Search

Background: Terpenes are one of the most diverse and abundant classes of natural biomolecules, collectively enabling a variety of therapeutic, energy, and cosmetic applications. Recent genomics investigations have predicted a large untapped reservoir of bacterial terpene synthases residing in the genomes of uncultivated organisms living in the soil, indicating a vast array of putative terpenoids waiting to be discovered. Results: We aimed to develop a high-throughput functional metagenomic screening system for identifying novel terpene synthases from bacterial metagenomes by relieving the toxicity of terpene biosynthesis precursors to the Escherichia coli host. The precursor toxicity was achieved using an inducible operon encoding the prenyl pyrophosphate synthetic pathway and supplementation of the mevalonate precursor. Host strain and screening procedures were finely optimized to minimize false positives arising from spontaneous mutations, which avoid the precursor toxicity. Our functional metagenomic screening of human fecal metagenomes yielded a novel ß-farnesene synthase, which does not show amino acid sequence similarity to known ß-farnesene synthases. Engineered S. cerevisiae expressing the screened ß-farnesene synthase produced 120 mg/L ß-farnesene from glucose (2.86 mg/g glucose) with a productivity of 0.721 g/L·h. Conclusions: A unique functional metagenomic screening procedure was established for screening terpene synthases from metagenomic libraries. This research proves the potential of functional metagenomics as a sequenceindependent avenue for isolating targeted enzymes from uncultivated organisms in various environmental habitats. [ABSTRACT FROM AUTHOR]

Objective: Many sections of the health care system are facing a major challenge making infectious disease problematic to treat; antimicrobial resistance (AMR). Identification and surveillance of the resistome have been highlighted as one of the strategies to overcome the problem. This study aimed to screen for AMR genes in an oral microbiota, a complex microbial system continuously exposed to antimicrobial agents commonly used in dental practice. Materials and methods: As a significant part of the oral microbiome cannot be conventionally cultured, a functional metagenomic approach was chosen. The human oral metagenomic DNA was extracted from saliva samples collected from 50 healthy volunteers in Norway. The oral metagenomic library was then constructed by ligating partially digested oral metagenome into pSMART BAC vector and introducing into Escherichia coli. The library was screened against antimicrobials in dental practices. All resistant clones were selected and analyzed. Results: Screening of the oral metagenomic library against different antimicrobials detected multiple clones with resistance against chlorhexidine, triclosan, erythromycin, tetracycline, and sodium hypochlorite. Bioinformatic analysis revealed both already known resistance genes, including msr, mef(A), tetAB(46), and fabK, and genes that were not previously described to confer resistance, including recA and accB conferring resistance to sodium hypochlorite and chlorhexidine, respectively. Conclusion: Multiple clones conferring resistance to antimicrobials commonly used in dental practices were detected, containing known and novel resistant genes by functional-based metagenomics. There is a need for more studies to increase our knowledge in the field. [ABSTRACT FROM AUTHOR]

Background: Sulfate modification of N-glycans is important for several biological functions such as clearance of pituitary hormones or immunoregulation. Yet, the prevalence of this N-glycan modification and its functions remain largely unexplored. Characterization of N-glycans bearing sulfate modifications is hampered in part by a lack of enzymes that enable site-specific detection of N-glycan sulfation. In this study, we used functional metagenomic screening to identify enzymes that act upon sulfated N-acetylglucosamine (GlcNAc). Using multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) -based glycoanalysis we proved their ability to act upon GlcNAc-6-SO4 on N-glycans. Results: Our screen identified a sugar-specific sulfatase that specifically removes sulfate from GlcNAc-6-SO4 when it is in a terminal position on an N-glycan. Additionally, in the absence of calcium, this sulfatase binds to the sulfated glycan but does not remove the sulfate group, suggesting it could be used for selective isolation of sulfated N-glycans. Further, we describe isolation of a sulfate-dependent hexosaminidase that removes intact GlcNAc-6-SO4 (but not asulfated GlcNAc) from a terminal position on N-glycans. Finally, the use of these enzymes to detect the presence of sulfated N-glycans by xCGE-LIF is demonstrated. Conclusion: The present study demonstrates the feasibility of using functional metagenomic screening combined with glycoanalytics to discover enzymes that act upon chemical modifications of glycans. The discovered enzymes represent new specificities that can help resolve the presence of GlcNAc-6-SO4 in N-glycan structural analyses. [ABSTRACT FROM AUTHOR]

Background: Expression screening of environmental DNA (eDNA) libraries is a popular approach for the identification and characterization of novel microbial enzymes with promising biotechnological properties. In such "functional metagenomics" experiments, inserts, selected on the basis of activity assays, are sequenced with high throughput sequencing technologies. Assembly is followed by gene prediction, annotation and identification of candidate genes that are subsequently evaluated for biotechnological applications. Results: Here we present A-GAME (A GAlaxy suite for functional MEtagenomics), a web service incorporating state of the art tools and workflows for the analysis of eDNA sequence data. We illustrate the potential of A-GAME workflows using real functional metagenomics data, showing that they outperform alternative metagenomics assemblers. Dedicated tools available in A-GAME allow efficient analysis of pooled libraries and rapid identification of candidate genes, reducing sequencing costs and saving the need for laborious manual annotation. Conclusion: In conclusion, we believe A-GAME will constitute a valuable resource for the functional metagenomics community. [ABSTRACT FROM AUTHOR]

Background: With the development of Next Generation Sequencing technologies, the number of predicted proteins from entire (meta-) genomes has risen exponentially. While for some of these sequences protein functions can be inferred from homology, an experimental characterization is still a requirement for the determination of protein function. However, functional characterization of proteins cannot keep pace with our capabilities to generate more and more sequence data. Results: Here, we present an approach to reduce the number of proteins from entire (meta-) genomes to a reasonably small number for further experimental characterization without loss of important information. About 6.1 million predicted proteins from the Global Ocean Sampling Expedition Metagenome project were distributed into classes based either on homology to existing hidden markov models (HMMs) of known families, or de novo by assessment of pairwise similarity. 5.1 million of these proteins could be classified in this way, yielding 18,437 families. For 4,129 protein families, which did not match existing HMMs from databases, we could create novel HMMs. For each family, we then selected a representative protein, which showed the closest homology to all other proteins in this family. We then selected representatives of four families based on their homology to known and well-characterized lipases. From these four synthesized genes, we could obtain the novel esterase/lipase GOS54, validating our approach. Conclusions: Using an in silico approach, we were able improve the success rate of functional screening and make entire (meta-) genomes amenable for biochemical characterization. [ABSTRACT FROM AUTHOR]

Background: Through functional screening of a fosmid library, generated from a phytopathogen-suppressive soil metagenome, the novel antifungal chitinase--named Chi18H8 and belonging to family 18 glycosyl hydrolases-- was previously discovered. The initial extremely low yield of Chi18H8 recombinant production and purification from Escherichia coli cells (21 μg/g cell) limited its characterization, thus preventing further investigation on its biotechnological potential. Results: We report on how we succeeded in producing hundreds of milligrams of pure and biologically active Chi18H8 by developing and scaling up to a high-yielding, 30 L bioreactor process, based on a novel method of mild solubilization of E. coli inclusion bodies in lactic acid aqueous solution, coupled with a single step purification by hydrophobic interaction chromatography. Chi18H8 was characterized as a Ca2+-dependent mesophilic chitobiosidase, active on chitin substrates at acidic pHs and possessing interesting features, such as solvent tolerance, longterm stability in acidic environment and antifungal activity against the phytopathogens Fusarium graminearum and Rhizoctonia solani. Additionally, Chi18H8 was found to operate according to a non-processive endomode of action on a water-soluble chitin-like substrate. Conclusions: Expression screening of a metagenomic library may allow access to the functional diversity of uncultivable microbiota and to the discovery of novel enzymes useful for biotechnological applications. A persisting bottleneck, however, is the lack of methods for large scale production of metagenome-sourced enzymes from genes of unknown origin in the commonly used microbial hosts. To our knowledge, this is the first report on a novel metagenome- sourced enzyme produced in hundreds-of-milligram amount by recovering the protein in the biologically active form from recombinant E. coli inclusion bodies. [ABSTRACT FROM AUTHOR]

Background: The metagenomic analysis of gut microbiomes has emerged as a powerful strategy for the identification of biomass-degrading enzymes, which will be no doubt useful for the development of advanced biorefining processes. In the present study, we have performed a functional metagenomic analysis on comb and gut microbiomes associated with the fungus-growing termite, Pseudacanthotermes militaris. Results: Using whole termite abdomens and fungal-comb material respectively, two fosmid-based metagenomic libraries were created and screened for the presence of xylan-degrading enzymes. This revealed 101 positive clones, corresponding to an extremely high global hit rate of 0.49%. Many clones displayed either β-D-xylosidase (EC 3.2.1.37) or α-L-arabinofuranosidase (EC 3.2.1.55) activity, while others displayed the ability to degrade AZCL-xylan or AZCL-β-(1,3)-β-(1,4)-glucan. Using secondary screening it was possible to pinpoint clones of interest that were used to prepare fosmid DNA. Sequencing of fosmid DNA generated 1.46 Mbp of sequence data, and bioinformatics analysis revealed 63 sequences encoding putative carbohydrate-active enzymes, with many of these forming parts of sequence clusters, probably having carbohydrate degradation and metabolic functions. Taxonomic assignment of the different sequences revealed that Firmicutes and Bacteroidetes were predominant phyla in the gut sample, while microbial diversity in the comb sample resembled that of typical soil samples. Cloning and expression in E. coli of six enzyme candidates identified in the libraries provided access to individual enzyme activities, which all proved to be coherent with the primary and secondary functional screens. Conclusions: This study shows that the gut microbiome of P. militaris possesses the potential to degrade biomass components, such as arabinoxylans and arabinans. Moreover, the data presented suggests that prokaryotic microorganisms present in the comb could also play a part in the degradation of biomass within the termite mound, although further investigation will be needed to clarify the complex synergies that might exist between the different microbiomes that constitute the termitosphere of fungus-growing termites. This study exemplifies the power of functional metagenomics for the discovery of biomass-active enzymes and has provided a collection of potentially interesting biocatalysts for further study. [ABSTRACT FROM AUTHOR]

Background: Functional metagenomic selections represent a powerful technique that is widely applied for identification of novel genes from complex metagenomic sources. However, whereas hundreds to thousands of clones can be easily generated and sequenced over a few days of experiments, analyzing the data is time consuming and constitutes a major bottleneck for experimental researchers in the field. Findings: Here we present the deFUME web server, an easy-to-use web-based interface for processing, annotation and visualization of functional metagenomics sequencing data, tailored to meet the requirements of non-bioinformaticians. The web-server integrates multiple analysis steps into one single workflow: read assembly, open reading frame prediction, and annotation with BLAST, InterPro and GO classifiers. Analysis results are visualized in an online dynamic web-interface. Conclusion: The deFUME webserver provides a fast track from raw sequence to a comprehensive visual data overview that facilitates effortless inspection of gene function, clustering and distribution. The webserver is available at cbs.dtu.dk/services/deFUME/and the source code is distributed at github.com/EvdH0/deFUME. [ABSTRACT FROM AUTHOR]

Background: New antibiotic resistance determinants are generally discovered too late, long after they have irreversibly emerged in pathogens and spread widely. Early discovery of resistance genes, before or soon after their transfer to pathogens could allow more effective measures to monitor and reduce spread, and facilitate genetics-based diagnostics., Results: We modified a functional metagenomics approach followed by in silico filtering of known resistance genes to discover novel, mobilised resistance genes in class 1 integrons in wastewater-impacted environments. We identified an integron-borne gene cassette encoding a protein that conveys high-level resistance against aminoglycosides with a garosamine moiety when expressed in E. coli. The gene is named gar (garosamine-specific aminoglycoside resistance) after its specificity. It contains none of the functional domains of known aminoglycoside modifying enzymes, but bears characteristics of a kinase. By searching public databases, we found that the gene occurs in three sequenced, multi-resistant clinical isolates (two Pseudomonas aeruginosa and one Luteimonas sp.) from Italy and China, respectively, as well as in two food-borne Salmonella enterica isolates from the USA. In all cases, gar has escaped discovery until now., Conclusion: To the best of our knowledge, this is the first time a novel resistance gene, present in clinical isolates, has been discovered by exploring the environmental microbiome. The gar gene has spread horizontally to different species on at least three continents, further limiting treatment options for bacterial infections. Its specificity to garosamine-containing aminoglycosides may reduce the usefulness of the newest semisynthetic aminoglycoside plazomicin, which is designed to avoid common aminoglycoside resistance mechanisms. Since the gene appears to be not yet common in the clinics, the data presented here enables early surveillance and maybe even mitigation of its spread.

Background: The built environment of the International Space Station (ISS) is a highly specialized space in terms of both physical characteristics and habitation requirements. It is unique with respect to conditions of microgravity, exposure to space radiation, and increased carbon dioxide concentrations. Additionally, astronauts inhabit a large proportion of this environment. The microbial composition of ISS particulates has been reported; however, its functional genomics, which are pertinent due to potential impact of its constituents on human health and operational mission success, are not yet characterized., Methods: This study examined the whole metagenome of ISS microbes at both species- and gene-level resolution. Air filter and dust samples from the ISS were analyzed and compared to samples collected in a terrestrial cleanroom environment. Furthermore, metagenome mining was carried out to characterize dominant, virulent, and novel microorganisms. The whole genome sequences of select cultivable strains isolated from these samples were extracted from the metagenome and compared., Results: Species-level composition in the ISS was found to be largely dominated by Corynebacterium ihumii GD7, with overall microbial diversity being lower in the ISS relative to the cleanroom samples. When examining detection of microbial genes relevant to human health such as antimicrobial resistance and virulence genes, it was found that a larger number of relevant gene categories were observed in the ISS relative to the cleanroom. Strain-level cross-sample comparisons were made for Corynebacterium, Bacillus, and Aspergillus showing possible distinctions in the dominant strain between samples., Conclusion: Species-level analyses demonstrated distinct differences between the ISS and cleanroom samples, indicating that the cleanroom population is not necessarily reflective of space habitation environments. The overall population of viable microorganisms and the functional diversity inherent to this unique closed environment are of critical interest with respect to future space habitation. Observations and studies such as these will be important to evaluating the conditions required for long-term health of human occupants in such environments.

Background: Recent metagenomic analyses of the human gut microbiome identified striking variability in its taxonomic composition across individuals. Notably, however, these studies often reported marked functional uniformity, with relatively little variation in the microbiome's gene composition or in its overall metabolic capacity., Results: Here, we address this surprising discrepancy between taxonomic and functional variations and set out to track its origins. Specifically, we demonstrate that the functional uniformity observed in microbiome studies can be attributed, at least partly, to common computational metagenomic processing procedures that mask true functional variation across microbiome samples. We identify several such procedures, including commonly used practices for gene abundance normalization, mapping of gene families to functional pathways, and gene family aggregation. We show that accounting for these factors and using revised metagenomic processing procedures uncovers such hidden functional variation, significantly increasing observed variation in the abundance of functional elements across samples. Importantly, we find that this uncovered variation is biologically meaningful and that it is associated with both host identity and health., Conclusions: Accurate characterization of functional variation in the microbiome is essential for comparative metagenomic analyses in health and disease. Our finding that metagenomic processing procedures mask underlying and biologically meaningful functional variation therefore highlights an important challenge such studies may face. Alternative schemes for metagenomic processing that uncover this hidden functional variation can facilitate improved metagenomic analysis and help pinpoint disease- and host-associated shifts in the microbiome's functional capacity.