomic DNA extraction methods were developed for different Persian oak tissues (leaf, stem, root, and rhizospheric) and soil samples. The new “omics” studies on the genus Quercus are of importance to help finding efficient strategies for overcoming environmental challenges, and to do this, presence of efficient DNA extraction protocols for different Quercus species are very critical. The objective of the present study was to develop new efficient methods for extraction of metagenomic DNA (mDNA) from of Persian oak (Quercus brantii Lindl.) tissues. The efficiency of two newly developed mDNA extraction methods, including indirect SDS-based (ISB or concentrate method) and one spin column-based method (SCB) were compared to that of two classical direct methods, including CTAB-based and SDS-based methods, and two commercial mDNA extraction kits. The maximum average yield of mDNA for all samples (leaf, stem, root, bulk, and rhizospheric soils) was obtained by SCB (258 ng/μl) and ISB (189 ng/μl) methods, respectively. Successful PCR amplification for 16SrRNA and ITS sequence was consistently observed for ISB, SCB, and kit-extracted mDNAs, which confirmed the high purity of mDNA extracted by these methods. The new methods showed more than 96% quantitative PCR efficiency, and partial restriction digestion and metagenomic library construction confirmed the high efficiency of the newly developed methods. It could be concluded that two new protocols enhanced efficiency (yield, purity, and cost) of mDNA extraction from different tissues of Persian oak.

Rapid and efficient method to extract metagenomic DNA from estuarine sediments.

Metagenomic DNA from sediments of selective estuaries of Goa, India was extracted using a simple, fast, efficient and environment friendly method. The recovery of pure metagenomic DNA from our method was significantly high as compared to other well-known methods since the concentration of recovered metagenomic DNA ranged from 1185.1 to 4579.7µg/g of sediment. The purity of metagenomic DNA was also considerably high as the ratio of absorbance at 260 and 280nm ranged from 1.88 to 1.94. Therefore, the recovered metagenomic DNA was directly used to perform various molecular biology experiments viz. restriction digestion, PCR amplification, cloning and metagenomic library construction. This clearly proved that our protocol for metagenomic DNA extraction using silica gel efficiently removed the contaminants and prevented shearing of the metagenomic DNA. Thus, this modified method can be used to recover pure metagenomic DNA from various estuarine sediments in a rapid, efficient and eco-friendly manner.

Library Construction from Subnanogram DNA for Pelagic Sea Water and Deep-Sea Sediments.

Shotgun metagenomics is a low biased technology for assessing environmental microbial diversity and function. However, the requirement for a sufficient amount of DNA and the contamination of inhibitors in environmental DNA leads to difficulties in constructing a shotgun metagenomic library. We herein examined metagenomic library construction from subnanogram amounts of input environmental DNA from subarctic surface water and deep-sea sediments using two library construction kits: the KAPA Hyper Prep Kit and Nextera XT DNA Library Preparation Kit, with several modifications. The influence of chemical contaminants associated with these environmental DNA samples on library construction was also investigated. Overall, shotgun metagenomic libraries were constructed from 1 pg to 1 ng of input DNA using both kits without harsh library microbial contamination. However, the libraries constructed from 1 pg of input DNA exhibited larger biases in GC contents, k-mers, or small subunit (SSU) rRNA gene compositions than those constructed from 10 pg to 1 ng DNA. The lower limit of input DNA for low biased library construction in this study was 10 pg. Moreover, we revealed that technology-dependent biases (physical fragmentation and linker ligation vs. tagmentation) were larger than those due to the amount of input DNA.

Construction of Small-Insert and Large-Insert Metagenomic Libraries.

The vast majority of the Earth's biological diversity is hidden in uncultured and yet uncharacterized microbial genomes. The construction of metagenomic libraries is a cultivation-independent molecular approach to assess this unexplored genetic reservoir. High numbers of novel biocatalysts have been identified by function-based or sequence-based screening of metagenomic libraries derived from various environments. Here, we describe detailed protocols for the construction of metagenomic small-insert and large-insert libraries in plasmids and fosmids, respectively, from environmental DNA.

Construction and Screening of Marine Metagenomic Large Insert Libraries.

The marine environment covers more than 70 % of the world's surface. Marine microbial communities are highly diverse and have evolved during extended evolutionary processes of physiological adaptations under the influence of a variety of ecological conditions and selection pressures. They harbor an enormous diversity of microbes with still unknown and probably new physiological characteristics. In the past, marine microbes, mostly bacteria of microbial consortia attached to marine tissues of multicellular organisms, have proven to be a rich source of highly potent bioactive compounds, which represent a considerable number of drug candidates. However, to date, the biodiversity of marine microbes and the versatility of their bioactive compounds and metabolites have not been fully explored. This chapter describes sampling in the marine environment, construction of metagenomic large insert libraries from marine habitats, and exemplarily one function based screen of metagenomic clones for identification of quorum quenching activities.

Functional Metagenomics to Study Antibiotic Resistance.

The construction and screening of metagenomic expression libraries has great potential to identify novel genes and their functions. Here, we describe metagenomic library preparation from fecal DNA, screening of libraries for antibiotic resistance genes (ARGs), massively parallel DNA sequencing of the enriched DNA fragments, and a computational pipeline for high-throughput assembly and annotation of functionally selected DNA.

Identification and characterization of a chitin deacetylase from a metagenomic library of deep-sea sediments of the Arctic Ocean

BackgroundThe chemical and biological compositions of deep-sea sediments are interesting because of the underexplored diversity when it comes to bioprospecting. The special geographical location and climates make Arctic Ocean a unique ocean area containing an abundance of microbial resources. MethodsA metagenomic library was constructed based on the deep-sea sediments of Arctic Ocean. Part of insertion fragments of this library were sequenced. A chitin deacetylase gene, cdaYJ, was identified and characterized. ResultsA metagenomic library with 2750 clones was obtained and ten clones were sequenced. Results revealed several interesting genes, including a chitin deacetylase coding sequence, cdaYJ. The CdaYJ is homologous to some known chitin deacetylases and contains conserved chitin deacetylase active sites. CdaYJ protein exhibits a long N-terminal and a relative short C-terminal. Phylogenetic analysis revealed that CdaYJ showed highest homology to CDAs from Alphaproteobacteria. The cdaYJ gene was subcloned into the pET-28a vector and the recombinant CdaYJ (rCdaYJ) was expressed in Escherichia coli BL21 (DE3). rCdaYJ showed a molecular weight of 43kDa, and exhibited deacetylation activity by using p-nitroacetanilide as substrate. The optimal pH and temperature of rCdaYJ were tested as pH7.4 and 28°C, respectively. ConclusionsThe construction of metagenomic library of the Arctic deep-sea sediments provides us an opportunity to look into the microbial communities and exploiting valuable gene resources. A chitin deacetylase CdaYJ was identified from the library. It showed highest deacetylation activity under slight alkaline and low temperature conditions. CdaYJ might be a candidate chitin deacetylase that possesses industrial and pharmaceutical potentials.

The hunt for original microbial enzymes: an initiatory review on the construction and functional screening of (meta)genomic libraries

Introduction. Discovering novel enzymes is of interest in both applied and basic science. Microbial enzymes, which are incredibly diverse and easy to produce, are increasingly sought by diverse approaches. Literature. This review first distinguishes culture-based from culture-independent methods, detailing within each group the advantages and drawbacks of sequence- and function-based methods. It then discusses the main factors affecting the success of endeavors to identify novel enzymes through construction and functional screening of genomic or metagenomic libraries: the sampled environment, how DNA is extracted and processed, the vector used (plasmid, cosmid, fosmid, BAC, or shuttle vector), the host cell chosen from the available prokaryotic and eukaryotic ones and the main screening steps. Conclusions. Library construction and screening can be tricky and requires expertise. Combining different strategies, such as working with cultivable and non-cultivable organisms, using sequence- and function-based approaches, or performing multihost screenings, is probably the best way to identify novel and diverse enzymes from an environmental sample.

Construction and screening of metagenomic library derived from soil for β-1, 4-endoglucanase gene

Abstract A metagenomic library consisting of 7500 clones was constructed from the soil continuously irrigated with effluents of paper and pulp mill. A cellulase positive clone H-10 was identified by function based screening. A β-1, 4- endoglucanase gene with an open reading frame of 1499 bp was identified from this positive clone. The deduced amino acid sequence analysis revealed that amplified β-1, 4-endoglucanase belongs to GH5 and shares 98% similarity with Bacillus subtilis (EF070194), 95% with Bacillus subtilis IARI-SP-1(KF240847) 96% with B. subtilis (AY183475) and 95% with uncultured bacterium clone (JX567739). Clone H-10 indicated higher CMCase activity of 1 IU/ml on the substrate CMC as compared to rice straw (0.5 IU/ml). The characterization of enzymes revealed its pH optima between 8.5–9 and retained 80% of activity at pH 11. The thermal profile of the enzyme indicated broad range of thermotolerance.

Functional Metagenomics: Construction and High-Throughput Screening of Fosmid Libraries for Discovery of Novel Carbohydrate-Active Enzymes.

Activity-based metagenomics is one of the most efficient approaches to boost the discovery of novel biocatalysts from the huge reservoir of uncultivated bacteria. In this chapter, we describe a highly generic procedure of metagenomic library construction and high-throughput screening for carbohydrate-active enzymes. Applicable to any bacterial ecosystem, it enables the swift identification of functional enzymes that are highly efficient, alone or acting in synergy, to break down polysaccharides and oligosaccharides.

Current and future resources for functional metagenomics.

Functional metagenomics is a powerful experimental approach for studying gene function, starting from the extracted DNA of mixed microbial populations. A functional approach relies on the construction and screening of metagenomic libraries—physical libraries that contain DNA cloned from environmental metagenomes. The information obtained from functional metagenomics can help in future annotations of gene function and serve as a complement to sequence-based metagenomics. In this Perspective, we begin by summarizing the technical challenges of constructing metagenomic libraries and emphasize their value as resources. We then discuss libraries constructed using the popular cloning vector, pCC1FOS, and highlight the strengths and shortcomings of this system, alongside possible strategies to maximize existing pCC1FOS-based libraries by screening in diverse hosts. Finally, we discuss the known bias of libraries constructed from human gut and marine water samples, present results that suggest bias may also occur for soil libraries, and consider factors that bias metagenomic libraries in general. We anticipate that discussion of current resources and limitations will advance tools and technologies for functional metagenomics research.

A Rapid and Economical Method for Efficient DNA Extraction from Diverse Soils Suitable for Metagenomic Applications.

A rapid, cost effective method of metagenomic DNA extraction from soil is a useful tool for environmental microbiology. The present work describes an improved method of DNA extraction namely “powdered glass method” from diverse soils. The method involves the use of sterile glass powder for cell lysis followed by addition of 1% powdered activated charcoal (PAC) as purifying agent to remove humic substances. The method yielded substantial DNA (5.87 ± 0.04 μg/g of soil) with high purity (A260/280: 1.76 ± 0.05) and reduced humic substances (A340: 0.047 ± 0.03). The quality of the extracted DNA was compared against five different methods based on 16S rDNA PCR amplification, BamHI digestion and validated using quantitative PCR. The digested DNA was used for a metagenomic library construction with the transformation efficiency of 4 X 106 CFU mL-1. Besides providing rapid, efficient and economical extraction of metgenomic DNA from diverse soils, this method’s applicability is also demonstrated for cultivated organisms (Gram positive B. subtilis NRRL-B-201, Gram negative E. coli MTCC40, and a microalgae C. sorokiniana UTEX#1666).

Targeted Metagenomics: Finding Rare Tryptophan Dimer Natural Products in the Environment

Natural product discovery from environmental genomes (metagenomics) has largely been limited to the screening of existing environmental DNA (eDNA) libraries. Here, we have coupled a chemical-biogeographic survey of chromopyrrolic acid synthase (CPAS) gene diversity with targeted eDNA library production to more efficiently access rare tryptophan dimer (TD) biosynthetic gene clusters. A combination of traditional and synthetic biology-based heterologous expression efforts using eDNA-derived gene clusters led to the production of hydroxysporine (1) and reductasporine (2), two bioactive TDs. As suggested by our phylogenetic analysis of CPAS genes, identified in our survey of crude eDNA extracts, reductasporine (2) contains an unprecedented TD core structure: a pyrrolinium indolocarbazole core that is likely key to its unusual bioactivity profile. This work demonstrates the potential for the discovery of structurally rare and biologically interesting natural products using targeted metagenomics, where environmental samples are prescreened to identify the most phylogenetically unique gene sequences and molecules associated with these genes are accessed through targeted metagenomic library construction and heterologous expression.